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Plant Physiol, May 2001, Vol. 126, pp. 210-221
Altered Middle Lamella Homogalacturonan and Disrupted Deposition
of (1
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Cnr (colorless non-ripening) is a pleiotropic tomato
(Lycopersicon esculentum) fruit ripening mutant with
altered tissue properties including weaker cell-to-cell contacts in the
pericarp (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour
[1999] Plant Physiol 120: 383-390). Whereas the genetic basis of the
Cnr mutation is being identified by molecular analyses,
here we report the identification of cell biological factors underlying
the Cnr texture phenotype. In comparison with wild type,
ripe-stage Cnr fruits have stronger, non-swollen cell
walls (CW) throughout the pericarp and extensive intercellular space in
the inner pericarp. Using electron energy loss spectroscopy imaging of
calcium-binding capacity and anti-homogalacturonan (HG) antibody probes
(PAM1 and JIM5) we demonstrate that maturation processes involving
middle lamella HG are altered in Cnr fruit, resulting in
the absence or a low level of HG-/calcium-based cell adhesion. We also
demonstrate that the deposition of (1
5)-
-L-arabinan is disrupted in Cnr pericarp CW and that this disruption
occurs prior to fruit ripening. The relationship between the disruption of (15)--L-arabinan deposition in pericarp CW and the
Cnr phenotype is discussed.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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The modification of cell walls (CW)
is an important aspect of plant cell development. During fruit ripening
the regulated swelling and dissolution of primary CW and the
modification of middle lamellae (ML) between adherent primary CW are
important factors contributing to tissue softening (Brady, 1987
;
Fischer and Bennett, 1991). The biochemistry and the spatial regulation of the dissolution of primary CW and ML are not fully understood, but
in all cases appear to involve modifications to the network of pectic polysaccharides.
The multi-functional pectic polysaccharides are the most complex class
of polysaccharides in primary plant CW (Jarvis, 1984). Core backbone
structures of contiguous 1,4-linked -D-galacturonic acid
(homogalacturonan, HG) or repeats of the disaccharide
[4)--D-GalA-(12)--L-Rha-(1] (rhamnogalacturonan, RG) are elaborated with a range of
modifications and substitutions. These include methyl-esterification,
acetylation, and the addition of neutral polysaccharide side chains.
Side chains may be attached to HG and RG to form the branched
polysaccharides RG-II and RG-I, respectively, the latter often rich in
(15)--L-arabinan and
(14)-
-D-galactan components (O'Neill et al., 1990;
Albersheim et al., 1996; Mohnen 1999). Several of these pectic
structures appear to be capable of being enzymatically modified in
muro. For example, the de-esterification of HG by pectin methyl
esterases (PMEs) influences its capacity to form calcium cross-linked
gels. The relationships of pectic polysaccharide domains within large polymer structures and their functional properties in relation to
factors such as porosity, cell adhesion and expansion, ionic and
hydration status, and cell signaling, are far from clear.
Ripening-related textural changes in the tomato (Lycopersicon
esculentum) fruit pericarp have been extensively studied and are
thought to be associated with alterations in CW properties due to
modifications of the polysaccharide components, including the
cellulose-xyloglucan network (Sakurai and Nevins, 1993; Maclachlan and
Brady, 1994; Rose and Bennett, 1999) and the pectic matrix (Gross and
Sams, 1984; Seymour et al., 1990). Several single-gene mutations have
pleiotropic effects on ripening, including an inhibition of fruit
softening, and these have been very useful for understanding the
molecular mechanisms associated with softening (DellaPenna et al.,
1989; Gray et al., 1994). A pleiotropic tomato ripening mutant,
Cnr (colorless non-ripening), has recently been described with altered physical properties of the pericarp, including reduced cell-to-cell adhesion (Thompson et al., 1999). Although the genetic basis of this mutation has not yet been elucidated, it has been instructive to examine aspects of the CW in the pericarp of
Cnr fruit to gain insights into processes involved in the
ripening of tomato fruit. In this report we focus on the pectic
polysaccharides of the CW matrix and the ML. We show that maturation
processes involving ML HG are altered in Cnr fruit and
result in reduced cell adhesion. Furthermore, we demonstrate that the
deposition of (15)--L-arabinan is disrupted
in Cnr fruit prior to ripening. This is likely to be a key
factor underlying CW properties in the Cnr phenotype.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Altered CW Properties in Cnr Pericarp
To investigate the molecular basis of the textural properties of Cnr fruit, pericarp CW of cv Ailsa Craig wild type (WT) and the near-isogenic mutant Cnr from red-ripe (RR) stage fruit (about 60 d post-anthesis [DPA]) and mature-green (MG) fruit (about 40 DPA) were examined. Staining of sections of resin-embedded pericarp with the cellulose-binding probe Calcofluor White indicated that the cells walls in ripe WT fruit were thicker than the CW of Cnr fruit of an equivalent age, as shown in Figure 1. This was most apparent in the outer pericarp region of RR fruit where expanded ML can be seen (Fig. 1B). The reduction of cell-cell adhesion in Cnr fruit during the ripening process was greatest in the inner pericarp where large intercellular spaces were observed in Cnr (Fig. 1, G and H), but not in the WT (Fig. 1, E and F). Estimation from sections indicated that there was approximately 50% more intercellular space in Cnr pericarp than in WT. The larger intercellular air spaces in Cnr pericarp are likely to account for the observation that RR Cnr fruit (or excised pieces of pericarp) float when placed in water, whereas RR WT fruit sink (data not shown).
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Different swelling capacities of isolated CW material (CWM) from WT and
Cnr fruit were observed following suspension in water. During ripening, the volume of an equivalent weight of WT CW, when
hydrated in vitro, increased approximately 4-fold, whereas CWM from
Cnr fruit showed no increase (Fig.
2A). The CW swelling that occurs during
the normal ripening process (Crookes and Grierson, 1983; Redgwell et
al., 1997b) does not occur in Cnr fruit and its CW and ML
are more compact at the ripe stage.
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Examination of fracture planes with a scanning electron microscope
indicated distinct adhesion properties in Cnr fruit, as shown in Figure 2B. At the MG stage, Cnr pericarp cells
ruptured in a manner equivalent to WT. In contrast, no cell rupture
occurred in Cnr fruit at the RR stage, and the cells
separated (Fig. 2B). This demonstrates that the increased capacity for
cell separation of RR Cnr pericarp does not require the
solubilization of any CW components, which may occur when CW separation
studies are carried out in water (see below, Thompson et al., 1999).
These observations provide further evidence that changes do occur in Cnr pericarp CW during ripening and that these changes
result in stronger CW, weaker intercellular connections, or both of
these factors.
To study the strength of CW, pericarp tissue was subjected to two types of mechanical analysis. One set of analyses involved the determination of the forces required for CW failure using a single-cell probe. The results (Fig. 3A) indicate that at the MG stage CW strength in Cnr is similar to that in WT. Although some loss of wall strength does occur during the ripening process, this is not as extensive in Cnr as in WT. The force required to induce CW failure drops by only approximately 25% in Cnr, compared with a reduction of approximately 80% in WT pericarp cells (Fig. 3A). In addition, compression stiffness modulus was used as a measure of tissue mechanical properties. Cnr pericarp had a significantly higher compression stiffness at the MG stage when compared with WT, and this declined during ripening by approximately 70% (Fig. 3B). In comparison, the compression stiffness modulus of pericarp tissue from WT declined by approximately 90% (Fig. 3B), resulting in significantly reduced stiffness at the RR stage.
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In summary, the pericarp of ripe Cnr has stronger, less swollen, less adherent CW than the WT pericarp at the ripe stage. Although the reduced cell adhesion characteristic of Cnr is manifest at the developmental stage equivalent to RR, it is of interest that a difference in compression stiffness was observed at the MG stage.
Compositional Analysis of Pericarp CW
Analysis of the monosaccharide composition of isolated CW from pericarp of Cnr and WT fruit indicated no major differences, although there was an indication of elevated levels of Gal in Cnr at the MG and RR stages (Table I). Determination of the level of methyl esterification of the galacturonan components in the CWM indicated similar levels and an equivalent decline during ripening in WT and Cnr fruit (Table I). These data indicate that the reduced CW swelling and reduced cell adhesion observed in Cnr pericarp are not due to a gross change in CW polysaccharide composition.
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Maturation of Pericarp ML Is Disrupted in Cnr Fruit
In WT pericarp, cell-to-cell adhesion is known to be altered
during the ripening process, and in ripe fruit cell-to-cell links are
largely mediated by calcium cross-linking of de-esterified regions of
HG, as evidenced by the capacity of calcium chelators to induce cell
separation (Thompson et al., 1999). To study ML maturation in
Cnr and WT pericarp the calcium-binding capacity of ML and
primary CW at MG and RR stages was examined using electron energy loss
spectroscopy (EELS), a method capable of measuring calcium-binding
capacity at high resolution (Huxham et al., 1999). EELS analysis of
resin-embedded material demonstrated that at the MG stage the CW and ML
of Cnr pericarp had a significantly reduced capacity to bind
calcium compared with WT, as shown in Figure
4. During ripening in the WT the CW shows
a marked decrease in relative calcium-binding capacity, with a less
marked change in the ML. In contrast, in Cnr, values for
relative calcium binding in CW and ML are lower than for WT and
increase slightly during ripening. Thus, the calcium-binding capacities
of Cnr CW and ML are lower than WT prior to ripening and are
not modified in the same way during ripening.
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The pectic HG network of pericarp cells was examined by immunolabeling
of resin-embedded sections with PAM1, a phage display monoclonal
antibody specific to large stretches or blocks of de-esterified HG
(Willats et al., 1999a). If PAM1 is used after sections have been
treated to remove methyl ester groups, its binding indicates the
presence of HG in CW independently of the extent or pattern of methyl
esterification (Willats et al., 1999a). PAM1 labeling of resin-embedded
sections of MG and RR pericarp of WT and Cnr indicated that
the overall abundance of the PAM1 epitope on untreated sections was similar in WT and Cnr. However, there were
distinctive spatial differences in the occurrence of the epitope at the
ripe stage, as shown in Figure 5. In RR
WT pericarp the PAM1 epitope was particularly abundant in the region of
the CW lining intercellular spaces in untreated sections (Fig. 5A) and
throughout the ML of chemically de-esterified sections (Fig. 5C). In
contrast, the PAM1 epitope was not so abundant at the lining of
intercellular spaces in Cnr and was generally absent from ML
(Fig. 5B). Most significantly, this was even the case after
de-esterification (Fig. 5D). In regions of the CW away from
intercellular spaces, the PAM1 epitope was variable in its distribution
and was generally absent from the ML. It was frequently observed to be
absent from large areas stretching across adherent CW, as shown in
Figure 5E. This was also apparent after chemical de-esterification of equivalent sections (Fig. 5F). However, some HG components were present
in the ML of Cnr pericarp because the anti-HG probe JIM5 (that binds to a much smaller epitope than PAM1; Willats et al., 2001)
bound to equivalent regions in serially related sections (Fig. 5, B,
inset, and G).
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An altered metabolism of pericarp HG during the ripening process in
Cnr fruit was also reflected in water-soluble and calcium chelator-soluble pectic components. When pieces of pericarp were incubated in water and soluble material was analyzed by immunodot assay, the PAM1 epitope was considerably more abundant and a
high-methyl-ester HG epitope (bound by JIM7) was less abundant in RR WT
pericarp in comparison with material solubilized from Cnr
pericarp (data not shown). This indicates that in Cnr fruit
a water-soluble subset of HG components (that may derive from the ML
and that would not be detected in the resin-embedded samples due to
high solubility) was altered in the extent of its de-esterified block
structure compared with WT. A range of de-esterified
oligoglacturonides, analyzed by high-performance anion-exchange
chromatography, were present in the calcium chelator-soluble fraction
from WT RR pericarp, but absent from Cnr (data not shown),
which is likely to reflect the absence of polygalacturonase (PG)
expression in ripe Cnr fruit (Thompson et al.,
1999).
Although the overall level of galacturonic acid and its methyl esterification are similar (Table I), EELS and analysis of HG components using antibody probes have indicated that significant differences exist in ML and soluble components of the HG network of ripe-stage Cnr when compared with WT. In short, although changes occur in the ML of Cnr pericarp CW during ripening as indicated by the EELS data, they do not result in the abundance of calcium cross-linkable HG capable of maintaining cell-to-cell adhesion seen in the WT.
Disrupted Deposition of (15)--Arabinan in Cnr
Pericarp CW
To investigate other components of the pectic network, sections of
resin-embedded pericarp material were probed with monoclonal antibodies
to (15)--arabinan and (14)--galactan. These polysaccharide domains occur in side chains of RG-I (Albersheim et al., 1996; Mohnen,
1999). In MG pericarp of WT and Cnr, (14)--galactan occurred throughout the CW, but was generally absent from the ML and
pit fields, as shown in Figure 6 and as
previously reported (Jones et al., 1997; Orfila and Knox, 2000). In WT
fruit pericarp the (15)--arabinan epitope also occurred evenly in
CW and was absent from the ML (Fig. 6, C and D). In serially related
sections of WT CW, it can be seen that the (15)--arabinan epitope
appeared at a reduced level in regions of CW lining intercellular
spaces in comparison with (14)--galactan (e.g. Fig. 6, B and D).
Most significantly, the (15)--arabinan epitope did not occur in
an equivalent manner in Cnr pericarp. In MG Cnr
pericarp the (15)--arabinan occurred at a reduced level in the CW
and was absent from an extended region of CW surrounding intercellular
spaces. In addition, the (15)--arabinan epitope appeared to be
abundant at the inner CW/plasma membrane and in vesicle-like structures
in the peripheral cytoplasm (Fig. 6, H and I). This location of the
(15)--arabinan epitope was consistent throughout the pericarp of
Cnr fruit. The aberrant location was present at the MG stage
and was even more apparent at the RR stage (Fig. 6J). The altered
distribution of (15)--arabinan in Cnr pericarp was
confirmed by immunogold electron microscopy of MG material. This
indicated that the epitope was associated with vesicles in the
peripheral cytoplasm in addition to some occurrence in the inner CW,
near the plasma membrane, as shown in Figure
7. The deposition of (15)--arabinan
appeared to be more disrupted in thinner regions of pericarp CW than in thicker regions.
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The disrupted deposition of (15)--arabinan occurred in
Cnr fruit, but was not seen in leaf or stem tissues of
Cnr plants. Furthermore, immunolabeling of resin-embedded
pericarp indicated that there was no equivalent alteration in the
distribution of the (15)--arabinan epitope in the RR fruit of
rin or Never-ripe non-ripening mutants (data not shown).
To explore the nature of the (15)--arabinan-containing material
in Cnr fruit, SDS-PAGE analysis and immunoblotting using the
LM6 antibody was performed. This indicated that a considerable amount
of material carrying the (15)--arabinan epitope from MG
Cnr entered the stacking and resolving SDS-PAGE gels,
resulting in a high Mr smears, in contrast
to WT (Fig. 8A). Loss of the LM6
reactivity after treatment with Pronase E prior to analysis indicated
that the component entering the resolving gel was associated with
protein (Fig. 8A). Figure 8B shows immunodot assays, using LM6, of the
same preparations as used for SDS-PAGE analysis, indicating the
presence of abundant (15)--arabinan-containing material in WT
pericarp that did not enter the stacking or resolving gel systems.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Tomato fruit ripening involves the swelling and softening of CW and decreased cell adhesion, resulting in changes in fruit mechanical properties. In Cnr, CW swelling does not take place, CW are stronger, and cell adhesion is weakened significantly.
The bonds that maintain cell-to-cell contacts at the MG stage must be
lost during the course of ripening (in Cnr and WT), as water
or calcium chelators cannot induce cell separation at the MG stage
(Thompson et al., 1999). From our EELS analysis, the calcium-binding
capacity of the WT primary CW decreases markedly upon ripening, whereas
that of the ML is maintained. In contrast, the relative calcium-binding
capacity in Cnr was always lower than WT, with little
difference between the ML and CW regions. The immunocytochemical
studies indicate that the basis of the reduced calcium binding by
Cnr ML is a modified pectic HG network and most
notably the absence of long de-esterified stretches or blocks of HG
recognized by PAM1. Although the precise structure of the
Cnr ML HG is not known, it is clear that it cannot function in maintaining cell-to-cell attachment.
Modifications to the pectic network of the primary CW appear to be
important for fruit softening during ripening, but they are poorly
understood (Fischer and Bennett, 1991). Studies on pectins isolated
from unripe tomato fruits have emphasized the importance of the
polyelectrolyte and calcium-binding nature of pectin in promoting
swelling (MacDougall et al., 1996; Tibbits et al., 1998). Studies have
indicated that pectin solubilization occurs at early stages of fruit
ripening (Seymour et al., 1990; Rose et al., 1998) and the swelling of
CW in a range of fruit has been reported to correlate with pectin
solubilization (Redgwell et al., 1997b). It has been reported
previously that Cnr fruit do not express PG (Thompson et
al., 1999) and preliminary evidence suggests that the activity of the
main fruit-related isoform of PME in Cnr is reduced
significantly (E. Eriksson, G. Tucker, and G.B. Seymour, personal
communication). However, it is likely that PG and PME are just two of
the many components that play a role in fruit softening. Transgenic
tomato fruits with significantly reduced levels of PME activity have an
increased level of pectin methyl esterification that correlated
with reduced levels of bound calcium, but this had little impact on
pericarp firmness during ripening (Tieman et al., 1992; Hall et al.,
1993; Tieman and Handa, 1994). Furthermore, antisense reduction of PG
mRNA in ripening tomato fruits did not disrupt softening (Smith et al.,
1988; Carrington et al., 1993). The expression of PG in the
rin mutant resulted in HG degradation, but not fruit
softening (Giovannoni et al., 1989).
Taken overall, the observations indicate subtle alterations to the HG
network in Cnr, and one consequence of this is reduced cell
adhesion. It is not clear, however, how the alterations relate to other
aspects of the Cnr phenotype. It is possible that pectic fragments generated during normal fruit ripening exert effects on cell
physiology (Dumville and Fry, 2000) and that these factors are absent
in Cnr. The relationship between the disrupted
(15)--arabinan, the altered HG network, and altered CW properties
are unknown. It is possible that (15)--arabinan has a direct role
in CW swelling and its disrupted deposition in Cnr
contributes to the lack of pericarp softening. Arabinans are known to
be highly flexible, water-soluble polymers (Renard and Jarvis, 1999)
and they may function directly in CW softening or by modulation of HG
properties. Changes in Gal-containing polymers are well documented, and
a decrease in Gal levels during ripening has been linked to fruit softening (Pressey, 1983; Gross and Sams, 1984; Seymour et al., 1990;
Redgwell et al., 1997a; Rose et al., 1998). However, galactosidase activity is only detected after the onset of fruit softening (Carey et
al., 1995; Smith et al., 1998). It is now apparent that
(15)--arabinan and (14)--galactan are extensively modulated
in CW in relation to CW architecture and cell development in a range of
systems and thus are likely to have diverse functions (Jones et al.,
1997; Willats et al., 1998, 1999b; McCartney et al., 2000; Orfila and Knox, 2000). For example, induction of cell elongation in a carrot cell
culture correlates with up-regulation of (14)--galactan and
decreased occurrence of (15)--arabinan (Willats et al., 1999b).
The appearance of (14)--galactan in pea cotyledons, relatively
late in development, correlates with an increase in tissue firmness
(McCartney et al., 2000). It may also be of significance that a greater
compression stiffness modulus, reduced calcium-binding capacity, and
the disrupted deposition of (15)--arabinan are all observed in MG
stage Cnr fruit. It is known that some ripening-related changes start prior to ethylene-triggered ripening processes (Gillaspy et al., 1993; Thompson et al., 1999).
Evidence suggests that the (15)--arabinan epitope in
Cnr is associated with protein. This may indicate that the
epitope is carried by a glycoprotein or proteoglycan that is just at or below the level of detection due to a high turnover or incorporation into non-detergent-soluble fractions in WT fruit, but accumulates in Cnr fruit. One possibility is that the epitope is carried
by a member of the arabinogalactan-protein (AGP) group of cell surface proteoglycans. AGPs are known to be a diverse and heterogeneous group (Nothnagel, 1997) and the presence of (15)--linked
arabinosyl side chains has been reported (Mollard and Joseleau, 1994).
However, the (15)--arabinan-carrying component in Cnr
did not react with anti-AGP monoclonal antibody LM2 or the synthetic
AGP-binding -glucosyl Yariv reagent (data not shown). Furthermore,
the build up of the (15)--arabinan epitope near the plasma
membrane coincides with a reduced occurrence in the CW, suggesting the
disruption of the major component of the CW with (15)--linked
arabinosyl residues, which is likely to be a RG-I fraction. These
observations suggest that attachment to a protein component may be an
aspect of (15)--arabinan deposition or its assembly into the
pectic matrix, although an artifactual association with protein cannot be ruled out. It may be of significance that
(15)--arabinan-containing material from WT fruit did not enter
the stacking gel during SDS-PAGE analysis, presumably due to it being
part of a large polymeric structure.
To our knowledge, this is the first report of a mutation known to result in the specific disruption of the secretion and/or deposition of a plant CW component. This disruption is likely to be a key factor leading to the Cnr texture phenotype. Moreover, the disruption of arabinan deposition is specific to Cnr in that it does not occur in non-ripening rin or Never-ripe fruits. The observations also provide a basis for the further dissection of secretion mechanisms and CW assembly.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Plant Material
Tomato (Lycopersicon esculentum Mill. cv Ailsa
Craig) fruit and the mutant line Cnr in a cv Ailsa Craig
background were grown in a heated greenhouse using standard cultural
practices with regular additions of N,P,K fertilizer and supplementary
lighting when required. The original mutant Cnr was
detected in an F1-hybrid cv Liberto (Thompson et al.,
1999), and a homozygous mutant line was subsequently produced after
selfing for four generations. This line was backcrossed to cv Ailsa
Craig five times, and then selfed twice to produce a homozygous
Cnr mutant line near-isogenic to cv Ailsa Craig. Plants
were grown to three trusses. Fruits were harvested at the following
stages: 20 DPA, MG, approximately 40 DPA; and RR, approximately 60 DPA.
Cnr fruit do not become red, but were harvested at
equivalent times to WT fruit and are designated RR in this report.
Seeds for the rin and Nr ripening mutants
were obtained from the Glasshouse Crops Research Institute collection
at Horticulture Research International (Wellesbourne, UK).
Analysis of Mechanical Properties
Compression stiffness modulus was measured by axially loading
pericarp tissue discs using an LRX materials testing machine (Lloyd
Ltd., Fareham, Hampshire, UK) fitted with a 500 N
load cell and controlled by Nexygen-Ondio 3.0 software. Fruit were sectioned so that tissue discs of 15 mm diameter could be excised with
a cork borer from the equatorial region. Areas containing columellar
tissue were avoided and placental tissue was removed. Two discs, from
opposite sides, taken from each of three fruit per treatment were
measured. The thickness of each tissue disc was measured using
calipers, the disc was then compressed between parallel plates at 5 mm
min
1, and the stiffness modulus calculated from disc
dimensions and the slope of the force/deformation curve (Jackman and
Stanley, 1992).
CW strength was determined as the force required to penetrate the CW of
individual cells with a 50-µm diameter flat-ended probe driven at 10 µm s1. Tissue was excised from the equatorial region
and sectioned to produce a sample approximately 4 mm thick by 15 mm
long with the width being determined by the thickness of the pericarp,
so that the long axis of the sample was parallel to the pedicel to blossom end axis. The probe was driven into the tissue sample to a
depth of 1.5 to 2 mm, such that the direction of travel was parallel to
the skin. Measurements were carried out at six positions on one sample
from each fruit used for the compression measurements and at each
position the failure force for up to eight cells was recorded.
EELS
Duplicate samples of tomato tissue samples were placed into 70%
(v/v) ethanol and were dehydrated using an alcohol series and
then embedded in LR White resin for 2 d before polymerization using UV light. Light gold (60-80 nm thickness) sections were prepared
from two locations for each group for EELS analysis (sampling: 2 × 3 × 2 = 12 locations). Sections were doped with 5 mM calcium acetate for 1 h at room temperature and
were washed three times with water before analysis. EELS analysis of
undoped sections revealed no detectable calcium present. Analysis was
carried out using a transmission electron microscope (902 Leo,
Zeiss, Jena, Germany) operating at 80 KV, using a 60-µm objective
aperture with a vacuum of 5 × 107 torr. All image
sequences were recorded at 12,000× magnification using a 3 eV exit
aperture containing nonoverlapping spectral images covering a range of
the energy spectrum from 240 to 400 eV for the carbon and calcium
ionization edges. Fourteen-bit, 1,024 × 1,024 pixel images were
collected using a slow-scan cooled charge-coupled device camera
(Proscan Elektronische Systeme, Scheuring, Germany) with fixed
gain and bias by integration over 5,000 ms for each image. Two spectral
ranges included electron energy loss information for both elements of
interest: carbon (288 eV) and calcium (335 eV). For each sample 12 data
sets were collected. Areas of interest were divided into the ML and the
CW. Electron energy loss image sequences were analyzed using EsiVision
and AnalySiS software specifically designed for EELS data by Zeiss/Leo. Integrated energy loss values per pixel for carbon and calcium were
determined using the least mean squares fit of the Ae-r power law model
of the immediate background of each spectrum by projection and
subtraction. Data were calculated in terms of the relative intensity
per unit area and summarized using EXCEL. The relative intensity values
for carbon and calcium were measured and the relative intensity ratios
for calcium/carbon (×100) calculated for each domain. This calculation
accommodated any natural variation between the received electron dose
and the section thickness from one sample to another.
Scanning Electron Microscopy
Tomato fruit were washed and the pericarp was cut into large cubes (1 cm3). The cubes were frozen under liquid nitrogen and fractured using a pestle so that small fragments (approximately 0.03 cm3) were obtained. The pericarp fragments were fixed in 2.5% (w/v) glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 2 h at 4°C, and then washed extensively with sodium phosphate buffer and subsequently post-fixed in 1% (w/v) osmium tetroxide in sodium phosphate buffer for 1 h at 4°C. The fragments were washed extensively in sodium phosphate buffer and dehydrated in an acetone series (10%-100%). Dehydrated fragments were critical-point dried, mounted onto metal studs, coated with colloidal gold, and viewed using a scanning electron microscope (CamScan, Leica, Cambridge, UK).
Preparation of Pericarp Tissue for Microscopy
Pericarp cubes (0.06 cm3) were fixed in 2.5% (w/v) glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 2 h at 4°C, and then washed extensively with sodium phosphate buffer. Cubes were post-fixed in 1% (w/v) osmium tetroxide in 0.1 M sodium phosphate buffer for 1 h at 4°C, washed extensively with sodium phosphate buffer, and then dehydrated in an ethanol series (70%-100%). Dehydrated cubes were infiltrated with LR White resin (London Resin, Reading, UK), then placed in gelatin capsules containing LR White resin and allowed to polymerize at 37°C for 5 d.
Immuno- and Cytochemical Labeling for Light Microscopy
Antibody probes for HG included PAM1, a phage display monoclonal
antibody that binds to large de-esterified blocks of HG (Willats et
al., 1999a), JIM5, and JIM7 (Knox et al., 1990; Willats et al., 2001).
Antibody probes for epitopes carried by RG-I included LM5 to
(14)--D-galactan (Jones et al., 1997) and LM6 to
(15)--L-arabinan (Willats et al., 1998).
Sections obtained from resin-embedded material (0.5-µm thickness)
were incubated in a 5% (w/v) solution of fat-free milk powder in
phosphate-buffered saline (PBS), pH 7.2, for 30 min. Sections were then
incubated in a solution containing anti-HG PAM1 phage monoclonal
antibody (approximately 1012 phage particles
mL1) in milk powder/PBS for 1.5 h. The sections were
then washed extensively with PBS and were post-fixed with 1% (w/v)
glutaraldehyde in PBS for 10 min. The sections were washed extensively
with PBS and then incubated with a solution containing mouse anti-M13
monoclonal antibody (Pharmacia, Uppsala) diluted 1:50 in milk
powder/PBS for 1.5 h. The sections were extensively washed in milk
powder/PBS and then incubated in a solution containing anti-mouse
monoclonal antibody linked to fluorescein isothiocyanate (Sigma, St.
Louis) diluted 1:100 in milk powder/PBS. The sections were washed
extensively with PBS, mounted in a glycerol/PBS/fluorescence anti-fade
solution (Citifluor AF3, Agar Scientific, Stansted, UK), and examined
with a microscope equipped with epifluorescence. Where specified,
sections were treated with 0.1 M
Na2CO3 for 30 min to chemically de-esterify pectin, then washed extensively with deionized water. For cytochemical staining of cellulose, sections were treated with a solution of 0.025% (w/v) Calcofluor White (fluorescent brightener 28, Sigma) in deionized water for 30 s, then washed extensively with
deionized water. Sections were mounted as described above and
observed using a microscope equipped with UV fluorescence.
Preparation of Enzyme-Free CW
Isolation of CWM was based on a method described previously
(Seymour et al., 1990). Fruit were washed, peeled, and the
pericarp was cut into small cubes (0.125 cm3), which were
then homogenized in 4 volumes of acetone at 
20°C using a
homogenizer (Polytron, Kinematica, Lucerne, Switzerland). The
homogenate was filtered through Miracloth (Calbiochem-Novabiochem, San
Diego) and washed with 80% and 100% (w/v) acetone (12.5 mL g1 tissue fresh weight). Acetone-insoluble solids were
suspended in a solution of phenol:acetic acid:water (PAW, 2:1:1, w/v,
10 mL g1 tissue fresh weight) and the mixture was stirred
for 15 min at 4°C. After PAW treatment, acetone was added to a final
concentration of 80% (v/v) and the mixture was filtered though a
sintered glass filter. The filtrate was washed with 100% (w/v) acetone
(200 mL) to remove traces of PAW. The obtained CWM was dried in
air at room temperature (for CW hydration studies) or dried over
P2O5. In both cases CWM was stored desiccated
at 20°C.
The in vitro hydration of CW was visualized as follows. CW (3 mg dry weight) were suspended in 5 mL of water and allowed to hydrate for 8 h with gentle rocking at room temperature. CW were then allowed to sediment at 4°C for 30 min.
Determination of Total Monosaccharide Composition
Neutral monosaccharide composition was determined by gas liquid
chromatography as described by Selvendran et al. (1979) with modifications by Blakeney et al. (1983). UA content was determined according to Blumenkrantz and Absoe-Hansen (1973) and the degree of
methyl esterification was determined according to Wood and Siddiqui (1971), with minor modifications (addition of 0.1 M osmium tetroxide to the 0.125 M potassium
permanganate solution and to the acidified 0.125 M arsenite solution).
SDS-PAGE and Immunoblotting
Tomato pericarp was frozen and ground to a fine powder using a
mortar and pestle. The powder (2 mL) was suspended in 50 mM Tris-HCl, pH 6.5, or 50 mM Tris-HCl, pH 6.5, containing
Pronase E (Sigma) at 1 mg mL1. Samples were vortexed and
boiled for 5 min or left at room temperature for 2 h. All
samples were then suspended in double-strength SDS-sample buffer,
vortexed, and boiled for 5 min. Protein concentration of the samples
were determined using a Bio-Rad (Hercules, CA; Bradford) protein
assay. Aliquots containing 10 µg of protein were loaded per
lane for SDS-PAGE analysis and immunoblotting with LM6 as described in
Smallwood et al. (1996) and 1-µL aliquots (0.4 µg of protein) were
directly assayed for LM6 binding by immunodot assays as described in
Willats et al. (1999b). Anti-AGP monoclonal antibody LM2 and
-glucosyl Yariv reagent were also used to probe the blots as
described previously (Smallwood et al., 1996).
Cell Separation and Isolation of Soluble Pectic Components
Pericarp was cut into small cubes (0.03 cm3) and
incubated in three volumes of deionized water or 0.1 M
calcium chelator
(cyclohexanediamine-N,N,N',N'-tetraacetic acid), pH 6.5, at room temperature, with gentle rocking. After 3 h
the supernatant was collected, filtered though glass fiber, and
analyzed by immunodot assays as described elsewhere (Willats et al.,
2001), or ethanol was added to a concentration of 70% (w/v),
carbohydrate and protein polymers were allowed to precipitate overnight
at 4°C, and the solution was centrifuged (4,000 rpm) and the pellet
was freeze-dried. The dry pellet was resuspended in deionized water to
a concentration of 1 mg mL1 and was analyzed by high
performance anion-exchange chromatography as described elsewhere
(Willats et al., 1999a).
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FOOTNOTES |
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Received November 2, 2000; returned for revision December 8, 2000; accepted February 6, 2001.
1 This work was supported by the United Kingdom Biotechnology and Biological Sciences Research Council (G.B.S., A.J.T.), including a Co-operative Award in Science and Engineering studentship with Horticulture Research International (to C.O.) and a grant (no. 17/DC09694 to I.M.H.).
* Corresponding author; e-mail j.p.knox{at}leeds.ac.uk; fax 44-113-2333144.
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LITERATURE CITED |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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-D-galactanase: isolation, changes during ripening in normal and mutant tomato fruit, and characterization of a related cDNA clone.
Plant Physiol
108: 1099-1107[Abstract]4)--D-galactan.
Plant Physiol
113: 1405-1412[Abstract]4)--D-galactan in cell walls of developing pea cotyledons: implications for mechanical properties.
Plant J
22: 105-113[CrossRef][Web of Science][Medline]-Galactosidases in ripening tomatoes.
Plant Physiol
71: 132-135-galactosidase II is expressed during fruit ripening.
Plant Physiol
117: 417-4235)--L-arabinan.
Carbohydr Res
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5)-
-L-Arabinan in the Pericarp of
Cnr, a Ripening Mutant of Tomato
