Scientia Horticulturae, 42 (1990) 269-275 269

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Cellulase Activity and Ethylene in Ripening

Strawberry and Apple Fruits*

FRED B. ABELES and FUMIOMI TAKEDA

USDA-ARS, Appalachian Fruit Research Station, 45 Wiltshire Road, Kearneysville, WV 25430
(U.S.A.)
(Accepted for publication 3 August 1989)

ABSTRACT

Abeles, F.B. and Takeda, F., 1990. Cellulase activity and ethylene in ripening strawberry and apple
fruits. Scientia Hortic., 42: 269-275.

A six-fold increase in cellulase activity, measured as the reduction in viscosity of carboxymethyl

cellulose, was associated with maturation and softening in strawberry fruit. Applied ethylene did
not increase cellulase levels in harvested strawberry fruit. This agrees with earlier findings that
ethylene does not appear to regulate strawberry ripening. In apple, an ethylene-sensitive fruit, a
decrease in cellulase activity was observed in fruit allowed to ripen on the tree.

Keywords: apple; cellulase; Fragaria ananassa; Malus sylvestris; ripening; strawberry.

Abbreviation: CMC = carboxymethyl cellulose.

INTRODUCTION

The softening of fruits during ripening is thought to be controlled by a cel-

lulase that degrades cell-wall cellulose and polygalacturonase (s) that degrade
middle lamella pectin. Cellulase plays a role in the ripening of avocado (Chris-
toffersen et al., 1984), blackberry (Abeles and Takeda, 1989), papaya (Paull
and Chen, 1983), peaches (Sterling, 1961), pears (Jermyn and Isherwood,
1956) and tomato (Brady et al., 1987). In the case of avocado (Bennett and
Christoffersen, 1986) and tomato (Babbitt et al., 1973) ethylene (C2H4) has
been shown to control the synthesis of cellulase.
Ethylene is thought to play no role in the growth, coloration or softening of
nonclimacteric fruits such as strawberries (Reid, 1983). Mason and Jarvis
(1970), Hoad et al. (1971) and Nestler (1978) reported that C2H4 had no

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the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion
of other products that may also be suitable.

0304-4238/90/$03:50 1990 Elsevier Science Publishers B.V.

270 F.B. ABELES AND F. TAKEDA

effect on anthocyanin formation. However, a few reports suggest that C2H 4

may play a role in strawberry development. E1-Kazzaz et al. (1983) reported
that C2H4 enhanced the softening and after 1 week's exposure, respiration of
strawberries. Janes et al. (1978) also observed a 10% increase in CO2 produc-
tion after treatment of white-stage strawberries with C2H4. However, it is im-
portant to note that C2H4-enhanced respiration occurs in non-ripening tissues
such as flowers, leaves and tubers (Abeles, 1973).
Earlier, Barnes and Patchett (1976) showed that cellulase activity increased
during the ripening of strawberries and the largest increase occurred when fruit
were overripe. Knee et al. (1977) reported that the development of the straw-
berry receptacle involved a period of cell division followed by softening and
increase in cell volume. It is plausible that, in strawberry, cellulase plays a role
in this process of cell expansion.
The apple is an example of a climacteric fruit whose softening is promoted
by C2H4. However, a review of the literature does not provide examples in
which changes in cellulase activity have been recorded for ripening apples. The
enzymes responsible for the comparatively slow loss of firmness exhibited by
the apple may be exo-polygalacturonases described by Bartley (1978) and Bar-
tley and Knee (1982) or the endo-polygalacturonase described by Liang et al.
(1982).
The purpose of the experiments described here was to examine changes in
cellulase levels in fruit known to be insensitive (strawberry) or sensitive (ap-
ple) to C2H4.

MATERIALSAND METHODS

Strawberry (Fragaria ananassa Duch. cultivar 'Tribute') fruits were grown

in the greenhouse and harvested at five maturity stages. The stages used and
approximate days after anthesis were: green, 7; white, 21; pink, 24; red, 30; and
dark red, 32. Apples (Malus sylvestris Mill cultivar 'Apex Spur Red Delicious' )
were harvested from the orchard at 20 and four days before, during and eight
days after the peak of Cell4 production by fruit on the tree.
Anthocyanin was measured by homogenizing four strawberry fruit in 50 ml
methanol, centrifuging at 15 000 g for 1 min, and measuring the optical density
of the supernatant at 520 nm.
Cell4 and CO2 production from strawberries was measured by placing four
fruits (five in the case of the smaller green fruits) in Erlenmeyer flasks sealed
with a stopper through which was inserted a sealed syringe needle. After a
vented, 4-h preincubation period used to minimize accumulation of wound C2H4,
the bottles were sealed, and the gases allowed to accumulate for an additional
18 h. Three millilitres of gas were removed and measured with a gas chroma-
tograph or infrared gas analyzer. Gas production data were expressed as amount
of gas produced per gram fresh weight per hour. Internal levels of C2Ht in
STRAWBERRY AND APPLE CELLULASE 2 71

apples were measured by inserting a 13 gauge (2.38 m m O.D.) needle into the
center of the fruit. The needle was removed and the core of tissue inside the
needle extruded with a canula. The needle was reinserted in the fruit and a 3-
ml gas sample removed for subsequent analysis.
Flesh firmness in apples was measured with a Magness-Taylor fruit pressure
tester equipped with an l l . 1 5 - m m probe (D. Ballauf Manf. Co., Washington
DC). A Wagner (Greenwich, CT) force dial calibrated in centinewtons (cN)
and equipped with a 5-mm diameter probe was used to measure flesh firmness
of strawberries.
Strawberries were harvested at four stages of maturity described above. Ber-
ries were dipped in 0.05 N NaOC1 to minimize possible fungal development,
air-dried and then exposed for 2 days to 100/tl l-1 C2H4 in 10-1 glass desicca-
tors. The desiccators were vented and C2H4 or air reinjected after 1 day.
Cellulase was extracted from strawberries by homogenizing two fruits with
buffer (0.1 M pH 6.8, potassium phosphate, 1 ml buffer per 2 g fresh weight
fruit) in a Brinkman Kinematic homogenizer for about 15 s. In the case of
apples, 10 g of tissue from fruit were collected and frozen on various dates in
September (see Fig. 2). The tissue was then thawed and homogenized with 10
ml of buffer. The slurry was placed on a premoistened 15-cm square of Mira-
cloth (Calbiochem Corp.) and a portion of the filtered homogenate collected.
Enzyme activity in the supernatant after centrifugation at 10 000 g for 10 min
was measured by mixing 1 ml enzyme solution or appropriate blanks with 2 ml
of 1.5% high viscosity Na salt carboxymethyl cellulose (CMC). In a similar
fashion, viscometric assays ofpolygalacturonase and pectinase were performed
with 2% solutions of citrus sodium polygalacturonate or pectin, respectively.
After incubation for 18 h at 37 C, the viscosity of 1-ml samples was measured
in a model LVT Wells-Brookfield microviscometer (Brookfield Engineering,
Stoughton, MA). Data are expressed as percent reduction in viscosity of CMC
solutions compared with controls without enzyme. Several experiments were
rechecked with the simpler, but less accurate, method of measuring the length
of time it took for the reaction mixture to flow from the 0 to the 0.9 ml mark
of a 1-ml pipette.
The data shown are the averages of three replications. The means of these
determinations were separated by least significant difference as calculated by
the Duncan's multiple range test. The experiments were repeated twice with
similar results.

RESULTS

Fig. 1 presents data illustrating some key features of strawberry fruit rip-
ening. During the increase in fresh weight and anthocyanin development, both
C2H4 and CO2 production were found to be minimal half way through devel-
opment and then to rise two-fold above the m i n i m u m at maturity. The high-
272 F.B. ABELES AND F. TAKEDA

8- Fresh Weight g.fruit - 1

4-
I =LSD 0.05
0
0.4- Anthocyanin OD 5 2 0 ~,je/u

0.2- I
0
80- Ethylene%l. gfw-I .h -I -
/ -
40-
I =LSD 0.05
0-
40-

20-
CO 2 /~1- gfw - 1 . h - 1 ~ I =L-SDo.o5
0-
60- Cellulose % Reduction in Viscosity / .
40-
I = LSDo.o5 j /
20-
0
IOO0
8OO -~ 0.05
6OO
4OO
2OO Flesh F i r m n e s s C e n t i n e w t o n s ~ P i n k_ _K e .y e d Dork
0 I I I
0 10 20 30

Days a f t e r a n t h e s i s
Fig. 1. Changes in fruitweight, anthocyanincontent, C2H4production,C02 production,cellulase
activity and fleshfirmnessduringthe courseof strawberryfruitdevelopment.

low-high pattern of C2H4 production observed here is similar to that reported

earlier by Knee et al. (1977). Cellulase activity of strawberry fruits increased
six-fold on a fresh weight basis as they grew and softened. Using flesh firmness
as a criterion, the primary loss in firmness occurred between the white and
pink stage and a secondary one at the overripe stage. This agreed with earlier
reports by Barnes and Patchett (1976). Cellulase activity increased during the
growth and ripening and the largest increase was associated with overripe fruit.
We also confirmed (data not shown) the earlier reports (Neal, 1965; Barnes
and Patchett, 1976), that there was no increase in polygalacturonase during
strawberry ripening using sensitive viscometric assays of polygalacturonase or
pectinase activity.
As shown in Table 1, cellulase activity in harvested strawberries at different
maturities from green to red was measured prior to and after a 2-day exposure
to air or C2H4 ( 100/tl 1-1 ). The 2-day C2H 4 treatment had no effect on cellulase
levels in strawberry fruit of different maturities. However, cellulase activity of
immature fruit decreased somewhat in the absence of added C2H4.
Cellulase activity in attached ripening apples was determined for a period of
STRAWBERRYAND APPLE CELLULASE 273

TABLE 1

Effect of C2H4 (2 days, 100 #l l - 1 ) on the cellulase activity of strawberries harvested at different
stages of maturity

Stage Cellulase activity1

Initial Air C2H4

Green 31 b~ Ia 35 ~d
White 37 cd 0" 11 "b
Pink 59 def 57 def 45 ode
Red 64 e~ 72 f 69 ef

1Cellulase values shown are the percent reduction in viscosity of CMC after 20 h.
Similar superscripts indicate that the means were similar at the 5 % level.

0
(J 8t ' T :
'
LSD 0.05
80
T- v
_= z~ 60
z
o @
.~40 0 40

~- 0--0 Cellul 20
~A
~" -- Firmness
b- / A--Z~ Ethylene / I = LSDO.O5~,~
0 ,A , ,~ ,
0 10 20 30
DATE SEPTEMBER
Fig. 2. Changes in C2H4 production, flesh firmness, and ceUulase activity during the course of
apple fruit ripening.

time preceding and following the climacteric. We observed a decline in cellu-

lase levels in apples as they went through the respiratory climacteric and par-
tial loss of flesh firmness (Fig. 2).

DISCUSSION

The initial high rate of C 2 H 4 production associated with green strawberry

fruit followed by a low point 20 days after anthesis and a subsequent increase,
is similar to that reported earlier by Knee et al. (1977). The high auxin levels
associated with developing achenes may be responsible for the initial high lev-
els of C2H4. Recently, Given et al. (1988) observed that anthocyanin formation
is under the control of auxin. They reported that the removal of achenes (a
source of auxin) induced anthocyanin formation, while externally applied auxin
delayed it. Since external applications of auxin delayed strawberry ripening, it
274 F.B.ABELESANDF. TAKEDA

is p o s s i b l e t h a t r i p e n i n g in t h i s f r u i t is c a u s e d b y i n c r e a s e d s e n s i t i v i t y t o C2H4
a l r e a d y p r e s e n t as t h e s u p p l y o f a u x i n f r o m t h e a c h e n e s d e c r e a s e s d u r i n g m a -
t u r a t i o n . K n e e et al. (1977) r e p o r t e d t h a t cell division i n c r e a s e d t h r e e - f o l d in
t h e first 7 d a y s a n d t h e r e a f t e r all g r o w t h w a s c a u s e d b y cell e x p a n s i o n . T h e s e
w o r k e r s also d e m o n s t r a t e d t h a t swelling a n d d i s s o l u t i o n o f cell walls o c c u r r e d
d u r i n g t h e l a t t e r s t a g e s o f d e v e l o p m e n t . T h e s e o b s e r v a t i o n s s u g g e s t t h a t cel-
lulase m a y p l a y a role in t h e l o o s e n i n g o f cells u n d e r g o i n g a 25-fold i n c r e a s e in
v o l u m e d u r i n g t h e l a s t 25 d a y s o f f r u i t m a t u r a t i o n .
O n t h e o t h e r h a n d , t h e slower s t e a d y loss o f flesh f i r m n e s s o f a p p l e s m a y b e
c a u s e d b y t h e c o n t i n u e d a c t i o n o f a cellulase a l r e a d y p r e s e n t in t h e fruit. Al-
t e r n a t i v e l y , t h e e n z y m e r e s p o n s i b l e for s o f t e n i n g in t h e a p p l e m a y b e a n exo-
or e n d o - p o l y g a l a c t u r o n a s e d e s c r i b e d earlier.

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