Plant Physiol. (1998) 117: 345-361
Temporal Sequence of Cell Wall Disassembly in Rapidly Ripening
Melon Fruit1
Jocelyn K.C. Rose,
Kristen A. Hadfield,
John M. Labavitch, and
Alan
B. Bennett*
Mann Laboratory, Department of Vegetable Crops (J.K.C.R., K.A.H.,
A.B.B.), and Department of Pomology (J.M.L.), University of California,
Davis, California 95616
 |
ABSTRACT |
The Charentais variety of melon
(Cucumis melo cv Reticulatus F1 Alpha) was observed to
undergo very rapid ripening, with the transition from the preripe to
overripe stage occurring within 24 to 48 h. During this time, the
flesh first softened and then exhibited substantial disintegration,
suggesting that Charentais may represent a useful model system to
examine the temporal sequence of changes in cell wall composition that
typically take place in softening fruit. The total amount of pectin in
the cell wall showed little reduction during ripening but its
solubility changed substantially. Initial changes in pectin solubility
coincided with a loss of galactose from tightly bound pectins, but
preceded the expression of polygalacturonase (PG) mRNAs, suggesting
early, PG-independent modification of pectin structure.
Depolymerization of polyuronides occurred predominantly in the later
ripening stages, and after the appearance of PG mRNAs, suggesting the
existence of PG-dependent pectin degradation in later stages.
Depolymerization of hemicelluloses was observed throughout ripening,
and degradation of a tightly bound xyloglucan fraction was detected at
the early onset of softening. Thus, metabolism of xyloglucan that may
be closely associated with cellulose microfibrils may contribute to the
initial stages of fruit softening. A model is presented of the
temporal sequence of cell wall changes during cell wall disassembly in
ripening Charentais melon.
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INTRODUCTION |
Ripening in many fruits is associated with textural changes that
are believed to result from disassembly of the primary cell wall. This
includes modifications of the structure and composition of the
constituent polysaccharides that have been correlated with the
expression of a range of hydrolases and transglycosylases (Fischer and
Bennett, 1991
; Lashbrook et al., 1997) and the potential alteration of
covalent and noncovalent interactions between polysaccharide classes. A
recent model of the plant primary cell wall described a network of
cellulose microfibrils that surrounds the cell and is enmeshed in
coextensive matrices of pectic and hemicellulosic polymers, with
additional minor components such as structural proteins (Carpita and
Gibeaut, 1993). During fruit softening, pectins (Brady, 1987; Fischer
and Bennett, 1991) and hemicelluloses (Lashbrook et al., 1997)
typically undergo solubilization and depolymerization that are thought
to contribute to wall loosening and disintegration, although the
relative extent and timing vary between species.
Extensive research has addressed the cell wall changes that occur in
ripening tomato fruit, and many reports have focused on the
considerable pectin degradation that coincides with softening and
expression of the pectin hydrolase PG (Huber, 1983; Hobson and
Grierson, 1993). Early models implied that PG-catalyzed pectin degradation represented the fundamental process underlying fruit softening (Crookes and Grierson, 1983); however, molecular genetic approaches subsequently revealed that PG-dependent pectin degradation is not essential for fruit softening (Smith et al., 1988; Giovannoni et
al., 1989), but may play a role in other aspects of fruit quality (Kramer et al., 1990; Schuch et al., 1991). This suggests that other
wall polymers contribute significantly to fruit firmness, and recent
studies have examined the extent of hemicellulose degradation during
fruit ripening (for review, see Lashbrook et al., 1997). The principal
hemicellulose in dicotyledons is xyloglucan, and it has been
demonstrated that xyloglucan undergoes substantial depolymerization in
many ripening fruit, including tomato (Sakurai and Nevins, 1993;
Maclachlan and Brady, 1994).
Xyloglucan coats and cross-links cellulose microfibrils (Hayashi and
Maclachlan, 1984; McCann et al., 1990), and disruption of the
cellulose/xyloglucan matrix may be a key element in regulating wall
integrity. Additional hemicellulosic polymers, including xylans,
arabinoxylans, mannans, and galactoglucomannans, have been detected in
different fruit species; however, these are typically minor components
and turnover of these polysaccharides during ripening remains
relatively unexplored.
In addition to the depolymerization of both pectic and hemicellulosic
polymers, a characteristic feature of ripening fruit is the loss of
neutral sugars from the cell wall, primarily Gal and Ara (Gross and
Sams, 1984). This has been associated with a decrease in neutral sugar
concentration of both pectic and hemicellulosic polysaccharides (Gross
and Wallner, 1979; McCollum et al., 1989; Kojima et al., 1994),
although this and the relative extent of neutral sugar loss appear to
vary between species.
Tomato has served as a model for cell wall disassembly during ripening
because of the extensive literature related to modification of
polysaccharide structure and composition, and because there are several
ripening mutants that can exhibit dramatically reduced softening and
have therefore been used as genetic tools to investigate ripening-related processes (Ng and Tigchelaar, 1977; Mitcham et al.,
1991; Maclachlan and Brady, 1994). Tomato fruit, however, generally
develop from the preripe to the overripe stage over a period of
approximately 10 to 18 d, depending on the variety. In contrast,
fruit of the Charentais variety of melon (Cucumis melo cv
Reticulatus F1 Alpha) undergo remarkably rapid softening; vine-ripened
fruit typically undergo an equivalent transition from the preripe to
overripe stages in 24 to 48 h. Therefore, Charentais melons may
represent an excellent model system in which to examine the temporal
sequence of cell wall disassembly.
The rapid softening of Charentais melons also raised the question of
whether it involved characteristic wall modifications that have been
reported in other fruit (including other melon varieties; Lester and
Dunlap, 1985; McCollum et al., 1989), but at a much accelerated rate,
or whether a unique pattern of wall degradation exists in this variety.
Furthermore, the large fruit provide an abundant source of material at
a uniform stage of development for extraction of substantial quantities
of cell wall material, nucleic acids, and protein from a single fruit.
We describe the process of wall disassembly in ripening Charentais
fruit in terms of cell wall composition and degradation of
hemicellulosic and pectic polymers, and identify features that are
associated with early and later stages of melon fruit softening.
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MATERIALS AND METHODS |
Charentais melon (Cucumis melo cv Reticulatus F1 Alpha)
flowers were tagged on the day of pollination and a developmental series of fruit was subsequently harvested at defined DAP or at specified ripening stages: IG, attaining full size between 20 and 26 DAP; MG, full size between 30 and 34 DAP, pale green rind, and no
detectable internal ethylene or discernible softening; R1, the onset of
ripening with detectable levels of ethylene production and < 10%
loss of flesh firmness; R2, approximately 30% loss of flesh firmness,
10 to 30 µL L
1 internal ethylene; R3, 50 to
75% of maximal softening, internal ethylene concentration in excess of
80 µL L1; and R4, overripe, orange rind,
disintegrated and waterlogged flesh, and fruit often split open at the
blossom end. The internal concentration of ethylene was determined with
the fruit attached to the vine, as described in Hadfield et al. (1995).
Equatorial sections were cut from the fruit and the flesh firmness was
measured at points 2 cm from the junction of the rind and the outer
pericarp using a 0.8-mm-diameter probe attached to a firmness tester
penetrometer (Western Industrial Supply, San Francisco, CA). The flesh
was then frozen in liquid N2 and stored at
80°C.
Isolation of Cell Walls and Determination of Autolytic
Activity
Approximately 50 g of frozen outer pericarp tissue from each
fruit developmental stage was boiled with 250 mL of 95% ethanol for 30 to 45 min, homogenized in a blender (Waring), and filtered through
Miracloth (Calbiochem). Insoluble material was washed sequentially with
500 mL of boiling ethanol, 500 mL of chloroform:methanol (1:1, v/v),
and 500 mL of acetone; oven-dried at 30°C; and stored in an
auto-desiccator (RPI, Mount Prospect, IL), yielding the crude cell wall
extract, alcohol insoluble solids. Subsequent assessment of autolytic
activity of the samples and sequential chemical extraction are
described below and shown in Figure 1.
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| Figure 1.
Sequential chemical-extraction protocol used for
the preparation and isolation of Charentais melon fruit cell wall
fractions.
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A variety of protocols for isolating autolytically inactive cell wall
material have been developed that rely on phenol-acetic acid:water
(2:1:1, v/v) or Tris-buffered phenol to inactivate endogenous cell
wall-bound enzymes (Huber, 1992; Redgwell et al., 1992; Huber and
O'Donohue, 1993). Other reports have found that extraction of tissue
in boiling ethanol can successfully prevent the autolytic generation of
reducing end groups during subsequent experiments with AIS preparations
(Carrington et al., 1993). The AIS residue isolated from melon,
described above, was assayed for autolytic activity to confirm the
effectiveness of the ethanol extraction in providing autolytically
inert material. Approximately 30 mg of AIS from both R1 and R4 stages
was resuspended in 5 mL of 40 mM
NaC2H3O2,
pH 5.0, several drops of toluene were added, and the mixture was
incubated at 30°C for up to 24 h. As a control, identical
experiments were performed using pericarp tissue homogenized in 40 mM
NaC2H3O2,
pH 5.0, or in ethanol at room temperature.
Aliquots (250 µL) were extracted over the 24-h time course, passed
through 0.45-µm filters (Uniflo, Schleicher & Schuell), and assayed
for TS using the phenol-sulfuric acid method (Dubois et al., 1956).
Whereas a linear increase in the solubilization of TS from the
buffer-homogenized material was observed over 24 h, a minimal
release (approximately 8-fold less) was detected from
room-temperature-ethanol-extracted AIS, and no increase was detected
from boiling-ethanol-extracted cell wall material (data not shown). It
was concluded that the melon AIS isolated using boiling ethanol was
autolytically inactive.
Cell Wall Chemical Extraction and Fraction Analysis
The AIS residue from each sample was homogenized in 75 mL of
0.02% NaN3 for 4 h at room temperature and
centrifuged at 6,000g for 20 min, and the pellet was
extracted twice with 25 mL of 0.02% NaN3 for 20 min. The supernatants were combined, lyophilized, resuspended, and
stirred in 15 mL of DMSO:water (9:1, v/v) for 24 h to solubilize
any starch (although it has been reported that melons accumulate
little, if any, starch [Pratt, 1971]), and then centrifuged at
20,000g for 20 min and filtered through Miracloth (Calbiochem). The pellet was washed with 50 mL of DMSO:water, followed
by three washes with 30 mL of 80% ethanol and three washes with 30 mL
of acetone, oven-dried at 30°C, and stored in a desiccator. This was
designated the water-soluble fraction. Aliquots of the supernatants
from the DMSO extraction were combined with 2 volumes of ice-cold 95%
ethanol to precipitate polymeric material, stirred at 1°C for 12 h, centrifuged at 6,000g for 30 min, and filtered, and any
pelleted material was washed with 150 mL of ice-cold 95% ethanol.
Virtually no polysaccharide was extracted with DMSO from any
developmental stage, and analysis of the sugar composition of any
detectable pellet by GC (for methodology, see below) showed a ratio of
neutral sugars similar to that of the total water-soluble fraction,
indicating that differential solubilization of specific cell wall
polymers by DMSO had not occurred.
The water-insoluble pellets were homogenized with 50 mM
CDTA and 50 mM
NaC2H3O2,
pH 6.5, stirred vigorously at room temperature for 12 h, filtered
and centrifuged as for the water-soluble fraction, and re-extracted
with an additional 50 mL of the same solution for 12 h. The
supernatants were combined and exhaustively dialyzed (Mr cut-off 6-8 kD) against distilled water for
2 d at 5°C, lyophilized, and stored in a desiccator. The
CDTA-insoluble pellet was washed once with 50 mL of the CDTA buffer
solution, twice with 100 mL of 80% ethanol, and twice with 100 mL of
acetone, and was then extracted with 50 mL of 50 mM
Na2CO3 and 20 mM NaBH4 at 1°C as for the CDTA
extraction. The 4 and 24% KOH fractions were obtained by sequential
chemical extraction of
Na2CO3-insoluble material, as described in Maclachlan and Brady (1994).
Samples of the crude cell wall (AIS) and sequentially extracted
fractions were assayed for total UA (Ahmed and Labavitch, 1977) and TS
using a modification of the phenol-sulfuric acid assay (Dubois et al.,
1956), in which the lyophilized sample was first dissolved in sulfuric
acid/water as for the UA assay above. Aliquots were removed and the
reagents for the phenol-sulfuric acid assay added in the following
order: sulfuric acid, phenol, and water, using Glc as a standard. For
GC analysis of the AIS and extracted fractions, aliquots were
hydrolyzed with 2 N trifluoroacetic acid at 121°C for
1 h, and the resulting hydrolysates were converted to alditol
acetates (Blakeney et al., 1983). Analysis of neutral sugar composition
used a gas chromatograph (model 8320, Perkin-Elmer) with a 30-m × 0.25-mm i.d. capillary column (model DB-23, J & W Scientific, Folsom,
CA) as described in Carrington et al. (1993).
Size-Exclusion Chromatography and Analysis of
Subfractions
Aliquots (approximately 5 mg) of lyophilized water-, CDTA-, or
Na2CO3-soluble polymers
were chromatographed on a size-exclusion column (1.0 × 90 cm) of
Sepharose CL-4B (Pharmacia) eluted in 200 mM
NH4-acetate, pH 5.0. Fractions were collected
(2.0 mL) at a flow rate of 17 mL h1 and assayed
for UA by the m-hydroxydiphenyl method (Blumenkrantz and
Asboe-Hansen, 1973) and for TS as described in Dubois et al. (1956).
Although the NH4-acetate buffer has been reported
to reduce aggregation of pectins during chromatography (Mort et al.,
1991), it was observed to cause interference with the UA assay. This was avoided by leaving the fractions at room temperature for 48 to
72 h before analysis, presumably allowing a portion of the buffer
to volatilize.
Size separation of 4 and 24% KOH-soluble polymers was with a Sepharose
CL-6B column (1.0 × 90 cm, Pharmacia) eluting with 0.1 N NaOH. Aliquots of lyophilized extract (2.0 mg) were
applied to the column and 2-mL fractions were collected at a flow rate of 18 mL h1, neutralized with glacial acetic
acid, and assayed for TS as above or for xyloglucan as described in
Maclachlan and Brady (1994). For neutral sugar compositional analysis,
subfractions were pooled, lyophilized, washed with 20 mL of ice-cold
95% ethanol to reduce the salt concentration, lyophilized again, and
then the component sugars were hydrolyzed, derivitized to alditol
acetates, and analyzed by GC as described above.
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RESULTS AND DISCUSSION |
Correlation of Flesh Softening with Internal Ethylene
Concentration
The large central cavity in Charentais melons allows sampling and
accurate determination of internal gas concentrations with no apparent
disturbance to normal fruit development. Using such techniques it has
previously been reported that Charentais melons exhibit a respiratory
climacteric during ripening that coincides with an abrupt increase in
internal ethylene concentration (Hadfield et al., 1995). Figure
2 shows a typical profile of increasing internal ethylene concentration concomitant with a loss of flesh firmness during ripening. No substantial softening was observed between
IG and R1. Rapid loss of firmness was initiated at R1, corresponding to
the first detectable rise in internal ethylene, and this trend
continued through R4.
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| Figure 2.
Flesh firmness (black bars) and internal ethylene
concentration (white bars) of Charentais melon fruit at defined
developmental stages. kgf, Kilograms force.
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Over the course of three seasons, it was observed that the fruit did
not ripen synchronously, based on the number of DAP, but that
individual fruit typically made the transition from MG to R4 within 24 to 48 h. Similarly, a dramatic loss of firmness was observed 24 to
48 h following treatment of preripe fruit with ethylene (data not
shown). As described in "Materials and Methods," the fruit shown in
Figure 2 were defined based on representative stages of development for
these experiments, rather than on DAP. As far as we are aware,
softening of vine-ripened Charentais melon fruit occurred more rapidly
than has been reported for any other species to date, and therefore
represent an excellent system in which to examine the temporal sequence
of cell wall modification. Furthermore, the large fruit size allowed
the use of a series of individual fruit at characterized uniform
developmental stages as sources of cell wall material.
Yield and Composition of Cell Wall Fractions
Autolytically inactive crude cell wall extracts, termed AIS, were
assayed for both UA and TS content and then subjected to sequential
chemical extraction (Fig. 1) designed to enrich for particular
subclasses of cell wall polymers. The water-soluble fraction is
typically thought to include polymeric material that has been
solubilized from the cell wall, whereas the CDTA- and Na2CO3-soluble fractions
are generally considered to be enriched for ionically and covalently
bound pectins, respectively. Polymers extracted with 4% KOH usually
contain a high proportion of hemicellulosic polysaccharides, whereas
24% KOH is necessary to extract hemicellulose-rich polymers that are
tightly bound to the cell wall, and to cellulose microfibrils in
particular. The residual material following 24% KOH extraction is
comprised of mainly cellulose and a small quantity of associated
polysaccharides. The total mass of AIS extracted from different
ripening stages did not change and comprised about 1.5% of the fresh
weight of the fruit. Fractions derived from the AIS were also assayed
for UA and TS (Table I).
GC analysis of the noncellulosic neutral sugar composition of both the
AIS and wall fractions (Table II) was
used to determine whether changes in the proportion of specific sugars
could be correlated with early or late stages of softening, and to
provide a basis for association of particular classes of polysaccharide with specific ripening stages or cell wall fractions.
Yield and Composition of Pectic Fractions
In common with previous analyses of other melon varieties
(McCollum et al., 1989; Simandjuntak et al., 1996), the total
polyuronide content of the cell wall showed little change either prior
to or during ripening. However, there was a substantial change in the
relative solubility of pectins among the cell wall fractions as
softening proceeded (Table I). At the IG stage only 19% of the total
UA was water soluble, whereas the CDTA- and
Na2CO3-soluble fractions
together accounted for 66% of the total polyuronides. The relative
proportion of pectins among these three fractions showed little change
between IG and R1, although there was a small increase in the UA
content of the
Na2CO3-soluble fraction and a corresponding decrease in the CDTA-soluble fraction.
Between R1 and R2, an increase in the proportion of CDTA-soluble
pectins was accompanied by a marked reduction in
Na2CO3-soluble UA, which
then decreased even more substantially between R2 and R3. The quantity
of water-soluble UA increased at R3 and again to a greater extent
between R3 and R4, when it comprised 40% of the total AIS. Similar
losses of polyuronides were detected in the 4 and 24% KOH-soluble
fractions between R2 and R3, although these comprised less than 6% of
the total AIS UA. Small quantities of polyuronides were detected in the
residual cell wall material, but the relative amounts did not change
during ripening.
Assay of TS using the phenol-sulfuric acid method is primarily to
quantify noncellulosic neutral sugars, although there is a low degree
of sensitivity to galacturonosyl residues, and this was used to monitor
TS in the cell wall fractions. TS was relatively consistent through the
R1 stage (Table I), followed by a consistent decline at the onset of
softening between R1 and R4. The relative amount of TS in the water-
and Na2CO3-soluble
fractions was greater than that in the CDTA-soluble fraction and showed
changes similar to those of UA, increasing and decreasing in the later
ripening stages, respectively. The ratio of TS to UA was considerably
greater in the
Na2CO3-soluble fraction
than in the CDTA-released polymers (1:1.2 and 1:5.3 at IG,
respectively), suggesting a greater degree of branching and neutral
sugar substitution of the covalently bound pectins. A high ratio of
neutral sugar to UA in the
Na2CO3-soluble polymers
suggests an origin in the primary cell wall, whereas a low ratio in the
CDTA-extracted fraction is indicative of pectins from the middle
lamella, as has been reported in other species (Seymour et al., 1990;
Cutillas-Itturralde et al., 1993). No changes in the relative
proportion of TS were apparent during ripening in the CDTA-soluble
fraction.
Analysis of the noncellulosic neutral sugar composition of the AIS
(Table II) revealed that the predominant neutral sugar was Gal at all
stages, although a 27% decrease of the Gal mol % concentration
occurred between the IG and R4 stages. Similarly, the concentration of
Ara decreased by 31% over the same developmental period. Conversely,
the mol % of Xyl increased primarily in the later ripening stages,
whereas Glc increased from IG to R2 and decreased between R2 and R3.
The loss of Gal and Ara from the AIS is typical of many fruit
species and has been reported in other melon varieties (Gross and Sams,
1984; McCollum et al., 1989; Simandjuntak et al., 1996), although
the polymeric origin has not been investigated and these sugars are
constituents of both hemicelluloses and pectins.
The pectin-rich (water-, CDTA-, and
Na2CO3-soluble) fractions
contained a high proportion of Ara and Gal. In the water-soluble fraction, Ara and Gal showed opposite patterns of relative abundance, increasing and decreasing, respectively, in the later softening stages.
In the water-soluble polymers, as in the CDTA-soluble fraction and the
AIS, Glc showed a transient increase from 11.6 mol % at IG to 13.7 mol
% at R1, before declining to comprise only 8 mol % at R4. Glc, if
present, is considered to be only a minor component of pectins,
suggesting that the reduction in Glc concentration represented a
relative increase in the proportion of water-soluble polyuronides at R3
and R4. This is supported by the higher mol % values of Ara and Rha,
sugars commonly found in the backbone and side chains of pectins, and
the increase in relative abundance of water-soluble UA at these later
ripening stages (Table I). The proportion of Xyl increased marginally prior to softening, thereafter remaining constant.
A similar distribution of neutral sugars was detected in the
CDTA-soluble fraction, with high concentrations of Ara and Gal, which
again decreased during ripening, particularly between R3 and R4.
Relative to the other pectin-rich fractions, there was a high ratio of
Rha to Gal and Ara, possibly indicative of a less substituted
rhamnogalacturonan pectic backbone, typical for pectins from the middle
lamella (Brett and Waldron, 1996). The proportions of Xyl, Man, and Fuc
all increased late in ripening.
A very high mol % of Gal was evident in
Na2CO3-soluble polymers,
which, together with Rha, declined through ripening, whereas the
relative concentrations of Xyl, Man, and Glc increased. This coincided
with a reduction in the relative amounts of UA (Table I) and
indicates a loss of pectins from this fraction at R3 and R4. The
relatively high ratio of Gal to Rha suggests a greater degree of
substitution of the pectin backbone with Gal-rich side chains.
Taken together, these data suggest that the relative solubility of
pectic polysaccharides exhibited no change between IG and R1. The loss
of covalently bound pectins between R1 and R2 coincided with an
increase in chelator-extractable pectins, whereas increases in
water-soluble polyuronides occurred in the last two softening stages,
R3 and R4.
Yield and Composition of Hemicellulosic and Residual Fractions
The 4 and 24% KOH and residue fractions together contained only
minor quantities of UA (Table I) and these were mostly associated with
the cell wall residue (
-cellulose). The greatest proportion of TS
was extracted by 24% KOH, and this fraction showed a two-step reduction, declining 10% from MG to R1, followed by a 10% reduction between R2 and R4, although the overall proportion of TS with respect
to the AIS showed a decline from MG through ripening. The TS detected
in the residue increased from IG to R1, showed a reduction at R2, and
remained constant through the overripe stage, although the proportion
of TS relative to the TS in the AIS increased until R1 and showed
little change through ripening. The 4% KOH-extracted material also
showed a transient increase in the concentration of TS, peaking at R1,
whereas the proportion of the overall TS remained relatively constant.
In the 4 and 24% KOH-soluble fractions, the principal neutral sugars
were Xyl, Gal, and Glc (Table II). The proportion of Xyl was especially
high in the 4% KOH-soluble fraction and decreased dramatically during
softening (46% reduction from IG to R4), particularly prior to and
coincident with the onset of softening. The levels of Glc increased
throughout ripening, whereas those of the other sugars remained
relatively constant or showed no clear pattern. Conversely, the Gal mol
% decreased throughout ripening in the 24% KOH-soluble polymers, and
values for Glc showed no apparent trend. The mol % of Xyl and Ara
increased and decreased, respectively, from IG through ripening. In the
residual cell wall material, Glc represented by far the predominant
neutral sugar, almost certainly the result of partial hydrolysis of
cellulose by the TFA treatment, with no change in relative abundance
during softening. However, high levels of Man were present and declined
from R2 to R4, in addition to smaller quantities of Gal, which showed
no change in the ripening series.
Gel Chromatography of Pectic Cell Wall Fractions and Compositional
Analysis of Subfractions
To examine changes in the molecular mass of the
constituent polysaccharides during softening, cell wall fractions were
subjected to size-exclusion chromatography. Column fractions were
assayed specifically for UA in the pectin-rich fractions and for
xyloglucan in the hemicellulose-rich fractions, as well as for TS to
assess the presence of additional polysaccharides representing other cell wall components. Following the identification of particular size
classes of polymers, indicated by the presence of distinct peaks,
subfractions were pooled and their neutral sugar compositions were
examined to establish whether particular polysaccharide types could be
assigned to specific size fractions.
Water-Soluble Fraction
Size fractionation of water-soluble polymers on a Sepharose CL-4B
column (dextran fractionation range of 30-5000 kD) gave a single broad
peak of UA with an average molecular mass close to the 260-kD dextran
marker (Fig. 3) and a trace amount of UA in the void volume, suggesting that the pectins had been well fractionated. The profile of TS was also within the fractionation range, and at least two additional peaks were detected at approximately 11 kD and in the total volume. Between MG and R1 there was an apparent
increase in the molecular size of the polyuronide peak, and an increase
in the proportion of high-molecular-mass pectins fractionating at or
near the void volume. From R2 to R3, a gradual decrease in
molecular mass occurred until the profile of polyuronides at R3
appeared similar to that at IG. However, in the overripe stage (R4) a
complete loss of high- and intermediate-molecular-mass pectins was
indicated by a single symmetrical peak of polyuronides, corresponding
to the 38-kD marker.
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| Figure 3.
Gel-filtration profiles of water-soluble
polysaccharides derived from six developmental stages of Charentais
melon fruit fractionated on Sepharose CL-4B. Column fractions (2.0 mL)
were assayed for UA content ( ) using the
m-hydroxybiphenyl method (Blumenkrantz and Asboe-Hansen,
1973) or for TS ( ) using the phenol-sulfuric acid method (Dubois et
al., 1956). Dextran molecular-mass markers (kD) used as a calibration
scale are shown at the top. i through iv, Subfractions described in the
text; Vo, void volume; Vt, total volume.
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Four peaks were identified based on TS and UA assays, and subfractions
were pooled for neutral sugar composition analysis (Fig. 3).
Subfraction i was comprised of polymers eluting in the void volume and
represented only a minor portion of the total polysaccharides in this
fraction, showing no change in relative prominence during ripening
until R4, when no high-molecular-mass polymer remained. The neutral
sugar composition was also similar between IG and R1, comprising mainly
Ara (60-70%), with smaller amounts of Glc, Gal, and Man (Table
III).
Subfraction ii corresponded to the major peak of the polyuronide and TS
profiles from IG, R1, and R4, but comprised lower-molecular-mass polymers at the latter stage following a large decrease in polymer size. A high relative ratio of UA to TS was evident, and neutral sugar
analysis revealed a large mol % of Gal, together with Ara and Glc. The
concentrations of Glc and Gal were greater at R1 than at IG, and,
although this subfraction at R4 also comprised mainly Gal, Ara, and
Glc, an increased proportion of Xyl was also detected.
Subfraction iii had a greater ratio of TS to UA and consisted of Glc,
Xyl, Man, and Gal. The proportion of Ara increased at R1, coincident
with a greater prominence of the peak, whereas the amount of Xyl
decreased. By R4, the concentration of Ara had decreased, whereas that
of Man had increased.
Subfraction iv represented relatively low-molecular-mass polymers that
were comprised mainly of Glc, Gal, and Man. Higher levels of Ara were
seen at R1, coincident with a substantial peak increase, whereas the
proportion of Xyl increased substantially from 5% at R1 to 43% at R4
as the peak showed a relative decline.
Assignment of subfraction numbers to particular fractions was
complicated by shifts in the elution profiles. For example, subfraction
ii spanned the peak of polyuronides at IG, the lower-molecular-mass side of the peak at R1, and the higher-molecular-mass polyuronides at
R4. Thus, the subfractions may represent the same classes of polysaccharides following changes in molecular mass or they may represent different classes of polymers. However, the neutral sugar
composition of subfraction iii at R4 was more similar to that of
subfraction iii of R1 than to ii of R1, suggesting that depolymerization of the principal polymers in subfraction iii was
substantially less than that indicated for the polyuronides.
CDTA-Soluble Fraction
This fraction was characterized by a peak of excluded
high-molecular-mass pectins, and a broad population of polymers peaking between 260 and 500 kD (Fig. 4). The
gel-filtration profile revealed an increase in the relative amount of
excluded material until R2, followed by a marked decrease through the
later ripening stages. A downshift in the polyuronide profile was seen
in the last two stages, resulting in an average molecular mass of
approximately 260 kD at R4 and a small proportion of polysaccharide in
the void volume. In general, the profiles of TS and polyuronides
paralleled each other, with no appearance of additional peaks.
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| Figure 4.
Gel-filtration profiles of CDTA-soluble
polysaccharides derived from six developmental stages of Charentais
melon fruit. Details are as described for Figure 3.
|
|
Na2CO3-Soluble Fraction
The chromatographic separations of
Na2CO3-soluble polymers in
common with those of the CDTA-solubilized material, resulted in similar
profiles when assayed for UA or TS (Fig.
5), although the ratios of UA to TS
varied across the fractionation range of the column, suggesting a
greater association of neutral sugars with particular molecular-size
classes. Four peaks (i, ii, iii, and iv) were identified, corresponding
to fractions in the void volume, between the void volume and the 500-kD
marker, close to the 260-kD marker, and between the 11- and 38-kD
markers, respectively. Subfractions spanning these peaks were pooled
for neutral sugar analysis (Table III).
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| Figure 5.
Gel-filtration profiles of
Na2CO3-soluble polysaccharides derived from six
developmental stages of Charentais melon fruit. Details are as
described for Figure 3.
|
|
The composition of subfraction i remained relatively consistent
throughout the series, comprising primarily Gal and Ara with some
additional Rha and Glc, and the profile exhibited no clear pattern of
increase or decrease through ripening. Subfraction ii had an especially
high ratio of TS relative to UA, and corresponded to
large-molecular-size polymers, which showed a decrease in molecular mass between R2 and R4. GC analysis revealed an extremely high proportion of Gal (more than 70 mol %) at IG and R1, whereas by R4 a
relative reduction of 20% had occurred. The proportion of Rha
increased coincident with the loss of Gal at R4.
The neutral sugar composition and large molecular size are consistent
with the profile of subfraction ii, depicting a loss of Gal-rich
neutral sugar side chains from rhamnogalacturonan I, a common pectic
polymer with a backbone of alternating -1,4-galacturonosyl and
-1,2-rhamnosyl residues, which has been described in other ripening
fruit (Seymour et al., 1990; Redgwell et al., 1992). The elution point
of the peak changed only slightly through ripening. Peak iii had a
similar composition to peak ii, comprising primarily Gal and Ara, but
with a ratio of TS to UA approximately one-half that of subfraction ii
and a greater proportion of Rha, suggesting the presence of
less-substituted pectins. High levels of Rha were detected at IG and
declined in later stages, coincident with an increase in the mol % concentrations of Gal and Xyl and the decreased relative prominence of
the peak.
Subfraction iv comprised lower-molecular-mass polymers and at IG,
exhibited a low ratio of TS to UA and a high proportion of Rha and Ara
(14 and 55 mol %, respectively). By R1 the ratio had increased, as had
the relative amount of Gal (approximately 9-50 mol %), although Ara
decreased to a similar degree, suggesting that depolymerization of
higher-molecular-mass Gal-rich polymers contributed to this
subfraction. The relative size and profile of peak iv changed little
during softening, although the proportions of Xyl and Ara increased and
decreased, respectively. The high level of Xyl in this subfraction
(Table III) and in the CDTA-extracted polymers (Table II), especially
at R4, has previously been reported in other fruit (Seymour et al.,
1990; Martin-Cabrejas et al., 1994), where it was suggested to
originate from xylans and proposed to reflect the existence of
xylan-pectin complexes (Martin-Cabrejas et al., 1994). In general, the
size distribution of
Na2CO3-extracted polymers
showed a relative downshift only in the last ripening stage.
During ripening the pectin concentration of the cell wall (AIS)
remained relatively constant, showing an approximately 10% decrease
between the pre- and overripe stages. However, there was clear evidence
of pectin solubilization and depolymerization during softening. At the
onset of softening, substantial solubilization of polyuronides from the
Na2CO3-soluble fraction
coincided with an increase in chelator-soluble pectins, suggesting that
once released from the wall, a proportion of the covalently bound
pectins became ionically associated. Subsequently, at the R3 stage,
continued losses from the
Na2CO3-soluble fraction
corresponded to large increases in polyuronides and TS in the
water-soluble fraction that continued until R4. This, in addition to
the neutral sugar composition of the fractions, suggests solubilization
of covalently bound pectins into the water-soluble fraction, as has
been reported in tomato (Carrington et al., 1993). At R4 the
water-solubilized pectins underwent a dramatic downshift in molecular
mass. Depolymerization of pectins in the
Na2CO3- and CDTA-soluble
fractions was more modest, but also occurred primarily late in
ripening.
The exact mechanisms underlying such pectin metabolism during ripening
remain unclear and may reflect the cumulative effects of both enzymatic
and nonenzymatic processes. In many fruits, including tomato, pectin
degradation has been associated with the action of endo-PG.
However, in melon a decrease in the molecular mass of polyuronides has
previously been reported to occur in the apparent absence of PG
activity (McCollum et al., 1989), and other melon cultivars are
described as lacking PG activity. In an accompanying paper (Hadfield et
al., 1998), we report the identification of a Charentais melon PG gene
family and describe the expression of three PG mRNAs, primarily in the
later stages of ripening fruit. This suggests that PG may participate
in pectin metabolism in melon, particularly in pectin depolymerization
evident in the late stages of fruit softening. However, solubilization
of covalently associated pectins was apparent prior to the detection of
PG mRNAs, suggesting that PG-independent as well as PG-dependent
processes may contribute to overall pectin disassembly.
It has been suggested that pectin solubilization may result from the
loss of galactosyl residues in the form of the neutral Gal-rich side
chains of rhamnogalacturonans (Seymour et al., 1990; Redgwell et al.,
1992). Such degalactosylation is typically seen in ripening fruit, and
although the structural relevance has not been demonstrated, galactans
may be integral to the formation of a cohesive pectin matrix,
cross-linking pectin molecules with each other, as well as with
hemicelluloses and other cell wall components.
The loss of Gal can occur independently of PG activity (Carrington et
al., 1993), suggesting the involvement of other classes of enzymes such
as 
-galactosidases/-galactanases, which have been associated with
many ripening fruits (Ross et al., 1993, 1994; Carey et al., 1995;
Lashbrook et al., 1997), and which may act on several classes of
polysaccharides, including the galactan side chains of
rhamnoglacturonans (Pressey, 1983). Loss of galactans has been
demonstrated to accompany or even precede increased solublization of
polyuronides (Gross and Wallner, 1979; Kim et al., 1991), and purified
or partially purified -galactosidase has been shown to catalyze an
apparent decrease in the molecular size of pectins in vitro in tomato
(De Veau, 1993) and melon fruits (Ranwala et al., 1992).
Gel Chromatography of Hemicellulosic Cell Wall Fractions and
Compositional Analysis of Subfractions
4% KOH-Soluble Fraction
Hemicellulosic material was size-fractionated on a Sepharose CL-6B
column (dextran fractionation range of 10-1000 kD), and fractions were
assayed for xyloglucan and TS. Size separation of 4% KOH-soluble
polymers (Fig. 6) revealed a large peak
of excluded polysaccharides (subfraction i) that showed a relative
decline from R2 to R4. Compositional analysis indicated that
at the IG and R1 stages, prior to its loss, subfraction i was
particularly rich in Gal (40 mol %), with smaller amounts of Ara and
Glc (Table III) and approximately 5 mol % Rha. The presence of
substantial amounts of Gal and Ara in discrete, high-molecular-mass
hemicellulosic polymers (> 1000 kD) has been described previously
(Talbott and Ray, 1992). This was attributed to the presence of large
arabinogalactan molecules, possibly comprised of glycosidically linked
arabinan and galactan subunits.
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| Figure 6.
Gel-filtration profiles of 4% KOH-soluble
polysaccharides derived from six developmental stages of Charentais
melon fruit and fractionated on Sepharose CL-6B. Column fractions (2.0 mL) were assayed for xyloglucan () as described in Maclachlan and Brady (1994) or for TS () using the phenol-sulfuric acid method (Dubois et al., 1956). Details are as described for Figure 3.
|
|
Arabinogalactans or arabinans and galactans have been identified as
side chains of rhamnogalacturonans or proposed to exist as free
macromolecules, possibly forming a separate layer surrounding the
xyloglucan/cellulose network (Talbott and Ray, 1992). A prominent peak
of xyloglucan co-eluted in the void volume and showed a similar pattern
during softening to that of the arabinogalactan-rich TS peak,
exhibiting apparent degradation in the last two ripening stages.
Relatively few reports exist of similarly extracted and fractionated
hemicellulosic polymers from fruit, since the hemicellulose fraction is
typically isolated using much higher concentrations of alkali in a
single step, rather than in two steps with an initial lower
concentration as described here.
Cutillas-Iturralde et al. (1994) assayed a similar wall fraction,
extracted with 0.5 M KOH from ripening persimmon, for
xyloglucan and TS and described an excluded peak of TS. However, this
peak was not coincident with appreciable quantities of xyloglucan, which generally fractionates within the column range. Hemicelluloses extracted from vegetative tissues with low concentrations of alkali and
size-fractionated have been reported to exhibit a similar pattern
(Nishitani and Masuda, 1983; Lorences and Zarra, 1987; Lorences et al.,
1987); xyloglucan and a population of high-molecular-mass polymers
assayed for TS, often rich in Ara and Gal, are resolved as discrete
peaks or with excluded xyloglucan representing a minor component of the
total xyloglucan in that fraction. Figure 6 shows that the xyloglucan
in the void volume comprised an atypically large proportion of the
total xyloglucan in this fraction, but that the ratio of excluded to
included xyloglucan decreased dramatically at R3 and R4. At R4, in
addition to a reduction in the relative size of the peak, the
proportion of Gal had decreased 10 mol %, whereas Glc showed an
equivalent increase.
Earlier models of the primary cell wall proposed a covalently linked
macromolecule of noncellulosic polysaccharides: xyloglucan to
arabinogalactans, arabinogalactans to pectins, and pectins to
structural cell wall proteins. (Keegstra et al., 1973). More recently,
the idea of predominantly noncovalent associations between matrix
polymers has received attention (Varner and Lin, 1989; Talbott and Ray,
1992; Carpita and Gibeaut, 1993). It has been pointed out that almost
all of the polyuronide component of the cell wall can be chemically
extracted under relatively mild conditions, without the concomitant
release of xyloglucan, arabinogalactan, or other hemicelluloses
(Talbott and Ray, 1992). However, the possibility of covalent bonds
between a small subset of the pectin, arabinogalactan, and xyloglucan
populations should not be excluded. The coelution of an unusually
high-molecular-mass xyloglucan with a Gal- and Ara-rich polymer and a
small amount of Rha following a chemical extraction expected to disrupt
alkali-labile linkages might suggest a covalent association between
large arabinogalactan polymers and xyloglucan. The formation of such a
complex could prevent a portion of the xyloglucan from chromatographing
within the column range, as is apparent in Figure 6.
A second broad peak of xyloglucan with an average molecular mass of
between 260 and 500 kD showed a large increase in abundance from IG to
R2, where a slight upshift was detected, followed by depolymerization
at R3 and R4. The neutral sugars in subfraction iii at the peak of this
xyloglucan profile were comprised of mainly Glc, Gal, and Xyl (Table
III), typical components of xyloglucan. The proportions of neutral
sugars were similar to those reported for weak-alkali-solubilized
xyloglucan from bean (Nishitani and Masuda, 1983), and the detectable
levels of Fuc suggest that the xyloglucan in this peak was fucosylated.
The ratio of sugars showed little variation during softening.
The TS profile at IG indicated the existence of two unique and
substantial populations of polysaccharides (Fig. 6, ii and iv) that did
not correspond to the xyloglucan profile. Both peaks declined
dramatically between IG and MG and were largely indistinguishable in
the later ripening stages. Analysis of the neutral sugar composition of
ii (Table III) showed a high proportion of Xyl, which fell from 70 to
35 mol % between IG and R1, coincident with an increase in the
proportion of Glc, Gal, and Fuc, suggesting the loss of a Xyl-rich
polymer and a relative increase in the proportion of xyloglucan in this
fraction prior to ripening. Subfraction iv was also rich in Xyl,
although the mol % did not change to a great extent during ripening.
Glc and Fuc increased between IG and R1, coincident with a slight
decrease in Ara and Xyl and a decline in the overall prominence of the
peak, suggesting an increase in the proportion of xyloglucan and a
relative decrease in xylans. A small amount of UA was detected in this
subfraction (data not shown), suggesting the presence of xylans,
glucuronoxylans, and/or glucuronoarabinoxylans.
Putative xylan-pectin complexes in ripening tomato fruit (Seymour et
al., 1990) and covalent associations between glucuronoxylans and
xyloglucan in olive pulp (Coimbra et al., 1995) have been described,
although little has been reported to suggest a role for xylan
metabolism in cell wall disassembly during fruit ripening.
24% KOH-Soluble Fraction
The polymers in this fraction mostly eluted as a symmetrical
population within the fractionation range of the gel, with a single
broad peak of xyloglucan near the 500-kD dextran marker (Fig.
7). The profile of TS paralleled that of
xyloglucan, however, additional peaks were detected in the void volume
and near the 38-kD marker. The ratio of TS to xyloglucan was greater in
fractions corresponding to lower-molecular-mass xyloglucans (below the
500-kD marker). Subfractions ii and iii consisted almost entirely of Xyl, Gal, and Glc and trace amounts of Ara and Man (Table III), suggesting a relatively pure xyloglucan fractionation. However, Fuc was
not present at detectable levels, suggesting an absence or a much lower
degree of fucosylation of this tightly bound xyloglucan population
compared with the more readily extracted (4% KOH) xyloglucan.
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| Figure 7.
Gel-filtration profiles of 24% KOH-soluble
polysaccharides derived from six developmental stages of Charentais
melon fruit. The superimposed profiles from the xyloglucan assay of R1
and R2 polymers are shown in the inset, top right. Details are as described for Figures 3 and 6.
|
|
A slight upshift in elution profile was seen between IG and R1,
followed by depolymerization of the entire spectrum of xyloglucan polymers at the onset of softening between R1 and R2, which continued throughout ripening. A similar size distribution of xyloglucan was seen
in hemicellulosic extracts from tomato pericarp and locule tissue, and
depolymerization was evident during ripening (Maclachlan and Brady,
1994). Comparisons of these data with other fruit and melon varieties
is difficult, because the equivalent of 4 and 24% KOH-soluble polymers
are typically not considered separately and are
co-chromatographed. Figure 7 demonstrates that the depolymerization of tightly bound xyloglucan was coincident with the early onset of
softening.
Subfraction i consisted primarily of Glc, Gal, and Xyl (Table III),
with a relatively high proportion of Ara (13 mol %) and Man (11 mol
%) at IG. UA was also primarily localized in this fraction (data not
shown). Apart from a slight decline at R4, the relative prominence of
the peak showed little change; however, there was an increase in the
relative abundance of Ara between IG and R1. Furthermore, it appeared
that a tightly bound, high-molecular-mass, Man-rich polymer or polymer
complex underwent depolymerization during ripening, since Man was
detected only in subfraction i at IG, showed distribution throughout
all the subfractions at R1, and was detected mostly in subfraction iv
at R4, where it comprised 26 mol % of the neutral sugars (Table III).
The overall proportion of Man in the AIS did not decrease during
ripening (Table II). It is likely that Man was a component of
glucomannans and/or galactoglucomannans such as those detected in the 4 M KOH hemicellulose fraction of tomato (Seymour et al., 1990) and pineapple (Smith and Harris, 1995) fruit. It has been suggested that glucomannans and/or galactoglucomannans are not degraded
during ripening in tomato (Sakurai and Nevins, 1993); however,
continued glucomannan synthesis during tomato fruit ripening has been
reported (Tong and Gross, 1988), and analysis of cell wall biosynthesis
during tomato ripening also indicated increased incorporation of Xyl
and Man residues into hemicellulosic polymers during ripening (Greve
and Labavitch, 1991). Therefore, it is possible that the apparent
depolymerization of glucomannans in the 24% KOH-soluble material
during ripening (Table III) actually reflects solubilization of
high-molecular-mass, Man-containing polysaccharides followed by de novo
synthesis of lower-molecular-mass glucomannans. Alternatively, since
the Man content of the residue fraction showed a major decrease between
R2 and R3, a population of lower-molecular-mass glucomannans may be
more readily extracted by 24% KOH in the later two ripening stages, R3
and R4.
| |
CONCLUSIONS |
A summary of cell wall changes and potential enzyme activities
during Charentais melon fruit ripening is shown in Figure
8. Pectinase activity is interpreted to
result from the cumulative action of a range of pectolytic enzymes,
including those targeting side-chain and backbone linkages of complex
pectin molecules. In Charentais melon PG mRNAs were abundant at
R2, whereas a peak of pectinase activity was apparent at R3 (see
accompanying paper, Hadfield et al., 1998), suggesting that this later
peak was the result of PG activity. Studies in tomato also suggest a
substantial time lag between the appearance of PG mRNA and either
protein or enzyme activity (Brady et al., 1982, 1985; Tucker and
Grierson, 1982; Speirs et al., 1989).
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| Figure 8.
Model of the temporal sequence of cell wall
changes, pectinase activity, and PG mRNA expression in ripening
Charentais melon fruit at defined developmental stages. XG,
Xyloglucan.
|
|
Previous reports have associated disassembly of the pectin
macromolecular matrix with loss of wall galactans, probably through the
action of -galactosidases/-galactanases (De Veau et al., 1993;
Carey et al., 1995; Lazan et al., 1995). Our data suggest that early
loss of Gal coincided temporally with an early peak of pectinase
activity in IG fruit, which showed a gradual decline through R2 and may
reflect the presence of -galactosidase/-galactanase activity. In
addition to the loss of Gal from pectic polymers, covalently bound
pectins appear to undergo solubilization prior to or coincident with
the appearance of PG mRNAs.
A model of pectin disassembly in Charentais melon can therefore be
proposed in which loss of Gal, possibly from pectin-associated galactan
side chains, contributes to early PG-independent solubilization of
covalently linked pectins prior to or coincident with the onset of
significant fruit softening. Solubilized pectins are subsequently subject to deglycosylation and depolymerization in the later stages of
ripening through the action of endo- and/or
exo-acting PG. The decreases in the molecular mass of
polyuronides in all of the pectic fractions in the later stages of
ripening also suggest the action of endo-PG.
The model summarizes the data obtained from the above experiments and
could be expanded to include other enzymic activities that have been
associated with pectin metabolism during ripening, such as pectin
methylesterase (Tucker et al., 1982; Harriman et al., 1991; Tieman et
al., 1992). A similar model of pectin degradation involving the
solubilization of the bulk of pectic polymers independent of both PG
and -galactosidase, which act to degrade the solubilized pectins
later in softening, has been used to describe cell wall changes in
ripening kiwifruit (Redgwell et al., 1992).
Hemicelluloses also show substantial changes in the degree of
solubility and molecular mass profiles during ripening. Xyl-rich polymers show substantial degradation prior to ripening (Fig. 8), and
it is possible that this is related to the coincident solubilization of
pectin-associated Gal, suggesting the existence of xylan-pectin
complexes. Of particular interest was a decrease in the molecular mass
of the tightly bound xyloglucan fraction coincident with the onset of
softening (Figs. 7 and 8). This component of the wall is presumed to
coat and cross-link the cellulose microfibrils, and thus may play an
important structural role. Disruption of this close association by
cleavage of the xyloglucan cross-links, perhaps catalyzed by
endo-1,4--glucanases or xyloglucan endotransglycosylases, both of
which have been associated with ripening (Lashbrook et al., 1997),
could allow cell wall loosening.
Alternatively, disruption of the noncovalent associations between the
xyloglucan sheath and the cellulose microfibril could also result in
textural changes, as well as an increase in the accessibility of the
substrate for enzymic attack by cell wall hydrolases. Expansins are
proteins that are thought to disrupt the noncovalent association
between cellulose and matrix polysaccharides and could act to regulate
noncovalent cell wall polymer associations in ripening fruit. Rose et
al. (1997) recently identified and characterized a highly abundant,
fruit-ripening-specific expansin gene from tomato, as well as homologs
from strawberry and melon. The role of expansins in fruit softening is
not clear and is currently under investigation. However, it is possible
that they act at the cellulose/xyloglucan interface at the onset of
fruit softening, disrupting a crucial structural component of the wall.
A consequence of this activity might also be to increase the extent of
wall swelling, allowing increased access of other cell wall-degradative enzymes to their substrates and reducing the degree of physical entanglement of cell wall polymers, as has been suggested to occur in
ripening kiwifruit (Redgwell et al., 1992; Redgwell and Fry, 1993).
Charentais melons show evidence of modification of both pectic and
hemicellulosic polymers during ripening, as do many other fruit
species. However, the exceptionally rapid softening allowed a clear
delineation of cell wall-disassembly events associated with the early
or later stages of ripening that had not been apparent in fruit that
undergo more gradual and subtle textural changes. This analysis has
suggested that modification of tightly bound hemicellulose,
specifically xyloglucan, may represent one of the early changes to the
cell wall at the onset of ripening. Research is now in progress, in
part through the use of transgenic plants, to determine whether genes
associated with hemicellulose metabolism, such as expansins,
endo-1,4--glucanases, and xyloglucan endotransglycosylases may play
an important and perhaps synergistic role in regulating early events in
fruit softening.
| |
FOOTNOTES |
1
This research was supported by a grant from
Zeneca Plant Science, Jealotts' Hill, UK.
*
Corresponding author; e-mail abbennett{at}ucdavis.edu; fax
1-530-752-4554.
Received July 2, 1997;
accepted January 8, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AIS, alcohol-insoluble solids.
CDTA, cyclohexane
diamine tetraacetic acid.
DAP, days after pollination.
IG, immature-green ripening stage.
MG, mature-green ripening stage.
PG, polygalacturonase.
R1 to R4, ripening stages 1 to 4.
Rha, rhamnose.
TS, total sugars.
UA, uronic acid.
| |
ACKNOWLEDGMENT |
The authors would like to thank Dr. L. Carl Greve for useful
discussion and assistance.
| |
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[CrossRef]
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[Abstract/Free Full Text]
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The respiratory climacteric is present in Charentais (Cucumis melo cv. Reticulatus F1 Alpha) melons ripened on or off the plant.
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[Abstract/Free Full Text]
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Polygalacturonase gene expression in ripe melon fruits supports a role
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Abstract
Introduction