Cellulase, Fruit Softening and Abscission in Red Raspberry Rubus idaeus L. cv Glen Clova
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Annals of Botany 80 : 371–376, 1997
Cellulase, Fruit Softening and Abscission in Red Raspberry Rubus idaeus L. cv
Glen Clova
R O Y S E X T O N*, J A N E M. P A L M E R, N I C H O L A A. W H Y TE and S U S A N L I T T L E J O H NS
* Department of Biological and Molecular Sciences, Stirling Uniersity, Stirling FK9 4LA, Scotland, UK.
Received : 13 February 1997 Accepted : 1 May 1997
The ripening of raspberry fruit (Rubus ideaus L. cv Glen Clova) is associated with a climacteric rise in ethylene
production. As the fruit pigments change from green to red there is a progressive softening, loss of skin strength and
a breakdown of cell walls in the mesocarp. An increase in cellulase (endo-1,4-β--glucanase) in both drupelets and
receptacles accompanies these changes. The localization of cellulase in the regions of the fruit associated with
abscission zones suggest the enzyme may be involved in fruit separation as well as softening.
# 1997 Annals of Botany Company
Key words : Rubus idaeus L, raspberry, fruit ripening, ethylene, abscission, cell wall breakdown, cellulase, endo-1,4-
β--glucanase.
Abeles and Takeda (1989) have shown that cellulase activity
INTRODUCTION
increases during the ripening of the closely related blackberry
The delicate nature of raspberry fruits is a major difficulty (Rubus fruticosus).
for growers and processors. The ripe fruit are easily ruptured In an attempt to obtain more basic information about the
during harvesting, transport and commercial operations nature of raspberry softening we have measured the loss of
(Reeve, Wolford and Nimmo, 1965 ; Jennings, 1988 ; Joles et skin strength which accompanies ripening of Rubus idaeus
al., 1994). Continued softening after harvesting exacerbates L. cv Glen Clova and related this to anatomical changes
this problem and is a contributory factor to their extremely within the fruit. A correlation has also been sought between
short shelf life (Barritt et al., 1980 ; Sjulin and Robbins, cell wall breakdown and the levels of cellulase.
1987).
The recent transfer of genes into raspberry plants MATERIALS AND METHODS
(Mathews et al., 1995) raises the prospect of being able to
manipulate raspberry softening. Such methods have been Selection of deelopmental stages
successfully employed in tomatoes although this was only Raspberry canes (Rubus idaeus L. cv Glen Clova) were
possible because the role of ethylene (Hamilton, Lycett and grown at Dollar Fruit Farm, Dollar, Perthshire, UK. Fruit
Grierson, 1990 ; Oeller et al., 1991) and wall degrading were categorized into five developmental stages according
enzymes (Smith et al., 1988 ; Sheehy, Kramer and Hiatt, to the criteria of Burdon and Sexton (1990). The major
1988 ; Tieman et al., 1992) was well established. Very little characteristics of these five stages are shown in Table 1.
research has been done into the nature of the corresponding Ethylene production rates were determined by methods
changes in raspberries. described previously (Burdon and Sexton, 1990).
There is an increase in ethylene production as raspberries
ripen until physiologically active concentrations are found
Measurement of fruit texture during deelopment
in red fruit (Burdon and Sexton, 1990 ; Perkins-Veazie and
Nonnecke, 1992). The softening of fruit appears to be a Fruit texture was measured using a penetrometric method
multicomponent process. Underneath the epidermis and to assess the skin strength. A JJ Lloyd Tensile Testing
hypodermis the thin walled mesocarp cells become distended Machine (Southampton, UK) fitted with a 5 N load cell was
during fruit expansion and the delicate nature of these cells used to drive a blunt ended steel probe, diameter 0±82 mm,
contributes to the textural changes (Reeve, 1954 ; Jennings, into individual drupelets at a speed of 8 mm min−". To
1988). It seems very likely that there is also extensive cell standardize, drupelets were chosen from the centre of the
wall breakdown since Duclos and Latrasse (1971) report a fruit, and the probe was driven down perpendicular to the
halving in the total pectin content of Malling Exploit fruit drupelet surface. After the probe made contact with the
during maturation. Wall degradation is usually accom- drupelet a gradual increase in applied force was recorded on
panied by increases in cell wall hydrolases such as a time¬force trace. This was followed by a sudden drop as
polygalacturonase and endo-1,4-β- glucanase (cellulase) it penetrated the surface, the peak force applied before
(Fischer and Bennett, 1991). The levels of these enzymes do rupture is referred to as skin strength. Fruit firmness was
not appear to have been measured in raspberries ; however also assessed by adapting the compression method of
0305-7364}97}09037106 $25.00}0 bo970465 # 1997 Annals of Botany Company372 Sexton et al.—Cellulase, Fruit Softening and Abscission in Raspberry
T 1. Characteristics of fie stages of fruit deelopment cloth (Calbiochem, Nottingham, UK). The pH was moni-
tored thoughout to ensure that it remained above 7±0 and
Approx. weight EPR was adjusted to 7±2 prior to assay. Cellulase activity was
Developmental stage Drupelet colour of fruit (g) nl g−"h−" measured using the viscometric assay of Durbin and Lewis
(1988). This method is based on the loss in viscosity of 1 %
Small green (SG) Bluish green 1±00 0±05 carboxymethylcellulose solution (CMC 7H3SF, Aqualon
Large green (LG) Pale yellow green 2±00 0±07
Mottled (M) Green and red 2±90 0±65 Ltd, Warrington, UK) in 20 m sodium phosphate buffer,
Red ripe (R) Red 3±90 2±15 pH 7±2. Cellulase units were calculated from intrinsic
Purple ripe (PR) Purple red 3±80 5±98 viscosity changes using the table in Durbin and Lewis
(1988).
The ethylene production rate (EPR) data are from the fruit used in The location of cellulase in fruit was initially determined
Table 4. in separated drupelets and receptacles. To establish if the
cellulase associated with the receptacles was found in the
Barritt et al. (1980). Compression is defined as the force ruptured abscission zones coating its surface they were
required to close the opening of a berry by applying a force dissected into the following parts : (1) the surface or outer
at right angles to the fruit’s long axis. This was measured by 1 mm portion of the receptacle which contains all the
driving a small flat plate at 8 mm min−" onto a fruit lying on abscission zones ; (2) the base or basal smooth section of the
its side until the opening had just closed. The point at which receptacle proximal to the attachment of the drupelets
this occurred was marked by hand on the force¬time trace which does not have abscission zones ; and (3) the core or
as no obvious change was recorded at closure. region within the surface area of the receptacle. The
abscission zones on the inner side of the cap of fresh ripe
drupelets were difficult to dissect, but if frozen fruit are
Microscopy
allowed to thaw slightly, the abscission zones can be shaved
Individual raspberry drupelets from fruit at each stage of off with a sharp scalpel.
development were fixed overnight at 4 °C in 2 % glutar-
aldehyde in 25 m sodium cacodylate buffer pH 7±2. The
drupelets were then dehydrated through an ethanol series RESULTS
and embedded in JB4 resin (Polysciences, Warrington,
Pennsylvania, USA) according to the manufacturer’s in- Measurement of fruit texture
structions. Longitudinal 4 µm sections running through the The force necessary to rupture the skin of fruit at the large
seed were cut with glass knives using a LKB Pyramitome green (LG), mottled (M), ripe (R) and purple ripe (PR)
(Bromma, Sweden), and stained with either ruthenium red, stages was measured by penetrometry (Table 2). As the fruit
periodic acid Schiff ’s stain (PAS) or calcofluor, according to start to change colour the skin strength drops progressively
O’Brien and McCully (1981). from 1±12 N to 0±055 N at the PR stage. Values within each
For electron microscopy apical portions of drupelets were stage were very consistent with small standard errors.
excised using a sharp razor and fixed as described above. The force required to close the opening of a detached fruit
These ‘ domes ’ were washed several times with distilled has been previously used as an index of softening (Robbins
water and fixed overnight with 1 % osmium tetroxide before and Sjulin, 1986). A progressive decrease in this ‘ compression
washing thoroughly and dehydrating through an ethanol force ’ was observed throughout development (Table 2)
series. The dehydrated samples were left uncovered over- similar to that reported by Robbins and Sjulin (1986). The
night in a 1 : 1 mixture of LR White (London Resin Co. values obtained in this compression test were more subjective
Ltd., Woking, UK) and absolute ethanol. This mixture was than the penetrometer determinations, since the point of
decanted off and the tissue infiltrated for a few hours in LR closure is not precise.
White before the resin was set by heating at 60 °C for 22 h.
Extraction and assay of cellulase T 2. Changes in fruit softness at different stages of
Enzymes can easily be inactivated during extraction from deelopment
raspberry fruit by coming into contact with the very acidic
vacuolar sap. Abeles and Takeda (1989) overcame this Skin strength (N) Compression (N)
Stage of development ³s.e. ³s.e.
problem in blackberries by precipitation of proteins with
acetone. However, preliminary trials showed that the
Large green 1±124³0±052a —
following method retained significantly more activity. Fruit Mottled 0±481³0±011b 4±84³0±243w
were frozen in liquid nitrogen and ground to a powder in a Ripe 0±202³0±009c 2±38³0±614x
pre-cooled coffee grinder. The powder was thawed into ice Purple ripe 0±055³0±003d 1±33³0±343z
cold 1 Tris}HCl, 0±5 NaCl, pH 8±0 (for drupelets) or
pH 7±2 (for receptacles) before filtration through two layers Skin strength was measured in Newtons with a penetrometer and
compression values determined in Newtons by adapting the method of
of muslin. Cell debris was removed by spinning for 20 min
Barritt et al. (1980). Means were separated by Duncan’s multiple range
at 27 000 g in a Sorvall Superspeed RC2-B centrifuge at 4 °C test P ! 0±05 ; n ¯ 15 berries for compression, n ¯ 45 drupelets for skin
and straining the supernatant through 50 µm mesh Mira- strength.Sexton et al.—Cellulase, Fruit Softening and Abscission in Raspberry 373
F. 1. Sections through surface of a large green (A), mottled (B), and purple ripe drupelet (C) stained with PAS. Note how the discrete mesocarp
walls (A) become swollen (arrows, B) and eventually separate (arrows, C) as the fruit ripens. Bar ¯ 50 µm. D, The separated mesocarp cells released
into water from a cut purple ripe fruit. Bar ¯ 100 µm. Sections from small green (E) and ripe fruit (F) stained with calcofluor and viewed using
epi-fluorescence. Note how the discrete mesocarp walls of green fruit (E) degrade and separate along the line of the middle lamella (arrows) in
ripe fruit (F). Bar ¯ 50µm.
Very few cells were released from large green and mottled
Morphological changes during ripening
drupelets, but there was a large increase in the number of
Changes in fruit texture can be due to a variety of single cells lost from cut surfaces of ripe fruit. Data for cell
different factors. Since cell wall breakdown is commonly clusters of various sizes followed the same trend (data not
implicated in the process, an anatomical investigation of shown). These observations indicate extensive cell sep-
changes in cell wall structure was undertaken. When a ripe aration is already complete at the ripe stage.
raspberry drupelet is ruptured in water, a large number of Changes in cell wall structure during development were
isolated cells are released, presumably because the in- investigated by light microscopy using a variety of stains for
tercellular cement has been degraded (D. L. Jennings, pers. different cell wall components (Fig. 1). The mesocarp cell
comm. ; see Fig. 1 D). To investigate when this process was walls were discrete and undegraded in small green (SG) and
initiated, we standardized the procedure and counted the large green (LG) fruit (Fig. 1 A). By the mottled stage (M),
number of single cells released from a cut drupelet (Table 3). cell wall swelling was apparent in some regions of the374 Sexton et al.—Cellulase, Fruit Softening and Abscission in Raspberry
T 3. Numbers of single isolated cells released into water thin and undegraded with distinct, darkly stained middle
from the seered apical domes from druplets at different lamellae. A similar cell from ripe fruit (Fig. 2 D) illustrates
stages of deelopment how the wall becomes diffuse and distended. It still retains
cellulose microfibrils but the middle lamella and the rest of
Average number of single the wall matrix have become swollen and diffuse.
Stage of development separated cells released
Large green 1 Cellulase actiity
Mottled 14
Red ripe 392 To investigate whether there was any relationship between
Purple ripe 535 cellulase activity and the breakdown of drupelet mesocarp
walls, the outer red drupelets and inner white receptacles
were assayed throughout development (Table 4). Surpris-
mesocarp (Fig. 1 B). This swollen material is PAS positive ingly most cellulase activity per gram of tissue was located
suggesting it is degraded wall. The extent of wall breakdown in the receptacle rather than the drupelets at each stage.
was even more apparent in ripe (R) and purple ripe (PR) Receptacle cellulase activity increased progressively from
fruit (Fig. 1 C). Calcofluor, which stains the cellulosic com- 43±0 units g−" in SG fruit to 548±77 units g−" in PR. In the
ponent of the wall, also revealed swelling and separation drupelets, the activity increased from 3±87 to 49±89 units g−"
of the wall of ripe fruit (Fig. 1 F). Ruthenium red staining over the same developmental stages.
for pectins produced quite strong staining of the mesocarp During the ripening of raspberry fruit there is a progressive
walls of green fruit (Fig. 2 A) which became paler and more weakening of the 70–80 abscission zones which attach the
diffuse as the fruit matured (Fig. 2 B). Cell wall degradation drupelets to the central receptacle (Burdon and Sexton,
was not so obvious in the epidermis and hypodermis (eg Fig. 1990) The force necessary to rupture these abscission zones
2 B) which was retained as an intact skin. decreases as the fruit ripen until ultimately the berry will
Figure 2 C shows a cell from the mesocarp of unripe SG often fall if touched (Table 4). Since cellulase is implicated
fruit examined by electron microscopy. The cell walls are in the wall breakdown associated with abscission (Brummell,
F. 2. Sections from green (A) and ripe (B) drupelets stained with ruthenium red. Note the loss of pectin staining in the mesocarp of the ripe
fruit (arrows) with the exception of the epidermis (ep) and hypodermal layer under it. Bar ¯ 50µm. Electron micrographs of mesocarp cells from
small green fruit (C) and ripe fruit (D). Note the swelling of the walls in the ripe fruit (arrows). Bar ¯ 10µm.Sexton et al.—Cellulase, Fruit Softening and Abscission in Raspberry 375
T 4. Cellulase actiity of maturing raspberry receptacles and drupelets expressed as units per gram fresh weight or per
fruit
Drupelets Receptacles
FRF Cellulase Cellulase Cellulase Cellulase
Stage (N)(s.d.) units g−" units per fruit units g−" units per fruit
SG " 9±8 3±87 3±64 43±07 1±55
LG " 9±8 6±16 9±42 97±25 9±34
M 5±09 (1±20) 14±27 37±53 169±98 22±10
R 3±09 (1±43) 17±77 57±86 321±01 86±67
PR 0±45 (0±88) 49±89 173±62 548±77 170±12
The fruit removal force (FRF) shows the mean force in Newtons necessary to separate the druplets from the receptacle (n ¯ 25) (typical values
from a 1990 sample)
T 5. Cellulase actiity expressed as units per gram of ‘ bleeding ’ of raspberries caused by skin lesions is a
fruit zone considerable problem during fruit handling and transport.
The softening and loss of skin strength in raspberries is
Activity almost certainly a multicomponent process. The mesocarp
Zone units g−" cells become extensively elongated (Reeve, 1954) during the
expansion phase of fruit development and this enlargement
Drupelets from Excised drupelet 168±50 probably explains the textural changes of fruit observed
ripe fruit abscission zones
Drupelets with abscission 8±30 prior to any colour change (Jennings, 1988). As the fruit
zones removed turn red and then purple the berries soften to the point
Intact whole drupelets 28±20 where they are so delicate that just handling them can cause
Receptacles from Excised surface containing 1855±00 damage. Associated with the start of softening there appears
purple ripe fruit abscission zones to be extensive breakdown of the mesocarp cell walls. This
Central core of receptacle 292±00 degradation involves all components of the wall with the
Excised basal surface containing 194±00
no abscission zones exception of the cellulose microfibrils. The wall must retain
some structural integrity since the isolated cells which are
released from ruptured ripe fruit still withstand the turgor
forces generated when they are suspended in water (Fig.
Lashbrook and Bennett, 1994) it seemed possible that this 1 D). The epidermal cell walls and particularly those at the
could be its role during raspberry fruit maturation. At each base of the trichomes are not degraded to the same extent
stage of development the total cellulase activity per raspberry and form an ensheathing skin around the fruit. Consistent
fruit (Table 4) was split roughly equally between drupelet with these observations is the spectacular loss of proto-
and receptacle, suggesting that when the abscission zone pectins from the walls of ripening soft raspberry varieties
breaks approximately half the cellulase is left on the two like Malling Exploit (Duclos and Latrasse, 1971). The levels
fracture faces. of soluble pectins are very high in expressed raspberry juice
An attempt was made to identify the location of cellulase producing viscous solutions which are a problem for the
activity within the receptacles and drupelets (Table 5). The food industry (Will and Dietrich, 1994).
surface of the purple ripe receptacles containing the In their SEM study of Botrytis infection of cryoprepared
abscission zones had six times more activity per gram of raspberry fruit, Williamson and Duncan (1989) did not
tissue than the inner core and five times the activity of the observe such extensive wall modification in uninfected ripe
base which carries no abscission zones. Excised drupelet fruit as we would have expected, but they did report
abscission zone tissue had 20 times the activity of the rest of reticulate material between all the cells in over ripe fruit.
the drupelet. A major component of the cellulase activity in Obtaining good, well fixed anatomical preparations of ripe
ripe fruit is found in regions where abscission is taking soft fruit is notoriously difficult (Williamson and Duncan,
place. 1989) and there is a possibility that our embedding
procedures have accentuated the extent of the wall changes.
DISCUSSION
Cellulase activity increases markedly during softening of
Skin strength, as measured by penetrometry, appears to be a raspberry drupelets. Similar changes have been found in
good index of fruit integrity or ‘ softness ’ despite the fact it ripening avocados (Awad and Young, 1979), strawberries
depends on several parameters including mesocarp and (Abeles and Takeda, 1990), blackberries (Abeles and
epidermal integrity, turgor and cuticle strength. In this Takeda, 1989), peaches (Hinton and Pressey, 1974), man-
study it was much less subjective than the compression goes (Abu-Sarra and Abu-Goukh, 1992) and tomatoes
measurements used previously (Barritt et al., 1980 ; Robbins (Brummell et al., 1994). The levels of cellulase in the
and Moore, 1990). It may be a useful method of assessing drupelets of ripe raspberries are similar to those in tomato,
the susceptibility to damage of different fruit since the about half those found in mango, but only 0±3 % of those376 Sexton et al.—Cellulase, Fruit Softening and Abscission in Raspberry
found in avocado (Saucedo and Sexton, unpubl. res.). In Fischer RL, Bennett AB. 1991. Role of cell wall hydrolases in fruit
spite of these correlations the precise role of cellulase in the ripening. Annual Reiew of Plant Physiology and Plant Molecular
Biology 42 : 675–703.
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clear (Brummell et al., 1994). expression of 2 endo-1,4-beta-glucanase genes in the pericarp and
High levels of cellulase were found in the receptacle which locules of wild type and mutant tomato fruit. Plant Physiology
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