Journal of Experimental Botany, Vol. 56, No. 413, pp. 1049–1060, March 2005
doi:10.1093/jxb/eri098 Advance Access publication 14 February, 2005
RESEARCH PAPER
Tomato (Lycopersicon esculentum Mill.) fruit growth and
ripening as related to the biomechanical properties of fruit
skin and isolated cuticle
Hendrik Bargel* and Christoph Neinhuis
Institut für Botanik, TU Dresden, Zellescher Weg 22, D-01062 Dresden, Germany
Received 9 August 2004; Accepted 3 December 2004
Abstract
The control of growth rate and the mechanical integrity
of the tomato (Lycopersicon esculentum Mill.) fruit has
been attributed to the exocarp. This study focused on
the biomechanics of the fruit skin (FS) comprising
cuticle, epidermis and a few subdermal cell layers, and
the enzymatically isolated cuticular membrane (CM)
during fruit growth and ripening. Morphology and
mechanical properties of the FS and the CM of three
cultivars were analysed separately at three distinct
ripening stages by scanning electron microscopy
(SEM) and one-dimensional tension testing, respectively. Both were subject to significant cultivar-specific
changes. Thickness of the CM increased during ripening from 7.8–8.6 to 9.9–15.7 lm and exceeded by far
that of the epidermal cell wall. The mechanical properties, such as modulus of elasticity, strength, and failure
strain, were highest in the FS for all cultivars at any
stage, with only one exception; however, the cuticle
largely mirrored these properties throughout fruit maturation. Stiffness of both isolated CM and FS increased
from immature to fully ripe fruits for all cultivars, while
failure stress and failure strain displayed a tendency to
decrease for two of them. Stress–strain behaviour of
the CM could be described as strain softening, mostly
linear elastic throughout, and strain hardening, and
was subject to growth-related changes. The FS displayed strain hardening throughout. The results indicate evidence for the cuticle to become increasingly
important as a structural component for the integrity of
the tomato fruit in addition to the epidermis. A supplementary putative model for tomato fruit growth is
proposed.
Key words: Cuticle, cracking, epidermis, fruit growth,
Lycopersicon esculentum, plant biomechanics, ripening,
stiffening, tomato.
Introduction
Fruit growth and ripening are complex developmental
processes that involve multiple metabolic changes. The
climacteric berry fruit type of tomato (Lycopersicon esculentum Mill.) is the most intensively studied model species
(Giovannoni, 2001), thus representing a good system for
biomechanical studies as related to growth and ripening.
The mechanical performance of the exocarp, or fruit skin,
including the cuticle, the epidermis, and a variable number
of hypodermal cell layers, is of considerable economic
significance for the integrity of the whole fruit. It affects not
only fruit appearance, handling, and storage (Chu and
Thompson, 1972), but also plays a prominent role in fruit
cracking (Sekse, 1995; Emmons and Scott, 1997; Bertin
et al., 2000). Moreover, it is generally accepted that the
growth rate of plant tissues is regulated by the interaction
of cell wall stress and cell wall mechanical properties
(Cosgrove, 1993), and it has been argued that tissue tension
in tomato fruits indicates that the fruit epidermis is of importance in determining the rate of expansion (Thompson
et al., 1998). During the ripening process, cell wallmodifying activity of several enzymes, including polygalacturonase, pectin-methyl-esterase, endo-b-mannase, a- and
b-galactosidases, and b-glucanases, causes softening of the
whole fruit by altering the texture due to degradation of the
structural components necessary to reinforce the cell wall
and the adhesion of cells (Fischer and Bennett, 1991; Rose
* To whom correspondence should be addressed. Fax: +49(0)351 463 37032. E-mail: [email protected]
Abbreviations: CM, cuticular membrane; E, modulus of elasticity; FS, fruit skin; ig, immature-green; mg, mature-green; mr, mature-red; SEM, scanning
electron microscopy.
ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please email: [email protected]
1050 Bargel and Neinhuis
et al., 1997; Seymour et al., 2002). Expansins have been
described as contributing significantly to ripening-related
fruit softening (McQueen-Mason, 1995), and may affect
both cell wall viscosity and elasticity of tomato fruit
epidermis (Thompson, 2001). On the other hand, peroxidase-mediated stiffening of the tomato fruit skin cell walls
has been proposed as a control mechanism for fruit growth
(Thompson, 2001; Andrews et al., 2002a, b), and may
counteract the softening of the inner fruit tissues. Thus,
knowledge of the mechanical properties of the outer fruit
envelope is of vital interest, and several studies have been
carried out, mostly by measuring the mechanical properties
of the tomato fruit skin without distinguishing its components (Batal et al., 1970; Voisey et al., 1970; Thompson,
2001; Andrews et al., 2002b).
By contrast, there is only a limited number of studies that
have aimed at characterizing the mechanical properties and
behaviour of the isolated tomato fruit cuticle (Petracek and
Bukovac, 1995; Wiedemann and Neinhuis, 1998; Bargel
et al., 2000). The plant cuticle is a natural composite that
covers the epidermis as a continuous extracellular membrane, consisting mainly of two components, the insoluble
biopolymer cutin and soluble lipids, collectively called
‘waxes’ (Kolattukudy, 1980). Minor amounts of cell wall
polysaccharides are gradually encrusted during development and link the cuticle to the epidermis (Holloway,
1994). A second, highly resistant biopolymer named cutan
originally found in fossilized cuticles has not been detected
in tomato fruit cuticle (Ramı́rez et al., 1992). The polymeric
plant coverage appears to be without uniform ultrastructure
and chemical composition, and differences across and
within species have been demonstrated (overviews in
Jeffree, 1996; Kolattukudy, 2001). Moreover, Baker et al.
(1982) have studied the chemical composition of the cuticle
in relation to tomato fruit development and ripening, and
have reported distinct dynamic changes of both cutin and
cuticular wax composition. The same was proposed for
phenolic compounds (Hunt and Baker, 1980). The plant
cuticle is a multifunctional interface that primarily prevents
the plant from uncontrolled water loss (Schönherr, 1982;
Riederer and Schreiber, 2001), but also serves as a protective layer against multiple biotic and abiotic environmental influences (Bargel et al., 2004a). From a mechanical
point of view, the location at the outer perimeter of the
tomato fruit indicates that the cuticle may function as an
external structural element that adds mechanical support
for tissue integrity, since engineering theory shows that
stresses are highest at the surface of a body (Niklas, 1992).
In this context, Wiedemann and Neinhuis (1998) studied
the mechanical properties of isolated cuticles from five
leaf species and mature tomato fruit by means of onedimensional tension. Recently, Matas et al. (2004) analysed
the mechanical properties of fruit peels and isolated cuticle
of ripe tomato fruits, and stated that the cuticle is a mechanically important component of the tomato fruit. As far as is
known, integrative studies of both the cuticle and underlying
cell wall of a plant organ related to growth and ripening are
not available.
This paper focuses on the tensile properties of both
enzymatically isolated cuticular membrane (CM) and
peeled fruit skin (FS) of tomato fruits as related to fruit
growth and ripening, i.e. at three distinct ripening stages.
Three cultivars were used which differed in cracking
susceptibility and fruit shape. The objectives were to
evaluate (i) changes of the mechanical properties of both
CM and FS, and (ii) the contribution of the CM to the
mechanics of the outer fruit envelope and, hence, the
integrity of the whole organ during fruit growth and
ripening. Concurrent scanning electron microscopy was
carried out for morphological characterization.
Materials and methods
Plant material
Three tomato cultivars, cv. Harzfeuer (eight plants), cv. Vanessa F1
(six plants; both oblate-spherical) and cv. Roma (five plants; oblong),
were grown in parallel rows on a 135 m patch from commercially
available seeds under greenhouse-conditions without supplemental
lighting during the summer period (Experimental Station, Botanical
Gardens, University of Bonn). Cultivar-specific differences were
expected by comparing three cultivars with different genetic backgrounds, while cv. Harzfeuer was chosen as a cracking-susceptible
cultivar. Plants were irrigated manually every second day, and
fertilization followed a regular scheme every second week throughout
the season with a standard mineral nutrition solution. Plants were
reduced to six to eight trusses, and trusses were pruned to a maximum
of six fruits. Fruits with diameter >30 mm were harvested at three
distinct ripening stages, i.e. immature-green (ig), mature-green (mg),
and mature-red (mr), following the nomenclature of Emmons and
Scott (1997). The first fruits that appeared per truss were left on the
plant until full maturity, and subsequent fruits were then harvested at
the appropriate stage during the course of 4 weeks. The classification
of the ripening stages was made on the relative parameters of size,
weight, and colour change after the ‘breaker’ stage, rather than by days
from anthesis. This classification may introduce inaccuracies due to
cross-cultivar variation of fruit maturation, however, with the cultivars
used, the classification system sufficiently matched uniform ripening
stages (Table 1). Fruit size dimensions were obtained using a gauge,
and fruit weight was measured individually with a digital laboratory
balance at 0.1 g resolution. A representative assembly of the three
cultivars at the corresponding ripening stages is shown in Fig. 1.
Parallel cut samples (sample size: 3340 mm) were obtained with a
custom-built double-blade device, with cutting direction from the
peduncle insertion to the tip. These peel samples were divided into two
batches of similar size. Cuticular membranes (CM) were then isolated
enzymatically using a modified cellulase/pectinase solution after
Wiedemann and Neinhuis (1998), containing cellulase (1.75% [w/
w], Celluclast; Novo Nordisk, Bagsvaerd, Denmark), pectinase (3.5%
[w/w], Fluka, Buchs, Swiss), sodium azide (NaN3, 2 mM; Merck
Schuchardt, Hohenheim, Germany), polyvinyl pyrrolidone (0.2%
[w/w]) and citric acid monohydrate (20 mM; Merck, Darmstadt,
Germany) at 30 8C. A pH of 5.0 was established for optimum enzyme
activity. Completely isolated CM were rinsed twice in distilled water,
air-dried on Teflon meshes, and stored in Petri dishes at room
temperature before testing. Corresponding fruit skin (FS) samples
were prepared from the peeled strips by carefully removing adhering
Biomechanics of tomato fruit cuticle 1051
Table 1. Mean fruit size dimensions measured as diameter (cm)
and weight (g) of tomato cv. Harzfeuer, Vanessa F1 (both oblatespherical), and Roma (oblong) as indicators of the ripening
classification system used in this study
Vertical diameter was measured from the region of peduncle insertion to
the tip, and horizontal diameter was measured at the fruit equator: ig,
immature-green; mg, mature-green; mr, mature-red.
Cultivar
Stage
Vertical ø
Horizontal ø
Weight
Harzfeuer
ig
mg
mr
ig
mg
mr
ig
mg
mr
3.2
4.0
4.1
3.1
4.1
4.0
4.4
5.1
5.4
3.8
4.8
4.6
3.6
5.0
4.9
3.5
4.4
4.1
22.6
47.9
48.8
22.1
55.3
55.5
19.4
41.2
44.4
Vanessa F1
Roma
Fig. 1. Compilation of representative tomato fruits of the cvs Vanessa F1
(top), Harzfeuer (middle), and Roma (bottom) at three distinct ripening
stages. From left to right: immature-green, mature-green, and mature-red.
parenchyma tissue with a razor blade. To eliminate physiological
stress reactions and an effect of turgor during mechanical testing, FS
were incubated for 1 min in ethanol (80%; Roth, Hamburg, Germany),
and subsequently washed for 5 min in distilled water, using polyvinyl
pyrrolidone/NaN3 (0.2% [w/w]/2 mM) as storage medium until
testing. The frequently used freeze–thaw technique in extensibility
studies was excluded since earlier preliminary studies on tomato and
cherry (Prunus avium L.) fruit indicated that freezing could produce
micro-cracks in the cuticle. In addition to the above, a preliminary
study including fruit material of cv. Vanessa and cv. Roma was
conducted in an earlier growth season. The CM was isolated similarly,
but the FS was tested fresh without eliminating physiological stress
reactions prior to mechanical characterization.
Scanning electron microscopy
Following the conclusions of Hill and Dilcher (1990), scanning
electron microscopy (SEM) combined with digital image analysis of
cross-sections was used for morphological characterization and
determination of sample thickness, from which the corresponding
cross-sectional area (thickness3width of the sample) necessary for
the calculation of stress was derived. FS samples were fixed
with methanol (99.9%; Fluka, Darmstadt, Germany) according to
Neinhuis and Edelmann (1996) for 1 min, and subsequently criticalpoint-dried (CPD 020; Balzers Union, Wiesbaden, Germany). Prior
to SEM, air-dried CM as well as FS samples were coated with gold
(;25 nm) in a sputter coater (SCD 040; Balzers Union, Wiesbaden,
Germany), and subsequently examined on a modified aluminium stub
at 15 kV (440i; LEO, Oberkochen, Germany). Digital image analysis
was carried out with the free NIH Image software (http://rsb.info.
nih.gov/nih-image/). For the FS, thicknesses of the cuticle and
underlying epidermal cell walls were determined by scanning
a selected range of each cross-section, excluding cell lumen as well
as the hypodermal cell layers. Wherever possible, the inner epidermal
part of the double wall was differentiated from the part belonging to
the subepidermal cells. In general, the distinction between the cuticle
and the epidermal cell wall was easily distinguished in the preliminary studies using SEM analysis. Thickness measurements of the
CM covered all cutinized layers of the enzymatically isolated
cuticular material. Measurements were carried out with at least eight
replicates of both CM and FS per fruit (n=3–5) and ripening stage of
each cultivar, and the results are given as means 6confidence
intervals (CI).
Mechanical tests
Mechanical characterization of fully hydrated samples was carried
out by means of one-dimensional tension testing with a custom-built
computer-controlled device featuring vice-type clamps, as has been
described by Wiedemann and Neinhuis (1998). Accordingly, the test
speed was set to 2 mm min1. CM samples were rehydrated, and both
CM and FS samples were kept moist during testing by adding small
amounts of distilled water with a hand pipette to mimic a fully
hydrated water status of both cell walls and cuticle. Effectiveness of
rehydration of the CM was not quantified, but was considered to
ensure fully hydrated CM in only a few minutes after adding water.
This has been shown in the authors’ laboratory for a large variety of
CM from different species, while the mechanical properties changed
immediately (H Bargel, C Neinhuis, unpublished results). From the
tensile tests, the modulus of elasticity E (stress r/strain e), conventional breaking stress r (force/cross-sectional area), and conventional
breaking strain e (change of length/initial length) were derived. Most
of the stress–strain curves displayed an initial and a second linear
slope, resulting in two different moduli of elasticity, as well as in
a transition point between the first and the second slope, which was
equivalent to the proportional limit for part of the CM measurements.
In addition, modulus, stress, and strain values were also calculated as
true stress rt and strain et, since plant polymers can change their form
to a great extend (>10%) over a relatively short period of time
(Vincent, 1990b). Hence, calculation of conventional stress and strain
could produce underestimated and overestimated values, respectively. This may be illustrated as follows: if the extension of any
material (with a known initial length l0) above 10% total length
increase is considered, the same incremental increase in length will
produce a smaller true strain than the conventional equivalent, since
the true strain provides a reference strain for each increment of
extension relative to the preceding total deformation. By contrast, the
conventional strain is always referred to l0, irrespective of the total
deformation. In a similar way, the cross-sectional area used for the
calculation of stress defines the differences between true stress and
conventional stress. Calculations of true stress and strain were related
to the conventional numbers for the starting conditions as has been
proposed by Biewener (1992), assuming isovolumetric deformation
(Shadwick, 1992).
1052 Bargel and Neinhuis
rt = ð1 + eÞr
ð1Þ
et = lnl=l0
ð2Þ
Measurements were carried out with at least eight replicates of both
CM and FS per fruit (n=3–5) and ripening stage of each cultivar. In
the Results section, means and confidence intervals (CI) are given for
modulus of elasticity and transition point. For strength and breaking
strain, trend values are given instead, since a large number of samples
fractured close to the clamps, which made reproducible results
difficult (Wiedemann and Neinhuis, 1998). This phenomenon is
commonly observed in tensile tests and is caused by shear stresses
perpendicular to the tensile stresses near the clamping site. While
stiffness is not affected, fracturing close to the clamps may occur at
lower stress and strain at failure. In this study, these measurements
were included, and trend values reported here do account for this
problem.
Statistical analysis
An unbalanced two-way ANOVA with Bonferroni post-test (Prism 4;
GraphPad, San Diego, California, USA) at a = 0.05 was performed
on thickness and modulus of elasticity in order to evaluate significant
changes of either morphology or stiffness related to fruit ripening. An
outlier test after Nalimov (Lozán and Kausch, 1998), as well as
a normality test, were carried out prior to the statistical analysis.
Results
Morphology
SEM analysis of both CM and FS cross-sections revealed
overall thicker cuticles for all cultivars compared with
epidermal cell walls at any stage of ripening. Representative cross-sections of the CM and FS at all three ripening
stages are shown in Fig. 2. No adhering cellular material is
visible on the inner fasciae of the CM, indicating complete
enzymatic isolation of the extracellular biopolyester membrane. Differentiation between the cuticular material and
the cell walls generally could be clearly depicted. The shape
of epidermal cells changed from more or less isodiametric
towards elongated during development. This was manifested not only in the FS, but also in the isolated CM. The
fracture surfaces of both CM and FS are of mostly uniform
texture, with slightly smoother surfaces at the earlier stages
of ripening for all cultivars. Cuticular encasing of epidermal
cells was typically absent in stage ig, but increasingly
abundant during ripening regardless of cultivar, resulting in
an overall thicker CM at ripening stages mg and mr. This
was particularly the case for cv. Vanessa. Gain in mean
thickness of CM from immature to fully mature fruits was
highly significant (P <0.001) for all cultivars, with considerable differences among the cultivars (Fig. 3). In detail, cv.
Harzfeuer increased from 8.760.3 lm to 10.260.6 lm, cv.
Vanessa almost doubled from 8.760.5 lm to 15.760.6
lm, and cv. Roma enlarged from 8.460.4 lm to 9.660.7
lm. However, thickness of CM of cv. Harzfeuer was
highest at stage mg and then dropped towards stage mr.
Further analysis of the CM in order to evaluate the
contribution of the outer solid part of the cuticle to overall
CM thickness revealed a highly significant increase
(P <0.001) of thickness from 4.660.1 lm at stage ig to
5.460.1 lm at stage mr for cv. Harzfeuer, and from
4.960.1 lm to 8.160.3 lm for cv. Vanessa F1. A nonsignificant increase from 5.360.1 lm to 5.560.1 lm was
measured for cv. Roma (Fig. 4).
By contrast, thickness of the epidermal cell walls showed
no uniform trend. While cv. Roma showed a very significant gain (P <0.01) from 1.060.04 lm at stage ig to
1.160.07 lm at stage mr, cell wall thickness of cv. Vanessa
dropped highly significantly (P <0.001) from 1.360.07 lm
to 1.160.04 lm, and cv. Harzfeuer remained constant at
1.3 lm (Fig. 3).
Biomechanical properties
Tensile measurements demonstrated considerable changes
in the mechanical properties of the outer fruit envelope
during fruit maturation in a cultivar-specific manner, as
well as differences between the CM and FS (Fig. 5). A
stress–strain analysis revealed three forms of stress–strain
behaviour for the tomato fruit CM with persistent patterns
worthy of further analysis. Representative examples are
given in Fig. 5A. The most obvious tendency was strain
hardening material behaviour (increasing modulus) at stage
mg for all cultivars, whereas stage ig could be described as
a mixture of strain softening (decreasing modulus) and
a mostly linear form. At maturity, stress-strain behaviour
was not that uniform, and cultivar-specific differences
could be noted. Here, cv. Harzfeuer showed a mixture of
strain softening and mostly linear form, while a mostly
linear form and strain hardening for a large part of the
stress–strain curves could be depicted for the majority of
CM samples of cv. Vanessa. For cv. Roma, a mostly linear
form dominated. By contrast, the stress–strain behaviour of
the FS could be described as strain hardening up to
approximately 6–9% strain throughout all ripening stages
(Fig. 5B), the exception being cv. Roma stage mr which
displayed linear behaviour. Two linear slopes were identified in all diagrams showing strain softening and strain
hardening and, consequently, transition points between
primary and secondary slopes could be defined (Köhler and
Spatz, 2002).
From the individual stress–strain diagrams of each
measurement, the mechanical properties of the CM and
the FS were calculated. The modulus of elasticity, a measure
for tensile resistance or stiffness, was found to be highest
for the FS of all cultivars throughout ripening, with the
exception of stage mr of cv. Harzfeuer (Fig. 6). However,
modulus changes of the FS as related to ripening had no
uniform trend. Stiffness of the FS of cv. Roma increased
highly significant (P <0.001) from 141.6617.7 MPa at
stage ig to 292.6636.0 MPa at stage mr, while that of cv.
Harzfeuer displayed a non-significant increase from
Biomechanics of tomato fruit cuticle 1053
Fig. 2. Representative SEM micrographs of cross-sections of isolated cuticular membranes (CM) and critical-point dried fruit skins (FS) of cv. Roma
at three different ripening stages. Cross-sections of the other cultivars were almost identical and differed only in the degree of cutinization and overall
thickness. (A, D) CM and FS at stage immature-green; (B, E) CM and FS at stage mature-green; (C, F) CM and FS at stage mature-red. Scale bars:
10 lm.
204.4623.4 MPa to 251.8647.6 MPa. The FS of cv.
Vanessa F1 showed a non-significant decrease in stiffness
from 296.7659.2 MPa to 258.3612.0 MPa (Fig. 6). In
contrast to the FS, the CM of all three cultivars displayed
a significant species-specific increase of the modulus of
elasticity from stage ig to mr, although generally at lower
values. The only exception was CM of cv. Harzfeuer at full
maturity, which superseded the modulus of the corresponding FS (Fig. 6). This ripening-related increase was highly
significant (P <0.001) for both cv. Harzfeuer from
117.0623.9 MPa to 258.0666.1 MPa, and for cv. Roma
from 96.767.7 MPa to 191.8650.7 MPa. A significant
increase (P < 0.05) could be determined for cv. Vanessa F1
from 175.2639.8 MPa to 209.3617.3 MPa, respectively.
1054 Bargel and Neinhuis
Fig. 3. Mean thickness 695% CI of the cuticular membrane (CM, top) and
epidermal cell wall (CW, bottom) of the FS of the three investigated tomato
fruit cultivars as related to growth and ripening. Stacked columns resemble
overall thickness of the fruit skin as measured for CM and CW based on
SEM and digital image analysis. 95% CI values are too small for CW to
show properly: ig, immature-green; mg, mature-green; mr, mature-red.
Fig. 5. (A, B) Representative examples of stress–strain diagrams of the
cuticular membrane (CM) and fruit skin (FS) as related to fruit growth
and ripening. (A) CM of cv. Harzfeuer at stage ig and mg, and CM of cv.
Roma at stage mr. (B) FS of cv. Vanessa F1 at stage ig, and FS of cv.
Harzfeuer at stage mg and mr. ig, immature-green; mg, mature-green; mr,
mature-red.
Fig. 4. Contribution of the outer solid part (bottom) to the overall
thickness of the cuticular membrane (stacked bars) of the three cultivars
as related to fruit growth and ripening. Values represent mean thickness
695% CI: ig, immature-green; mg, mature-green; mr, mature-red.
A small decrease in CM stiffness of immature to maturegreen fruits was common for all cultivars.
As far as stress and strain at failure are concerned, both
the CM and the FS displayed a trend to have the lowest
values at full maturity in all cultivars, with the notable
exception of FS of cv. Roma. Conversely, the CM of
mature-green fruits of all cultivars displayed the highest
strength as well as strain, with the sole exception of cv.
Vanessa F1, where the highest strain was found at stage
ig (Table 2). Comparison of the conventional values with
the true analogues revealed moderate differences (Table
3). The modulus of elasticity of both CM and FS was 10–
15 MPa higher when calculated as true stress versus
strain. True stress at failure of the CM exceeded that of
the conventional values up to 2 MPa, and that of the FS
about 2–4 MPa, while true failure strain of the CM was
up to 1% lower, and that of the FS about 1–3%,
respectively.
Discussion
Morphological changes related to fruit ripening
Morphology of the fruit exocarp, in particular of the cuticle,
of all three cultivars was subject to significant changes
during fruit maturation, resulting in extensive cutinization
of epidermal cell walls at full maturity. The change in
cellular shape indicated that higher tensile stresses were
exerted on the exocarp at the later stages of maturity,
according to the stress distribution during fruit growth
(Considine and Brown, 1981; Thompson et al., 1998). CM
thickness at maturity of the three cultivars studied here are
consistent with other reports, for example, 6.7–15.3 lm for
ripe tomato fruit (Petracek and Bukovac, 1995). In this
latter study, thickness of the solid outer CM region ranged
between 4.4–10.7 lm, which is fairly consistent with the
range of 4.6–8.1 lm determined here.
An increase in thickness has to be compensated by
biosynthesis of material throughout growth and ripening, as
has been demonstrated for tomato fruit cuticle by Baker
et al. (1982). The gain in thickness of the CM as related to
fruit growth could mainly be assigned to the accumulation
of cuticular compounds at the outer solid part, as well as
Biomechanics of tomato fruit cuticle 1055
material. By contrast, thinner cell walls at stage mr, as was
the case with cv. Harzfeuer and cv. Vanessa, might be due
to growth-related stress during rapid expansion, while accumulation of wall material is persistent at different activities
until the final stages of ripening, where growth generally
ceases (Monseline et al., 1978; Thompson et al., 1998).
Fig. 6. Mean modulus of elasticity E695% CI of the cuticular membrane
(CM, squares) and fruit skin (FS, triangles) of the three cultivars as related
to fruit growth and ripening. The modulus is highest for FS at all ripening
stages, except for cv. Harzfeuer mr, and increases from immature to
mature fruits for both CM and FS, with the exception of FS of cv. Vanessa
F1. However, values of CM mirror, in large part, the stiffness of the fruit
skins throughout growth.
excessive cutinization of epidermal (and hypodermal) cell
layers. This led to the sponge-like morphology at full
maturity observed in all cultivars. The contribution of the
outer solid part to overall CM thickness was generally high,
indicating no preferred site of material accumulation (Fig.
4). The relation remained constant at 53% in cv. Harzfeuer,
dropped slightly from 56% to 52% in cv. Vanessa F1, and
from 63% to 57% in cv. Roma. The observations are
consistent with transport concepts proposed for the hydrophobic cuticular compounds in the plant cuticle (Baur et al.,
1996; Neinhuis et al., 2001; Bargel et al., 2004a). The
increase in overall CM thickness appeared not to be linear,
for example, cv. Roma showed only a small change of
about 1% from stage ig to stage mg, but a further gain of
about 14% to full maturity. This could be attributed to the
sigmoidal growth pattern of the tomato fruit, where at the
phase of rapid expansion the fruit exocarp constituents are
stretched to a great extent (Monseline et al., 1978). To
explain the differing results reported for cv. Harzfeuer,
where stage mg displayed the highest thickness, it is
possible that the biosynthesis of cuticular constituents is
down-regulated before the volumetric increase declines at
the onset of ripening. Otherwise, it could also represent
a sampling artefact, and a classification system based on the
number of days after anthesis might probably diminish this
effect.
Concerning the changes in cell wall thickness of the FS,
the results were within the range of 0.66–4.40 lm reported
for five different tomato cultivars by Borowiak and Habdas
(1988), but indicated a certain degree of variability among
the cultivars tested. The same mechanisms as stated for the
CM are very likely to determine thickness changes of the
cell wall of the FS. On the one hand, a growth-related
increase in thickness as shown for cv. Roma might reflect
a more or less continuous biosynthesis of new cell wall
Mechanical properties related to fruit ripening
The mechanical properties of the CM and FS were also
subject to changes for all cultivars during fruit growth and
ripening. The modulus of elasticity of both the CM and the
FS generally increased during ripening, while strength and
strain at failure showed a tendency to decrease. The
reported moduli of all cultivars were in the upper range
compared with other studies. Wiedemann and Neinhuis
(1998) reported a modulus of elasticity between 60–100
MPa for the CM of fully ripe tomato fruits, depending on
the hydration status, and Matas et al. (2004) noted stiffness
values of ripe tomato fruit CM of cv. Inbred 10 and cv.
Sweet 100 of 70 MPa and 53 MPa. A similar growth-related
increase in tomato fruit skin tensile resistance as observed
in this study was recently reported with values ranging from
about 30–110 MPa (Andrews et al., 2002b). Other available studies on fruit skins reported modulus values between
10–20 MPa for different tomato cultivars (Batal et al.,
1970), and 600 MPa for tomato cv. Beefsteak (Vincent,
1990a). Concerning strength and strain at failure, values for
skins between 0.9–1.7 MPa and 7.8–13.9% (Batal et al.,
1970) as well as 9.6–15.5 MPa and 28.5–29.2% (Voisey
et al., 1970) for different tomato cultivars, and 2.5 MPa and
23.5% for cv. Beefsteak (Vincent, 1990a) can be found in
the literature. Andrews et al. (2002a) noted stress and strain
values at maximum load during the course of fruit growth
of cv. Espero to increase from about 5 MPa to 10 MPa and
to decrease from about 24% to 10%, respectively. However, Matas et al. (2004) reported comparably low breaking
stress values between about 1–1.2 MPa for the CM of ripe
tomato fruits. The compliance of isolated CM from mature
tomato cv. Pik Red in a load-creep test has been analysed,
with a strain at failure of about 3% (Petracek and Bukovac,
1995), which is in very good agreement with strain of 3–5%
at full maturity for the three tested cultivars in this study. A
possible reason for the rather large discrepancies between
the modulus and strength values of the FS in this study and
the literature might be the determination of sample thickness. If the cell lumens of the cross-sectional areas were
included, i.e. by measuring thickness crudely with a pair of
callipers, lower stress and stiffness values would be the
outcome. In this study, cross-sectional area of the FS was
based on the thickness of the epidermal cell walls excluding
the hypodermal cells, since the epidermis is likely to be
mechanically more important for tensile resistance, breaking stress and breaking strain. There are studies available
that give some support for this assumption (Thompson,
1056 Bargel and Neinhuis
Table 2. Strength as normal breaking stress (MPa), strain as normal breaking strain (%), and transition point (TP; %) of the
cuticular membrane (CM) and the fruit skin (FS) of three tomato cultivars as related to fruit growth and ripening
TP represents the transition point from the first into the second slope of the stress–strain curves. Values for strength and strain represent trends, while
means 695% CI are given for the TP: ig, immature-green; mg, mature-green; mr, mature-red.
Cultivar
Harzfeuer
Vanessa F1
Roma
Stage
ig
mg
mr
ig
mg
mr
ig
mg
mr
Strength (MPa)
Strain (%)
TP (%)
CM
FS
CM
FS
<11
<11
<8
<17
<15
<12
<5
<14
<5
<37
<29
<28
<43
<35
<26
<20
<23
<25
9
12
3
10
10
5
6
14
3
16
13
10
14
15
9
13
12
9
CM
FS
3.062.7
5.060.7
2.260.7
4.460.7
4.660.3
3.260.8
4.762.9
6.560.3
–
3.460.4
3.160.3
3.260.3
3.260.4
2.660.2
2.560.3
4.860.8
3.560.2
4.161.4
Table 3. Modulus of elasticity (MPa), strength (MPa), and strain (%) of the cuticular membrane (CM) and the fruit skin (FS) of three
tomato cultivars as related to fruit growth and ripening
Calculations are based on true numbers after equations (1) and (2). Although the absolute values differed, changes in the mechanical properties as related
to fruit maturation remained stable. Means 695% CI are given for the modulus, while values for strength and strain represent trends: ig, immature-green;
mg, mature-green; mr, mature-red.
Cultivar
Harzfeuer
Vanessa F1
Roma
Stage
ig
mg
mr
ig
mg
mr
ig
mg
mr
Modulus (MPa)
Strength (MPa)
Strain (%)
CM
FS
CM
FS
CM
FS
131.6629.1
102.0610.4
267.2667.9
186.6642.2
141.767.7
224.6620.7
100.768.0
99.5619.1
199.5652.1
215.6637.2
182.6624.0
263.8649.9
310.7661.9
229.2624.7
268.5613.2
153.0618.6
173.8612.9
315.8641.5
<12
<13
<8
<18
<16
<12
<5
<16
<5
<35
<33
<30
<46
<39
<28
<22
<26
<28
2001; Matas et al., 2004). However, the hypodermal cell
layer(s) may support the skin on the fruit, for example, by
energy absorption (Bargel et al., 2004b; Matas et al., 2004).
The methanol fixation technique used in conjunction with
critical-point-drying has been shown to produce very few
shrinking artefacts of tissues and cell wall components
(Neinhuis and Edelmann, 1996), making it a favourable
method for SEM and image analysis of the fruit exocarp
components, as has been also successfully demonstrated for
cherry fruit by Bargel et al. (2004b).
As far as true stress and strain values are concerned, only
very few data for biological materials exist so far (Niklas,
1992). As far as is known, no study on tomato fruit skins is
available, making comparison with other data somewhat
difficult. The differences between stiffness calculated from
conventional as well as true stress and strain were obviously small in the range exhibited by the cultivars studied,
and only a little higher for stress and strain at failure.
Although it may be a counsel of perfection, true stress and
strain could provide a more accurate picture of what is
happening in a material, particularly far above 10% strain
(Vincent, 1990b). It would be even more favourable to
9
11
3
10
10
5
5
13
3
13
12
10
13
13
8
11
11
8
calculate these numbers without relating them to the
starting conditions, as was done in this study.
Ripening-related changes of the mechanical properties
of the FS
Not surprisingly, the FS displayed a higher tensile stiffness
as well as strength, but overall it also showed higher
extensibility than the CM. Clearly, the epidermal cell walls
contribute to a large extent to the mechanical properties of
the tomato fruit exocarp, where the cellulose network is the
load-bearing component (Vincent, 1990b; Niklas, 1992). It
has been proposed that the epidermis plays a major role in
resistance to turgor-driven tomato fruit growth (Ho, 1992;
Thompson, 2001; Andrews et al., 2002b), since it is altered
during the ripening process by cell wall-specific enzymatic
activity. For example, the interplay between xyloglucanendo-transglycosylase (XET) and peroxidase, their pattern
of appearance, and their weakening and stiffening effects
on the epidermal cell wall, respectively, have been described (Thompson et al., 1998; Andrews et al., 2000).
Moreover, peroxidase-mediated stiffening of exocarp cell
Biomechanics of tomato fruit cuticle 1057
walls have been described (Andrews et al., 2002a, b). This
has also been proposed for maize (Zea mays L.) coleoptiles
after hydrogen peroxide treatment (Schopfer, 1996). These
ripening-related physiological modifications appear largely
to account for the observed changes during fruit maturity in
fruit skin tensile stiffness, but also to some extent of
strength and strain at failure. However, the changes of the
mechanical properties are not uniform for the tested
cultivars, and thus cannot be categorically generalized.
Whereas the results of cv. Roma agree well with the
proposed model of epidermal cell wall strengthening, stress
at failure values of cv. Harzfeuer and cv. Roma decreased
during maturity. It is very likely that the peroxidase content
and hence the stiffening effect varies in a cultivar-specific
way. In addition, the ripening-induced activity of expansins
and other enzymes such as endo-b-mannanase and polygalacturonase suggested to be involved in fruit softening
(McQueen-Mason, 1995; Bewley et al., 2000; Seymour
et al., 2002), may be higher in certain cultivars, thus
resulting in a less strong fruit exocarp.
However, most strikingly, the CM inevitably covered
a large range of the mechanics of the skin, at least at lower
strains, and thus mirrored the mechanical properties of the
FS. It can also be concluded from the transition points (Table
2) that the tomato fruit cuticle inherits a large contribution to
the mechanical demands up to 3–5% total strain. At these
strain levels, the mechanical behaviour and properties of the
FS, in particular the modulus of elasticity, seemed to be
determined to a high degree by the cuticle, whereas at higher
levels they were governed mostly by the cell layers beneath.
Under physiological conditions, which may fall into the
3–5% total strain, it is assumed that skin tensile resistance is
more important than stress or strain at failure, which are only
likely to be relevant under exceptional conditions. Consequently, the mechanical importance of the tomato fruit
cuticle as an integrative structural component becomes
evident. The same observations were made in the preliminary study, although with different absolute values (Fig. 7).
In this study, the FS was tested fresh, whereas the isolation of
the cuticle followed a similar protocol. A similar trend of
ripening-related increase in stiffness of both cuticle and fruit
skin can be depicted. Again, the cuticle mirrored the
mechanical properties of the skin to a high degree. Despite
this, there were considerable differences between the behaviour of the FS found in the preliminary study and the
‘main’ study that may primarily result from some preparation effects on the cell wall, whereas the mechanics of the
cuticle was virtually identical. In the preliminary study, cell
metabolic influences as well as changes in turgor during the
course of mechanical testing, i.e. mechanical stress, could
have directly affected the physical wall properties, resulting
in a generally higher stiffness of the FS. It has been shown
that water stress by a decreased water potential in the cell
wall of rye coleoptiles (Secale cereale L.) caused a decrease
in extensibility (Edelmann, 1995a). The possible effect of
Fig. 7. Mean modulus of elasticity 6SE of isolated cuticle (squares) and
fruit skin (triangels) of cv. Vanessa F1 and cv. Roma of a preliminary
study. Fruit skins were prepared fresh without eliminating physiological
stress reactions prior to testing. A similar ripening-related increase of
stiffness of both cuticle and fruit skin can be depicted, whereas the cuticle
mirrors the mechanical properties of the skin to a high degree.
physiological stress reactions during testing has been eliminated in the ‘main’ experiments by incubation in ethanol,
and, moreover, cytoplasmic solutes are supposed to have
been thoroughly extracted during the washing procedure and
incubation in the storage medium. Considering possible
artefacts by ethanol on the cell wall physical properties,
Edelmann (1995b) has reported a stiffening effect of ethanol/
water solutions on rye coleoptiles, but this effect was fully
reversible upon washing with distilled water, and thus, the
preparation used for the FS is believed not to have significantly affected the mechanical properties of the cell walls.
Ripening-related changes of the mechanical properties
of the CM
The observed ripening-related changes of the mechanical
properties of the CM seem to be determined mainly by
modifications of the chemical composition of the CM. In
their study, Baker et al. (1982) showed that the amount of
C18 tri-hydroxy-fatty acids decreased from immature to
mature fruits, which would give rise to a relative gain of C16
cutin monomers in the cuticle of tomato fruit at stage mr.
More recently, Marga et al. (2001) have undertaken
chemical analysis of cuticles from different organs of
a thistle (Cirsium horridulum Michx.), each having different biomechanical properties, and were able to correlate
these properties with the predominant cutin monomer type,
i.e. rigid cuticles can be classified by C16 cutin monomers,
while more elastic cuticles correspond to mixed C16/C18.
Thus, hydroxyl groups may enhance the hydrophilic
character and the hydration state of the cutin matrix, which
would result in a higher elasticity at immaturity. Graca
et al. (2002) have also characterized tomato fruit cutin from
ripe fruits as a C16 type, and reported small amounts of
glycerol as an esterified constituent in the cutin-matrix that
possibly reinforces the polymeric structure. Whether glycerol is abundant only in the case of maturity or also at
1058 Bargel and Neinhuis
immature stage remains to be established. Moreover,
abundance and increase of phenolic compounds in the
cutin matrix of the CM during tomato fruit ripening, for
example, p-coumaric acid as well as the flavonoids naringenin and chalconaringenin, has been reported by several
authors (Hunt and Baker, 1980; Baker et al., 1982; Laguna
et al., 1999). The presence of ‘lignin-like’ phenolics in the
tomato fruit exocarp has been speculated to be involved in
the regulation of fruit growth (Andrews et al., 2002a, b).
Available data indicate that the phenolic constituents form
molecular clusters and/or bind covalently to cutin (Riley
and Kolattukudy, 1975; Laguna et al., 1999), most reasonably at free available secondary hydroxyl groups proposed
to be only partly esterified (Deas and Holloway, 1977;
Heredia, 2003). Given this, the increase of flavonoids in the
cutin network during ripening seems to reduce the flexural
rigidity of the cuticle, either by reducing free available
hydroxyl groups in the cutin matrix, or by a decrease of the
free volume necessary for motional constraints being in
a molecular domain of a mobile category in the membrane.
The observed increase of cutinization of the CM during
ripening as a possible structural stiffening factor need not
necessarily be involved in the reported changing mechanical properties, as has been proposed by Matas et al. (2004).
Although it might be plausible that a sponge-like structure
of the cutinized CM at later stages of maturity stabilizes the
cuticle mechanically, the corresponding mechanical properties at stage mg did not indicate such a structural support.
More plausible would be a stiffening influence on the CM
by an increase in the cell wall polysaccharide content as
reported for the tomato fruit cuticle (Baker et al., 1982).
Differences in cell wall content of the cuticle have been
proposed to affect the mechanical performance of fruit
exocarp of ripe tomato fruits (Matas et al., 2004). Biologically even more important, cutinization of cell walls
could result in a fruit exocarp composite with the potential
to be stronger than either component individually. This
could be due to structural, but also chemical causes, since
non-covalent interactions between cell wall polysaccharides are likely to be strengthened in the non-polar environment of the cuticle. In addition, tissue tension exerted on the
outer fruit coverage may also account for the ripeningrelated mechanical changes, since this prestress could
restrict the extensibility of the CM as well as the FS,
resulting in higher stiffness.
A putative model of tomato fruit growth
From the results presented here, a generalized putative model
of tomato fruit growth is proposed. Since rapid fruit
expansion is predominant in the earlier stages of growth
(Monseline et al., 1978; Thompson et al., 1998), equivalent
to stage ig, the outer fruit coverage has to adapt to this
expansion with a more ductile nature. As far as the cuticle is
concerned, this is consistent with the majority of CM
samples of immature fruits, which were characterized by
strain softening or linear behaviour and high extensibility.
A comparable low modulus of elasticity and high extensibility was revealed for both CM and FS. Strain softening
biphasic behaviour has been reported for other plant material
during early ontogeny, including Aristolochia stems (Köhler
et al., 2000), whereas strain hardening material behaviour as
displayed by FS throughout growth and maturation is more
typical for the fibrous polymer network of cell walls
(Vincent, 1990a, b; Niklas, 1992). At the mature-green
ripening stage, equivalent to the phase where fruit growth
declines, the material behaviour of the cuticle changed
towards strain hardening uniformly for all three tomato fruit
cultivars. Thus, extensibility may be restricted even at the
apparently higher strains. At the fully ripe stage, stress–strain
behaviour of the CM again underwent changes, but no
uniform pattern could be observed. The overall predominant
curve form was linear material behaviour, but strain softening and strain hardening also occurred. At this stage, fruit
growth generally ceases, and coinciding higher tensile
stiffness for both CM and FS for all three cultivars could
be observed. Thus, the tensile measurements reported here
indicate that the fruit exocarp provides a mechanical constraint to turgor-driven growth, while the cuticle inherits
a large contribution to the overall tensile resistance. The
described changes in material behaviour and properties agree
well with the altered chemical composition and physiological modifications of the fruit exocarp as shown.
Conclusion
During tomato fruit growth and ripening, morphology and
the mechanical properties of the cuticle as well as the
epidermis were subject to considerable changes. Pronounced cultivar-specific differences could be also noted.
The mechanical properties of the FS exceeded that of the
CM, but were largely mirrored by the biopolyester membrane at all ripening stages of the three cultivars tested.
Thus, evidence is high for the cuticle to become increasingly important as a structural component for the integrity of
the ripening tomato fruit. It is even possible to speculate that
the cuticle impacts on morphogenetic processes, and may be
involved in the determination of fruit growth rate in addition
to the epidermis, since the mechanisms responsible for fruit
growth regulation are likely to be multiple. The results are
also of vital interest for breeders and the fruit industry, since
the reported mechanical properties, in particular the decrease of extensibility of the tomato fruit cuticle at the final
stages of ripening, also have implications concerning cracking susceptibility, harvesting, transportation, and extended
storage. Future investigations are required to analyse further
the relationship between the mechanical properties and the
dynamic changes of the chemical composition of the tomato
fruit cuticle throughout maturation, as well as comparative
studies of other cultivars.
Biomechanics of tomato fruit cuticle 1059
Acknowledgements
The authors would like to thank Gerald Elsen and the Botanical
Gardens, University of Bonn (Germany), for substantial help,
Antonio Heredia, University of Málaga (Spain), for critical discussions, and also two reviewers whose comments improved this paper.
Funding of the Deutsche Forschungsgemeinschaft (DFG), grant No.
NE 681/1-1 and NE 681/1-3, is gratefully acknowledged.
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