ETHYLENE-INDUCED TISSUE BREAKDOWN IN FRUIT OF WATERMELON
[Citrullus lanatus (Thunb.) Matsum and Nakai]

BY

MOHAMED ELHAG ELKASHIF

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to Dr. D. J.
Huber, chairman of his supervisory committee, for his guidance,
unlimited help, valuable suggestions, and encouragement throughout the
course of this study. Much appreciation is extended to the members of
the supervisory committee, Drs. D. D. Gull and Mark Sherman, for their
help and advice with some experimental procedures, and Drs. C. B. Hall
and R. C. Smith, for their assistance during the preparation of this
manuscript.

The help and cooperation of the faculty and staff of the
Vegetable Crops Department, the staff of the Horticultural Unit, and
the graduate student colleagues also are very much appreciated.

The author is grateful to his wife, Asma B. Abdelrahman, for her
patience, endurance, continuous help, and encouragement throughout the
duration of this study.

Indebtedness to the People of the Democratic Republic of the
Sudan for financial support is also acknowledged.

ii

TABLE OF CONTENTS

PAGE

ACKNOWLEDGMENTS ii

ABSTRACT v

CHAPTER I INTRODUCTION 1

CHAPTER II REVIEW OF THE LITERATURE 3

Respiration and Ethylene Production of Fruits in

Response to Exogenous Ethylene or Propylene 3

Respiration and Ethylene Production of Fruit as

Affected by Microorganisms 10

Membrane Permeability Changes During Ripening and

Senescence 12

The Role of Calcium in Delaying Senescence 18

Activity of Hydrolytic Enzymes During Fruit

Ripening 21

Ultrastructural Changes in Fruit Cells During

Ripening 32

CHAPTER III RESPIRATION AND ETHYLENE PRODUCTION IN WATERMELON

FRUIT EXPOSED TO ETHYLENE OR PROPYLENE 37

Introduction 37

Materials and Methods 39

Results 41

Discussion 45

CHAPTER IV ELECTROLYTE LEAKAGE, FIRMNESS, AND SCANNING

ELECTRON MICROSCOPIC STUDIES OF WATERMELON FRUIT
TREATED WITH ETHYLENE 51

Introduction 51

Materials and Methods 53

Results 55

Discussion 64

CHAPTER V CELL WALL HYDROLASES AND ULTRASTRUCTURE OF

WATERMELON FRUIT AS INFLUENCED BY ETHYLENE 75

Introduction 75

Materials and Methods 77

Results 81

Discussion 90

ili

PAGE

CHAPTER VI SUMMARY AND CONCLUSIONS 103

LITERATURE CITED 106

BIOGRAPHICAL SKETCH 117

»

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ETHYLENE-INDUCED TISSUE BREAKDO\VN IN FRUIT OF WATERMELON
[Citrullus lanatus (Thunb.) Matsum and Nakai]

By

MOHAMED ELHAG ELKASHIF
August 1985

Chairman: Dr. D. J. Huber

Major Department: Horticultural Science

Although watermelon [Citrullus lanatus (Thunb.) Matsum and Nakai]
fruit have been classified as climacteric, there are reports that they
exhibit cellular breakdown when exposed to ethylene gas. This study
comprises an investigation of the postharvest behavior of watermelon
fruit under normal conditions and in response to ethylene or
propylene. Parameters measured included the effects of ethylene on
ripening and respiratory activity, electrolyte leakage, cell wall
ultrastructure, and cell wall hydrolases. All experiments included a
comparison of the changes which occurred during both ethylene-induced
breakdown and natural ripening of fruit.

Respiratory rates in harvested melons showed little change
throughout ripening. Respiratory activity was enhanced in the
presence of ethylene but returned to normal levels upon removal of the
gas. Endogenous ethylene production was not initiated by exposure of
fruit to propylene and was detected only in fruit exhibiting symptoms

V

of decay. The results supported the hypothesis that watermelon fruit
was nonclimacteric.

Ethylene-treated tissue exhibited extensive cell separation, high
activity of D-galacturonase, and enhanced electrolyte leakage. These
ethylene responses did not appear to represent merely an acceleration
of normal ripening since they were not observed in fruit not exposed
to ethylene.

The sequence of events which resulted in watermelon tissue
breakdown emphasized the predominant role of the cell wall. The
increase in D-galacturonase activity observed after the first day of
ethylene treatment coincided with the development of a distinct zone
of separation in the middle lamella of the cell wall. Following
longer periods of ethylene treatment, extensive pectin degradation was
apparent from the increased quantities of polymers fractionating on
Ultrogel AcA 34. Ultrastructural studies revealed that cell walls
started to show signs of deterioration after the first day of ethylene
treatment.

The membrane system did not seem to play a major role in
initiating watermelon tissue breakdown, since no change in electrolyte
leakage was noted during the first 3 days of ethylene treatment. The
increase in electrolyte leakage observed after the 3rd day of ethylene
treatment was apparently due to cell wall degradation and weakening
which led to the rupture of the membrane.

vi

CHAPTER I
INTRODUCTION

Wat6rmelons [Citrullus lanatus (Thunb) Mats, and Nak.] ara among
the most important vegetable crops grown in Florida. A total area of
64,000 acres was planted during the 1983-84 crop year. Production was
placed at 10 million cwt., and the total value of the crop was
estimated at $62.1 million (Florida Agric. Stat., 1984).

Early in the season, watermelons were shipped in mixed loads with
other commodities that produce ethylene or stored in warehouses near
ethylene-producing products. Exposure of watermelons to ethylene
emanations adversely affected their quality and rendered them unfit
for consumption. This observation prompted Risse and Hatton (1982) to
investigate the postharvest response of watermelons to ethylene. They
found that exposure of watermelons to various ethylene concentrations
for 3 or 7 days accelerated their deterioration. Ethylene-treated
watermelons had thinner rinds and were softer than untreated fruit.

The melons were spongy, watery, and had developed off-flavors.

Ethylene treatment had a harmful effect on preripe watermelons as
well. Earlier, Shimokawa (1973) observed that watermelon tissue
exhibited maceration when exposed to ethylene emanations evolved by
'Prince' melons. Enzymic studies of macerated tissue revealed higher
activities of cell wall hydrolytic enzymes as compared to healthy
tissues. These results are seemingly contradictory to the data
presented by Mizuno and Pratt (1973), who reported that watermelons

1

2

produced ethylene in a manner characteristic of climacteric fruits.

It has been well established (McGlasson et al., 1978; Rhodes, 1980a;
Biale and Young, 1981) that ethylene treatment of preripe climacteric
fruits will shorten the time required for the induction of the
climacteric. Ethylene treatment will also trigger autocatalytic
ethylene production and enhance normal fruit ripening to aesthetically
acceptable quality. On the other hand, a ripe climacteric fruit which
is in the climacteric or postclimacteric phases is insensitive to
further applications of ethylene (Biale and Young, 1981; Solomos,
1983). Hence, the fact that watermelons are deleteriously affected by
ethylene treatment casts doubt on their climacteric categorization.

The objectives of this research are

1) to study the effects of ethylene treatment on respiration and
endogenous ethylene production in watermelon fruit at various stages
of ripening;

2) to determine the nature of the ethylene-induced breakdown in

watermelon tissue: is it due to cell wall breakdown, increased

membrane permeability, or both?

3) to study the effect, if any, of ethylene on the structural
polymers of the cell wall, and in particular the pectins, a) examining
tissue for presence of D-galacturonase, and b) examining tissue for
evidence of pectin degradation;

4) to carry out ultrastructural studies of ethylene-treated and

control tissues.

CHAPTER II

REVIEW OF LITERATURE

Respiration and Ethylene Production of Fruits in Response
to Exogenous Ethylene or Propylene

Introduction

Studies of the respiratory activity of fruits indicate that the
climacteric pattern of respiration is not shown by all fruits. The
respiration rate of some fruits was found to follow a slow downward
drift after detachment. Hence, Biale (1960) classified fruits as
climacteric or nonclimacteric on the basis of their respiratory
patterns. Examples of climacteric fruits are tomatoes, mangoes,
bananas, apples, and avocadoes. Nonclimacteric fruits are typically
represented by citrus fruit, grapes, cherries and others (Biale,

1960). The ripening of climacteric fruits was found to be accompanied
by rapid compositional changes that include hydrolysis of storage
polysaccharides, breakdown of cell wall materials and softening,
changes in organic acids, increases in aroma, increased ethylene
production, loss of astringency, and changes in color. When
climacteric fruits are picked at relatively immature stages, they will
undergo the same physiological changes as mature fruits, but with an
inferior quality (McGlasson et al., 1978). Nonclimacteric fruits,
however, are characterized by a slow utilization of soluble sugars
rather than the hydrolysis of polysaccharides. The capacity of many

3

4

nonclimacteric fruits to ripen after picking is very limited (Rhodes,
1980b).

One of the most important criteria used to differentiate between
climacteric and nonclimacteric fruits is their specific response to
ethylene. Highly sensitive techniques have revealed that ethylene is
produced throughout the growth and development of both climacteric and
nonclimacteric fruits. Nonclimacteric fruits produce ethylene at a
very low rate throughout their growth and maturation and the
attainment of full maturity is not associated with increased ethylene
production. However, the rate of ethylene production increases
dramatically during the ripening of climacteric fruits (McGlasson et
al., 1978; Rhodes, 1980a).

Fruit Response to Applied Ethylene

Climacteric and nonclimacteric fruits behave quite differently in
their response to exogenously applied ethylene. A treatment of 0.1 to
1.0 ppm ethylene for one day is sufficient to initiate ripening in
most climacteric fruit. The respiration rate of the fruit increases
to a peak value, the magnitude of which is independent of the
concentration of ethylene applied. Once ripening is initiated,
removal of exogenous ethylene does not affect the progress of ripening
and the fruit becomes insensitive to further applications of ethylene
(Biale and Young, 1981; Solomos, 1983). Ethylene treatment shortens
the period after harvest required for the induction of the climacteric
rise without affecting the respiratory peak (Rhodes, 1980a).

Treatment of climacteric fruits with ethylene at concentrations higher

5

than a threshold level of 0.1 ppm will, depending on fruit maturation,
induce autocatalytic ethylene production (McGlasson et al., 1978).

The stage of fruit maturity affects the time lag to the induction
of the respiratory climacteric. Wang and Hansen (1970) treated
immature pears with 500 ppm ethylene for 24 hrs and reported a
decrease in flesh firmness, an increase in soluble pectins, and the
fruits attained full ripeness without a concomitant change in the
respiratory activity. However, in fully mature, ethylene-treated
pears, the climacteric rise in respiration developed together with
ripening. The induction of the climacteric rise in respiration of
immature fruits required at least 48 hrs of exposure to ethylene.
Kosiyachinda and Young (1975) reported that ethylene was not the
single control factor in the induction of the climacteric. The effect
of ethylene on respiration seemed to be independent from its effect on
ripening. Wang et al. (1972) studied the effects of ethylene
treatment on the respiratory rate and chemical composition of pears
harvested at different stages of maturation. Respiration rate of
immature fruits varied according to ethylene concentration. Ethylene
production in response to exogenously applied ethylene occurred only
in fully mature fruits during the development of the respiratory
climacteric. Ethylene-induced softening started before the
climacteric rise in respiration. It was suggested that ripening as
measured by softening was not dependent on the development of the
respiratory climacteric. Akamine and Goo (1979) studied respiration
and ethylene production in fruits of species and cultivars of Psidium

and Eugenia . Fruits of Psidium species exhibited typical climacteric

6

type respiration with the increase in ethylene production preceding
the respiratory rise by one day. In contrast, the respiration rate of
ethylene-treated fruits of Eugenia species increased and then
decreased to the control level upon removal of the gas. Those fruits
showed a response typical of nonclimacteric fruits to ethylene. The
climacteric rise in respiration was shown to occur even when the
fruits were still attached to the tree, except avocado fruits which do
not ripen on the tree (Biale and Young, 1981).

Gazit and Blumenfeld (1970) reported that unpicked avocado fruits
did not respond to 50 ppm ethylene for 48 hrs. Ethylene treatments
immediately after harvest also resulted in no response. Fruits
responded to ethylene only when the treatment was initiated at least
25 hrs after harvest. The lack of response to ethylene was attributed
to the presence of endogenous inhibitory substances (Gazit and
Blumenfeld, 1970). Pauli (1982), working with soursop fruits,
concluded that fruit response to ethylene was dependent upon the
sensitivity of the tissue to the initiation of ripening brought about
by an increase in ethylene receptor sites, a decrease in inhibitors,
or an increase in activators.

Treatment of nonclimacteric fruits with ethylene causes a
respiratory rise, the peak value of which is proportional to the
logarithm of the ethylene concentration applied (Rhodes, 1980b).
Removal of the exogenous ethylene leads to a fall in the rate of
respiration to its value before the onset of treatment. The enhanced
respiration can be repeatedly stimulated by successive ethylene
treatments (McGlasson et al., 1978; Rhodes, 1980b). Application of

7

ethylene to nonclimacteric fruits has no effect on the normal low rate
of basal ethylene evolution. Burg and Burg (1962) earlier suggested
that the essential difference between climacteric and nonclimacteric
fruits lies in their relative abilities to produce ethylene
autocatalytically in response to the accumulation of threshold levels
of ethylene within their tissues.

Ethylene has been used for many years for citrus degreening
(Grierson and Newhall, 1960), and is known to increase respiration
rate as well as stem end rot (Kusunose and Sawamura, 1980). Vines et
al. (1965) reported that exposure of 'Valencia’ oranges to 50 ppm
ethylene for 24 hrs caused a 400% increase in respiration rate. After
withdrawal of ethylene, respiration rate declined but did not return
to the control levels within 7 days. The relationship between
climacteric and nonclimacteric fruits was further clarified by studies
of mutant strains of tomato. Herner and Sink (1973) showed that rin
tomato mutant fruits had low rates of respiration and ethylene
production and showed very limited degree of ripening. Exogenous
ethylene treatments caused a rise in respiration but without
autocatalytic ethylene production or ripening. The continuous
presence of ethylene was necessary for a sustained rise in respiration
rate and the fruit was capable of repeated stimulation by successive
ethylene treatments (Herner and Sink, 1973). Frenkel and Garrison
(1976) reported on the initiation of lycopene synthesis in rin tomato
mutant fruits. Detached rin fruits synthesized no lycopene in the
absence of ethylene. However, lycopene synthesis was initiated at 10
ppm ethylene and elevated levels of oxygen.

8

Internal ethylene levels in nonclimacteric fruits usually range
from 0.02 to 0.2 ppm (Burg and Burg, 1962). Studies on postharvest
respiration and ethylene production in jujube fruits (Kader et al.,
1982) showed that internal ethylene production rate ranged from 0.05
to 0.25 1/kg-hr. Their data indicated that Chinese jujube had a
nonclimacteric pattern of respiration and did not exhibit accelerated
ethylene production during ripening.

Initiation of a respiratory rise in response to ethylene
treatment is not restricted to fruits. Reid and Pratt (1972) compared
the respiratory response of potato tubers to ethylene treatment with
that of oranges. Ethylene treatment increased the respiration rate of
potato tubers 5 to 10 times that of the control. Ethylene
concentrations of 2, 20, and 90 ppm resulted in the same respiratory
response. Tubers' response to applied ethylene was similar to that of
nonclimacteric fruits such as oranges. Reid and Pratt (1972) proposed
that the respiratory rise in climacteric fruits and that of wounded
plant tissue was induced by a rise in endogenous ethylene
concentration.

Fruit Response to Applied Propylene

Experiments demonstrating autocatalytic ethylene production by
climacteric fruits in response to exogenously applied ethylene were
made possible by the use of propylene (McMurchie et al . , 1972).
Propylene is a biologically active analogue of ethylene and although
it is not as active as ethylene, it also initiates fruit ripening.

The equivalent concentration of propylene required to give a

9

comparable response was found to be 130 times that of ethylene (Burg
and Burg, 1967). By applying propylene to fruits it is possible to
measure endogenous ethylene evolution accurately, since propylene and
ethylene are readily separable by gas chromatography. McMurchie et
al. (1972) treated climacteric bananas and nonclimacteric oranges and
lemons with 500 ppm propylene. In bananas, propylene initiated a
typical climacteric pattern of respiration, ripening, and
autocatalytic production of ethylene. Propylene-treated lemons and
oranges had increased respiratory rates without being accompanied by
autocatalytic ethylene production. The authors proposed that two
systems were involved in the responses of fruits to ethylene. In
system I, ethylene biogenesis was initiated by factors involved in the
regulation of aging processes. This brought the concentration of
ethylene to a sufficient level to induce system II which led to the
respiratory climacteric, ripening changes, and further ethylene
production. Hence, nonclimacteric fruits lacked system II but
possessed system I. The effect of propylene treatment on the stage of
fruit maturation was investigated by Sfakiotakis and Dilley (1973) in
apples. The ability of propylene to stimulate ethylene production
increased progressively with fruit maturation. The lag time to the
onset of autocatalytic ethylene production decreased with increased
maturation, thereby reflecting increased tissue sensitivity.

McGlasson et al. (1975) found that continuous application of 300 to
1000 ppm propylene advanced ripening in normal tomato fruits of all
maturation stages by at least 50%. Exposure to propylene stimulated
respiration in non-ripening mutant fruits but without a change in

10

endogenous ethylene production. Eaks (1980) and Brecht and Kader
(1984a, 1984b) also reported on the relationships between propylene
concentration and stage of fruit maturation. Immature fruits usually
responded by showing an increased respiration rate. Upon termination
of the treatment, the respiration rate fell to the untreated control.
Propylene did not induce ethylene production in immature fruits, but
did so in mature ones. Propylene concentrations of 1250 to 12,500 ppm
advanced the onset of ethylene production, stimulated ripening, and
enhanced softening of slow-ripening nectarine fruits (Brecht and
Kader, 1984b). Propylene-treated avocado fruits (Eaks, 1980) had a
progressively shorter time to the climacteric and to softening as they
matured .

Respiration and Ethylene Production of Fruits as Affected by
Microorganisms

Few studies have been carried out on the contribution of
microbial activity to respiration and ethylene production of fruits.

In some cases, many fruits which have been classified as climacteric
were found to be decayed and owed much of their high respiration rates
and ethylene production to microorganisms (Oslund and Davenport,

1983). An increase in respiratory activity was found in lemons and
oranges naturally infected with Penicillium digitatum. Diplodia
natalensis, and Phomopsis citri (Vines et al., 1965), and in lemons
artificially infected with Penicillium digitatum (Eaks, 1955).

Studies on respiration and ethylene production of carambola fruits
revealed that exogenously applied ethylene had no significant effects

on either respiration or autocataly tic ethylene production (Oslund and

11

Davenport, 1983). Higher levels of CO2 and ethylene produced by fully
ripe fruits were mainly attributed to microbial activity. The
respiratory and ripening patterns of healthy fruits were typical of

those for nonclimacteric fruits. Barkai-Golan and Kopeliovitch (1983)
reported on ethylene evolution and respiration of Rhizopus-inf ected
tin and nor tomato mutant fruits. Rhizopus stolonif er infection
stimulated ethylene and CO2 production by fruits of the two non-
ripening tomato mutants, and caused the climacteric-like patterns of
respiration typical of normal fruits. The rates of respiration and
ethylene production of uninfected mutant fruits remained low and
constant. When normal fruits were inoculated in the postclimacteric
(red) stage, new climacteric peaks with rates higher than those of
uninfected fruits followed fungal infection. Similarly, Zauberman and
Barkai-Golan (1975) studied changes in respiration and ethylene
evolution induced by Diplodia natalensis in orange fruit. The
respiration rate and ethylene evolution were higher in infected than
in healthy fruits. In uninfected fruits, however, very low and
constant levels of ethylene evolution were recorded. Accelerations in
respiration and ethylene evolution were also induced in avocado fruits
inoculated with Fusarium solani (Zauberman and Schif fmann-Nadel ,

1974). Increases in respiration and ethylene production rates started
earlier than in healthy fruits, but their pattern and intensity were
the same. In vitro studies revealed that when the fungus was cultured
on potato dextrose agar under optimal growth conditions, it evolved no
ethylene during 14 days of growth. Early studies by Biale and
Shepherd (1941) and Biale (1948) showed that citrus fruits inoculated

12

with P. digitatum evolved considerable amounts of ethylene and the
authors suggested that the fungus, by producing ethylene, induced the
increased fruit respiration. However, natalensis did not seem to
produce ethylene (Zaubermann and Barkai-Golan , 1975) and therefore it
appeared that ethylene evolution in infected fruits was a direct
response of the fruit to fungal attack. Studies by Barkai-Golan and
Kopeliovitch (1983) on tomato fruits revealed that the highest rates
of ethylene production occurred in the healthy tissue at the margin of
the rot and little ethylene was produced by rotted tissues with
actively growing fungus. Hence, ethylene appeared to be a product of
the host rather than the fungus.

Membrane Permeability Changes During Ripening and Senescence

The semipermeable nature of biological membranes is of crucial
importance in maintaining compartmentation. Early investigators
(Sacher, 1966; Simon, 1977a) have suggested that the respiratory rise
during ripening of certain fruits was due to membrane permeability
changes. They postulated that the loss of permeability barriers could
lead to changes in protoplasmic compartmentation, leading to access
between enzymes and substrates, and result in the metabolic changes
associated with the climacteric.

Effect of Ethylene on Membrane Permeability

Ethylene has been shown by many workers (Hanson and Kende, 1975;
Kende and Hanson, 1976; Suttle and Kende, 1978, 1980; Ferguson and
Watkins, 1981; Borochov and Faragher, 1983) to be involved in the

13

regulation of senescence in a wide variety of plant organs.

Senescence in plant tissues is known to be accompanied by changes in
membrane permeability (Sacher, 1973). Leakage of electrolytes, sugars,
or pigments is a commonly used method to assay membrane permeability
in plant tissues. Ethylene treatment prematurely induced senescence
and induced endogenous ethylene production in flowers of Ipomoea
tricolor (Kende and Baumgartner, 1974). The authors proposed a model
to explain the mechanism by which ethylene can induce ethylene
synthesis or autocatalysis. They suggested that low, system I levels
of ethylene caused loss of cellular compartmentation which caused
intermixing of previously sequestered components of the ethylene-
generating system. Hanson and Kende (1975) reported that exogenous
ethylene treatment induced senescence and enhanced efflux of solutes
from the cells of morning glory flower tissue. Electrolyte leakage
was a consequence of increased permeability of either the tonoplast or
the plasraalemma or both. The view that ethylene increases the
permeability of the tonoplast is consistent with observations made by
Suttle and Kende (1978, 1980). Working with isolated petals of
Tradescantia , they showed that exogenous ethylene treatment hastened
the onset as well as the increase in anthocyanin and electrolyte
leakage from the petals. The sensitivity to applied ethylene
increased as the petals matured. They supported the hypothesis put
forward by Kende and Baumgartner (1974) to explain autocataly tic
ethylene production.

The loss of tonoplast integrity would allow the mixing of
vacuolar hydrolases with their cytoplasmic substrates resulting in

14

autocatalytic ethylene production. It was concluded that ethylene
regulated senescence by determining the rate of loss of
compartmentation which played a central role in the deteriorative
changes occurring during senescence.

Ferguson and Watkins (1981) studied cation leakage from apple
fruits during development and ripening. Leakage of K"*" exceeded that
of Ca^'*' and Mg^"^ and was not affected by the external presence of
divalent cations. Leakage of Ca ^ and Mg^^ was markedly increased by
the external presence of either ion. Whereas K'*' and Mg^'*’ leakage
increased with fruit senescence, Ca^"*” leakage decreased. Ion leakage
was found to increase during the climacteric rise in apple fruit. A
close relationship had been reported (Berard and Lougheed, 1982)
between an increase in membrane permeability and a rising production
of ethylene in apples. Apple fruits held under low pressure storage
had reduced membrane permeability due to delayed ethylene production.
Similar results were reported by Kobayashi et al. (1981) with water-
stressed plum leaves.

Changes in Membranes During Senescence

Beutelmann and Kende (1977) correlated changes in membrane lipid
content with aging symptoms in morning glory flowers. They found that
the level of phospholipid had already started to decline before
visible signs of senescence were evident. The rate of phospholipid
loss accelerated sharply with ethylene production whereas synthesis of
new phospholipids fell by 40%. Exogenously applied ethylene
accelerated phospholipid loss and senescence of flower tissues whereas

15

benzyladenine retarded both of these processes. Likewise, Suttle and
Kende (1980) found that the increase in membrane permeability of
Tradescantia flowers was accompanied by a massive loss of
phospholipids. Their results suggested that the observed increase in
membrane permeability was a direct result of an increased phospholipid
degradation presumably caused by an increase in the activity of pre-
existing phospholipases.

The compositional changes that take place in membranes during
senescence play a significant role in their permeability. It has been
shown that sterol to phospholipid ratio increased with senescence and
this increase was proportional to the decrease in membrane fluidity
(Borochov et al., 1978, 1982; Borochov and Faragher, 1983; Borochov
and Weinberg, 1984; Thompson et al., 1982). Borochov et al. (1978)
studied senescence in rose petals and reported that the microviscosity
of the plasmalemma increased during senescence due to an increase in
sterol to phospholipid ratio. The increased ratio was presumably
brought about by a decrease in the amount of phospholipids caused by
the action of endogenous phospholipases. Further studies (Thompson et
al., 1982) indicated that lipid microviscosity of membranes from
senescing carnation flowers increased with advancing senescence and
coincided with the climacteric-like rise in ethylene production.
Exogenous ethylene treatment of the flowers increased the
microviscosity and accelerated rigidif ication of membranes. Membrane
rigidif ication was accompanied by an increased sterol to phospholipid
ratio that reflected selective phospholipid loss with senescence.
Studies by Borochov et al. (1982) on rose petals revealed that there

16

was no quantitative change in the level of free sterols. The content
of phospholipids decreased without any significant change in their
composition. They concluded that the fluidity of rose petal membranes
decreased with age as a result of a decrease in phospholipid content
brought about by both reduced synthesis and enhanced degradation.

Although it has been accepted that membrane lipid phases are
dominated by the liquid crystalline phase, recent evidence (Legge et
al., 1982a) indicates the presence of a gel phase at physiological
temperatures in senescent tissues. The presence of lipids in the gel
phase in biological membranes is known to induce an increase in
membrane permeability (Borochov and Weinberg, 1984). Studies with
rose flowers (Legge et al., 1982b) revealed that microsomal membranes
from the tight bud stage contained liquid crystalline lipid and only
traces of gel phase lipid. With advancing senescence, however, the
proportion of gel phase lipid increased.

It has also been observed that changes that occur in the fatty
acid chain saturation of membrane phospholipids could affect membrane
permeability (Wade and Bishop, 1978; Wade et al., 1980). Generally,
an increase in saturation would make the lipids less fluid at any
given temperature. Wade and Bishop (1978) studied changes in lipid
composition of ripening banana fruits. They found that the relative
proportions of the different lipids remained constant but the fatty
acid composition changed during ripening. The change was confined to
the phospholipid fraction in which there was an increase in total
unsaturation of the fatty acids. In contrast to the information
presented above, the authors suggested that increased lipid

17

unsaturation resulted in increased membrane fluidity which
consequently resulted in increased membrane permeability. Further
studies by Wade et al. (1980) suggested that increased passive
permeability of ripening banana fruit was due to increased
polyunsaturation of membranes. They concluded that the increased
electrolyte leakage observed in banana fruit tissue treated with
propylene could be correlated either with changes in membrane lipid
composition or with changes in tissue water potential.

Lipid peroxidation might also contribute to the modification
processes in membranes, presumably through generation of a gel phase
domain in membrane lipids. The presence of such domains might cause
an increase in membrane permeability (Borochov and Weinberg, 1984).
Data presented by Dhindsa et al. (1981) showed a close relationship
between increased solute leakage and increased level of lipid
peroxidation in senescent tobacco leaves.

Electrolyte leakage from fruit tissues may, however, depend upon
factors other than membrane permeability. Of particular importance,
once other ripening changes such as soluble solids accumulation have
begun, the gradient in water potential between fruit tissues and the
bathing solution may result in measurement artifacts (Sacher, 1973;
Simon, 1977a). Incubation of tissues in hypotonic media can lead to
membrane damage particularly in the relatively fragile cells of
ripening fruit (Wade et al . , 1980). Simon (1977b) studied electrolyte
leakage from apple fruits and found that when the tissues were placed
in water, 90% of their electrolytes and soluble carbohydrates leaked
out in 2-3 hours. Leakage was greatly reduced when tissues were

18

placed in 1 M glycerol or 0.5 M KCl. It was suggested that apple
fruit cells burst in water but not in isotonic media. Leakage from
tissues of other soft fruits followed similar trends and became more
extensive with advancing maturation.

The Role of Calcium in Delaying Senescence

Calcium has long been known to play a significant role in
numerous physiological processes in plant tissues. The maintenance of
relatively high calcium concentration in fruit tissues delayed
ripening in tomatoes (Wills et al., 1977; Wills and Tirmazi, 1979;
Buescher and Hobson, 1982), and avocados (Tingwa and Young, 1974);
reduced CO^ and ethylene production (Tingwa and Young, 1974; Wills and
Tirmazi, 1982); maintained firmness of apple fruits (Sams and Conway,
1984); and inhibited abscission (Poovaiah and Leopold, 1973a).

Ripening of green tomatoes (Wills and Tirmazi, 1979) was inhibited
when Ca content of the fruit was raised to greater than 40 mg/ 100 g
fresh weight. The fruits showed no signs of ripening even after 6
weeks' storage at 20°C, and the application of 1000 ppm ethylene for 3
weeks had no effect. Inhibition of ripening was not restricted to Ca
as other divalent ions such as Mn, Co, and Mg were also effective.
Monovalent metal ions were less effective than Ca.

The breakdown of pectic substances in the middle lamella and cell
wall may result in loss of wall integrity in ripening fruits (Sams and
Conway, 1984; Ferguson, 1984). Free carboxyl groups on D-
galacturonase polymers play an important role in stabilizing and
maintaining wall integrity through the cooperative binding of Ca ions

19

(Demarty et al., 1984). Sams and Conway (1984) reported a negative
relationship between soluble polyuronide content and Ca concentration
in apple fruits. The binding of Ca is crucial for strengthening fruit
tissue and making it more resistant to hydrolytic enzyme attack
(Buescher and Hobson, 1982). Sawamura et al. (1978) reported that the
presence of Ca decreased hydrolytic enzyme activity in tomato fruits,
and Ca was found to inhibit the natural decline in resistance to D-
galacturonase activity in the cell wall.

The nonenzymic mechanism of fruit softening has been explained on
the basis of the important role played by Ca in pectin stabilization.
Removal of Ca destabilizes the pectic matrix resulting in the
weakening of the cell wall (Huber, 1983b).

The role of Ca in membrane structure and function is well
established (Rousseau et al., 1972; Poovaiah and Leopold, 1973b;
Lieberman and Wang, 1982; Ferguson, 1984). The ability of Ca to delay
senescence is a direct result of its role in maintaining membrane
integrity and hence cellular compartmentation (Hecht-Buchholz , 1979;
Poovaiah, 1979; Legge et al., 1982a). During senescence, the most
obvious indication of membrane change is an increase in electrolyte
leakage which indicates the loss of selective permeability. Poovaiah
(1979) demonstrated that Ca decreased membrane permeability during
tomato fruit senescence. Levels of bound Ca in mutant fruits were
high at advanced stages of maturation whereas in normal fruits bound
Ca decreased about 3-fold during maturation. Changes associated with
senescence were suppressed by Ca as a consequence of its role in
maintaining cell wall structure and membrane integrity. Lieberman and

20

Wang (1982) reported that protein leakage from apple tissue slices was
significantly reduced when the incubation medium contained 100 mM Ca
plus Mg. Their results indicated a membrane preservation effect of
Ca, Mg, or Ca plus Mg during rapid aging of apple fruit tissues.

Calcium reduces membrane permeability presumably through tighter
packing of lipids which is brought about by binding of Ca to
negatively charged phospholipids resulting in aggregation (Ferguson,
1984). Studies by Legge et al. (1982a) revealed that Ca rigidified
and stabilized membranes mainly at their surfaces, whereas its effect
was less pronounced deeper within the lipid bilayer. The ability of
Ca to tightly pack lipid domains with a subsequent reduction in
membrane permeability also results in reduced membrane fluidity.
Borochov et al. (1978) showed that the presence of 5 to 25 mM Ca ions
in the incubation medium of rose petals increased the microviscosity
of the plasmalemma.

The inhibition of senescence by Ca may also involve protection of
membranes from free radical or peroxidative attack. It was found that
apple fruit tissues incubated in 100 mM Ca or Mg showed considerably
less lipid peroxidation as compared to the untreated control
(Lieberman and VJang, 1982).

One of the main evidences supporting the role of Ca in
maintaining membrane integrity is made possible by the use of electron
microscopy. Ultrastructural studies (Hecht-Buchholz , 1979) of Ca-
deficient potato sprout cells revealed extensive disintegration of the
plasmalemma and the tonoplast.

21

Activity of Hydrolytic Enzymes During Fruit Ripening
Introduction

The cell wall of fruits is basically composed of cellulose,
hemicelluloses , polyuronides, and associated glycoproteins and
enzymes. These major components are extensively associated to form
strong yet flexible matrices. The cell wall also includes a
morphologically distinct layer known as the middle lamella, which is
located between the primary cell walls of adjoining cells and forms a
continuous intercellular matrix. Since this layer is particularly
rich in pectic polysaccharides, it is believed to be the region of the
cell wall most affected during fruit softening (Huber, 1983b).

Softening of many fruits during ripening has long been attributed
to enzymic action that results in the solubilization of cell wall
polysaccharides (Hobson, 1964, 1965; Pressey and Avants, 1971, 1973a,
1973b, 1975; Zauberman and Schiffmann-Nadel , 1972; Buescher and
Tigchelaar, 1975; Pauli and Chen, 1983). Enzymes reported to be
involved in fruit softening are the D-galacturonases, cellulases, and
pectinmethylesterases (Rexova-Benkova and Markovic, 1976; Pressey,
1977; Huber, 1983b). In this context, discussion will focus on the D-
galacturonases (the polygalacturonases) and cellulases only, since
they are the major carbohydrases associated with fruit cell wall.

D-Galacturonases

Rexova-Benkova and Markovic (1976) suggested that pectolytic
enzymes be referred to as D-galacturonases on the basis of their

22

preferred substrates — the D-galacturonans. D-Galacturonases were
first detected in ripe tomato fruit (Hobson, 1964, 1965; Buescher and
Tigchelaar, 1975). These enzymes were found in a wide range of fruits
and are generally associated with fruit ripening and softening.

Usually there is little or no D-galacturonase activity in immature
fruits and activity increases during ripening (Hobson, 1964; Sawamura
et al., 1978; Ahmed and Labavitch, 1980). Hobson (1964) studied
blotchy ripening in tomato fruits and found low D-galacturonase
activity in the apparently normal red area and a severe decrease in
activity in the abnormal, non-red areas. He concluded that blotchy
ripening was due to a failure in D-galacturonase synthesis. Buescher
and Tigchelaar (1975) compared D-galacturonase activity in normal and
mutant tomato fruits and found that D-galacturonase activity increased
during ripening of normal fruits. However, in mutant fruits, D-
galacturonase activity \<ias not detected and the authors concluded that
the lack of softening of mutant fruits was due to the lack of D-
galacturonase . Hobson (1965) concluded that softening of tomato
fruits during ripening was directly associated with the activity of D-
galacturonase. Studies on D-galacturonase synthesis (Tucker and
Grierson, 1982) could not detect D-galacturonase activity in green
tomatoes. D-galacturonase appeared on the onset of ripening and was
one of the major cell-wall-bound proteins in ripe fruit. The data of
Tucker and Grierson indicated that during tomato ripening, D-
galacturonase was synthesized ^ novo .

Pressey et al. (1971) studied D-galacturonase activity, water-
soluble pectin, molecular weight distribution of water-soluble pectin.

23

and fruit firmness during ripening of freestone peaches. D-
galacturonase activity was absent in unripe fruits but it developed
during ripening and was accompanied by increasing amounts of water-
soluble pectins and a loss of firmness. Gel-filtration studies
revealed that the molecular weights of pectins decreased progressively
during ripening, indicating a role for D-galacturonase in pectin
solubilization .

D-galacturonases are classified on the basis of their mode of
action as endo-D-galacturonases (E.C. 3.2.1.15) and exo-D-
galacturonases (E.C. 3.2.1.67) to designate a random or terminal
action pattern, respectively (Rexova-Benkova and Markovic, 1976;

Huber, 1983b).

Many research workers have reported on the occurrence of one or
more forms of D-galacturonases (Pressey and Avants, 1971, 1973a,

1973b, 1975; Hunter and Elkan, 1974; Tucker et al., 1980; Huber,
1983a). Pressey and Avants (1973a) separated two D-galacturonases
from extracts of ripe tomatoes and referred to them as PG I and PG II.
In all of the samples examined, PG II was the predominant enzyme. The
enzymes differed in stability with respect to both temperature and pH,
and their molecular weights were 84,000 and 44,000 for PG I and PG II,
respectively. Polygalacturonase II was found to be more effective
than PG I in reducing the viscosity of pectic acid (endo-D-
galacturonase) . Similarly, Pressey and Avants (1973b) separated two
D-galacturonases from ripe peach fruits and named them PG I and PG II.
Polygalacturonase I hydrolyzed polygalacturonic acid from the
nonreducing ends of the molecules and released galacturonic acid as

24

the product. It required Ca^'*’ for activity, functioned optimally at
pH 5.5 and hydrolyzed low molecular substrates more rapidly (exo-D-
galacturonase) . In contrast, PG II cleaved substrate molecules at
random, with a pH optimum of 4.0, and was most reactive with
intermediate molecular weight substrates — all characteristics of endo-
D-galacturonases .

Pressey and Avants (1978) investigated the textural differences
between clingstone and freestone peach fruits by comparing their
pectolytic activities and pectin solubility. Preripe fruits from both
types of peaches had very low levels of water soluble pectins and no
detectable D-galacturonase activity. Whereas ripe clingstone peaches
had exo-D-galacturonase and insoluble pectin, ripe freestone peaches
had both exo and endo-D-galacturonases and high levels of water-
soluble pectin. The textural differences between the two peach types
were basically due to enzyme composition rather than levels of total
D-galacturonase .

Hunter and Elkan (1974) identified only endo-D-galacturonase from
ripe tomatoes possibly due to less sensitive extraction techniques.

In contrast, Pressey and Avants (1975) were successful in resolving
only an exo-D-galacturonase from cucumber fruits. They reported that
cucumber exoenzyme was similar to peach exoenzyme (Pressey and Avants,
1973a) in pH optimum, cation activation, molecular weight, and in its
stepwise removal of monomer units from the non-reducing ends of the
substrate molecules. The participation of exo-D-galacturonase in
cucumber softening was not through random cleavage of pectin but
rather through specific hydrolysis of terminal linkages.

25

Huber (1983a) examined polyuronides from cell wall preparations
of ripening tomato fruits using gel-filtration chromatography. The
increase in the quantity of polymers that fractionated on the gel
indicated that polyuronides were extensively degraded during ripening.
Low molecular weight polyuronides were first observed in fruit
harvested at the turning stage and continued to increase with the
advancement of ripening. There was a close relationship between the
appearance of degraded polyuronides and the activity of endo-D-
galacturonase which seemed to be solely responsible for the
degradation of the wall polymers.

Research workers could not agree as to whether it was D-
galacturonase or ethylene that led to the initiation of ripening in
fruits. Tigchelaar et al. (1978) suggested that D-galacturonase
played a key role in fruit ripening. They found that ethylene was not
evolved unless D-galacturonase activity increased and ethylene had no
effect on D-galacturonase activity. It had also been suggested
(Tigchelaar and McGlasson, 1977) that the rise in the activity of D-
galacturonase was an early or even the primary event in fruit
ripening. These suggestions are in disagreement with other findings
(Babbitt et al., 1973; Poenicke et al., 1977; Awad and Young, 1979;
Pauli and Chen, 1983). The data presented by Sawamura et al. (1978)
indicated that exposure of tomato fruits to different ethylene
concentrations resulted in an increased D-galacturonase activity. The
activity of the enzyme was found to increase rapidly at considerably
higher ethylene concentrations. Saltveit and McFeeters (1980)
reported increased D-galacturonase activity in cucumber fruits after a

26

burst of ethylene production. D-galacturonase activity in harvested
mature or immature cucumber fruits could be induced by exogenously
applied ethylene. A close relationship was found to exist between D-
galacturonase activity and the rise in respiration and ethylene
production (Babbitt et al., 1973; Avrad and Young, 1979). Babbitt et
al. (1973) studied the effects of growth regulators on pectolytic
enzymes and ripening of tomato fruits. Growth regulators markedly
influenced the rate of the normal sequence of changes during ripening.
Softening, color development, and D-galacturonase activity were
accelerated by ethephon and delayed by gibberellic and indole acetic
acids.

Pauli and Chen (1983) studied the relationship between hydrolytic
enzymes and ethylene production during the ripening of papaya fruits.
D-galacturonase was not detectable in the preclimacteric stage but it
increased during the climacteric rise and declined during the
postclimacteric stage. The increase in D-galacturonase activity
occurred simultaneously with increased ethylene production and the
peak of D-galacturonase activity was correlated with maximum ethylene
production. No D-galacturonase activity was detected prior to the
rise in ethylene production indicating a possible role of ethylene in
initiating D-galacturonase activity.

Emanations from ethylene-producing fruits could be effective in
initiating hydrolytic enzymes activity in other fruits, not only in
the same storage room, but even in the field. Shimokawa (1973)
observed that Vvratermelon tissue was macerated by emanations evolved by
'Prince' melons. The presence of ethylene in the emanations was

27

demonstrated by gas chromatography. Enzymic studies of macerated
tissue revealed higher activities of pectinase as compared to healthy
tissue not exposed to ethylene.

One of the recent tools used to demonstrate the crucial role of
D-galacturonase in pectin degradation is isolated cell wall material
(Gross and Wallner, 1979; Themmen et al., 1982). Cell wall
preparations from green normal and mutant tomato fruits were equally
degraded vitro by a cell wall-bound protein extract from ripe
normal tomatoes. Similar cell wall-bound protein extracted from
mutant fruits was not effective in in vitro cell wall degradation
because it lacked D-galacturonase. The fact that purified D-
galacturonase was capable of vitro degradation of cell wall
material indicated that D-galacturonase was the major enzyme in cell
wall-bound protein extracts responsible for wall degradation.

Controlled atmosphere storage has been shown to influence
pectolytic activity remarkably. Goodenough et al. (1982) reported
that the appearance of D-galacturonase and red pigmentation in mature
green tomato fruits was prevented by storage conditions of 5% O2, 5%
CO2 , and 90% N2* The authors speculated that the reduced respiration
rate under these conditions might have supplied less ATP to the fruit
biosynthetic systems resulting in the retardation of D-galacturonase
synthesis. Upon removal of fruits to ambient conditions, D-
galacturonase was synthesized and fruits changed color and ripened
normally .

There are causes of cell wall modifications during ripening other
than enzymic mechanisms. Huber (1984) reported on the roles of

28

polyuronides and hemicelluloses in strawberry fruit softening. He
found that soluble polyuronides were unchanged during early
development of strawberry fruits but increased markedly throughout
ripening. Polyuronide solubility was not accompanied by molecular
weight changes indicative of enzymic action. This observation was
supported by the fact that D-galacturonase activity was not detected
in strawberry fruits. The author suggested that increased levels of
soluble polyuronides were due to the synthesis of more freely soluble
forms during ripening. Hemicelluloses were also found to be altered,
showing an increased proportion of low-molecular weight polymers
during ripening.

Cellulase

The degradation of cell wall components other than the pectic
substances may also contribute to softening. Considerable evidence
indicates that changes in cellulose may occur during ripening in some
fruit. Hall (1964) and Dickinson and McCollum (1964) were among the
first workers to report on cellulase in fleshy fruits. They observed
an increase in cellulase activity during tomato ripening and suggested
that the enzyme might be involved in the softening process. A close
relationship exists between cellulase activity, the climacteric rise
in respiration, ethylene production, and fruit softening (Sobotka and
Watada, 1971; Pesis et al., 1978; Awad and Young, 1979). In contrast,
Pauli and Chen (1983) detected significant cellulase activity in
papaya fruit at harvest. Cellulase has been suggested to play a role
during fruit growth (Hall, 1964). Cellulase facilitates cell

29

enlargement through breaking cellulose fibrils and allowing cell wall
expansion.

Hinton and Pressey (1974) found low cellulase activity in
immature peach fruits but it increased during ripening. The greatest
increase in cellulase activity occurred before a significant change in
fruit firmness. Since cellulase was observed to appear before fruit
softening, it might be involved in the initiation of the processes
leading to tissue softening and disintegration. The large quantity of
cellulase found in avocado fruit mesocarp and the changes in its
activity during ripening suggests that it plays a critical role in the
ripening process (Awad and Young, 1979; Awad and Lewis, 1980). In
avocado fruits, it appeared that the initial phase of softening was
correlated with cellulase activity whereas D-galacturonase was
involved in the later stages of ripening (Awad and Young, 1979).
Sobotka and Watada (1971) reported a close association between
increased cellulase activity and softening of ripening tomato fruits.
Tomato lines with firmer fruit exhibited lower cellulase activity than
those with softer fruit. In contrast to these findings, Hobson (1968)
reported that cellulase activity was not correlated with fruit
firmness. His data showed that cultivars with firmer fruit had higher
cellulase activity than those with softer fruit. Likewise, Buescher
and Tigchelaar (1975) compared cellulase activity in normal and mutant
tomato fruits. Cellulase activity increased during ripening and
softening in normal fruits, whereas in mutant fruits, cellulase
activity increased during "ripening" yet the firmness of the fruit was
not affected. The authors concluded that the lack of softening in

30

mutant fruits was associated with the lack of D-galacturonase
activity.

The difference in opinion concerning the role of cellulase in
fruit softening can be partly explained by the lack of detailed
information regarding the different types of cellulases and their
specific roles in fruit ripening. The difficulty in detecting
cellulolytic enzymes in fruits is attributed to the insolubility of
cellulose. Researchers have circumvented this problem by using
carboxymethylcellulose (CMC), which is a soluble derivative of
cellulose, as a substrate. The high viscosity of aqueous solutions of
CMC makes it ideal for viscometric methods used for the determination
of cellulase activity. Most of the cellulases reported in ripening
fruits have been of the C^ or carboxymethylcellulase types, which
alone have little capacity to degrade insoluble forms of cellulose
(Huber, 1983b). Microorganisms have been reported to produce at least
four different types of enzymes which have distinctly different roles
in the degradation of cellulose (Li et al., 1965). Few reports have
provided information on the multiple enzymic nature of the cellulase
complex of higher plants (Lewis and Varner, 1970; Pharr and Dickinson,
1973; Sobotka and Stelzig, 1974; Pesis et al., 1978). Pharr and
Dickinson (1973) extracted two enzymes from ripening tomatoes. One
enzyme reduced the viscosity of CMC and generated reducing groups,
oligosaccharides, and glucose. The other enzyme hydrolyzed cellobiose
rapidly but the rate of cleavage decreased with increased substrate
chain length. No evidence was found for the presence of exocellulase
which attacked insoluble cellulose. Lewis and Varner (1970)

31

demonstrated the existence of two forms of cellulase in abscission
zones of aging bean explants. Awad and Lewis (1980) established the
optimum conditions for extraction and purification of avocado

cellulase. The enzyme was purified to a single protein with a
molecular weight of 49,000. Studies of cellulase activity in peaches
(Hinton and Pressey, 1974) revealed low activity in immature peaches,
but it increased considerably during ripening. Peach cellulase
degraded CMC and was therefore a C^ cellulase. Sobotka and Stelzig
(1974) separated four enzyme-containing fractions by (NH^)2S0^
fractionation from postbreaker tomatoes. Two enzymes were
endocellulases that degraded both CMC and insoluble cellulose. The
third enzyme was identified as a nonspecific g -glucosidase and the
fourth was an exo-g-1, 4-glucanase. Collectively, the enzymes were
capable of completely degrading insoluble cellulose to short chain
cellodextrins and glucose.

Cellulase is believed to be involved in cell wall breakdown in
abscission zones in a wide range of plant material (Lewis and Varner,
1970; Lewis and Koehler, 1979; Sexton et al., 1980). It has also been
demonstrated that ethylene treatment increases cellulase activity in
abscission zones. Lewis and Koehler (1979) reported that ethylene
treatment promoted leaf abscission and enhanced cellulase activity in
kidney bean seedlings. Sexton et al. (1980) induced abscission of
bean leaves by treating them with 50 ppm ethylene. Total cellulase in
the abscission zones increased dramatically after 18 hours and the
force necessary to break the abscission zone declined. Abscission

32

zones treated with cellulase antiserum failed to break, confirming the
central role played by cellulase in the abscission process.

Ethylene has also been shown to induce cellulase activity in
fleshy fruits. Ethylene treatment of mutant tomato fruits increased
cellulase activity (Poovaiah and Nukaya, 1979). Pesis et al. (1978)
reported that ethylene treatment of avocado fruits induced cellulase
activity and the response increased with degree of maturation. The
enzyme appeared to be an endocellulase , the activity of which seemed
to be controlled by ethylene. Babbitt et al. (1973) studied the
effects of growth regulators on cell wall hydrolases, respiration, and
ripening of tomatoes. They found that growth regulators significantly
influenced cellulase activity. Ethephon markedly accelerated
cellulase activity whereas gibberellic and indole acetic acids delayed
it. The authors suggested that softening was initiated by
cellulolytic enzymes and that pectolytic enzymes were involved in
subsequent changes in texture.

Ultrastructural Changes in Fruit Cells During Ripening

Fleshy fruits are usually composed of parenchymatous cells of
very large size (150 to 700 pm dia.), with large air spaces between
them. Ultrastructural studies of fruit cells during ripening have
proven difficult due to their large size and fragile nature. The high
water content of fruit cells dilutes the fixatives and hinders the
penetration of embedding resins. The development of suitable fixation
procedures to preserve the fine structure of the cell organelles and
the improvement of the embedding resins for better penetration are

33

vital for minimization of artifacts in sectioning. Moreover, the
choice of a suitable staining procedure to increase electron density
and achieve sufficient contrast is equally important. The
distribution of chemical components within a cell can be achieved by
the development of appropriate specific stains. Albersheim and
Killias (1963) reported that the treatment of onion root tip tissue
with alkaline hydroxy lamine prior to staining with ferric chloride
increased electron density throughout the cell wall and particularly
the region of the middle lamella. The distribution of iron indicated
that pectin existed throughout the primary wall as well as in the
middle lamella.

The ripening process of fruits had been considered to be a
senescence phenomenon involving loss of membrane integrity and
resulting in the breakdown of compartmentation and death (Bain and
Mercer, 1964). However, later studies (Simpson et al., 1976)
employing improved fixation procedures revealed no obvious
degenerative changes in all non-plastid cytoplasmic structures in
cells of ripe tomato fruits. Of all the cellular organelles,
mitochondria retain their ultrastructure throughout the ripening
period and until late in senescence.

Most of the research work on fruit ultrastructural changes during
ripening was done on avocado. Pesis et al. (1978) studied cellulase
activity and softening in avocado fruits and reported that the middle
lamella of the cell wall of soft fruits was dissolute and sparse as
compared to the orderly tight fibrils evident in hard fruits. The
authors suggested that cellulase, in addition to the pectic enzymes,
played an important role in avocado fruit softening.

34

Platt-Aloia and Thomson (1981) studied the mesocarp tissue of
ripening avocado fruits using thin section electron microscopy. When
ripening began, one of the most obvious changes was a loosening and
eventual breakdown of the cell wall. In post-climacteric soft fruit,
all of the organelles and membranes appeared whole and intact, but the
cell wall had almost completely disappeared. Although ripening in
avocadoes involved the degradation of the cell walls, it did not
involve the loss of compartmentation.

Extensive ultrastructural studies were carried out at different
ripening stages of avocado fruits (Platt-Aloia et al., 1980).
Ultrastructural changes in the cell wall were correlated with changes
in the activity of wall hydrolytic enzymes. Initial wall breakdown
involved degradation of pectins in the matrix and in the middle
lamella and corresponded with increased polygalacturonase activity.

The loss of fibrillar components of the cell wall indicated a possible
role for cellulase in fruit softening. No correlation existed between
location of wall degradation and the presence of plasraodesmata.

Ben-Arie et al. (1979) followed the ultrastructural changes in
the cell walls of ripening apple and pear fruits. Structural
alterations in the cell wall of apple fruits became evident at
advanced stages of softening, and involved dissolution of the middle
lamella. Likewise, softening in pears uas associated with the
dissolution of the middle lamella, accompanied by a gradual
disintegration of fibrillar material throughout the cell wall.
Application of polygalacturonase and cellulase enzymes to tissue discs
from firm pear fruit led to ultrastructural changes observed in

35

naturally ripening pears. In apple fruits, polygalacturonase alone
was sufficient to dissolve the middle lamella of the cell wall. In
contrast to the findings of Platt-Aloia et al. (1980), these workers
found that the cell wall areas containing plasmodesmata maintained
their structural integrity throughout the ripening process in both
apple and pear fruits.

Electron microscopic studies on non-climacteric fruits did not
show cell wall degradation. Ultrastructural studies on the epicarp of
ripening oranges (Thomson, 1969) did not show any significant changes
in the fine structure of the cells that could be correlated with
ripening, except for the transformation of chloroplasts to
chromoplasts.

Simpson et al. (1976) conducted comparative ultrastructural
studies of fruits of mutant and normal tomatoes to determine whether
possible ultrastructural differences could be related to the lack of
normal ripening in the mutants. They did not note any unique
differences between normal and mutant tomato fruits. However,
recently Crookes and Grierson (1983) studied ultrastructural changes
in the pericarp of normal tomato fruits during ripening. They found
that changes in cytoplasmic ultrastructure were not consistent with
the speculation that ripening was a senescence phenomenon. In ripe
fruit, a high degree of ultrastructural organization of the
mitochondria, chromoplasts, and rough endoplasmic reticulum was
retained. The most striking changes were noted in the structure of
the cell wall, which started with the dissolution of the middle
lamella and ended with the eventual disruption of the primary cell
wall. Changes in the cell

36

wall were correlated with the appearance of polygalacturonase
isoenzymes. Those changes were duplicated by the application of
purified tomato polygalacturonase isoenzymes to mature green fruit
tissue.

Most work on fruit abscission has generally dealt with
chemically-induced fruit drop. Few authors have reported on
anatomical and ultrastructural studies of abscission-promoting regions
of fruit (Nelson et al., 1984; Jerie, 1976; Reed and Hartmann, 1976).
Nelson and co-workers (1984) reported that the application of the
ethylene releasing compound, CGA 15281, enhanced the formation of
abscissional zones in peach fruits. In treated fruits, the formation
of abscissional zones preceded that in controls by 3 days. The
development of discrete separation layers at the base of fruits was
attributed to enzymic action resulting in cell wall degradation.

CHAPTER III

RESPIRATION AND ETHYLENE PRODUCTION IN WATERMELON
FRUIT EXPOSED TO ETHYLENE OR PROPYLENE

Introduction

Fruits differ greatly in their response to applied ethylene
(Biale and Young, 1981). Treatment of climacteric fruits with
ethylene promotes ripening as evidenced by the initiation of the
respiratory climacteric and autocatalytic ethylene production. Once
ripening is initiated, removal of exogenous ethylene does not affect
the progress of ripening and the fruit is apparently insensitive to
further ethylene applications (Wang and Hansen, 1970; Akamine and Goo,
1979; Solomos, 1983). The response with nonclimacteric fruit is
variable but generally does not include ripening initiation. Ethylene
applied to nonclimacteric fruit increases the respiration rate, and
upon removal of the gas, respiration rate returns to basal levels
(McGlasson et al., 1978; Rhodes, 1980a). Respiration in
nonclimacteric fruit can be repeatedly stimulated by ethylene
treatments administered in succession (Herner and Sink, 1973).
Application of ethylene to nonclimacteric fruit has no effect on
endogenous ethylene production (Burg and Burg, 1962; Rhodes, 1980b).

Propylene is an active analogue of ethylene and can also initiate
fruit ripening. The equivalent concentration of propylene required to
give a comparable response is 130 times that of ethylene (Burg and
Burg, 1967), Propylene has been employed to demonstrate that

37

38

autocatalytic ethylene production by climacteric fruit can indeed
occur in response to exogenously applied ethylene (McMurchie et al.,
1972; Sfakiotakis and Dilley, 1973; Eaks, 1980; Brecht and Kader,
1984a, 1984b). Propylene facilitates accurate measurement of
endogenous ethylene production since they are readily separated by gas
chromatography (McGlasson, 1975; Eaks, 1980).

Microorganisms can significantly contribute to increased
respiratory activity and ethylene production in decayed or otherwise
wounded fruits (Eaks, 1955; Vines et al., 1965). High rates of CO2
and ethylene evolution were reported for decayed lemons (Eaks, 1955),
lemons and oranges (Vines et al., 1965), tomatoes (Barkai -Golan and
Kopeliovitch, 1983), and avocados (Zauberman and Schiffman-Nadel ,
1974). Microbial infection caused climacteric-like patterns of
respiration and ethylene production in nonclimacteric fruits (Biale,
1948; Biale and Shepherd, 1941). Oslund and Davenport (1983) reported
that lychee fruit, reportedly climacteric (Vines and Grierson, 1966),
were nonclimacteric and showed enhanced CO2 and ethylene only when
infected by microorganisms.

On the basis of postharvest CO2 and ethylene production, Mizuno
and Pratt (1973) classified the watermelon as a climacteric fruit.
Shimokawa (1973) reported that ethylene enhanced the respiration rate
of watermelon fruit, but upon withdrawal of ethylene, respiration rate
returned to control levels. He also reported that applied ethylene
did not trigger autocatalytic ethylene production. Recently, Risse
and Hatton (1982) showed that exposure of watermelon fruit to various
concentrations of ethylene accelerated tissue deterioration.

39

Placental tissue in ethylene-treated fruits was soft, watersoaked, and
unacceptable in taste. Ethylene treatments had an adverse effect on
preripe fruits as well. Shimokawa (1973) also observed that
watermelon tissue was macerated by exogenously applied ethylene. It
is evident from numerous studies that ethylene treatment of fruit
exhibiting an orthodox climacteric ripening pattern does nc.,t induce
adverse effects beyond those normally observed during the latter
stages of ripening . The more general response is ripening initiation
(McGlasson et al., 1978; Rhodes, 1980b; Biale and Young, 1981). The
data considered together present an unsolved dilemma. The objective
of this study is to determine the postharvest respiratory and ethylene
production characteristics in watermelon fruit in response to
exogenously applied ethylene or propylene.

Materials and Methods

Plant Material

'Charleston Gray' watermelons [Citrullus lanatus (Thunb.) Matsum
and Nakai] were grown at the Horticultural Unit near Gainesville,
Florida. Fruits were harvested immature (white flesh), preripe
(pink), and ripe (red) in June-July, 1983. During the 1984 season,
watermelon fruits were harvested at the ripe stage only. Criteria
used as harvesting indices included yellowing of the ground spot,
withering of the tendril adjacent to the stem, and the audible
response of tapping the fruit with knuckles. Some fruits were
sacrificed to verify uniformity of development. Fruits in all
treatments were washed with sodium hypochlorite solution (150 ppm) and

40

then dipped for 30 sec. in an aqueous suspension of Botran (2,6-
dichloro-4-nitroaniline, Upjohn) at the rate of 0.8 g/liter for
postharvest decay control. Individual fruits were placed in air-tight
24 liter plastic containers at 18°C and ventilated with a constant
flow of air or the desired gas mixture. At appropriate intervals
during the experiments, watermelon samples were cut longitudinally and
rind and placental tissue characteristics noted. The firmness of the
rind and the placental tissue was determined using an Instron
universal testing instrument (Model 1132, Instron Corporation, 100
Royall Street, Canton, Mass. 02021). Instron probe size was 1 cm in
diameter, and tissue sample size was 5x5x2 cm.

Gas Treatments

Endogenous ethylene production by ethylene-treated fruit could
not be determined separately because it could not be differentiated
from that used in the treatment. Therefore, propylene, which is an
active analogue of ethylene, was employed to trigger endogenous
ethylene production (if any) by watermelon fruit.

Air, air and ethylene, or air and propylene combinations were
mixed at the desired concentrations by means of a flow through system
equipped v^rith flow meters and regulating valves (Gull, 1981). A
constant flow rate of the different gas treatments was maintained at
12 liters.hr ^.container Ethylene or propylene gas mixtures were
administered to the individual fruits at concentrations of 50 and 6500
1. liter respectively. Relative humidity ranged between 80% and
90%. For ethylene and CO2 measurements, the containers were sealed

41

for 4 hrs. and gas samples withdrawn through rubber septa using
gastight syringes. Levels of ethylene and propylene were determined
with a gas chromatograph equipped with an activated alumina column and
flame ionization detector. Levels of CO2 were determined with an
infrared gas analyzer (Beckman 215 A).

Results

Effect of Ethylene Treatment and Stage of Maturation on Respiration

The respiratory patterns of immature, preripe, and ripe
watermelon fruit are shown in Fig. 3-1 (A-C). Immature fruit (A)
exhibited higher respiratory rates as compared to fruit from the other
two maturation stages (B and C) which exhibited comparable respiration
rates and respiratory drifts which decreased during storage. Ethylene
induced a climacteric-like rise in the respiration rates of fruits
from all maturation stages (Fig. 3-1, A-C). Respiration rates of
ethylene-treated watermelon fruit from all maturation stages decreased
during storage and then within several days increased again. This
increase was associated with ethylene-induced tissue damage and
subsequent pathogen proliferation. Respiration rates of untreated
fruit slowly declined with time. Untreated fruit did not produce
detectable amounts of ethylene (Fig. 3-1, A-C). When ethylene-treated
fruits of all maturation stages were examined internally, they
appeared leaky and soft (Table 3-1), and had off odors and an
unacceptable taste. Ethylene-induced breakdown of watermelon fruit
tissue was visible on the third day of treatment, and on the sixth day
the rind became very soft (Table 3-1) and decay symptoms appeared.

Fig. 3-1. Respiration and ethylene production by harvested
’Charleston Gray’ watermelon fruit. Fruit were
harvested and treated with air (OA) or air +

50 ppm ethylene (•) as described in Materials
and Methods. (*0), respiration (CO^ .kg“^.hr~^) .

, ethylene production (pl.kg“^.hr~^) .
Watermelons harvested immature (A), preripe (B) ,
and ripe (C) . Vertical bars represent standard
error of the mean of five replications.

43

Days

44

Table 3-1. Firmness of ripe watermelon fruit exposed to 0 or 50 ppm
ethylene during 0, 3, 6, and 9 days’ storage at 18°C.^

Treated

Control

Days

Rind

Center

Rind

Center

(Newton)

0

144.0 ±

4.7

6.3 ± 0.6

144.9 ±

5.8

7.0 ± 0.7

3

127.2 ±

8.4

5.8 ± 0.2

158.1 ±

2.1

9.3 ± 0.9

6

123.2 ±

3.1

3.1 ± 0.5

156.6 ±

8.3

9.7 ± 0.9

9

78.7 ±

5.5

1.1 ± 0.1

150.3 ±

5.1

6.7 ± 0.5

2

Each value is the mean of 5 replications plus or minus the standard
error of the mean.

45

The respiratory response of ripe watermelon fruit to intermittent
ethylene treatment is shown in Fig. 3-2. Ethylene twice stimulated
the respiration of fruit, and upon removal of the gas, respiration
rate returned to control levels.

Effect of Propylene Treatment on Respiration and Ethylene Production

Propylene treatment of ripe watermelon fruit stimulated
respiration in a manner similar to that caused by ethylene (Fig. 3-3).
Respiration rate of propylene-treated watermelon fruit declined close
to control level upon removal of the gas and then increased steadily
after the 10th day. Respiration rate of untreated fruit slowly
declined and was typical of trends observed in other experiments (Fig.
3-1). Propylene treatment at 6500 yl.liter”^ did not trigger ethylene
production during the first 10 days of treatment. The rise in
ethylene production observed after the 10th day of propylene treatment
was always accompanied by the widespread appearance of decay on the
surface of fruits. This rise in ethylene production during fruit
decay was also accompanied by a rise in CO2 production. The bacterium
Erwinia herbicola and the fungus species Paecilomyces (identified by
J. A. Bartz, Dept, of Plant Pathology, Univ. of Florida) were
recovered from decayed watermelon fruit.

Discussion

Ethylene induced a climacteric-like rise in the respiration rate
of watermelon fruit of all maturation stages (Fig. 3-1, A-C). Since
immature (white) and preripe (pink) fruit also exhibited this response

46

Days

Fig. 3-2. Respiration of harvested 'Charleston Gray' watermelon
fruits in response to exogenous ethylene. Fruit were
treated with air (o) or air + intermittent 50 ppm
ethylene (•) as described in Materials and Methods.
Vertical bars represent standard error of the mean of
five replications. Arrows indicate when ethylene
was applied or withdrawn.

47

8

6,

4

2

0

Days

Fig. 3-3. Respiration and ethylene production by harvested
'Charleston Gray' watermelon fruit in response to
propylene. Fruit were treated with air (OA) or air
+ 6500 ppm propylene («a) . («o) ^ respiration

(C02*kg“l.hr“l) . , ethylene production (pi. kg .hr ).

Vertical bars represent standard error of the mean of
five replications. Arrov/s indicate when propylene was
applied or withdrawn.

pi C2H^« kg • hr

48

yet did not ripen during the treatment period, the increase in CO2
production observed did not represent a climacteric rise in
respiration. This is consistent with the findings of Vines et al.

(1965) and Herner and Sink (1973) who reported that exposure of
oranges and mutant tomato fruits, respectively, to ethylene resulted
in increased respiration rates without enhancing ripening.

It has long been known that ethylene treatment of preripe
climacteric fruit will shorten the period of time required for the
induction of the climacteric (Rhodes, 1970; McGlasson et al., 1978).

In this study, ethylene did not induce the respiratory climacteric
rise and ripening in watermelon fruit at any of the maturation stages.
The rise in the respiration rate of ethylene-treated fruit which
started around the 8th day of ethylene treatment was solely attributed
to decay of the fruit. Chlorination and fungicide treatments
(disinfection) delayed but did not prevent microbial infection in
ethylene-treated watermelon fruit. Microorganisms isolated from
decayed fruit were identified as the bacterium Erwinia herbicola and
the fungus, Paecilomyces. In untreated fruit, however, disinfection
prevented microbial infection throughout the duration of the
experiment. It appeared that ethylene treatment significantly reduced
the resistance of watermelon fruit to microbial attack. The fruits
were discarded on the 12th day of the ethylene treatment because of
advanced symptoms of decay. The respiration rates of untreated fruit
of all maturation stages slowly declined with time, typical of
nonclimacteric respiratory drifts (Fig. 3-1, A-C). Untreated preripe
(pink) fruit did not continue ripening during storage. Also,

49

untreated fruit of all stages of maturation did not produce detectable
amounts of ethylene. These results support the conclusion that
watermelon fruit is nonclimacteric.

Upon internal examination of ethylene-treated fruits, they
appeared leaky and soft (Table 3-1), and developed unpleasant odors
and an unacceptable flavor. These results are consistent with those
reported by Shimokawa (1973) and Risse and Hatton (1982) and provide
more evidence in favor of the nonclimacteric nature of the fruit.

Ethylene stimulates respiration of nonclimacteric fruit and upon
its removal, the respiration rate declines to its value before
treatment (McGlasson et al., 1978). Also, respiration of
nonclimacteric fruit can be repeatedly stimulated by successive
ethylene treatments (Vines et al., 1965; Rhodes, 1980a). Respiration
rate of watermelon fruit was stimulated by two successive ethylene

treatments (Fig. 3-2) and upon removal of the gas, respiration rate

<

returned close to control level. These results are typical of
nonclimacteric fruit and concur with the findings of Herner and Sink
(1973) and Shimokawa (1973).

Propylene treatment at 6500 yl.liter”^ stimulated respiration in
watermelon fruit in a manner similar to that caused by ethylene, but
did not trigger ethylene production during the first 10 days of
treatment (Fig. 5-3), McMurchie et al. (1972) reported increased
respiration rates but not ethylene production in lemons and oranges
treated with propylene. After prolonged exposure, propylene-treated
fruit developed decay symptoms characteristic of those observed with

50

9

ethylene treatment. The rise in ethylene production observed after
the 10th day of propylene treatment was associated with decay.

The effect of pathological infection on respiration and ethylene
production in the watermelon fruit is similar to that reported by
other workers for various pathogenic infections of nonclimacteric
fruits (Vines et al. 1965; Zauberman and Barkai -Golan, 1975). Barkai-
Golan and Kopeliovitch (1983) reported that Rhizopus infection
stimulated both ethylene and CO2 production and induced the
development of climacteric-like respiratory patterns in tomato mutant
fruit (rin) . Studies on respiration and ethylene production in
carambola fruit (Oslund and Davenport, 1983) revealed that the high
levels of CO2 and ethylene produced by the fruits were mainly
attributable to fruit decay. Similarly, in this study, the rise in
ethylene produced by propylene-treated watermelon fruit was a direct
consequence of microbial activity and was not related to autocatalytic
ethylene production in response to propylene. Untreated fruits were
decay-free and produced no detectable amounts of ethylene throughout
the duration of the experiment.

McMurchie et al. (1972) reported that propylene stimulated a
typical respiratory climacteric and a rise in ethylene production in
bananas. In contrast, lemons and oranges (nonclimacteric fruits)
showed only a climacteric-like rise in respiration in response to
propylene. Judging by the similarity of the responses of watermelon
and citrus fruit to propylene, and from the evidence presented in this
context, we conclude that watermelon fruit is nonclimacteric.

CHAPTER IV

ELECTROLYTE LEAKAGE, FIRMNESS, AND SCANNING ELECTRON
MICROSCOPIC STUDIES OF WATERMELON FRUIT TREATED WITH ETHYLENE

Introduction

Exposure of various plant tissues to ethylene has been reported
to cause an increase in membrane permeability (Hanson and Kende, 1975;
Kende and Hanson, 1976; Suttle and Kende, 1978, 1980; Ferguson and
Watkins, 1981). Leakage of electrolytes, sugars, or pigments has been
commonly used as an index of membrane permeability in plant tissues
(Sacher, 1973; Borochov and Faragher, 1983). Kende and Baumgartner
(1974) and Hanson and Kende (1975) found that ethylene induced
senescence and enhanced efflux of solutes from the cells of morning

glory petal tissue. Suttle and Kende (1978, 1980) similarly

<

demonstrated that ethylene hastened the onset as well as the rate of
leakage of anthocyanin and electrolytes from isolated petals of
Tradescantia. A close relationship was reported between increased
membrane permeability and ethylene production in apple fruit (Berard
and Lougheed, 1982).

Electrolyte leakage from fruit tissues in particular depends upon
factors other than membrane permeability. As fruits mature and ripen,
they accumulate sugars and acids leading to a high osmotic potential,
and at the same time their cell walls are weakened by hydrolytic
action and/or other means including calcium fluxes (Simon, 1977a).
Incubation of fruit tissues in hypotonic media such as distilled

51

52

water or weak buffers may lead to membrane rupture and therefore the
potential for overestimation of membrane permeability (Sacher, 1973;
Wade et al., 1980).

Calcium is involved in numerous physiological processes in
plants. Calcium binds to pectins resulting in stabilization and
maintenance of cell wall integrity (Ferguson, 1984; Demarty et al.,
1984). Sams and Conway (1984) reported a negative relationship
between soluble polyuronide content and Ca concentration in apple
fruit. The role of Ca in membrane structure and function stems from
its ability to stabilize membranes (Paliyath et al., 1984). Poovaiah
(1979) concluded that Ca suppressed changes associated with senescence
in tomato fruit through its function in maintaining cell wall
structure and membrane integrity. Lieberman and Wang (1982) reported
that electrolyte leakage from apple fruit tissue discs was
significantly reduced when incubated in a solution containing 100 mM
Ca and Mg .

It has been reported that watermelon fruits are adversely
affected by exposure to ethylene (Shimokawa, 1973; Risse and Hatton,
1982). Placental tissue of ethylene-treated fruit appear macerated
and watersoaked. We have observed that watermelon fruit allowed to
overripen (either attached or postharvest) do not show any symptoms of
watersoaking (Chapter III). Furthermore, small underdeveloped fruit
become watersoaked in response to ethylene. There would thus seem to
be a close relationship between ethylene and the disorder, independent
of ripening or fruit maturity. Shimokawa (1973) reported that
pectinase increased in watermelon fruit exposed to ethylene and this
may well contribute to the disorder. However, many fruit types

53

contain D-galacturonase but do not exhibit watersoaking until perhaps
the terminal stages of senescence, long after the period of maximum
ethylene production. The objectives of this research are to study the
effects of ethylene on electrolyte leakage, firmness, and
ultrastructure in watermelon fruit.

Materials and Methods

Plant Material

'Charleston Gray' watermelons [Citrullus lanatus (Thunb) Matsum
and Nakai] were grown at the Horticultural Research Farm near
Gainesville, Florida. Medium sized (7 to 9 kg) fruits were harvested
at the ripe stage. Criteria used as harvesting indices included
yellowing of the ground spot, withering of the tendril adjacent to the
stem, and the audible response of tapping the fruit with the knuckles.
Some melons were sacrificed to verify uniformity of development.

Fruits were washed with sodium hypochlorite (150 ppm) and then dipped
for 30 seconds in an aqueous suspension of Botran (2 , 6-dichloro-4-
nitroaniline, Upjohn) at the rate of 0.8 g liter~^ for postharvest
decay control.

Gas Treatments

Individual fruits were placed in air-tight 24 liter plastic
containers at 18°C and ventillated with a constant flow of air or a
mixture of air and ethylene. Air and ethylene (50 ppm) combinations
were mixed by means of a flow through system and delivered to the
fruit at a flow rate of 12 liters hr ^ container Relative humidity

54

was between 80% and 90%. Fruit were treated with 0 or 50 ppm ethylene
for 0, 3, 6, or 9 days.

Electrolyte Leakage

From each treatment, three watermelon fruits were cut
longitudinally and visually inspected for symptoms of ethylene damage
(watersoaking) . Tissue plugs (1.0 cm thick and 1.8 cm in diameter)
were prepared from the central part of the placental tissue using a
sterilized cork borer. The plugs were briefly rinsed with distilled
water and then placed in 10 ml of either distilled water, 0.4 M
mannitol, or 0.4 M mannitol containing 2.5 mM CaCl2, and shaken gently
throughout the experiment. Electrolyte content of the bathing
solutions was measured at hourly intervals using a conductivity bridge
(YSI model 31, Yellow Springs Instruments Co., Inc., Yellow Springs,
Ohio 45387). Firmness of both the rind and the placental tissue was
determined using an Instron universal testing instrument (model 1132,
Instron Corporation, 100 Royall Street, Canton, Massachusetts 02021).

Scanning Electron Microscopy

Sections of placental tissue (2x4 mm) were fixed for 2 hrs in
half-strength Karnovsky's fixative (Karnovsky, 1965) at 22°C. The
samples were then transferred to 1% OsO^ (buffered in 0.2 M sodium
cacodylate buffer, pH 7.2) for 2 hrs at 22°C, and then washed three
times with distilled water. The samples Mere dehydrated in a standard
ethanol series, critical point dried (Balzers Union CPD 010), and
sputter-coated with gold (Giko IB-2 Ion Coater, Giko Engineering

55

Co., Japan). The samples were viewed in the scanning electron
microscope (Hitachi S-450).

Results

The effects of storage duration and 50 ppm ethylene treatment on
firmness is presented in Table 4-1. Firmness of the rind and the
placental tissue decreased with increased duration of exposure to
ethylene. However, firmness of untreated tissue remained unchanged
throughout the duration of the experiment. Table 4-2 shows the values
for electrolyte leakage rates calculated over the first 2 hrs of
incubation. Initial rates of leakage increased markedly with
increased duration of exposure to ethylene. Rate of leakage was
highest in tissues incubated in distilled water, followed by 0.4 M
mannitol and least in 0.4 M mannitol containing 2.5 mM CaCl2.

Preliminary studies showed that 0.4 M mannitol was isotonic with ripe

(

watermelon fruit tissue. The effects of ethylene treatment and
duration (averaged over incubation media) on electrolyte leakage is
shown in Fig. 4-1 (A). Leakage from all tissues increased with time
of incubation. Exposure of watermelon fruit to ethylene for 6 and 9
days resulted in significantly higher electrolyte leakage as compared
to the untreated controls. The effect of incubation medium on
electrolyte leakage is shown in Fig. 4-1 (B-D). Leakage in all
incubation media followed a trend similar to that observed in Fig. 4-1
(A) and described in Table 4-2. Electrolyte leakage in 0.4 M mannitol
and 0.4 M mannitol containing 2.5 mM CaCl2 was 65.3 and 38.5%,
respectively, of leakage in distilled water (Fig. 4-2). Total leakage
values are shown in Table 4-3. Leakage values with respect to

56

Table 4-1.

Firmness of ripe watermelon fruit exposed to 0 or 50 yl
liter" ethylene and stored at 18°C.

Storage ^2^4 Firmness

period treatment Rind Center

(days) (Newton)

0

—

150.113.5

6.410.3

3

_

132.212.5

6.510.2

+

107.512.5

5.110.2

6

_

130.312.1

7.010.3

+

86.417.9

3.710.3

9

_

135.013.6

6.810.1

+

64.414.3

1.010.1

'Each value is the

mean of five

replications plus or

minus the

standard error of

the mean .

57

Table 4-2. Rates of electrolyte leakage from watermelon tissue discs
prepared from fruit exposed to 0 or 50 yl liter” ethylene
and stored at 18°C.^

Ethylene Conductance

Days treatment H2O Mannitol Mannitol + CaCl2

( mhos)

0

—

90

18

10

3

_

93

38

13

+

98

45

15

6

_

105

15

10

+

163

90

57

9

_

150

55

25

+

175

120

58

2

Values represent
first two hrs of

the mean initial change in conductance during the
incubation.

58

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Fig. 4-1. — Continued.

60

Fig. 4-1. — Continued

CD

Cl

in

CO

if)

L_

JZ

(D

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62

(OL|Lur1) A;iAi;3npuoD

q; □

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of 'Charleston Gray' watermelon fruit. Fruit were treated as describi
Materials and Methods and incubated in distilled water (o) , 0.4 manni
or 0.4 mannitol +’2.5 mM CaCl2 (a). Vertical bars represent standard
of the mean of six replications.

63

Table 4-3. Total electrolyte leakage values for watermelon tissue
discs prepared from fruit exposed to 0 ot 50 pi liter”
ethylene and stored at 18°C.

Ethylene Conductance

Days treatment H2O 0.4 M Mannitol 0.4 M Mannitol+CaCl2

( mhos)

0

496117

237121

164118

3

518140

402133

175115

+

565119

429126

345134

6

599135

168121

152114

+

732133

551110

4051 5

9

627133

317120

185114

+

768135

6351 8

350131

'Each value is the
standard error.

mean of four

replications plus or

minus the

64

ethylene treatment and incubation media followed trends described
earlier (Fig. 4-1, B-D; Table 4-2).

Scanning electron micrographs of placental tissue from fruit
exposed to 0 or 50 ppm ethylene for 0, 3, 6, and 9 days are shown in
Fig. 4-3. Cell walls of tissues not exposed to ethylene [Fig. 4-3 (A,
B, D, F)] appeared upright, rigid, and the cells were well defined.

In contrast, cell walls of tissues exposed to ethylene for 3 days
exhibited signs of structural weakening and were slightly collapsed
[Fig. 4-3 (C)]. Longer exposures to ethylene resulted in essentially
complete cellular collapse [Fig. 4-3 (E and G)].

Discussion

The results of these experiments clearly demonstrate that one
consequence of exposure of watermelon fruit to ethylene is altered
membrane permeability, as indicated by the significant increases in
the rate and magnitude of electrolyte leakage. Leakage increased
progressively with increased time of exposure to ethylene [Fig. 4-1
(A)]. This enhanced leakage in response to ethylene, particularly in
isotonic bathing solution [Fig. 4-1 (C)], suggests an ethylene-induced
increase in membrane permeability. These results are consistent with
the findings of Hanson and Kende (1975) and Suttle and Kende (1978,
1980) for morning glory and Tradescantia flowers.

Extensive leakage of electrolytes occurred from tissues incubated
in distilled water [Fig. 4-1 (B) and Fig. 4-2]. Watermelon fruits
have particularly large cells with thin primary walls and relatively
high solute contents, which results in high osmotic potential. The
(abnormally) high rate of electrolyte loss from tissues incubated in

Fig. 4-3. Scanning electron micrographs of placental tissue from
ethylene treated or untreated watermelon fruit. Tissue
from freshly-harvested fruit (A) , stored for 3 days (B) ,
ethylene treated for 3 days (C) , stored for 6 days (D) ,
ethylene treated for 6 days (E) , stored for 9 days (F) ,
and ethylene treated for 9 days (G) . Magnification
was SOX for all plates. ,

66

(A)

67

(B)

Fig, 4-3. — Continued.

68

(C)

Fig. 4-3. — Continued.

69

(D)

Fig. 4-3. — Continued

(E)

Fig. 4-3. — Continued

71

(F)

Fig. 4-3. — Continued

72

(G)

Fig, 4-3. — Continued

73

distilled water was undoubtedly a consequence of cell rupture, Simon
(1977b) reported that when apple fruit tissue was incubated in water,
90% of its electrolytes leaked out in 2 hrs, whereas leakage was
greatly reduced when tissues were incubated in 1 M glycerol or 0.5 M
KCl. The author suggested that apple fruit cells burst in water but
not in isotonic media. In this study, when tissues were incubated in
0.4 M mannitol (isotonic), leakage from untreated tissue still
occurred but was significantly reduced [Fig. 4-1 (C)].

Leakage was further reduced in all treatments, including the
controls when tissues were incubated in 0.4 M mannitol containing 2,5
mM CaCl2 [Fig. 4-1 (D)]. This was most likely due to the capacity of
Ca to stabilize the cell wall and membrane. The cross-linking role of
Ca is crucial for stabilization and maintenance of cell wall integrity
(Demarty et .al., 1984). The ability of Ca to reduce leakage is
probably a direct result of tighter packing of membrane lipids made
possible by its binding to negatively charged phospholipids (Ferguson,
1984). Poovaiah (1979) demonstrated that Ca suppressed the loss in
membrane permeability during tomato fruit senescence. Leakage from
apple fruit tissue (Lieberman and Wang, 1982) was significantly lower
when the bathing solution contained 100 mM Ca and Mg.

Ultrastructural studies showed that placental tissue of
watermelon fruit consisted of large (^ 0.5mm) parenchyma cells with
thin walls. Untreated tissue from all storage durations had well-
defined cells with intact, well-preserved cell wall [Fig. 4-3 (A, B,

D, and F)]. Although tissues of fruit exposed to ethylene for 3 days
showed obvious signs of deterioration, [Fig. 4-3 (C)], however, their
electrolyte leakage values did not differ significantly from those of

74

the control [Fig. 4-1 (A, B, and C)]. The data suggest that ethylene
exerts its effect first on the cell wall and then on the membrane
system. Cell walls of fruit exposed to ethylene for 6 and 9 days
[Fig. 4-3 (E and G)] appeared totally collapsed. This collapse was
closely paralleled by a progressive decrease in firmness of fruit as
the duration of exposure to ethylene increased (Table 4-1). These
results collectively indicate an ethylene-induced weakening and
degradation of the cell wall. A close relationship exists between
increased electrolyte leakage, reduced tissue firmness, and collapsed
cell v/alls of ethylene-treated tissue. It appears from these results
that the increase in electrolyte leakage and reduction in firmness of
fruit exposed to ethylene is a consequence of a deteriorative
influence of ethylene on both the cell wall and the membranes.

Further experiments should be focused towards elucidating the effects
of ethylene on the activity of cell wall hydrolytic enzymes and the
magnitude of wall degradation.

CHAPTER V

CELL WALL HYDROLASES AND ULTRASTRUCTURE OF
WATERMELON FRUIT AS INFLUENCED BY ETHYLENE

Introduction

Softening of fruits during ripening is mainly attributed to
enzymic hydrolysis of cell wall polysaccharides, largely the pectins
(Pressey, 1977; Huber, 1983b). Although a number of hydrolases have
been implicated in softening, the D-galacturonases (endo-D-
galacturonases, E.C. 3.2.1.15, and possibly exo-D-galacturonases , E.C.
3.2.1.67) are believed to be of major importance (Huber, 1983b).

Numerous reports have indicated a role of ethylene in enhancing
the appearance of D-galacturonase activity in ripening fruit.

Sawamura et al . (1978) reported that exposure of tomato fruit to
ethylene resulted in an increase in the activity of D-galacturonase.

A close relationship was found to exist between D-galacturonase and
ethylene evolution in tomato (Babbitt et al., 1973) and avocado fruit
(Awad and Young, 1979). Pauli and Chen (1983) showed that D-
galacturonase activity was not detected in papaya fruit prior to the
rise in ethylene production. They concluded that the development of
D-galacturonase activity was initiated by ethylene.

There is some evidence, mostly indirect, that C^-cellulase (E.C.
3. 2.1. A) participates in fruit softening (Hall, 1964; Awad and Young,
1979). Even so, to date there have been no conclusive reports that
C^-cellulase hydrolyses native cell wall cellulose. Ethylene has been

75

76

shown to induce C^-cellulase activity in fleshy fruits (Babbitt et
al., 1973; Pesis et al., 1978) and abscission zones (Lewis and
Koehler, 1979). Poovaiah and Nukaya (1979) reported that exposure of

mutant tomato fruit (rin) to ethylene for 2 days resulted in increased
C^-cellulase activity.

Ultrastructural studies have provided supporting evidence that
changes occur in the cell wall during softening (Simpson et al., 1976;
Pesis et al., 1978; Ben-Arie et al., 1979; Platt-Aloia and Thomson,
1981). These studies reveal a progressive breakdown and dissolution
of the middle lamella and in some cases, a gradual separation of wall
fibrils. Initial degradation of the middle lamella is generally
correlated with increased D-galacturonase activity (Platt-Aloia et
al., 1980; Crookes and Grierson, 1983). This view is supported by the
results of Ben-Arie et al. (1979), who duplicated naturally occurring
ultrastructural changes in soft apple fruit by exogenous application
of D-galacturonase to firm fruit. Another example of the potential
role of D-galacturonase in fruit softening may be found in the
watermelon fruit. It has been reported that watermelons are adversely
affected by exposure to ethylene (Risse and Hatton, 1982). Placental
tissue of ethylene-treated fruit became soft and watersoaked.

Similarly, Shimokawa (1973) observed that watermelon fruit tissue was
macerated by exposure to ethylene. The author reported higher
activity of pectinase in ethylene-treated, macerated tissue. We have
observed that the ethylene-induced watersoaking syndrome bears little
relationship to natural ripening in watermelon fruit. This
observation raises questions regarding the precise role of ethylene
and wall hydrolases, particularly D-galacturonase, in natural ripening

77

of watermelon fruit. The objectives of these studies were to compare,
ultrastructurally and biochemically, changes occurring during both
ethylene-induced breakdown and natural ripening in watermelon fruit.

Materials and Methods

Plant Material

Fruit of 'Charleston Gray' watermelon [Citrullus lanatus (Thunb)
Matsum and Nakai] were harvested at the ripe stage from plants grown
at the Horticultural Research Farm near Gainesville, Florida.

Criteria used as harvesting indices included yellowing of the ground
spot, withering of the tendril adjacent to the stem, and the audible
response of tapping the fruit with knuckles. Some melons were
sacrificed to verify uniformity of development. Fruit were
disinfected with a solution of sodium hypochlorite (150 ppm) and then
dipped for 30 seconds in an aqueous suspension of Botran (2,6-
dichloro-4-nitroaniline, Upjohn) at the rate of 0.8 g liter ^ for
postharvest decay control.

Ethylene Treatment

Individual fruits were placed in air-tight 24-liter plastic
containers at 18°C and ventilated with the desired gas mixture. Air
or air and ethylene (50 ppm) combinations were mixed by means of a
flow-through system and administered to the containers at a flow rate
of 12 liters container ^ hr ^ . Relative humidity ranged between 80%

and 90%.

78

Fruit from all treatments were cut longitudinally at 0, 1, 2, 3,
6, and 9 days and visually examined for ethylene damage. Placental
tissue samples were collected at the previously mentioned sampling
periods and stored in polyethylene bags at -20°C. Samples were also
collected from fruit harvested overripe and from ripe fruit stored for
6 months at 15°C.

Preparation of Ethanol Insoluble Solids

Partially-thawed placental tissue (100 g) was homogenized in 400
ml of 100% ethanol for 2 min in a Counter Craft blender. The
homogenate was refluxed in a boiling water bath for 20 min to
inactivate endogenous enzymes and then stored overnight at -20°C. The
suspension was filtered through Miracloth (Biochemical Corp., La
Jolla, CA) and washed with 500 ml of 80% acetone followed by 1 liter
of 100% acetone. The powder was squeezed to remove excess acetone and
air dried at 24°C. *

Determination of Total Pectins

Total pectins were measured in the manner described by Ahmed and
Labavitch (1977) for cell walls. Samples of 8 mg of alcohol insoluble
solids were placed in 30-ml beakers in an ice bath. To these samples,
2.5 ml of chilled concentrated H2S0^ were added with continuous
stirring. Aliquots of distilled water (0.8 ml) were added twice, each
followed by a 5 min interval. After a final 5 min period, 21 ml of
distilled water were added. Acid sugar content in the hydrolysates
was determined using the procedure described by Blumenkrantz and
Asboe-Hansen (1973).

79

Gel-Filtration Chromatography

Gel chromatography of pectins was carried out according to the
procedure described by Huber (1983a). Alcohol insoluble solids (20
mg) were incubated in 2.5 ml of Na-acetate buffer (50 mM, pH 5.0)
containing 5 mM Na^EDTA and incubated at 23°C with stirring, for 6 hr.
The suspension was filtered through Miracloth and washed with 1.5 ml
of the extraction buffer. The filtrate was centrifuged in a table top
clinical centrifuge for 5 to 10 min at maximum speed. The supernatant
was filtered through glass filter paper using a light vacuum, and used
for gel chromatography.

Gel filtration of pectins was performed on a bed (60 cm high, 1.5

cm wide) of Ultrogel AcA 34 (LKB) packed in Na-acetate buffer (50 mM,

pH 5.0) containing 5mM Na2EDTA and 200 mM NaCl. Approximately 3 mg of

pectin in a volume of 2.5 ml buffer were applied to the column and

gravity eluted with the acetate-EDTA buffer. Fractions of 2 ml were

-2 -1

collected at a flow rate of 10 ml cm hr and 0.5 ml aliquots of
these used for the determination of acid sugars using the

procedure of Blumenkrantz and Asboe-Hansen (1973).

Preparation and Assay of C^-Cellulase and D-Galacturonase

Partially-thawed placental tissue (100 g) and 6.0 g NaCl were
homogenized for 2 min using a Counter Craft blender and the homogenate
stored overnight (generally 10 hrs) at 1°C. The homogenate was
centrifuged for 20 min at 12,000 g and the supernatant brought to 80%
saturation with solid (NH^)2 SO^. Following overnight storage at 1°C,
the salt-saturated supernatant was centrifuged for 20 min at 15,000 g.

80

The pellet was resuspended in 10 ml of Na-acetate buffer (60 mM, 100
mM NaCl, pH 5.0) and dialysed overnight against 10 mM Na-acetate
buffer containing 100 mM NaCl. The enzyme extract was filtered
through Miracloth and used for the determination of C^-cellulase and
D-galacturonase activities.

The assay mixture for the determination of C^-cellulase activity
consisted of 0.5 ml (active or boiled) enzyme extract and 3 ml of 1%
(w/v) carboxymethylcellulose (CMC) (Hercules Powder Co., type S-710)
in Na-acetate buffer (30 mM, pH 5.0) containing 100 mM NaCl and 0.02%
Thimerosal. Relative viscosity was calculated from the change in
drainage time of the mixture through a calibrated upper portion of a
1.0 ml pipette at 2A°C. Total protein content was determined
according to the procedure described by Bradford (1976). Bovine serum
albumin was used as the protein standard. C^-cellulase activity was
expressed as percent change in viscosity . mg protein ^.hr ^
calculated from readings made over a 6.5-hr period.

Reaction mixtures for the determination of D-galacturonase
activity consisted of 0.1 ml of the enzyme extract (active or boiled)
and 0.5 ml of 0.1% (w/v) polygalacturonic acid (Sigma) in Na-acetate
buffer (30 mM, pH 5.0) containing 100 mM NaCl and 0.02% Thimerosal.

The reaction mixture was incubated for 1 hr in a water bath at 34°C.
Activity of D-galacturonase was assayed reductometrically using the
method described by Milner and Avigad (1967). Total proteins were
determined according to the procedure of Bradford (1976). Activity
was expressed as g reducing equivalents . mg protein”^ .hr~^ .
Galacturonic acid (Sigma) was used as the standard.

81

Transmission Electron Microscopy (TEM)

After the desired ethylene treatment periods, watermelon fruit
were cut longitudinally. Sections (1x2 mm) of placental tissue were
dissected from the fruit and immediately fixed in half-strength
Karnovsky's fixative (Karnovsky, 1965) for 2 hrs at 22°C. The samples
were then transferred to 1% OsO^ (buffered in 0.2 M Na-cacodylate
buffer, pH 7.2) for 2 hrs at 22°C and then washed three times with
distilled water. The samples were dehydrated in a standard ethanol
series, followed by 100% acetone, and embedded in Spurr's resin
(Spurr, 1969). Ultrathin sections were cut with glass knives using an
LKB Ultrotome III microtome. Sections were stained in succession with
1% uranyl acetate, followed by 0.5% lead citrate, and then 1% BaMNO^.
Sections were examined with a Hitachi HU-1 IE electron microscope.

Results

Exposure of watermelon fruit to ethylene resulted in a
significant reduction in placental tissue firmness (Fig. 5-1). Fruit
showed obvious symptoms of watersoaking as time of exposure to
ethylene increased.

C^-cellulase activity in fruit exposed to 0 or 50 ppm ethylene is
presented in Table 5-1. Activity was highest in fruit exposed to
ethylene for 3 days and lowest in freshly harvested and overripe
fruit. Duration of exposure to ethylene had no significant effect on
C^-cellulase activity. Furthermore, activity in freshly harvested
fruit (0 days) was not significantly different from that in 9-day
ethylene-treated fruit.

82

CO

>>

cu

Q

Fig. 5-1. Firmness of the placental tissue of 'Charleston Gray' watermelon fruit. Fruit

were harvested and treated with air (o) or air + 50 ppm ethylene (•) as described
in Materials and Methods. Vertical bars represent standard error of the mean of

83

Table 5-1. Cx-cellulase^activity in watermelon fruit exposed to 0 or
50 pi liter” ethylene.

Treatment Change in viscosity

(% change . mg protein”^ .hr”^ )

0 days

7.5

c^

1 day (treated)

16.2

ab

2 days (treated)

15.3

ab

3 days (treated)

18.4

a

3 days (control)

11.8

be

6 days (treated)

16.4

ab

6 days (control)

13.7

abc

9 days (treated)

12.1

abc

Overripe melon

8.6

c

F value^

**

2

F values were significant at the 1% level.

%ean separation by Duncan's multiple range test, 5% level.

8A

Data for D-galacturonase activity are shown in Table 5-2.

Activity increased with increased exposure to ethylene and peaked at 6
days. D-galacturonase activity in control or overripe fruit showed no
change.

Analysis of alcohol insoluble powders showed that total pectins
content did not change in melons not exposed to ethylene, even in
fruit stored for periods of up to 180 days (Table 5-3). In contrast,
pectin levels decreased markedly in ethylene-treated fruit, showing a
20 and 33% decrease after 2 and 6 day exposure periods, respectively.

Gel-Filtration Chromatography

Gel-filtration profiles of pectins from ripe watermelon fruit
exposed to 0 or 50 ppm ethylene are presented in Fig. 5-2. These
profiles indicate a significant effect of ethylene on pectin molecular
size. Pectins obtained from freshly harvested and untreated fruit
[Fig. 5-2 (A); Fig. 5-3] were of apparent high molecular weight and
did not fractionate on Ultrogel AcA 34. Even after 180 days of
storage [Fig. 5-4 (B)], at which time the melons were by all accounts
"overripe", pectin degradation was not nearly as extensive as in fruit
treated with ethylene for short periods. Changes were first apparent
in pectins as early as 1 day of exposure of fruit to ethylene [Fig. 5-
2 (B)], as indicated by the appearance of small quantities of polymers
that fractionated on the gel. As time of exposure of fruit to
ethylene increased, the molecular weight of pectins progressively
decreased, and degradation was quite extensive after 6 and 9 days of
ethylene treatment.

85

Table 5-2. Activity of D-galacturonase extracted from watermelon
fruit exposed to 0 or 50 yl liter"'^ ethylene.

Treatment

Activity

0 days

( g galacturonic acid . mg protein"^ .hr"^ )

27.5^

1 day (treated)

35.5

2 days (treated)

70.2

3 days (treated)

68.6

3 days (control)

21.8

6 days (treated)

355.5

6 days (control)

22.7

9 days (treated)

257.9

Overripe melon

44.7

Values are means of three replications.

86

Table 5-3.

Total
50 yl

pectin^

liter

content of watermelon fruit exposed
ethylene.

to 0 or

Ethylene

Days

0 ppm

50 ppm

/o icCLxllfc> — —

0

30. 6^

—

1

28.3

30.0

2

30.6

24.7

3

29.1

22.5

6

27.2

20.0

9

30.3

20.3

20

30.3

—

180

31.3

—

2

Data expressed as percentage of alcohol-insoluble solids.
^Values are means of three replications.

520 nm Abs 520 nm Abs 520 nm

87

Fig. 5-2. Gel-filtration profiles of polyuronides from 'Charleston
Gray' watermelon fruit. Approximately 3 mg of pectin in
2.5 ml of the elution buffer were applied to Ultrogel AcA
34 column and eluted with Na-acetate-EDTA buffer. Fractions
were analyzed for acid (A^20^ sugars. Polyuronides were
prepared from freshly harvested fruit (A) , ethylene-treated
for 1 day (B) , 2 (C) , 3 (D) , 6 (E) , and 9 (F) . Arrows
correspond to elution positions of (left to right) blue
dextran 2,000,000, dextran 70,000, dextran 10,500, and
glucose .

Abs 520 nm Abs 520

88

Fig. 5-3. Gel-filtration profiles of polyuronides from ’Charleston
Gray' watermelon fruit not exposed to ethylene. Details
as described in Fig. 5-2. Polyuronides were prepared
from fruit stored for 1 day (A), and 9 (B) . Arrows
correspond to elution positions described in Fig. 5-2.

Abs 520 nm Abs 520 nm

89

20 30 40 50 60 70

Fraction No.

Fig- 5-4. Gel-filtration profiles of polyuronides from overripe
'Charleston Gray' watermelon fruit. Details of prepa-
ration were described in Fig. 5-2. Polyuronides were
prepared from field-overripe fruit (A) , and fruit
stored for 180 days at 15°C (B) . Arrows correspond to
elution positions described in Fig. 5-2.

90

Ultrastructural Changes in Watermelon Fruit Cell Wall

The placental tissue of ripe watermelon fruit is composed of
large, isodiametric , thin-walled parenchyma cells of approximately 500
m in diameter . The cells have a large central vacuole and the
cytoplasm is confined to a thin region adjacent to the cell wall.

Cell walls from freshly harvested and control fruit displayed darkly
stained, tightly packed fibrillar material [Fig. 5-5 (A-D)]. The
middle lamellar region in most cases was indistinguishable from the
primary walls of adjacent cells. Even in overripe fruit [Fig. 5-5
(E)], the cell wall was composed of densely packed fibrils with no
apparent evidence of disintegration. The first change in the
appearance of the cell wall was noted 1 day after the onset of
ethylene treatment [Fig. 5-5 (F)], and was represented by a thin zone
of separation along the middle lamella. Progressive dissolution of
the middle lamella continued to occur in tissues exposed to ethylene
for 2 days [Fig. 5-5 (G)]. Essentially complete cell separation was
observed in tissues exposed to ethylene for 3 days [Fig. 5-5 (H)]. At
6 and 9 days of ethylene treatment, the tissues were not able to
withstand the fixation procedures.

Discussion

Watermelon fruit exposed to ethylene exhibited significant tissue
damage and cellular breakdown. The data indicated that C^-cellulase
activity was not significantly influenced by ethylene, since activity
in freshly-harvested fruit was not significantly different from that
in 9-day ethylene-treated fruit. The lack of a clear relationship

Fig. 5-5. Transmission electron micrographs of placental tissue
from ethylene-treated or untreated watermelon fruit.
Fruit treated with air (A-E) or air + 50 ppm ethylene
(F-H) were prepared for microscopy as described in
Materials and Methods. Sections were prepared from
freshly harvested fruit (A), stored for 1 day (B) ,

2 (C) , 3 (D) , field overripe (E) , ethylene-treated
for 1 day (F) , 2 (G) , and 3 (H) . All plates
X40,000, except D (X32,000).

92

(A)

93

94

(C)

Fig. 5-5. — Continued

95

(D)

Fig. 5-5. — Continued.

96

(E)

Fig. 5-5. — Continued

97

(F)

Fig. 5-5. — Continued

98

(G)

Fig. 5-5. — Continued

99

(H)

Fig. 5-5. — Continued

100

between C^-cellulase activity and tissue degradation indicates that
C^-cellulase is not directly involved in the ethylene-induced
phenomenon. This is consistent with the view that C^-cellulase
appears to play a minor role in fruit softening (Hobson, 1968;

Hatfield and Nevins, 1985).

D-galacturonase activity in placental tissue increased
progressively with increased exposure to ethylene (Table 5-2).
Increased activity was apparent after 24 hrs and had doubled after 48
hrs of ethylene treatment. This increase was accompanied by visual
symptoms of watersoaking and loss of firmness (Fig. 5-1). Total
pectin content did not change during the first 24 hrs of ethylene
treatment but showed a 20% decrease after 48 hrs (Table 5-3). The
decrease in total pectins possibly reflects their extensive
depolymerization to products which remain soluble in 80% ethanol.

These products would be lost during preparation of the powders.
Clearly, the molecular weight of pectins from ethylene-treated fruit
decreased progressively with increased duration of ethylene treatment
(Fig. 5-2). The appearance of low molecular weight polymers and the
decrease in total pectin content followed the increase in D-
galacturonase activity. Pectins from untreated fruit showed no
evidence of extensive depolymerization. Also, extensive hydrolysis of
pectins was not observed in field overripe and in harvested fruit
stored for as long as 180 days. These observations indicate that
increased D-galacturonase activity is not a characteristic of normal
ripening and senescence of watermelon fruit. Enhanced activity of D-
galacturonase and extensive depoiymerization of pectins were observed
only in fruit exposed to ethylene. Saltveit and McFeeters (1980)

101

showed that D-galacturonase activity in harvested mature or immature
cucumber fruit could be induced by exogenously applied ethylene.
Shimokawa (1973) observed that watermelon tissue was macerated by
exposure to ethylene. He showed higher activity of pectinase in
macerated as compared to untreated tissue. In vitro studies by the
same author revealed that the maceration symptoms could be reproduced
by pectinase but not cellulase. Our results are in agreement with
these findings and suggest that watermelon tissue breakdown was
attributed solely to the activity of D-galacturonase.

Ultrastructural studies of fruit exposed to ethylene revealed an
early disintegration of the middle lamella, followed by separation of
the adjacent primary walls [Fig. 5-5 (F-H)]. Cell wall of untreated
fruit did not show any sign of disintegration, even in overripe fruit
[Fig. 5-5 (A-E)]. The development of a thin line of separation in the
middle lamellar region after 24 hrs of ethylene treatment [Fig. 5-5
(F)] coincided with the first notable increase in D-galacturonase
activity. The lack of change in total pectin as well as molecular
size of pectins during the first 24 hrs of ethylene treatment
indicates that extensive degradation of pectins, at least of a
magnitude to produce products fractionating on Ultrogel, was not
necessary for preliminary cell wall separation to occur. The decrease
in total pectins and the appearance of low molecular size polymers
were evident 2 and 3 days, respectively, after the first notable
increase in D-galacturonase activity. Hence, the evident separation
of the primary cell walls after 3 days of ethylene treatment was
apparently due to the progressive depolymerization of pectins from the
middle lamellar region as a result of D-galacturonase action.

102

Structural alterations in the cell wall of apple fruit (Ben-Arie et
al., 1979) and tomatoes (Crookes and Grierson, 1983) involved the
dissolution of the middle lamella which was attributed to increased D-
galacturonase activity. The authors further demonstrated that the
application of exogenous D-galacturonase to tissue discs from firm
fruit led to a dissolution of the middle lamella similar to that
occurring in naturally ripening fruit. In this context, it is
necessary to differentiate between two systems: in avocado and tomato
fruit, D-galacturonase is normally synthesized as an integral part of
the ripening process. However, in watermelon fruit, increased D-
galacturonase activity is observed only in fruit exposed to ethylene.
Furthermore, activity was extremely low and showed no increase in ripe
(or overripe) untreated fruit. These results thus indicate that
initiation of D-galacturonase activity in fruit treated with ethylene
is independent of the normal ripening process.

CHAPTER VI

SUMMARY AND CONCLUSIONS

The objectives of this research were to study the effects of
ethylene or propyplene on respiration and endogenous ethylene
production in watermelon fruit of different maturation stages; to
determine effects of ethylene on electrolyte leakage, firmness, and
ultrastructure; and to compare, ultrastructurally and biochemically,
the changes occurring during both ethylene-induced breakdown and
natural ripening in watermelon fruit.

Ethylene-treated watermelon fruit became watersoaked and
developed unpleasant odors and unacceptable flavor. Exposure of fruit
to ethylene or propylene neither enhanced ripening in preripe fruit
nor induced a respiratory climacteric. Propylene failed to trigger
endogenous ethylene production in watermelon fruit. Increased
production of CO2 or ethylene were observed only in fruit exhibiting
obvious symptoms of decay. Respiration rates of untreated fruit
followed trends characteristic of nonclimacteric fruit. Additionally,
respiration rate of fruit could be repeatedly stimulated by successive
ethylene treatments. These results collectively support the
conclusion that watermelon fruit is nonclimacteric.

Electrolyte leakage studies were conducted to investigate the
influence of ethylene on the membrane system. The high leakage
observed from tissues exposed to ethylene as compared to the controls
indicated ethylene-induced changes in membrane permeability. Studies
on the effect of incubation media showed that the highest leakage

103

l04

occurred when the tissues were incubated in distilled water due to the
bursting of cells. Leakage was significantly reduced when tissues
were incubated in isotonic media. Further reduction in leakage
occurred when the incubation medium contained CaCl2, most likely due
to the role of Ca^^ in maintaining cell wall and membrane integrity.
Ultrastructural studies revealed that cell walls of fruit exposed to
ethylene showed obvious signs of damage. The collapse in cell walls
and the increase in electrolyte leakage were apparently the outcome of
a deleterious effect of ethylene on both the cell wall and the
membrane system.

Further studies were conducted to determine the effects of
ethylene on cell wall hydrolases, pectin degradation, and cell wall
ultrastructure. Enzymic studies indicated that C^-cellulase was not
involved in watermelon tissue maceration. D-galacturonase activity
increased by’ 24 hrs and peaked after 6 days of ethylene treatment.

The increase in D-galacturonase activity was followed by the
appearance of low molecular weight polymers and a decrease in total
pectins. Hence, it was apparent that watermelon tissue breakdown was
attributed solely to D-galacturonase activity. Enhanced activity and
extensive depolymerization of pectins were observed only in fruit
exposed to ethylene. Ultrastructural studies of ethylene-treated
fruit revealed an early disintegration of the middle lamella, followed
by separation of the adjacent primary walls. Cell wall of untreated
fruit did not show any sign of disintegration. The development of a
thin line of separation along the middle lamella during the first day
of ethylene treatment coincided with the first notable increase in D-

105

galacturonase activity. The results thus indicated that initiation of
D-galacturonase activity in response to ethylene treatment was not a
characteristic phenomenon of normal ripening and senescence in
watermelon fruit.

The data considered together permit a tentative mechanism to be
proposed regarding the sequence of events resulting in watermelon
tissue breakdown. Firstly, the data emphasize a predominant role of
the cell wall, rather than the membrane, in this breakdown. Although
the first notable increase in D-galacturonase activity was observed
after the first 24 hrs of ethylene treatment, the appearance of low
molecular weight pectic polymers was not observed until after the 3rd
day of ethylene treatment. However, ultrastructural studies showed a
distinct separation of the middle lamella after 24 hrs of ethylene
treatment. Scanning electron microscopic studies revealed that cell
walls of fruit exposed to ethylene for 3 days started to show signs of
deterioration. ‘

Secondly, the membrane system did not seem to play a major role
in watermelon fruit breakdown. No change in electrolyte leakage was
noted during the first 3 days of ethylene treatment, but leakage
significantly increased with extended exposure to ethylene. The fact
that the increase in electrolyte leakage did not occur until after the
3rd day of ethylene treatment indicates that the increase in leakage
was due to cell wall degradation and weakening and consequently the
rupture of the membranes.

LITERATURE CITED

Ahmed, A. E., and J. M. Labavitch. 1977. A simplified method for
accurate determination of cell wall uronic content. J. Food
Biochem. 1:361-365.

Ahmed, A. E. , and J. M. Labavitch. 1980. Cell wall metabolism in

ripening fruit: II. Changes in carbohydrate-degrading enzymes in

ripening 'Bartlett' pears. Plant Physiol. 65:1014-1016.

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BIOGRAPHICAL SKETCH

Mohamed Elhag Elkashif was born in Mograt Island, Sudan, in
November 20, 1949. He graduated from the College of Agriculture,
University of Khartoum, Sudan, with a B.Sc. Agric. (honours) in
October, 1974. Upon graduation, he joined the Rahad Agricultural
Corporation as a horticulturist. In December, 1978, he joined the
University of Gezira as a teaching assistant in the Faculty of
Agricultural Sciences. This institution is sponsoring his graduate
training in the Department of Vegetable Crops, University of Florida,
where he earned the degree of Master of Science in May, 1982, and is
currently a candidate for the degree of Doctor of Philosophy.

Mr. Elkashif is married to Asma B. Abdelrahman and has a 2-year-
old son, Omar. ‘

117

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.

D. J.^uber, Chairman
Associ^e Professor of
Horticultural Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.

Associate Professor of
Horticultural Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.

M. Sherman

Associate Professor of
Horticultural Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.

C. B. Hall

Professor of Horticultural
Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.

This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Dj^ctor of
Philosophy .

August 1985

Dean, Collie of Agriculture

Dean, Graduate School