Plant Cell Physiol. 38(5): 603-610 (1997)
JSPP © 1997
Relationship between Fruit Softening and Wall Polysaccharides in Avocado
(Persea americana Mill) Mesocarp Tissues
Naoki Sakurai' and Donald J. Nevins 2
1
2
Environmental Studies, Faculty of Integrated Arts & Sciences, Hiroshima University, Higashi Hiroshima, 739 Japan
Department of Vegetable Crops, University of California, Davis, California 95616, U.S.A.
with the massive breakdown of the cell walls (Platt-Aloia et
al. 1980). An array of cell wall hydrolytic enzymes has been
identified with putative roles in softening based simply on
their presence in the ripening avocado fruit, these include
cellulase (Awad and Young 1979, O'Donoghue and Huber
1992), polygalacturonase (Zauberman and Schiflfman-Nadel
1972), xylanase and xylosidase (Ronen et al. 1991).
The elevated cellulase activity, assayed using the substrate carboxymethyl cellulose, has been of particular interest with respect to avocado softening (Pesis et al. 1978, Bennett and Christoffersen 1986, Kanellis et al. 1989, 1991). An
increase in the mRNA responsible for expression of Cx-cellulase has been demonstrated (Christoffersen et al. 1984),
and the members of the cellulase gene family in ripening
avocado fruit have been isolated and characterized (Cass et
al. 1990). Nevertheless, the physiological significance of
avocado cellulase is obscure, because, based on biochemical evidence, there is no clear relationship between the capacity of the enzyme for hydrolysis of glucosyl bonds and
the degradation of cellulose in vitro. Hatfield and Nevins
(1986) reported that the partially purified cellulase from
avocado fruits was incapable of liberating fragments from
cellulose when the enzyme was administered to avocado
cell walls, although the enzyme did, in fact, release other
sugar moieties. The release of these other sugars was interpreted as hydrolysis of limited glucosyl bonds within a
putative xyloglucan which, in effect, released sugars that
were components of side chains or other closely associated
polymers.
On the other hand, O'Donoghue et al. (1994) reported
Key words: Avocado (Persea americana Mill) — Cell walls — depolymerization of cellulose by the action of Cx-cellulase.
Fruit texture — Ripening — Stress relaxation — Xyloglu- A more complete interpretation of the degradation pattern
can.
is not possible because there is only limited information
about the basic wall structure available. Clear documentation of the changes in polysaccharides that occur during
avocado fruit ripening has also been lacking.
Pectin degradation during fruit ripening has been
Avocado fruit ripens only after detachment from the
tree and the process is characterized predominantly by more extensively studied (Fischer and Bennett 1991), but remesocarp softening. Microscopic observation of ripening cent studies (Giovannoni et al. 1989, Smith et al. 1990,
Cutillas-Iturralde et al. 1993) have de-emphasized the role
avocado tissue suggested that the softening was associated
of pectin degrading enzymes in fruit ripening.
These findings fueled a reassessment of the relationAbbreviations: To, minimum stress-relaxation time; Tm, maxship
between cell wall degradation and fruit softening. The
imum stress-relaxation time; R, relaxation rate; HA, hemicellulose A; HB, hemicellulose B; NS, neutral sugar; TS, total sugar; hemicellulose degradation has also been suggested as the
UA, uronic acid.
primary determinant of softening of tomato (Sakurai and
Changes in avocado (Persea americana) fruit texture
during ripening were evaluated by stress-relaxation analysis. A conical probe was imposed into the mesocarp tissue
to a depth of 0.6 mm and the initial stress and the stress
relaxation over 60 s were determined. The initial stress, an
elastic parameter, was substantially reduced within one day
when ripening was initiated by transferring the fruit from
15 to 25°C. The minimum and maximum relaxation time,
parameters which reflect viscosity, were also reduced within one day. Mesocarp cell walls were fractionated into
water-soluble (WS), hot EDTA-soluble (EDTA), alkaline
soluble (hemicellulose) and the residual (cellulose) fractions. The amount of cellulose did not change during ripening. Rhamnose, arabinose and uronic acids in the WS fraction increased during the initial day of ripening; those same
components decreased in the EDTA fraction. A molecular
weight downshift in the WS acidic polysaccharides was detected within one day, while only slight changes were observed in the molecular weights of the EDTA fraction. The
quantities of individual sugar components of major hemicellulose fraction were unchanged, but there was a prominent molecular weight downshift in the xyloglucan components within one day. These results clearly revealed that
both elastic and viscous properties of avocado mesocarp
tissues were substantially altered during ripening, and
that the solubility changes in acidic polysaccharides and
decreases in the average molecular weight of cell wall xyloglucan components were associated with significant changes
in fruit texture.
603
604
Fruit softening and wall changes in avocado
Nevins 1993), persimmon (Cutillas-Iturralde et al. 1994),
and avocado (O'Donoghue and Huber 1992).
Fruit softening and other aspects of texture are functions of changes in the physical properties of the tissue,
probably caused by developmental modifications of the
chemical structure of the cell walls (Brady 1986, Huber
1983). Physical changes that specifically contribute to fruit
texture have recently been measured by the adaptation of a
protocol for measuring stress-relaxation (Kojima et al.
1991, 1992, Sakurai and Nevins 1992, 1993). The parameters derived from stress-relaxation analysis clearly indicate
that both viscosity and elasticity in fruit tissues change as a
function of fruit ripening (Sakurai and Nevins 1992). In
tomato fruit, metabolic changes in both pectin and xyloglucans in the wall polysaccharides parallel changes in these parameters (Sakurai and Nevins 1992). If cellulose or other
polysaccharide constituents in avocado fruit cell walls were
degraded by wall hydrolyzing enzymes during ripening,
such action could be reflected by changes in the structure of
putative polysaccharide substrates. In addition, measurement of reliable physical parameters representative of fruit
tissues allows the association of textural changes with specific modification of cell wall polysaccharides. The present
experiments were undertaken to reconcile developmental
changes in cell wall polysaccharides with physical properties of mesocarp tissues of avocado.
Materials and Methods
Plant materials—Unripe avocado (Persea americana Mill, cv.
Haas) fruits were purchased in case lots from the local wholesale
distributor. The fruits were maintained at 15°C. To induce ripening, the fruits were transferred to 25°C.
Fruit firmness measurement—Fruit firmness was determined
by a stress-relaxation method (Kojima et al. 1991, 1992, Sakurai
and Nevins 1992, 1993). A tissue slice (7-8 mm in thickness) of
mesocarp was excised at the equatorial plane. The slice was placed
on a stage. A conical probe (3 mm in diameter) was introduced
into the slice by lowering the load arm of a Vitrodyne material
tester (Liveco Inc., Burlington VT). When the probe penetrated
the tissue to a depth of 0.6 mm, it was halted, and the subsequent
response of the tissue to the imposed stress was recorded. The
stress data were acquired by the computer at 1 s intervals over a
total of 60 s. The accumulated stress decay data were used for calculating four separate stress relaxation parameters by a leastsquare method programmed in C language. The value for To represents the time when the stress decay begins, R the progression in
stress decay, and Tm the time when the decay ceases. The initial
stress at t = 0 was also recorded. Immediately after the stress-relaxation analysis, the tissue was stored at — 80°C until cell wall analysis.
Cell wall fractionation—Diced avocado mesocarp tissues
were heated in boiling 95% EtOH for 30 min, and then homogenized with a Polytron homogenizer (Brinkmann Instrument, Type
PT10/35). The homogenate was centrifuged for 20 min at 3,000 x
g. The residue was dispersed in acetone at room temperature,
again homogenized with the Polytron homogenizer, and centrifuged for 20 min at 3,000 x.g. This residue was dispersed in
MeOH : CHC13 (1 : 1, v/v), homogenized once again, and then centrifuged for 20 min at 3,000 xg. The residue was again treated
with acetone as above, then the residue was dispersed at room temperature in distilled water, homogenized, and centrifuged for 20
min at 10,000 xg. The supernatant was designated as the watersoluble fraction. The precipitate was treated for 2 h with 300 units
of an a-amylase (hog pancreatic a-amylase, Sigma) in Na-acetate
buffer (pH 6.5, 50 mM). Then the residue was treated three times
with EDTA (50 mM, pH 6.8) for 15 min at 90°C to extract pectic
substances, and three times with 17.5% NaOH containing 0.02%
NaBH 4 for a total of 24 h. The alkaline extract was neutralized
with glacial acetic acid, dialyzed, and centrifuged. The supernatant was designated as hemicellulose B (HB) and the precipitate,
as hemicellulose A (HA). The residue was washed with diluted
acetic acid and EtOH : diethylether ( 1 : 1 , v/v), dried, and designated as the cellulose fraction.
Sugar analysis and gel-filtration chromatography—Total
sugar (TS) content was estimated by a phenol-sulfuric acid
method using glucose as a standard (Chaplin 1986), and uronic
acid (UA) by the /n-hydroxydiphenyl method (Blumenkrantz and
Asboe-Hansen 1973). The m-hydroxydiphenyl method reacts
slightly with NS. By measuring the color intensity of a mixture of
galacturonic acid and glucose at varying ratios, an equation was
derived which corrected the net amount of UA in the mixture as
follows: UA <ji%mr')=l 12.4x(A520-0.0273 x A490)
where A490 is the absorbency at 490 nm derived from the phenolsulfuric acid method, and A520, the absorbency at 520 nm for the
m-hydroxydiphenyl method. Xyloglucans were determined by the
iodine method (Wakabayashi et al. 1991). Xyloglucan content was
estimated by using the following relationship; one unit of absorbency at A640 is equivalent to 155 jug ml" 1 xyloglucan (Wakabayashi et al. 1991).
The dry cellulose fraction was dissolved in 0.2 ml of 72% of
sulfuric acid and kept for 1 h at room temperature, and then
diluted with 2.8 ml of distilled water. Sugar content of the solution was measured by phenol sulfuric acid method.
An aliquot (200 /t\) of the water-soluble fraction or dialyzed
pectin or HB was introduced into an HPLC system (Dionex BioLC, Dionex Co., CA). The gel-filtration column was a Bio-Sil
TSK-125 (Bio Rad). The sample was eluted with 50 mM Na-phosphate buffer (pH 7.2) at a flow rate of 1 ml min" 1 . Fractions were
collected at 0.5 min intervals.
Sugar composition analysis—Sugar composition of an aliquot (0.5 ml) of each fraction or the eluant from the gel filtration
column was assayed as previously reported (Sakurai and Nevins
1993). Briefly, the polysaccharides were hydrolyzed by 2 M
trifluoroacetic acid. Liberated monosaccharides were reduced
and acetylated by acetic anhydride with 1-methylimidazole as a
catalyst (Chaplin 1986). The acetylated sugars were dissolved in
200/il of acetone. A portion of the acetone solution was introduced into a GLC (Model 5890, Hewlett Packard) equipped with a
capillary column (SP-2380, Supelco, 0.21 mm i.d.x30m). The
oven temperature was raised from 260 to 270° C at the rate of 2°C
Results
Stress-relaxation analysis of avocado mesocarp tissue
during ripening—Avocado fruit maintained firmness for
at least one week when stored at 15°C. When the fruit was
transferred to 25°C, softening was initiated and progressed
rapidly. At the prescribed times after transferring the fruit
Fruit softening and wall changes in avocado
605
Water-soluble fraction
Vo 250 73
Ve
<u
8
so
on
Rt (min)
Fig. 1 Stress-relaxation parameters of avocado mesocarp tissues
associated with ripening. A transverse slice (7-8 mm thick) of
avocado mesocarp tissue was placed on the testing stage. The conical probe was inserted into the tissue to the depth of 0.6 mm.
Stress perceived by the probe was monitored from 0 to 60 s at 1 s
intervals. The initial stress was based on the force required at 0 s.
The time dependent decay in the stress (R) was used for the calculation of stress-relaxation parameters. Minimum stress relaxation
time (To) and maximum stress-relaxation time Tm are plotted
against a logarithmic scale. Vertical bars represent standard errors
(n=20).
Fig. 2 Molecular distribution profile of water soluble fraction
from avocado mesocarp tissues at different ripening stages. A portion of the water-soluble fraction was introduced into an HPLC
equipped with a gel-filtration column. The sample was eluted with
50 mM Na-phosphate buffer at a flow rate of 1 ml min" 1 . Fractions were collected at 0.5 min intervals. The total sugar content
was determined by the phenol sulfuric acid method. The m-hydroxydiphenyl method was used for uronic acids. Total peak area represents the amount of sugar extracted from one g FW of tissue.
The column was calibrated with dextrans with known molecular
mass (250 and 73 kDa) as shown in the upper panel. Vo and Ve
were calibrated with blue dextran and Glc. 0, day 0; • , day 1; D,
day 2.
Table 1 Sugar content of cell wall polysaccharides in avocado mesocarp tissues at different ripening stages
NS
UA
NS
Sugar content (mg (g FW)" ')
HB
HA
UA
NS
UA
NS
UA
0
4.21
7.0
2.14
5.38
6.29
0.69
0.17
1
4.54
11.2
1.56
3.79
6.75
0.87
2
4.93
10.5
1.36
1.37
5.76
3
9.06
12.1
1.26
1.40
6
6.76
10.3
1.05
0.80
Day
Pectin
WS
Cellulose
Total
0.06
10.1
36.0
0.23
0.07
11.7
40.5
0.74
0.15
0.05
9.8
34.6
5.83
0.88
0.19
0.06
12.3
43.1
6.67
0.81
0.51
0.04
13.2
39.8
Avocado fruits stored at 15°C were transferred to 25°C on day 0. Mesocarp tissues were excised at equatorial plane and stored at
- 80°C. Wall fractions were extracted successively with water (WS), hot EDTA (pectin) and alkaline solution (hemicelluloses). The
hemicellulose fraction was divided into soluble (HB) and insoluble (HA) fractions. The cellulose fraction is the residue. Cellulose content was determined by a phenol sulfuric acid method. Neutral sugar (NS) content was determined by GLC. Uronic acids (UA) were determined by a /n-hydroxydiphenyl method (n = 3). The ratio of standard errors to means was less than 3.6%.
606
Fruit softening and wall changes in avocado
Table 2 Sugar composition of water-soluble fraction from avocado mesocarp tissues at diflferent ripening stages
Rha
Fuc
1
Sugar composition (mg (g FW)- )
Xyl
Man
Ara
0
0.14
0.11
0.96
0.84
0.27
0.77
1.12
1
0.23
0.10
1.40
0.83
0.25
0.75
0.99
2
0.26
0.11
1.79
0.86
0.27
0.74
0.89
3
0.41
0.26
3.52
1.54
0.39
1.17
1.78
6
0.50
0.14
2.41
1.24
0.36
1.23
0.89
Gal
Glc
The water soluble fraction (WS) was subjected to GLC analysis. At each sampling, the fraction was hydrolyzed with 2 M trifluoroacetic
acid containing m-inositol as an internal standard for 1 h at 121 "C. Monomeric sugars were reduced, acetylated, and introduced into
GLC. The amount of each sugar constituent was determined by comparing with the peak area of m-inositol (internal standard).
to the higher temperature, the fruit was sliced and subjected to analysis of the physical parameters. We observed
that the initial stress, To, and Tm values declined rapidly
(Fig. 1), while the change in R increased. All of the parameters tended to stabilize after three days.
Sugar analysis of cell walls during avocado ripening—
Net changes in total amount of cell wall polysaccharides
recovered during avocado fruit ripening were found only in
water-soluble and pectic fractions (Table 1). The UA content of the WS fraction increased by 38% on the first day.
The WS neutral sugar content gradually increased in the
first 2 days, and then increased markedly. Conversely, the
UA content of pectin decreased by 75% and NS content by
36% within 2 days. The net sugar contents of hemicellulose
and cellulose fractions and the total amount of the cell wall
polysaccharide changed little during the ripening period
through day 6. The ratio of A490 to A480 within the cellulose fraction was 1.06 to 1.10, suggesting that pentose was
not a significant component of this fraction.
The NS composition of the WS fraction is shown in
Table 2. Rha and Ara content increased after day 0, and
Xyl and Gal content increased from day 3. The NS composition of the pectin fraction is shown in Table 3. In con-
trast to the data for the WS fraction, the pectic Rha, Ara,
and Gal contents decreased following day 0. Xyl, Man, and
Glc content remained relatively constant throughout ripening. The NS composition of HB is shown in Table 4. The
amounts of the monomeric sugar components changed little throughout the experiment, including those that were
components of the HA fraction (Table 5).
Molecular weight distribution of soluble polysaccharides—It is generally more difficult to detect depolymerization of polysaccharides if they are degraded by endo-type
hydrolytic enzymes; cleavage of bonds can occur without
the solubilization of significant amounts of the component
sugars. Figure 2 compares the molecular distribution of
WS fraction of avocado mesocarp tissues, extracted on
day 0, 1 and 2, by gel-filtration chromatography. The total
area of this chromatogram represents the amount of WS
fraction extracted from 1 g FW of the fruit. The upper
panel shows the molecular distribution of TS estimated by
phenol-sulfuric acid assay and the lower panel, that of UA
estimated by the /w-hydroxydiphenyl assay. The WS fraction at day 0 contained a major TS peak at low mol wt (Rt
13 min) and a small polysaccharide peak with high mol wt
(Rt 7 min). As the fruit ripened, the peak that previously
Table 3 Sugar composition of pectic fraction from avocado mesocarp tissues at different ripening stages
Rha
Fuc
Sugar composition (mg (gFW)- 1 )
Ara
Xyl
Man
0
0.32
0.04
1.13
0.07
0.13
0.32
0.13
1
0.21
0.02
0.79
0.06
0.11
0.22
0.15
2
0.17
0.00
0.64
0.05
0.12
0.19
0.18
3
0.17
0.00
0.60
0.05
0.12
0.19
0.15
6
0.16
0.00
0.48
0.05
0.11
0.17
0.08
Gal
Glc
Pectic fraction extracted with hot EDTA was dialyzed against water, and then hydrolyzed with 2 M trifluoroacetic acid. Each monomeric sugar was determined as described in Table 2.
Fruit softening and wall changes in avocado
Table 4
607
Sugar composition of HB from avocado mesocarp tissues at different ripening stages
Sugar composition (mg (gFW)-')
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
0
0.13
0.24
0.64
2.10
0.65
0.72
1.82
1
0.17
0.23
0.80
2.43
0.68
0.75
1.70
2
0.15
0.22
0.74
1.98
0.60
0.67
1.40
3
0.12
0.17
0.68
2.02
0.63
0.70
1.51
6
0.17
0.67
2.52
0.71
0.72
1.66
0.23
Soluble hemicellulose fraction (HB) was subjected to GLC analysis. The fraction was hydrolyzed with 2 M trifluoroacetic acid, and each
monomeric sugar constituent was determined as described in Table 2.
eluted at a low mol wt region shifted slightly toward the
high mol wt region. The elution pattern of UA within the
Pectin
V0
250 73
WS fraction differed from that of TS. On day 0, the main
peak eluted near the void volume. On day 1, the amounts
of intermediate sized polymers increased. On day 2, the
main peak eluted at lower mol wt. The TS peak in low mol
wt region did not coelute with the UA peak on day 0 or 1,
and the ratio of TS to UA in the fraction eluting at 13 min
was very high, suggesting that the TS peak in the low mol
wt region consists mainly of NS.
Figure 3 illustrates the molecular distribution pattern
of the pectin fraction of the mesocarp tissues on day 0, 1,
and 2. Elution patterns of TS and UA components of the
pectin fraction revealed that the uronides eluted near the
c
<u
bfj
00
Rt (min)
Fig. 3 Molecular distribution profile of the pectin fraction extracted from avocado mesocarp tissues at different ripening
stages. A portion of the pectin fraction was introduced into an
HPLC equipped with a gel-filtration column. The sample was
eluted with 50 mM Na-phosphate buffer at a flow rate of 1 ml
min" 1 . Fractions were collected at 0.5 min intervals. Sugar content was determined by a phenol sulfuric acid method for total
sugar and /n-hydroxydiphenyl method for uronic acids. Total
peak area represents the amount of sugar extracted from one FW
of tissue. The column was calibrated with dextrans with known
molecular mass (250 and 73 kDa) as shown in the upper panel. Vo
and Ve were calibrated with blue dextran and Glc. Q, day 0; • ,
day 1; O, day 2.
10
12
Rt (min)
Fig. 4 Molecular distribution profile of soluble hemicellulose
fraction from avocado mesocarp tissue at different ripening
stages. A portion of the soluble hemicellulose fraction was introduced into an HPLC equipped with a gel-filtration column.
The sample was eluted with 50 mM Na-phosphate buffer at a flow
rate of 1 ml min" 1 . Fractions were collected at 0.5 min intervals.
Sugar content was determined by a phenol sulfuric acid method.
Total peak area represents the amount of sugar extracted from
one g FW of tissue. The column was calibrated with dextrans with
known molecular mass (250 and 73 kDa) as shown in the upper
panel. Vo and Ve were calibrated with blue dextran and Glc. H,
day 0; • , day 1; O, day 2.
608
Fruit softening and wall changes in avocado
Table 5
Sugar composition of HA from avocado pericarp tissues at different ripening stages
Sugar composition (mg (gFW)- 1 )
Day
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
0
0.016
0.004
0.048
0.023
0.016
0.026
0.034
1
0.025
0.006
0.066
0.033
0.023
0.030
0.046
2
0.018
0.004
0.044
0.024
0.016
0.019
0.026
3
0.020
0.002
0.051
0.037
0.023
0.024
0.033
6
0.020
0.005
0.034
0.026
0.019
0.018
0.025
Insoluble hemicellulose fraction (HA) was subjected to GLC analysis. After neutralization of the alkaline extract with glacial acetic acid
and dialysis against water, the solution was centrifuged. The precipitate (HA) was dissolved in 2 M trifluoroacetic acid and hydrolyzed.
Each monomeric sugar constituent was determined as described in Table 2.
250
73
Ve
void volume contained higher amounts of NS than the intermediate sized uronides. As the fruit ripened, the amounts
of high and intermediate sized polymers in the pectin fraction decreased.
The molecular wt distribution of HB on day 0, 1, and
2 (Fig. 4) revealed that there was a slight decrease in the recovery of polysaccharides during ripening. The NS composition of each fraction eluted from the column was determined by GLC. The several sugar components of the
fractions were considered to be components of xyloglucan.
The elution patterns of Fuc and Glc in the profile were
shifted toward the lower mol wt region by day 2 (Fig. 5).
The shoulder peaks of Xyl and Gal eluted in the high mol
|
Vo 250 73
8
I
on
FW)
500
400-
fib
300-
•
i
i
ve
i
c
8
200-
"Hb
100X
Rt (min)
Fig. 5 Molecular distribution profile of sugar components of
soluble hemicellulose fraction from avocado mesocarp tissue at
different ripening stages. Soluble hemicellulose fraction extracted
from avocado mesocarp tissue on day 0 and 2 was introduced into
an HPLC system equipped with a gel-filtration column. Eluant
from the HPLC column was collected at 0.5 min intervals as described in Fig. 4. Each sample was subjected to GLC analysis. The
sample was hydrolyzed with 2 M trifluoroacetic acid, then reduced
and acetylated. The column was calibrated with dextrans with
known molecular mass (250 and 73 kDa) as shown in the upper
panel. Vo and Ve were calibrated with blue dextran and Glc. o,
day 0; • , day 2.
o-
Fig. 6 Molecular distribution profile of iodine-staining material
in soluble hemicellulose fraction from avocado mesocarp tissue at
different ripening stages. Soluble hemicellulose fraction extracted
from avocado mesocarp tissue on day 0, 1 and 2 was introduced
into an HPLC system equipped with a gel-filtration column.
Eluant from the column was collected at 0.5 min intervals. The
eluant (0.5 ml) was mixed with 1 ml of 15% sodium sulfate and
0.125 ml of 0.5% I 2 +1.0% KI. After 1 h at 4°C in the dark the absorbency at 640 nm was determined. The column was calibrated
with dextrans with known molecular mass (250 and 73 kDa) as
shown in the upper panel. Vo and Ve were calibrated with blue
dextran and Glc. a, day 0; • , day 1; D, day 2.
Fruit softening and wall changes in avocado
wt region also decreased. However, the Xyl that eluted
within the intermediate range of molecular weights may
not be a component of xyloglucan. In this region, the
amount of Glc was lower than that of Xyl. Nevertheless,
the results suggested that the high mol wt xyloglucan was
converted into smaller mol wt components during ripening.
To confirm this possibility, an iodine staining method
which is diagnostic for xyloglucan was applied to the eluant
of HB from the HPLC column. Fig. 6 clearly indicates that
the mol wt distribution of these iodine positive materials
shifted to the lower mol wt region in 2 days. The total
amount of stained materials was somewhat less on day 2.
Discussion
The results clearly revealed that the net cellulose content of avocado mesocarp cell walls did not change during
ripening. The measurement of physical properties of the
fruits by a stress-relaxation analysis demonstrated substantial softening of the fruit over the same 3 day period. These
results, however, do not negate the possibility that the molecular weights of glucan polymers comprising cellulose do
in fact decrease without a change in the absolute content.
O'Donoghue et al. (1994) confirmed the slight downshift in
the M r of unbranched cell-wall polymer, presumably cellulose, in avocado.
A putative avocado cellulase has been extensively investigated, because the fruit produces prodigious quantities of
this enzyme as a specific response during ripening (Awad
and Young 1979, Awad and Lewis 1980). In virtually all
cases, the method used to detect this putative cellulase activity is the measurement of change in viscosity or release of reducing sugar in the presence of carboxymethyl cellulose
(Awad and Lewis 1980, Hatfield and Nevins 1986, Pesis et
al. 1978, Ronen et al. 1991); therefore, the enzyme should
be designated as Cx-cellulase.
Hatfield and Nevins (1986) originally raised questions
relative to the role of Cx-cellulose in softening process of
avocado fruits, because the enzyme they purified from ripe
fruits was incapable of releasing Glc from immature fruit
cell walls. If the activity is instrumental in ripening processes, one possibility is that it is related to the breakdown
or depolymerization of xyloglucans which are degraded
by endo-type cellulase activity as shown in pea segments
(Hayashi et al. 1984). O'Donoghue and Huber (1992), however, claimed that the Cx-cellulase activity did not change
the molecular weight of xyloglucan in vitro. Nevertheless,
cellulase activity determined using carboxymethyl cellulose
as substrate is often reported as an integral part of fruit
softening mechanisms (Kanellis et al. 1991). In other tissues such as pear fruit, neither endo-type cellulase activity nor degradation of cellulose has been detected (Ahmed
and Labavitch 1980a, b). Besides the action of cellulase,
the degradation of xyloglucan during the process of avo-
609
cado ripening was suggested (O'Donoghue and Huber
1992), although the relationship between such degradation
and tissue firmness was not clear.
The results of this study clearly show that the molecular weight downshift of xyloglucan within the HB fraction,
a component determined by both the presence of specific
sugars and iodine staining, reflects the consequences of
depolymerization. Such changes in mol wt distribution of
xyloglucans have been previously reported in auxin-induced segment elongation (Nishitani and Masuda 1983,
Wakabayashi et al. 1991). Nakamura and Hayashi (1993)
purified endo-l,4-/?-glucanase from the culture medium
of poplar cells. It hydrolyzed xyloglucan, but the activity
was only 2% of that toward CM-cellulose. The activity of
endo-xyloglucan transferase increases during the softening
process of kiwifruit (Redgwell and Fry 1993) and tomato
(Maclachlan and Brady 1994). If the xyloglucan molecules
attach to the cellulose microfibrils and serve as cross-links
as proposed (Hayashi 1989), degradation or depolymerization of xyloglucans by endo-glucanase or transferase activity would facilitate sliding of the two adjacent microfibrils.
Cellulose molecules have a very high Young's modulus
(Nobel 1983). Hence, the elastic properties of cell walls are
likely due to properties of a cellulose microfibril network interconnected by xyloglucan molecules. The rapid decrease
in the initial stress detected by stress-relaxation analysis
may have been due to the loss of elastic properties of the cellulose microfibril network mediated by endo-type hydrolytic enzymic cleavage of xyloglucan molecules.
In addition to the xyloglucan breakdown, we found
significant degradation of pectic polysaccharides during
avocado fruit ripening. Pectic polysaccharide degradation
in avocado was also reported by Huber and O'Donoghue
(1993). They found that uronic acid content extractable by
CDTA increased during avocado fruit ripening. The increase they report appears to correspond to the increase in
UA that we observe in the WS fraction. The incremental increase of uronides in the WS fraction as a function of ripening corresponds to the reduction of amount of pectin fraction extracted by hot EDTA which is more effective for
extraction than CDTA at 23°C. The high ratio of UA content in the WS also supports the relationship between WS
and pectin fractions. It seems that the breakdown of pectic
polysaccharides facilitates the solubilization of oligomeric
sugar constituents from the cell wall; these are recovered in
the aqueous extraction. A summation of WS and pectin
fraction reveals nearly constant values throughout ripening. These results suggest that there was little actual loss of
acidic polysaccharide during the avocado ripening, but that
depolymerization does occur.
A decrease in T o has frequently been reported as a
reflection of wall changes in auxin-induced elongation in
many plant systems (Masuda 1990, Sakurai 1991), but a
decrease in Tm has also been noted in softening of tomato
610
Fruit softening and wall changes in avocado
fruit (Kojima et al. 1991) and banana (Kojima et al. 1992).
Both parameters are thought to reflect viscosity within cell
walls (Sakurai 1991). Decreases in To and Tm found in the
process of avocado ripening suggest a decrease in wall
viscosity. R was reported to decrease in auxin-induced elongation in oat coleoptiles (Sakurai et al. 1982). The increase
in R in the process of avocado ripening implies that the
wall softening process in avocado fruits differs from that induced by auxin in stems.
Supported in part by Grant DCB-9106136 from the National
Science Foundation (to D.J.N.). We gratefully acknowledge the
technical assistance of Hisae Kamakura.
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(Received December 13, 1996; Accepted March 5, 1997)