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Plant Physiol. (1998) 118: 783-792
Changes in Cell Wall Composition during Ripening of Grape
Berries1
Kylie J. Nunan,
Ian M. Sims2,
Antony Bacic,
Simon P. Robinson, and
Geoffrey B. Fincher*
Department of Plant Science, University of Adelaide, Waite Campus,
Glen Osmond, SA 5064, Australia (K.J.N., G.B.F.); Cooperative Research
Centre (CRC) for Viticulture, Plant Research Centre, Hartley Grove,
Urrbrae, SA 5064, Australia (K.J.N., S.P.R.); CRC for Industrial Plant
Polymers, School of Botany, University of Melbourne, Parkville,
Victoria 3052, Australia (I.M.S., A.B.); and Commonwealth Scientific
and Industrial Research Organization, Plant Industry, Horticulture
Unit, Urrbrae, SA 5064, Australia (S.P.R.)
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ABSTRACT |
Cell
walls were isolated from the mesocarp of grape (Vitis
vinifera L.) berries at developmental stages from before
veraison through to the final ripe berry. Fluorescence and light
microscopy of intact berries revealed no measurable change in cell wall
thickness as the mesocarp cells expanded in the ripening fruit.
Isolated walls were analyzed for their protein contents and amino acid compositions, and for changes in the composition and solubility of
constituent polysaccharides during development. Increases in protein
content after veraison were accompanied by an approximate 3-fold
increase in hydroxyproline content. The type I arabinogalactan content
of the pectic polysaccharides decreased from approximately 20 mol % of
total wall polysaccharides to about 4 mol % of wall polysaccharides
during berry development. Galacturonan content increased from 26 to 41 mol % of wall polysaccharides, and the galacturonan appeared to become
more soluble as ripening progressed. After an initial decrease in the
degree of esterification of pectic polysaccharides, no further changes
were observed nor were there large variations in cellulose (30-35 mol
% of wall polysaccharides) or xyloglucan (approximately 10 mol % of
wall polysaccharides) contents. Overall, the results indicate that no
major changes in cell wall polysaccharide composition occurred during
softening of ripening grape berries, but that significant modification
of specific polysaccharide components were observed, together with large changes in protein composition.
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INTRODUCTION |
Softening is an important part of the ripening process in most
fruit, and it is widely recognized that changes in cell walls accompany
fruit softening. Gross changes in wall composition may not always
occur, and indeed more subtle structural modifications of constituent
polysaccharides are often observed during softening (Brady, 1987 ;
Fischer and Bennett, 1991). For example, molecular mass, solubility,
and the degree of substitution (or branching) of an individual wall
polysaccharide may be altered without any large change in the total
amount of that polysaccharide.
Although localized alterations in pH or ion concentration can produce
detectable, noncovalent changes in cell wall properties (Carpita and
Gibeaut, 1993; Seymour and Gross, 1996), covalent modifications of wall
polysaccharides generally result from enzymatic processes (Fischer and
Bennett, 1991; Fry, 1995). In particular, an increase in water-soluble
pectic polysaccharides and the loss of Gal and/or Ara from the wall
occur in many fruit during softening (Huber, 1983; Gross and Sams,
1984), and this has been attributed in part to the action of PGs and
PMEs. However, in nonripening mutants and in antisense experiments in
tomatoes, it has become clear that these enzymes are not the only
contributing factors in the observed modification of pectin solubility
(Seymour et al., 1987; Smith et al., 1990). The softening process is
complicated by the fact that breakdown or modifications of different
components are usually accompanied by the incorporation of newly
synthesized components into the wall (Gibeaut and Carpita, 1994;
Seymour and Gross, 1996). The synthesis of cell wall polymers is
probably continuous throughout ripening, and a change in the turnover
rate of a particular component will affect the overall wall composition (Lackey et al., 1980).
Modifications of wall components might also be expected in ripening
grape (Vitis vinifera L.) berries, but little is known about
cell wall composition in grapes during ripening or of the mechanism of
softening in this fruit. The grape berry is a nonclimacteric fruit that
exhibits a double-sigmoidal growth curve characteristic of berry fruits
(Coombe, 1976). The first growth phase is initially due to cell
division and subsequently to cell enlargement (Harris et al., 1968).
Thereafter, the grape goes through a period of little or no growth.
This is followed by a second growth phase, in which the increase in
berry volume is entirely due to cell expansion within the berry. The
grape berry is somewhat unusual in that it softens at the same time as
it expands during the second growth, or ripening, phase. The onset of
the second growth phase is referred to as "veraison," which is a
viticultural term that describes the point at which a number of
developmental events are initiated, including the accumulation of
sugars, a decrease in organic acids, color development, berry
expansion, and softening (Coombe, 1973).
The monosaccharide compositions and structures of specific pectic
polysaccharide fractions from grape berries have been reported (Saulnier and Thibault, 1987; Saulnier et al., 1988), as have changes
in pectin solubility as the berry ripens (Silacci and Morrison, 1990),
but there have been no comprehensive analyses of intact walls during
ripening. The increasing commercial importance of the wine industry
internationally and the recognition that polysaccharides of cell wall
origin, in particular the pectic polysaccharides, create problems
during juice extraction, during filtration steps required to clarify
grape extracts, and during the formation of storage hazes that decrease
the shelf-life of the wine, all suggest that detailed studies of cell
wall composition might reveal important changes in the walls and might
point to key enzymes that mediate the process.
In the present study a recently developed procedure for the preparation
of cell walls from the mesocarp of grape berries (Nunan et al., 1997)
has been used to isolate walls at various stages during berry
development and ripening. The appearance of the walls during ripening
has been examined by light and fluorescence microscopy. The
monosaccharide and polysaccharide compositions of the walls have been
monitored and changes in solubility of specific polysaccharides have
been observed. Significant changes in protein content and amino acid
composition are also seen.
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MATERIALS AND METHODS |
Plant Material
Grape (Vitis vinifera L. cv Muscat Gordo Blanco,
hereafter referred to simply as Gordo) berries were harvested every 2 weeks from January to March, 1994, at the South Australian Advisory Centre (Nuriootpa, South Australia). The time of anthesis was taken to
be the point at which anthers were observed on 50% of the flowers
(November 28, 1993). At this time, bunches that were at mid-flowering
were tagged for subsequent sampling. For each sample time, a number of
tagged bunches were picked and all berries were removed. A
representative sample of 50 berries was used for measurements described
below. The remaining grapes were rinsed thoroughly with water and
stored at  80°C until required for wall isolation.
Berry Measurements
The lengths and widths of the 50 berries were recorded for the
calculation of berry volume, and berry weight was also recorded. Using
juice squeezed from each berry, °Brix (percent soluble sugar) was
measured using a hand-held refractometer (Leica) that had been
calibrated against standard Suc solutions. Deformability (the degree of
softness) was measured using skin-fold calipers according to the method
of Coombe and Bishop (1980).
Microscopy
Berries collected at each stage of development were prepared for
microscopy, essentially as described by O'Brien and McCully (1981).
Freshly picked berries were cut into small pieces and fixed in 3%
(v/v) glutaraldehyde, 0.05% (w/v) caffeine, and 25 mM
sodium phosphate buffer, pH 7.0, at 4°C for 16 h. The berry pieces were dehydrated for 2 h in each of two changes of
methoxyethanol, ethanol, propanol, and butanol. The dehydrated berries
were infiltrated with a solution of GMA containing 7% (w/v) PEG 400, 0.6% (w/v) benzoyl peroxide, and butanol (1:1, v/v) for 2 h at
4°C. The solution was removed and the berry pieces were infiltrated
with the GMA mixture for a further 2 d at 4°C. The berry pieces
were resuspended in fresh GMA mix and stored at 4°C prior to
embedding. Each infiltrated berry piece was placed in a small plastic
capsule containing GMA mixture, and the GMA was polymerized at 60°C
for 2 d. Berries were sectioned to a thickness of approximately 3 µm using a Reichert-Jung Microtome 2050 Supercut (NuBloch, Germany).
The sections were stained with 0.05% TBO in 10 mM sodium
benzoate buffer, pH 4.4, or 0.01% (w/v) Calcofluor in water for 3 min
and rinsed with water. The sections were viewed using an Axiophot Pol
photomicroscope (Zeiss).
Preparation of Cell Walls
During thawing of frozen berries, skin and seeds were removed and
mesocarp cell walls were isolated as described by Nunan et al. (1997).
Skin and seeds were removed manually and the remaining mesocarp tissue
was homogenized in 4 volumes of absolute ethanol using a household
blender. The homogenate was filtered sequentially through nylon mesh
(Swiss Screens, Moorabbin, Victoria, Australia) with pore sizes of 350, 280, and 71 µm. The material retained on the 71-µm mesh was stirred
in phenol that had been saturated with 500 mM Tris-HCl
buffer, pH 7.0, for 45 min and washed with ethanol and acetone to
remove the phenol. The retained material was stirred in
chloroform:methanol (1:1, v/v) twice for 1 h each, and in acetone
for 1 h. The isolated cell wall preparations were dried in a
vacuum oven at 40°C and stored over silica gel in a vacuum
desiccator.
Fractionation of Cell Walls
Wall preparations were fractionated essentially as described by
Redgwell et al. (1988). The walls (200 mg) were extracted twice in 20 mL of water at 40°C for 3 h with continuous stirring. After each
solvent extraction, the wall suspension was centrifuged for 10 min at
8000g. The supernatants were combined, dialyzed exhaustively
against water at 4°C, reduced in volume by rotor evaporation at
45°C, and freeze-dried. The water-insoluble pellet was suspended in
20 mL of 0.05 M CDTA and extracted twice, as described
above. The combined supernatant was dialyzed against 2 changes of 0.1 M NaCl, pH 6.5, before dialyzing against water. The
remaining cell walls were extracted with 0.05 M
Na2CO3 at 4°C for 16 h with continuous stirring. The supernatant was neutralized with
glacial acetic acid before dialysis. The pellet was extracted with 4 M KOH at room temperature for 24 h with continuous
stirring. Insoluble material was washed with water and freeze-dried.
Freeze-dried cell wall fractions were stored over silica gel in a
vacuum desiccator.
Protein and Amino Acid Estimations
Protein in the wall preparations and amino acid compositions were
determined as described by Nunan et al. (1997). Hyp in hydrolysates was
measured colorimetrically by the procedure of Kivirikko and Liesmaa
(1959).
Polysaccharide Linkage Analysis
Linkage analyses of cell wall preparations and extracted wall
fractions were performed as described in Nunan et al. (1997). All
methylation analyses were performed in duplicate, and where significant
variation was observed, the analyses were repeated again in duplicate.
For the reduction of uronic acid and esterified uronic acid residues,
the cell wall preparations were first treated with
NaBD4 (Sigma), which reduces esterified uronic
acid residues (Nunan et al., 1997). Free-uronic acid residues were
subsequently derivatized with 1-cyclo-3-(2-morpholinoethyl)
carbodiimide metho-p-toluenesulphonate (Aldrich), and
the samples were reduced either with NaBD4 for the determination of total uronic acids or with
NaBH4 to determine esterified uronic acids (Sims
and Bacic, 1995).
Carboxyl-reduced wall preparations and fractions were methylated using
the NaOH/CH3I method of Ciucanu and Kerek (1984)
as described previously (Nunan et al., 1997). The methylated
polysaccharides were hydrolyzed with trifluoroacetic acid, reduced with
NaBD4, and acetylated using perchloric acid as a
catalyst (Harris et al., 1984). Partially methylated alditol acetates
were separated on a bonded-phase BPX70 capillary column (SGE,
Melbourne, Australia) in a MAT 1020B GC-MS (Finnigan, San Jose,
CA). Neutral sugar and uronic acid derivatives were identified
and quantitated as described previously (Sims and Bacic, 1995).
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RESULTS |
Berry Development
Berry weight and volume, deformability, and the accumulation of
sugars during berry development are shown in Figure
1. There was a steady increase in berry
weight and volume throughout the developmental period. Berry
deformability and sugar content remained low until 58 dpa, when both
parameters began to increase markedly (Fig. 1B). Total soluble solids
remained at 5 to 6 oBrix until 58 dpa, but
thereafter increased markedly and reached a value of 18 oBrix when the last sample was taken at 114 dpa
(Fig. 1B). Deformability, which is a measure of berry softening, was
only 0.1 to 0.2 mm until 58 dpa, but increased rapidly to 1.8 mm at 114 dpa (Fig. 1B). Based on the rapid increase in soluble solids and
deformability from 58 dpa, we concluded that the inception of ripening
(veraison) occurred at that time.
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| Figure 1.
Growth curves for Gordo grapes showing change in
berry fresh weight ( ) and volume ( ) during development (A), and
change in berry °Brix ( ) and deformability ( ) (B). FW, Fresh
weight.
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Microscopy
The appearance of cell walls in intact berries was monitored
throughout development by fluorescence and light microscopy (Fig. 2). Examination of the walls of the skin
and hypodermis and the outer mesocarp (Fig. 2, A and B) revealed few
obvious differences between berries at veraison (58 dpa), with a
deformability of 0.2 mm, and those at 86 dpa, with a deformability of
1.3 mm. It should be noted that the outermost layers of cells, which
make up the skin and hypodermis, were removed before cell walls were prepared. It is also noteworthy that TBO, which stains polysaccharides purple and polyphenolics green, revealed a high content of phenolics in
the skin cells (Fig. 2B).
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| Figure 2.
Microscopy of the ripening grape berry. Sections
of the outermost region of the grape were prepared at 58 dpa (A) and 86 dpa (B). The sections were stained with Calcofluor and TBO,
respectively, and show the epidermis to the left and the mesocarp to
the right (bars = 100 µm). The sections in C and D are from
approximately the same location in the central mesocarp at 58 and 114 dpa, respectively, and are stained with TBO (bars = 200 µm).
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Comparison of the mesocarp tissue of a grape at 114 dpa, with a
deformability of 2 mm (Fig. 2D), and the mesocarp tissue of a grape at
58 dpa, with a deformability of 0.2 mm (Fig. 2C), showed that cells
were generally larger in the berry at 114 dpa compared with the 58 dpa
berry. No apparent change in cell wall thickness could be detected
during ripening (Fig. 2, C and D).
Cell Wall Isolation
Based on the developmental patterns observed in Figure 1, six
specific times of berry development were chosen for the isolation of
cell walls. These were: preveraison (44 dpa), veraison (58 dpa), and
four time points after veraison when berry softening was occurring (72, 86, 100, and 114 dpa). Because of the limited amount of available plant
material, the low yields of cell walls from grapes generally (Nunan et
al., 1997), and the time required for wall preparation, all of the
grapes at each developmental stage were pooled and a single cell wall
preparation was made. Nevertheless, yields for replicated wall
isolations with different samples of ripe grapes varied by less than
7% (data not shown). The yields of cell walls from Gordo berries at
each stage of development are shown in Figure
3. On a per gram fresh weight basis, the
amount of cell wall isolated decreased steadily throughout development. However, on a per berry basis, there was an initial increase in the
cell wall yield until 72 dpa, after which yields decreased to
preveraison levels. A similar decrease in cell wall yield was observed
when walls were isolated during the development of Ohanez, another
grape cultivar (data not shown).
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| Figure 3.
Cell wall yield from Gordo berries during
ripening, expressed on a per gram fresh weight basis () and on a per
berry basis (). The walls were isolated at six stages of development
from preveraison (44 dpa) through veraison (58 dpa) to the fully ripe
berry (114 dpa). FW, Fresh weight.
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Protein and Amino Acid Content
The changes in protein content of the isolated cell walls during
development are shown in Figure 4. Phenol
treatment has been shown to remove most adventitious proteins, and the
remaining protein is assumed to be an integral component of the cell
walls. The protein content of walls from young berries was less than 8% but as the berries matured, the amount of protein associated with
the cell walls increased to almost 12% by weight.
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| Figure 4.
Protein () content (% by weight) and Hyp ()
content (µg/mg cell wall) of purified cell walls isolated from
developing Gordo berries.
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The amino acid composition of isolated cell walls is shown in Table
I. The major amino acids were Glx, Arg,
Hyp, and Gly. The amino acids that showed the largest changes were Arg,
which decreased from 23.8 mol % to 14.5 mol % of wall protein, and
Hyp, which increased from 3.6 mol % to 9.9 mol % from preveraison to 100 dpa. Over the same period the amount of Hyp increased from 1.7 to
7.4 µg per mg cell wall (Fig. 4).
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Table I.
Amino acid composition of the proteins associated
with cell walls isolated from developing berries from 44 to 114 dpa
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Wall Fractionation
The yields of various fractions of walls isolated from different
stages of development are shown in Figure
5. The largest change was the 2-fold
increase in the water-soluble component after veraison. A temporary
decrease in the CDTA-soluble fraction at 58 dpa was observed in two
separate fractionations, but otherwise the amounts stayed relatively
constant throughout ripening. The Na2CO3-soluble fraction
increased somewhat before veraison but decreased as the berries
softened. Similarly, the KOH-soluble fraction also showed a small
increase before veraison and thereafter decreased steadily. The final
alkali-insoluble residue decreased throughout development, although the
overall decrease in this fraction was relatively small.
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| Figure 5.
Changes in solubility of cell wall material
isolated from developing Gordo berries, expressed on a dry weight
basis. The cell walls were fractionated into water-soluble (), 0.05 M CDTA-soluble ( ), 0.05 M
Na2CO3-soluble (), and 4 M
KOH-soluble () components, and a KOH-insoluble residue ().
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Linkage Analysis and Major Polysaccharides in the Cell Walls
The monosaccharide compositions and linkage types of mesocarp cell
wall polysaccharides isolated at the six stages of development were
analyzed by standard methylation procedures. Linkage analyses were
performed in duplicate for each of the six wall preparations, and the
values were averaged. The monosaccharide compositions are shown in
Table II. The major change was the
decrease in 4-linked galactopyranosyl (4-Galp) residues from
19 mol % before veraison to 1 mol % in fully ripe berries. The
unesterified, 4-linked galacturonic acid (4-GalAp) increased
during this ripening period from 10 to 20 mol %, and the esterified
4-GalAp increased from 14 to 18 mol %. The 4-linked
glucopyranose (4-Glcp) showed an initial small increase,
followed by a larger decrease later in development. The other
derivatives constitute 5% or less of the total polysaccharide in the
cell wall and did not vary significantly during ripening (Table II).
The most abundant polysaccharide types found in the developing grape
berry cell walls were subsequently deduced from the linkage composition
(Table III), based on the totals of
individual glycosyl residues that are characteristic of well-defined
wall polysaccharides. The mol % values for polysaccharides referred to
below and in Tables III and IV are calculated by the addition of mol % values of the appropriate methylated alditol acetates, and are
expressed as mol % of total polysaccharide content of the wall;
protein and other wall components are not included. These calculations embody a number of structural assumptions, but are widely used as a
good indicator of trends in contents of specific polysaccharide types
(Bacic et al., 1988; Shea et al., 1989; Gorshkova et al., 1996; Nunan
et al., 1997). Thus, xyloglucan content was calculated from the sum of
4,6-Glcp, 4-Glcp equivalent to one-third of
4,6-Glcp, terminal and 2-Xylp, and
2-Galp and terminal L-fucopyranose. The remaining 4-Glc was assumed to be cellulose. Xylan was estimated from
4-Xylp and 2,4-Xylp, and terminal Araf
or terminal GlcAp equal to the 2,4-Xylp.
Arabinogalactan II was calculated from the sum of 3,6-Galp,
3-Galp, 6-Galp, and terminal Araf
equivalent to the value for 3,6-Galp. Arabinogalactan I was
estimated from the sum of 4-Galp, 3,4-Galp, and
terminal Galp equivalent to 3,4-Galp. Arabinan
was estimated from 5-Araf, mannan from the sum of
4-Manp, 4,6-Manp, and terminal Galp
equivalent to 4,6-Galp. Galacturonan, which includes both
rhamnogalacturonan and homogalacturonan, was the sum of
4-GalAp, 4-GalAp ester, 2-Rhap, and
2,4-Rhap (Bacic et al., 1988; Shea et al., 1989; Gorshkova
et al., 1996).
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Table IV.
Distribution of major polysaccharides in cell wall
fractions at different stages of grape berry development
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On a mol % basis, AGI decreased from 21% of total polysaccharides in
preveraison walls to 1% in ripe, 100 dpa berries. Galacturonan increased from 26% preveraison to 41% in ripe berries; the DE decreased from 58% to 48% in the preveraison period and thereafter remained approximately constant. The cellulose content increased slightly early in development and decreased later. Xyloglucan was
present but did not change significantly during ripening, whereas the
smaller amounts of arabinan and mannan that were present remained
approximately constant. Heteroxylan levels were low generally, but
increased during ripening. Small amounts of material could not be
readily assigned to well-characterized polysaccharides from plant cell
walls and are therefore shown in Table III as "other."
Because comparisons of mol % data are complicated by the possibility
that a change in one component actually reflects a change in another,
attempts were made to express the contents of specific polysaccharides
on an absolute basis. From the relative proportion (mol % of total
polysaccharides) of each polysaccharide in Table III and the cell wall
yields of the same samples (Fig. 3), we were able to calculate the
amounts of each polysaccharide on a per gram fresh weight basis and on
a per berry basis. These data are presented in Figure
6, but should be viewed with some caution because changes in wall yields may be influenced not only by the absolute amount of wall material present, but also by factors such as
changes in wall fragility.
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| Figure 6.
Changes in levels of specific cell wall
polysaccharides (dry weights) during grape berry development, expressed
on a tissue fresh weight basis (A) and a per berry basis (B). The four
most abundant polysaccharides found in the cell walls are shown; these
account for 85% to 90% of total wall polysaccharides. , Cellulose;
, galacturonan; , AGI; and , xyloglucan. FW, Fresh weight.
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All polysaccharides decreased on a fresh weight basis (Fig. 6A), but on
a per berry basis (Fig. 6B) galacturonan and cellulose contents
increased up to and just after veraison before showing a slight
decrease. AGI content decreased during ripening, but xyloglucan content
per berry was relatively constant throughout development.
Fractionated Polysaccharides
Changing solubilities of polysaccharides, as indicated by their
movement between fractions, are shown in Table
IV. The increase in the water-soluble
fraction was due to the shift of galacturonan from the CDTA- and
Na2CO3-soluble fractions to
the water-soluble fraction. AGII became completely water soluble as
ripening progressed. The increase in water-soluble arabinan came from
the KOH-soluble and residue fractions. The remaining AGI in the cell
walls at 114 dpa was associated with the
Na2CO3-soluble fraction,
whereas it was found in the CDTA-,
Na2CO3-, and KOH-soluble
and KOH-insoluble residue fractions when the berries were young.
Heteroxylans became slightly more soluble as the berry ripened; there
was a decrease in the amount associated with the KOH-insoluble residue
fraction and a corresponding increase in the
Na2CO3-soluble fraction.
The solubilities of xyloglucan and cellulose changed very little during ripening.
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DISCUSSION |
The onset of ripening in grape berries varies between bunches and
even between individual berries in the same bunch (Coombe and Bishop,
1980; Coombe, 1992). To minimize the effects of this variation
on physical and chemical analyses, bunches that flowered at the same
time were tagged and used to provide samples at different stages of
berry development. At each time point, a subsample of 50 berries was
taken to measure berry characteristics; the remainder was used to
isolate cell walls. The double-sigmoidal pattern normally seen during
the development of individual berries was not obvious here (Fig. 1A)
and was probably masked not only by the averaging of values for 50 berries, but also because berries were sampled only once every 2 weeks
(Coombe and Bishop, 1980; Coombe, 1992). Nevertheless, the onset
of ripening, or veraison, was clearly indicated by the relatively
sudden increase in soluble solids and by the concomitant increase in
berry deformability, which occurred at 58 dpa (Fig. 1B).
Following veraison, the size of central mesocarp cells appeared to
increase (Fig. 2). The cell walls of many fruit swell as the fruit
ripens (Redgwell et al., 1997). Swelling in kiwifruit cell walls during
ripening is pronounced, and in some tissues a 3- to 4-fold increase in
cell wall thickness has been observed (Redgwell et al., 1997). No
swelling of the grape cell walls was visible by light microscopy during
development (Fig. 2), in agreement with a previous study by Hardie et
al. (1996).
The yield of isolated cell walls on a per-berry basis appeared to
increase before veraison and into the start of the second growth phase,
but decreased throughout the remainder of berry development (Fig. 3).
It should be emphasized, however, that the procedure for wall isolation
is not quantitative (Nunan et al., 1997) and that the decrease in yield
could be explained in several ways. If there were a net decrease in
cell wall synthesis, an increase in hydrolysis of wall polysaccharides,
or an increase in solute accumulation relative to wall deposition, then
yields would be expected to fall. Alternatively, if the walls become more fragile during ripening, they may be fragmented into smaller pieces during homogenization of the berry and be lost through the
71-µm nylon mesh.
The most significant change in the composition of grape cell walls
during ripening was the dramatic decrease in the amount of
(1 4)-linked Galp residues (Tables II and III). AGI is a
major component of the neutral side chains of pectic polysaccharides and the loss of these side chains has been observed in other ripening fruit (Huber, 1983; Gross and Sams, 1984). The loss of AGI, which constituted 21 mol % of total polysaccharides in grape cell walls before veraison (Table III), began well before softening was detected (Table III compared with Fig. 1B), suggesting that the loss of Type I
AG could be a crucial step that is associated with the initiation of
softening. However, Redgwell and Harker (1995) suggested that cell
wall-associated Gal loss and softening are separate processes, because
inhibiting Gal loss in kiwifruit discs did not inhibit fruit softening
and inhibiting the softening process did not prevent Gal loss. The
enzyme  -galactosidase has been shown to degrade galactans in fruit
such as apples and pears (Ross et al., 1993; Kitagawa et al., 1995),
although its activity was thought not to be high enough to account for
all of the degradation seen in the fruit nor was the extent of Gal loss
considered high enough when isolated walls were treated with purified
-galactosidase. Additional enzymic or nonenzymic processes might
therefore be involved in AGI degradation (Ross et al., 1993; Kitagawa
et al., 1995).
The other significant changes in the cell walls of developing grapes
were increases in galacturonan and heteroxylan content. Proportions
(mol % of total wall polysaccharides) of the other major
polysaccharides, cellulose and xyloglucan, remained at similar levels
throughout the ripening process (Table III). The cell wall preparations
were subsequently extracted with a series of increasingly harsh
solvents, in the expectation that polysaccharides recovered at each
stage would provide an indication as to the nature of intermolecular
interactions and as to how tightly individual polysaccharides were
associated with one another and with other wall components. The
increase in the water-soluble fraction was attributed to an increased
solubility of galacturonan that had required CDTA and Na2CO3 to extract it early
in development, together with increases in AGII and arabinan that had
been found in all fractions before the grapes began to soften (Table
IV). An increase in water-soluble polysaccharides has also been
reported in other fruit (Gross and Wallner, 1979; Ahmed and Labavitch,
1980). The movement of particular polysaccharides from one fraction to
another presumably results from modifications to the fine structure of
polysaccharides, which affect the way they aggregate with each other
or which simply increase the inherent solubilities of the
polysaccharides.
In other fruit, polyuronides that become more soluble during ripening
have lower apparent Mr (Yoshioka et al., 1992;
Huber and O'Donoghue, 1993). This may result from the removal of
neutral side chains, as discussed above, or it may result from other
modifications. Enzymes that have been implicated in these
modifications, and therefore in softening, include PME, which removes
the methyl groups from esterified galacturonans, and PG, which is an
endo-hydrolase that cleaves unesterified galacturonans (Seymour and
Gross, 1996). The removal of methyl groups by PME affects interactions
of the polysaccharide with calcium and therefore changes the way it
aggregates (Jarvis, 1984). The removal of some methyl groups may be
required before PG can act. Hydrolysis of long polygalacturonans by PG would decrease the Mr of pectic polysaccharides
and presumably increase their solubility.
In contrast to the relatively large decreases in the DE observed during
ripening in tomatoes (Koch and Nevins, 1989) and pears (Martin-Cabrejas
et al., 1994), it was found that there was a smaller decrease from an
initial DE of 58% in the grape polygalacturonans before veraison to
48% at veraison, and thereafter there was no further decrease (Table
III). The level of esterified 4-linked galacturonic acid residues
increased slightly during ripening (Table II). This result suggests
that the increased solubility of pectic polysaccharides as the grape
softens is not due to changes in their overall DE.
As mentioned above, cellulose and xyloglucan levels remained
approximately the same during ripening of grapes (Table III) and there
was no apparent increase in their solubility (Table IV). This may be
compared with the decrease observed in the Mr of
avocado xyloglucan (O'Donoghue and Huber, 1992) and a decrease found
in the Mr of alkali-soluble polysaccharides in
tomatoes (Huber, 1983). It should be noted that xyloglucans might also
be modified by xyloglucan endotransglycosylases (Fry, 1993), which
hydrolyze and transglycosylate xyloglucans. Modification of grape cell
wall xyloglucan in this way by xyloglucan endotransglycosylase might not be detected in the analyses of overall polysaccharide composition performed here.
In addition to the changes seen in polysaccharide composition and
solubility in walls of ripening grape berries, levels of wall-associated proteins were found to increase from approximately 7%
(w/w) at veraison to almost 12% later in ripening (Fig. 4). Although
it might be argued that the increase could be attributed simply to
increasing contamination of wall preparations with protein of
cytoplasmic origin (Nunan et al., 1997), the concomitant increase in
Hyp content from 2 to 7 µg/mg cell wall (Fig. 4) and from 2.6 to 3.6 µg/berry (data not shown) suggests that wall proteins such as the
Hyp-rich glycoproteins (extensins) are being deposited in the walls as
the berries develop (Fig. 4). Hyp-rich glycoproteins are believed to
form a fibrillar network in plant cell walls that is independent of the
cellulosic network, and this second, Hyp-rich glycoprotein network is
likely to considerably strengthen the cell wall (Varner and Lin, 1989;
Carpita and Gibeaut, 1993). It is possible that expansion of mesocarp
cells in the ripening berry (Fig. 2, C and D) necessitates the
reinforcement of the walls with Hyp-rich glycoproteins so that cellular
integrity can be maintained during the softening process. The changes
in amino acid composition during ripening, such as the decrease in
Arg content, suggest that other modifications are also occurring (Table I).
In summary, the changes in cell wall polysaccharides during grape berry
ripening suggest that a limited number of enzymes play an important
role in the softening process. In particular, these would include endo-
or exo-hydrolases capable of depolymerizing the (14)--galactan
constituents of pectic polysaccharides. Low but significant levels of
PG or PME, which could be responsible for the partial solubilization of
galacturonans (Table IV), might also be present, but only low levels of
cellulase, xyloglucanase, and xylanase would be expected. An
understanding of the role of these enzymes in the modification of
grape berry cell wall polysaccharides could provide opportunities to
improve grape and wine quality through genetic engineering.
| |
FOOTNOTES |
1
This work was supported by the Cooperative
Research Centre for Viticulture.
2
Present address: Industrial Research Limited,
Gracefield Research Centre, Lower Hutt, New Zealand.
*
Corresponding author; e-mail gfincher{at}waite.adelaide.edu.au;fax
61-8-8303-7109.
Received April 20, 1998;
accepted August 3, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AGI and AGII, type I and type II
arabinogalactan, respectivelyAraf,
L-arabinofuranose.
CDTA, trans-1,2-diaminocyclohexane-N,N,N ,N-tetraacetic
acid.
DE, degree of esterification.
dpa, days postanthesis.
GalAp, D-galacturonic acid.
Galp, D-galactopyranoseGlcAp, D-glucuronic acid.
Glcp, D-glucopyranose.
GMA, glycol
methacrylateManp, D-mannopyranose.
PG, endo-polygalacturonase.
PME, pectin methylesterase.
Rhap, L-rhamnopyranose.
TBO, Toluidine Blue
O.
Xylp, D-xylopyranose.
| |
ACKNOWLEDGMENTS |
We thank Dr. Robert Redgwell, Mr. Jelle Lahnstein, and Dr.
Meredith Wallwork for their assistance with various aspects of the
work.
| |
LITERATURE CITED |
Ahmed AE,
Labavitch JM
(1980)
Cell wall metabolism in ripening fruit I. Cell wall changes in ripening "Bartlett" pears.
Plant Physiol
65:
1009-1013
[Abstract/Free Full Text]
Bacic A, Harris PJ, Stone BA (1988) Structure and function of
plant cell walls. In J Preiss, ed, The Biochemistry of
Plants: a Comprehensive Treatise. Vol 14, Carbohydrates. Academic
Press, New York, pp 297-371
Brady CJ
(1987)
Fruit ripening.
Annu Rev Plant Physiol
38:
155-178
[CrossRef][ISI]
Carpita NC,
Gibeaut DM
(1993)
Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth.
Plant J
3:
1-30
[CrossRef][ISI][Medline]
Ciucanu I,
Kerek F
(1984)
A simple and rapid method for the permethylation of carbohydrates.
Carbohydr Res
131:
209-217
[CrossRef]
Coombe BG
(1973)
The regulation of set and development of the grape berry.
Acta Hortic
34:
261-273
Coombe BG
(1976)
The development of fleshy fruits.
Annu Rev Plant Physiol
27:
207-228
[ISI]
Coombe BG,
Bishop GR
(1980)
Development of the grape berry. II. Changes in diameter and deformability during veraison.
Aust J Agric Res
31:
499-509
[CrossRef]
Coombe BG
(1992)
Research on development and ripening of the grape berry.
Am J Enol Vitic
43:
101-110
[Abstract/Free Full Text]
Fischer RL,
Bennett AB
(1991)
Role of cell wall hydrolases in fruit ripening.
Annu Rev Plant Physiol Plant Mol Biol
42:
675-703
[CrossRef][ISI]
Fry SC
(1993)
Loosening the ties: a new enzyme, which cuts and then re-forms glycosidic bonds in the cell wall, may hold the key to plant cell growth.
Curr Biol
3:
355-357
Fry SC
(1995)
Polysaccharide-modifying enzymes in the plant cell wall.
Annu Rev Plant Physiol Plant Mol Biol
46:
497-520
[CrossRef][ISI]
Gibeaut DM,
Carpita NC
(1994)
Biosynthesis of plant cell wall polysaccharides.
FASEB J
8:
904-915
[Abstract]
Gorshkova TA,
Wyatt SE,
Salnikov VV,
Gibeaut DM,
Ibragimov MR,
Lozovaya VV,
Carpita NC
(1996)
Cell-wall polysaccharides of developing flax plants.
Plant Physiol
110:
721-729
[Abstract]
Gross KC,
Sams CE
(1984)
Changes in cell wall neutral sugar composition during fruit ripening: a species survey.
Phytochemistry
23:
2457-2461
[CrossRef]
Gross KC,
Wallner SJ
(1979)
Degradation of cell wall polysaccharides during tomato fruit ripening.
Plant Physiol
63:
117-120
[Abstract/Free Full Text]
Hardie WJ,
O'Brien TP,
Jaudzems VG
(1996)
Morphology, anatomy and development of the pericarp after anthesis in grape, Vitis vinifera L.
Aust J Grape Wine Res
2:
97-142
Harris JM,
Kriedemann PE,
Possingham JV
(1968)
Anatomical aspects of grape berry development.
Vitis
7:
106-119
Harris PJ,
Henry RJ,
Blakeney AB,
Stone BA
(1984)
An improved procedure for the methylation analysis of oligosaccharides and polysaccharides.
Carbohydr Res
127:
59-73
[CrossRef][Medline]
Huber DJ
(1983)
Polyuronide degradation and hemicellulose modifications in ripening tomato fruit.
J Amer Soc Hort Sci
108:
405-409
Huber DJ,
O'Donoghue EM
(1993)
Polyuronides in avocado (Persea americana) and tomato (Lycopersicon esculentum) fruits exhibit markedly different patterns of molecular weight downshifts during ripening.
Plant Physiol
102:
473-480
[Abstract]
Jarvis MC
(1984)
Structure and properties of pectin gels in plant cell walls.
Plant Cell Environ
7:
153-164
Kitagawa Y,
Kanayama Y,
Yamaki S
(1995)
Isolation of -galactosidase fractions from Japanese pear. Activity against native cell wall polysaccharides.
Physiol Plant
93:
545-550
[CrossRef]
Kivirikko KI,
Liesmaa M
(1959)
A colorimetric method for determination of hydroxyproline in tissue hydrolysates.
Scand J Clin Lab Invest
11:
128-133
Koch JL,
Nevins DJ
(1989)
Tomato fruit cell wall. I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins.
Plant Physiol
91:
816-822
[Abstract/Free Full Text]
Lackey GD,
Gross KC,
Wallner SJ
(1980)
Loss of tomato cell wall galactan may involve reduced rate of synthesis.
Plant Physiol
66:
532-533
[Abstract/Free Full Text]
Martin-Cabrejas M,
Waldron KW,
Selvendran RR
(1994)
Cell wall changes in Spanish pear during ripening.
J Plant Physiol
144:
541-548
Nunan KJ,
Sims IM,
Bacic A,
Robinson SP,
Fincher GB
(1997)
Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera).
Planta
203:
93-100
[CrossRef]
O'Brien TP, McCully ME (1981) In: The study of plant structure
principles and selected methods. Termarcarphi Pty., Ltd., Melbourne,
Australia
O'Donoghue EM,
Huber DJ
(1992)
Modification of matrix polysaccharides during avocado (Persea americana) fruit ripening: an assessment of the role of Cx-cellulase.
Physiol Plant
86:
33-42
[CrossRef]
Redgwell RJ,
Harker R
(1995)
Softening of kiwifruit discs; effect of inhibition of galactose loss from cell walls.
Phytochemistry
39:
1319-1323
[CrossRef]
Redgwell RJ,
MacRae E,
Hallet I,
Fischer M,
Perry J,
Harker R
(1997)
In vivo and in vitro swelling of cell walls during fruit ripening.
Planta
203:
162-173
[CrossRef]
Redgwell RJ,
Melton LD,
Brasch DJ
(1988)
Cell-wall polysaccharides of kiwifruit (Actinidia deliciosa): chemical features in different tissue zones of the fruit at harvest.
Carbohydr Res
182:
241-258
[CrossRef]
Ross GS,
Redgwell RJ,
MacRae EA
(1993)
Kiwifruit -galactosidase: isolation and activity against specific fruit cell-wall polysaccharides.
Planta
189:
499-506
Saulnier L,
Brillouet J-M,
Joseleau J-P
(1988)
Structural studies of pectic substances from the pulp of grape berries.
Carbohydr Res
182:
63-78
[CrossRef]
Saulnier L,
Thibault J-F
(1987)
Extraction and characterization of pectic substances from pulp of grape berries.
Carbohydr Poly
7:
329-343
[CrossRef]
Seymour GB,
Gross KC
(1996)
Cell wall disassembly and fruit softening.
Postharvest News and Information
7:
45N-52N
Seymour GB,
Harding SE,
Taylor AJ,
Hobson GE,
Tucker GA
(1987)
Polyuronide solubilization during ripening of normal and mutant tomato fruit.
Phytochemistry
26:
1871-1875
[CrossRef]
Shea EM,
Gibeaut DM,
Carpita NC
(1989)
Structural analysis of the cell walls regenerated by carrot protoplasts.
Planta
179:
293-308
Silacci MW,
Morrison JC
(1990)
Changes in pectin content of Cabernet Sauvignon grape berries during maturation.
Am J Enol Vitic
41:
111-115
[Abstract/Free Full Text]
Sims IM,
Bacic A
(1995)
Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia.
Phytochemistry
38:
1397-1405
[CrossRef]
Smith CJS,
Watson CF,
Morris PC,
Bird CR,
Seymour GB,
Gray JE,
Arnold C,
Tucker GA,
Schuch W,
Harding S,
and others
(1990)
Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes.
Plant Mol Biol
14:
369-379
[CrossRef][ISI][Medline]
Varner JE,
Lin L-S
(1989)
Plant cell wall architecture.
Cell
56:
231-239
[CrossRef][ISI][Medline]
Yoshioka H,
Aoba K,
Kashimura Y
(1992)
Molecular weight and degree of methoxylation in cell wall polyuronide during softening in pear and apple fruit.
J Am Soc Hort Sci
117:
600-606
[Abstract/Free Full Text]
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Top
Abstract
Introduction