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Journal of Experimental Botany, Vol. 53, No. 378, pp. 2185±2191, November 2002
DOI: 10.1093/jxb/erf070
Subcellular localization of peroxidase in tomato fruit skin
and the possible implications for the regulation of fruit
growth
J. Andrews1, S. R. Adams, K. S. Burton and C. E. Evered
Horticulture Research International, Wellesbourne, Warwickshire CV35 9EF, UK
Received 6 March 2002; Accepted 27 June 2002
Abstract
The cessation of tomato fruit growth has been associated with the appearance of three `wall-bound' peroxidase isozymes in the skin of tomato fruit.
However, the presence of these isozymes in the ionically eluted `wall-bound' fraction may be an artefact
of either non-speci®c binding of symplastic peroxidase to the cell wall, or isozymes bound to membranes included in the `wall-bound' fraction.
Therefore, subcellular localization of peroxidase in
both immature and mature tomato fruit skins was
studied. Immature fruits showed intense peroxidase
activity associated with the tonoplast and pro-vacuolar membranes, but little or no activity associated
with the cell wall. However, the presence of peroxidase activity within the cell wall of mature green
fruits was con®rmed. Furthermore, peroxidase activity was also observed associated with the plasma
membrane and large vesicles allied to the plasma
membrane. While cross-linking in cell wall
components was previously assumed to be the
mechanism by which peroxidase might control fruit
growth, the incorporation of `lignin-like' phenolics
may also play a part. Isoelectric focusing (IEF) of
both symplastic and apoplastic peroxidase extracted
from immature and mature tomato fruit skin showed
that all peroxidase isozymes present were highly
anionic. In this current study, histochemical techniques are used to demonstrate a developmental
increase in `lignin-like' phenolics within the sub-cuticular cell walls of the fruit skin. The localization of
peroxidase within tomato fruit skin is discussed in
relation to its potential role in the regulation of
tomato fruit growth.
1
Key words: Fruit growth, localization,
esculentum, peroxidase, tomato.
Introduction
In tomato, as in many other horticultural crops, fruit size is
one of the key determinants for both quality and yield, as
fruits are size-graded (Adams et al., 2001). The regulation
of tomato fruit growth has attracted much research interest,
which is re¯ected in the extent of information available on
environmental, nutritional and photoassimilate regulation
of fruit growth (Monselise et al., 1978; de Koning, 1984;
Ho et al., 1987; BussieÁres, 1993; Grange and Andrews,
1994).
The pericarp of tomato fruit ¯esh is composed of three
distinct tissue types: the endocarp, a unicellular layer
encasing the locular cavity, the mesocarp, a multicellular
layer of large, thin-walled parenchyma cells (>500 mm
diameter) and vascular tissue, and the exocarp (fruit skin)
which is composed of an outer epidermis with an
ingressing thick waxy cuticle (Osman et al., 1999) and
two to three layers of thick-walled hypodermal cells (Ho
and Hewitt, 1986). Thompson et al. (1998) demonstrated
that turgor-driven expansion of parenchyma cells within
the mesocarp creates tissue pressure, which is expressed on
the exocarp layer as tissue tension. They suggested that the
mechanical properties of the exocarp were important in
controlling fruit growth, and linked the cessation of fruit
growth to a developmental increase in peroxidase activity
associated with the exocarp. Andrews et al. (2000)
demonstrated the developmental appearance of three new
`wall-bound' peroxidase isozymes that may be responsible
for this increase in peroxidase activity.
To whom correspondence should be addressed: Fax: +44 (0)1789 470552. E-mail: [email protected]
ã Society for Experimental Biology 2002
Lycopersicon
2186 Andrews et al.
Peroxidase (EC 1.11.1.7) is an oxidoreductase that is
known to catalyse the oxidation of numerous substrates
through the associated reduction of hydrogen peroxide
(Dawson, 1988; Wallace and Fry, 1999). Given the
ubiquitous presence of peroxidase throughout nature
(VaÂmos-VigyaÂzoÂ, 1981) and the number of substrates
that may potentially be oxidized in the presence of
peroxidase, it seems unlikely that a single peroxidase
will catalyse a single speci®c reaction in vivo. In
plants, the main roles of peroxidase are attributed to
ligni®cation and suberization (Espelie et al., 1986;
Roberts et al., 1988; MuÈsel et al., 1997; Quiroga et al.,
2000), cutin deposition in outer aerial epidermal layers
(Ferrer et al., 1991), defence against pathogen attack
(Lagrimini et al., 1993) and the possible cross-linking
of cell wall components (Fry, 1986; Brownleader et al.,
1999; Hat®eld et al., 1999). Peroxidase catalysed crosslinking in cell walls is thought to result from the
formation of diferuloyl bridges between pectin residues,
and isodityrosine bridges between hydroxyproline-rich
extensin molecules (Fry, 1986; Brownleader et al.,
1999; Hat®eld et al., 1999).
The use of high ionic strength buffers to elute `wallbound' peroxidases is a standard procedure used to
examine ionically bound cell wall enzymes (Ferrer
et al., 1991; Andrews et al., 2000; Quiroga et al.,
2000). However, this procedure may not distinguish
between peroxidases bound to the cell wall and those
bound to other membranes such as the plasma
membrane or tonoplast, or symplastic peroxidases that
may bind non-speci®cally to exposed cell walls when
released from the cytosol during sample maceration.
The in vivo function of a particular peroxidase is likely
to be determined by its location in a particular tissue,
cellular or subcellular compartment, which, in turn,
may depend in part on its ionic nature (Chibbar and
van Huystee, 1984; MaÈder et al., 1986; GarcõÂaFlorenciano et al., 1991; Ros Barcelo et al., 1991;
Carpin et al., 2001). Here, using transmission electron
microscopy (TEM), the subcellular localization of
peroxidase in the exocarp from both immature and
mature green tomato fruit is shown, together with the
characterization of the isoelectric point (pI) of
peroxidase isozymes from a developmental range of
fruit. The ionic nature of individual peroxidase
isozymes may indicate possible binding sites within
the cell wall, whilst also allowing comparison with
peroxidase isozymes characterized in other systems
(Espelie et al., 1986). Furthermore, ¯uorescent histochemical techniques are used to highlight the presence
of `lignin-like' phenolics in the tomato fruit exocarp
and their possible roles in defence and the rheological
properties of the cell wall are discussed (Lulai and
Morgan, 1992; Schopfer et al., 2001).
Materials and methods
Plant material
Plants of Lycopersicon esculentum Mill. cv. Espero were grown as a
conventional long-season glasshouse crop following normal commercial practice (Adams et al., 2001).
Peroxidase cytochemistry
To establish the subcellular origin of the `soluble' and `wall-bound'
peroxidase fractions from tomato fruit exocarp (Andrews et al.,
2000), 3,3,-diamino benzidine (DAB) an electron-dense peroxidase
stain was employed (Bestwick et al., 1998). Fruit exocarp samples
(234 mm) from three replicate fruits at each of two developmental
stages, 14 d and 45 d post-anthesis (dpa), were excised directly into
®xative, 1% (v/v) glutaraldehyde and 1% (v/v) paraformaldehyde in
buffer A (50 mM sodium cacodylate buffer pH 7.0) at room
temperature for 60 min. This was followed by two subsequent 10 min
washes in buffer A, and a 30 min rinse in buffer B (50 mM potassium
phosphate buffer containing 20 mM 3-amino-1, 2, 4-triazole (ATZ)).
ATZ, was included in all subsequent buffers to inhibit peroxidative
artefacts resulting from any in vivo catalase activity (EC 1.11.1.6
H2O2:H2O2 oxidoreductase). Fixed samples were transferred to a
peroxidase activity staining solution containing buffer B, with 0.5
mg ml±1 DAB and 5 mM hydrogen peroxide or one of two-control
solutions (I) buffer B, without DAB, (II) buffer B, with 0.5 mg ml±1
DAB. While potassium cyanide (KCN) is commonly used as an
inhibitor in DAB-stained peroxidase localization studies, it is not a
speci®c peroxidase inhibitor and speci®c peroxidases may vary in
their sensitivity to KCN ( Barcelo et al., 1991). Therefore, KCN was
not used as a control in the current study. Preliminary studies showed
45 min to be an optimal staining time; however, a 15 min staining
time was included as a precaution against over-staining. All staining
procedures were conducted in the dark under mild vacuum
conditions to exclude auto oxidation of DAB and improve DAB
penetration into the tissue. Staining was followed by two 10 min
washes in buffer B, prior to ®xation in 1% osmium tetroxide in
buffer A, for 45 min. Samples were then washed for 10 min, twice in
buffer B, and twice in double distilled water. They were then
sequentially dehydrated for 15 min in each of 30%, 50%, 70%, 80%,
and 90% ethanol, followed by three 20 min washes in 100% ethanol.
Spurr's resin embedding
Spurr's embedding requires complete tissue dehydration (Spurr,
1969). Therefore, two further changes of 100% ethanol were
conducted, each for 24 h. Samples were in®ltrated through an
ascending concentration of resin diluted in ethanol: 25%, 50%, 75%,
100%. The ®rst three steps took 1.5 d the ®nal step lasted 2±3 d with
a change into fresh resin after ~8 h. The samples were embedded in
freshly prepared 100% Spurr's resin, and polymerized at 80 °C for
8 h. Ultrathin sections (~70 nm) were cut on a Reichert Ultracut E
ultramicrotome, stained with uranyl acetate followed by Reynolds'
lead citrate (Reynolds, 1963) and viewed in a JEOL 100CX TEM at
80 kV. Spurr sections (~2 mm) were also prepared for light
microscopy, and were stained with aqueous 0.5% toluidine blue.
Histochemical staining of `lignin-like' phenolics within tomato
fruit exocarp
Strips of pericarp tissue were excised from each of two green stages
of tomato fruit development (14 dpa and 50 dpa). The strips were
blotted on ®lter paper to remove excess moisture and mounted in a
cryostat embedding compound (Tissue-Tek OCT; Agar Scienti®c,
Stansted, UK) and sectioned at ±30 °C using a cryostat microtome
(Bright Ltd, Huntingdon, Cambridgeshire, UK). Sections were
mounted on glass slides and air-dried prior to staining with 0.1%
berberine which stains `lignin-like' phenolics, 0.02% ruthenium red
Peroxidase localization in tomato 2187
was employed as a counter stain (Lulai and Morgan, 1992). Sections
were visualized using an Leitz Dialux 20 ¯uorescence microscope
(with a leitz ®lter block `G') and images were captured and analysed
using an image analyser (Optimas version 6.1, Optimas Corporation,
Seattle, Washington, USA).
Ionic elution of peroxidase from cell walls
Exocarp strips were excised from the equatorial region of a
developmental range of proximal fruit 14, 21, 28, 35, 42, 49, and
56 dpa, using a razor blade. Approximately 50 mg was taken from
each fruit. This, together with an equal weight of acid-washed quartz
silica sand (Sigma, Poole, Dorset, UK), was ground thoroughly in a
pestle and mortar in the presence of liquid nitrogen. The macerated
sample was suspended in ice-cold 10 mM sodium acetate/citric acid
buffer, pH 6.0, using 100 ml mg±1 of the original fresh weight (OFW)
of tissue. The suspension was centrifuged at 3000 g for 15 min at
3 °C. The resulting supernatant represented the soluble (symplastic)
fraction. The pellet was resuspended in the same buffer and again
thoroughly mixed prior to centrifugation. This process was repeated
eight times to ensure all the soluble peroxidase activity had been
eluted from the sample. Supernatant from the ®nal wash was assayed
for residual peroxidase activity using 3,3¢,5,5¢-tetramethylbenzidine
(TMB) as previously described (Andrews et al., 2000).
The washed pellet was resuspended in ice-cold 100 mM sodium
acetate/citric acid buffer, pH 6.0, containing 1 M NaCl (100 ml mg±1
OFW). The suspension was mixed thoroughly and incubated on ice
for 60 min with periodic mixing. The resulting supernatant contained
the salt-extractable peroxidase which may represent in vivo
peroxidase ionically bound to the cell wall; the `wall-bound'
fraction (Thompson et al., 1998; Andrews et al., 2000). Following
dialysis against water for 48 h (desalting step), the `wall-bound'
fraction was freeze-dried (Edwards Modulyo 4K) for 72 h. The
lyophilized samples were resuspended in 100 ml of 50 mM MES
buffer pH 6.0 prior to isolectric focusing (IEF) and native PAGE.
Isoelectric focusing
Preliminary isoelectric focusing (pI range 3.5±9.3) showed that all
symplasic and apoplastic peroxidase isozymes extracted from a
developmental range of tomato fruit exocarp are highly anionic with
pI values less than 3.5. However, pI values could not be determined
accurately as they were below the resolution of the pre-cast gel
(Amersham Pharmacia Biotech pH 3.5±9.5). Therefore, an IEF gel
was prepared using Pharmalyte ampholytes ranging from pI 2.5±5.0
(Sigma, Poole, Dorset, UK), as per the manufacturer's instructions
(Amersham Pharmacia Biotech, Bucks, UK). Dialysed `wall-bound'
extracts were prepared (as above) from seven developmental stages
of tomato fruit exocarp 14, 21, 28, 35, 42, 49, and 56 dpa. To con®rm
the developmental appearance of three new peroxidase isozymes,
10 ml samples of each extract were run on native PAGE as previously
reported (Andrews et al., 2000), prior to application on the IEF gel.
Resolution of the isoelectric points of four peroxidase isozymes was
conducted at 7.5 °C on a Multiphor II ¯atbed electrophoresis unit,
using a low range (pI 2.8±6.5) pI marker kit as per the manufacturer's instructions (Amersham Pharmacia Biotech, Bucks, UK).
Peroxidase activity and the low range pI markers were visualized
using freshly prepared chloronaphthol followed by 0.1% Coomassie
blue R-250, respectively (Andrews et al., 2000).
Results
Peroxidase cytochemistry
TEM observations from immature tomato fruit exocarp
showed a lack of peroxidase activity associated with the
cell wall (Fig. 1A, B). However, intense peroxidase
Fig. 1. Immature fruit exocarp cells (14 dpa) (A) showing intense
DAB staining of the vacuolar (V) side of the tonoplast (T), some provacuolar vesicles (PR), but little or no DAB staining of the cell wall
(CW), or cell organelles such as chromoplasts (CH). (B) DAB staining
of the tonoplast (T) and a few particulates (PA) within the vacuole
(V). Calibration bars=1 mm.
activity was associated with the tonoplast, pro-vacuolar
membrane and a few particulates within the vacuole
(Fig. 1A, B). Mature fruit sections con®rmed the presence
of peroxidase activity within the inner edge of the cell wall
(Fig. 2) and occasionally near the middle lamella (Fig. 3).
No activity was observed in the cuticle of the outer
epidermal layer. By contrast to immature fruit, mature fruit
exocarp cells have proportionally larger vacuoles with
numerous particulates, less cytoplasm, no pro-vacuolar
vesicles, and little or no activity associated with the
tonoplast (Fig. 3). Mature fruit also had intense peroxidase
activity staining associated with the plasma membrane,
particulates and `vesicle-like' structures with membranes
that sometimes appeared continuous with the plasma
membrane (Fig. 3). Endoplasmic reticulum was allied to
the majority of `vesicle-like' structures at the plasma
membrane (Fig. 3). Image analysis of TEM photomicrographs showed that the `vesicle-like' structures had
diameters ranging from 1±2 mm. Neither peroxidase
staining of the plasma membrane nor the presence of
`vesicle-like' structures were observed in sections from
2188 Andrews et al.
Fig. 2. Mature fruit exocarp cell (45 dpa), showing intense DAB
staining of the inner edge of the cell wall (CW), and the plasma
membrane (PM). The cytoplasm, shows little or no DAB staining, with
no activity associated with organelles such as mitochondria (M),
chromoplasts (CH) and the nucleus (N). The tonoplast (T) bordering
the vacuole (V) shows little or no DAB staining compared with the
tonoplast of immature tomato fruit (Fig. 1A, B). Calibration bar=1 mm.
Fig. 4. Mature fruit exocarp (45 dpa) stained with both DAB and
0.1% toluidine blue and viewed under a light microscope. The
ingressing cuticle (CU) as distinct from the cell wall (CW) can clearly
be seen within both the outer epidermis (EP) and the hypodermal layer
(HY). Calibration bar=10 mm.
Fig. 3. Mature fruit exocarp cells (45 dpa). DAB staining of
membranes of `vesicle-like' structures (VE) associated with the plasma
membrane (PM) and the vacuole (V). DAB staining is also located in
areas of the plasma membrane (PM), cell wall (CW) allied to the
`vesicle-like' structures (VE) and tentatively in the middle lamella
(ML). The vacuole (V) also contains partially degraded vesicle-like
structures (PVE), and particulates (PA), which show residual amounts
of DAB staining. Little or no DAB staining was oberved in organelles
such as the chromoplasts (CH). Calibration bar=1 mm.
immature tomato fruits (Fig. 1A, B). Control sections with
and without DAB showed no electron-dense staining due
to auto-oxidation of DAB (images not presented).
Histochemical localization of `lignin-like' phenolics
within tomato fruit exocarp
Light microscope sections prepared from mature tomato
fruit exocarp were stained with 0.5% toluidine blue
(Fig. 4). The section illustrates the cellular organization
of the exocarp and provides a reference point for the
¯uorescence observed in subsequent sections (Fig. 5A, B).
Furthermore, the section demonstrates the extent of
cuticular ingress from the outer epidermis into the
hypodermal layers of the fruit exocarp. Fruit exocarp
Fig. 5. (A) Immature green fruit (14 dpa) exocarp stained for `ligninlike' phenolics with berberine/ruthenium red. The intensity of
¯uorescent staining in the outer epidermis is low and speci®c detail
cannot be observed. (B) Mature green fruit (50 dpa) exocarp stained
for `lignin-like' phenolics with berberine/ruthenium red. The
¯uorescent staining is intense within the outer epidermal cell wall
(CW), but not in the cuticle (CU). Little or no ¯uorescent staining was
observed in the underlying hypodermal layer (HY). Calibration
bars=10 mm.
Peroxidase localization in tomato 2189
Fig. 6. `Wall-bound' peroxidase isozymes from the exocarp of tomato
fruit of different ages (14±56 dpa), separated by isoelectric focusing
(A) and native PAGE (B). Marker pI values are shown to the left of
the IEF gel (A) and isozyme bands A, B, C, and D are shown to the
right of the native PAGE gel (B).
sections stained for the aromatic (lignin) domain of suberin
using berberine and counter-stained with ruthenium red,
showed ¯uorescence in the epidermal layer of the exocarp
(Fig. 5A, B). The degree and intensity of ¯uorescence from
berberine staining was greater in exocarp sections from
mature fruit (Fig. 5B) compared with those from immature
fruit (Fig. 5A). In mature fruits, staining was largely
associated with the sub-cuticular cell walls of the epidermis, in comparison to the limited staining of the cuticle
(Figs 4, 5A). The low intensity of staining within the
exocarp of immature fruit (Fig. 5A) made further localization of stain speculative.
Isoelectric focusing
Figure 6A illustrates isoelectric focusing of `wall-bound'
peroxidase extracted from seven developmental stages of
tomato fruit exocarp on an IEF gel pI range 2.5±5.0.
Samples from immature fruit (14±21 dpa) show a single
peroxidase isozyme with an apparent pI value of 3.1. At 28
dpa a further peroxidase isozyme is detected, with an
apparent pI value of 3.9. Further peroxidase isozymes are
detected at 35 dpa and 49 dpa with pI values of 2.9 and 3.5,
respectively. Native PAGE of the same developmental
range of wall-bound peroxidase (Fig. 6B) also illustrates
the appearance of four peroxidase isozymes at fruit
maturation. By correlating the age of fruit at which these
bands ®rst appeared with those detected on the IEF gel, it
seems likely that bands A, B, C, and D (Fig. 6B) have
isoelectric points of 3.1, 3.9, 2.9, and 3.5 (Fig. 6A),
respectively.
Discussion
`Soluble' and `wall-bound' peroxidase activity was previously reported in the skin of immature tomato fruits
(Andrews et al., 2000), both fractions contained a single
isozyme (58 kDa). However, these localization studies
have shown that peroxidase activity was associated with
the tonoplast and pro-vacuolar membranes in the exocarp
of immature fruits, with little or no obvious `wall-bound'
activity. This apparent contradiction may be due to either
non-speci®c binding of liberated symplastic peroxidase to
exposed cell walls during sample preparation, which
appears unlikely (Andrews et al., 2000), or peroxidase
binding to the tonoplast or pro-vacuolar vesicle membrane,
that pelleted on centrifugation with the cell wall fraction.
Peroxidase isozymes present in the `soluble' fraction may
represent precursors readily released from the tonoplast or
cell periphery on tissue homogenization, and more
strongly bound isozymes may be liberated on ionic elution
and may represent the `wall-bound' fraction. Similar
observations and conclusions were drawn for peroxidase
found in maize root tips. Furthermore, peroxidase activity
associated with the tonoplast and pro-vacuolar membranes
was thought to have a role in the formation of the tonoplast
and vacuole (Parish, 1975).
Peroxidase activity was observed in the cell walls of
mature tomato fruit exocarp cells. It was apparent that
peroxidase activity was mainly located in the inner regions
of the cell wall with occasional activity observed within
the middle lamella. The presence of `wall-bound'
peroxidase activity in mature fruit con®rms earlier ®ndings
and supports the notion that peroxidase mediated `stiffening' of the exocarp cell walls leads to the cessation of fruit
growth (Thompson et al., 1998; Andrews et al., 2000).
Furthermore, the lack of peroxidase activity associated
with the cuticle strengthens the hypothesis that peroxidase
is not involved in cuticle formation in tomato fruit
(Andrews et al., 2000). Peroxidase activity associated
with particulates, `vesicle-like' structures and the plasma
membrane, may contribute either to the `soluble' or the
`wall-bound' fraction, or both. The origin of both `soluble'
and `wall-bound' activity can only be speculated upon,
except when peroxidase activity is clearly identi®ed within
the cell walls of mature fruit exocarp.
Whilst it is generally accepted that both exocytosis and
endocytosis occur within plant cells, it is not possible to
distinguish between these processes through examination
of TEM photomicrographs (Battey et al., 1999). However,
the observation that both intact and partially degraded
`vesicle-like' structures appear to exist within the vacuole
may indicate endocytotic traf®cking from the plasma
membrane into the vacuole as it seems unlikely that the
vacuole will possess the `machinery' for vesicle production. A similar phenomenon involving the transport of
degradative enzymes by multivesicular bodies into the
vacuole has previously been reviewed (Battey et al., 1999).
However, the possibility that these `vesicle-like' structures
are an artefact of tissue preparation cannot be excluded.
Con®rmation of wall-bound peroxidase activity within
the cell walls of mature tomato fruit exocarp does not
reveal the in vivo function. However, the presence of
2190 Andrews et al.
highly anionic peroxidase isozymes within the exocarp cell
walls (pI 2.9±3.9) may indicate binding to positively
charged components, for instance, the Ca2+ pectin matrix,
which provides an integral component of the cell wall
(Penel and Greppin, 1979; Ros Barcelo et al., 1989; Carpin
et al., 2001). These results contradict previous tentative
measurements of a single pI value for all four peroxidase
isozymes of 4.6 (Andrews et al., 2000). The results
presented here have been consistent throughout ®ve
repeats, and it is not possible to explain why an earlier
preliminary measurement gave a pI value of 4.6.
The role of a highly anionic peroxidase in suberization
of wounded potato tubers has been previously reported and
appears to mediate deposition of aromatics in the cell wall
(Espelie et al., 1986), but is also present in wounded
tomato fruits (Roberts et al., 1988; Sherf and Kolattukudy,
1993). Two linked genes, tap1 and tap2, were cloned and
sequenced and shown to encode for this peroxidase activity
(Roberts et al., 1988). However, gene expression of tap1
and tap2 has also been shown to increase 2-fold throughout
development until the onset of the climacteric in tomato
(Sherf and Kolattukudy, 1993). There are remarkable
similarities when comparing this peroxidase (45 kDa, pI 3)
with some of the isozymes reported here (pI 2.9±3.9, with
bands A±D having apparent molecular weights of 43±58
kDa (Andrews et al., 2000).
The developmental increase of aromatics associated
with tomato fruit cuticle development was reported
previously (Hunt and Baker, 1980). The major component
of the fruit cuticle is cutin, composed of hydrophobic
aliphatic fatty acids (Osman et al., 1999). Previous studies
have demonstrated the deposition of speci®c ¯avonoids
into the cuticular membranes of the fruit epidermis during
fruit ripening (FernaÂndez et al., 1999). However, this study
has shown the presence of subcuticular `lignin-like'
phenolics within the epidermal cell walls of green tomato
fruit exocarp. The presence of such an aromatic domain
normally associated with lignin and suberin deposition
may also provide resistance to pathogen infection (Lulai
and Corsini, 1998). Relatively few pathogens affecting the
fruit of tomato appear to gain entry into the fruit through
the fruit skin. Those that do, appear to enter when the fruit
are immature and the skin is thin and poorly developed or
through wound or growth cracks in older fruit. Therefore,
the protective barrier presented by the fruit skin may be
fundamental to resistance against localized pathogen
attack (Watterson, 1986).
In conclusion, the developmental localization of highly
anionic peroxidase isozymes within the cell walls of the
exocarp layer supports earlier biochemical observations on
the activity of peroxdase during fruit development
(Thompson et al., 1998; Andrews et al., 2000).
Peroxidase activity was localized throughout the exocarp
tissue layer, whereas `lignin-like' phenolics were restricted
to the epidermis. This suggests that it is unlikely that the
isozymes present are involved solely in the deposition of
aromatics. It is suggested that peroxidase isozymes located
within the outer fruit exocarp may have a dual role in
restricting fruit expansion through cross-linking of cell
wall components and producing a protective barrier in the
epidermis.
Acknowledgements
This work was funded by DEFRA (previously MAFF) grant
HH1323. The authors are grateful to Drs LC Ho, D Gray,
S Clifford, R Napier, G Grif®ths, V Valdes, Mr C Clay, and
Mr K Manning of HRI Wellesbourne, Drs W Davies and J Taylor of
Lancaster University and Dr N Battey of Reading University for the
provision of plant material, expertise and helpful discussion.
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