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Texture and ripening physiology of tomato (Lycopersicon esculentum mill.) fruit

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Title:
Texture and ripening physiology of tomato (Lycopersicon esculentum mill.) fruit
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Ahrens, Milton Joseph
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English
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vi, 126 leaves : ill., photos ; 28 cm.

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Subjects / Keywords:
Apples ( jstor )
Calcium ( jstor )
Cell walls ( jstor )
Cells ( jstor )
Enzymes ( jstor )
Ethylene production ( jstor )
Fruits ( jstor )
Pericarp ( jstor )
Ripening ( jstor )
Tomatoes ( jstor )
Polygalacturonase ( lcsh )
Tomatoes -- Physiology ( lcsh )
City of Milton ( local )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
Bibliography:
Includes bibliographical references (leaves 112-125).
General Note:
Typescript.
General Note:
Vita.
General Note:
Includes abstract.
Statement of Responsibility:
by Milton Joseph Ahrens.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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AHG0610 ( NOTIS )
22450079 ( OCLC )
AA00004772_00001 ( sobekcm )

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TEXTURE AND RIPENING PHYSIOLOGY OF
TOMATO (LYCOPERSICON ESCULENTUM MILL.) FRUIT

















By

MILTON JOSEPH AHRENS


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


1989




TEXTURE AND RIPENING PHYSIOLOGY OF
TOMATO fLYCOPERSICON ESCULENTUM MILL.) FRUIT
By
MILTON JOSEPH AHRENS
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
1989


ACKNOWLEDGMENTS
I will ever be indebted to the postharvest group at the
Citrus Research and Education Center in Lake Alfred for
providing me the opportunity to begin my graduate studies.
Appreciation is further extended to the Vegetable Crops
Department (especially the postharvest group) for allowing me
to continue study toward the Doctor of Philosophy degree. I
would like to thank the members of my supervisory committee
for the strong support and encouragement they provided during
the course of my studies at the University of Florida.
Especially, I am grateful and honored to have been directed
by my supervisory chair, Dr. Donald J. Huber, who, to say the
least, provided an interesting, challenging, and rewarding
environment in which to pursue these studies. Of course, most
of my appreciation and admiration is directed at my wife,
Lynne, to whom I owe the most for my having successfully
completed this graduate program.
11


TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ii
ABSTRACT v
CHAPTERS
1 INTRODUCTION AND REVIEW OF LITERATURE 1
Introduction 1
Cell Wall Structure and Composition 5
Fruit Softening 8
Models of Softening in Fruits 10
Calcium Content of Pericarp and Fruit Firmness... 17
Polygalacturonase Activity and Ripening in Tomato
Fruit 20
Fruit Mealiness 26
2 PREAMBLE 3 2
Discussion 32
Objectives of this Study 35
3 FIRMNESS AND MEALINESS: ATTRIBUTES OF TEXTURE IN
TOMATO FRUIT AND THEIR MEASUREMENT 37
Introduction 37
Materials and Methods 41
Results and Discussion 47
4 POLYGALACTURONASE ACTIVITY, RESPIRATION, AND
ETHYLENE PRODUCTION IN RIPENING TOMATO FRUIT... 72
Introduction 72
Materials and Methods 75
Results and Discussion 79
5 HEMICELLULOSE MODIFICATIONS, POLYURONIDE CONTENT
AND CALCIUM CONCENTRATION IN MEALY TOMATO
FRUIT 91
Introduction 91
Materials and Methods 93
Results and Discussion 98
iii


6 SUMMARY AND CONCLUSIONS 109
REFERENCES 112
BIOGRAPHICAL SKETCH 126
IV


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
TEXTURE AND RIPENING PHYSIOLOGY OF
TOMATO (LYCOPERSICON ESCULENTUM MILL.) FRUIT
By
Milton Joseph Ahrens
May 1989
Chairman: Donald J. Huber
Major Department: Horticultural Science
Methods for determining texture in tomato fruit were
evaluated and an objective test for mealiness was developed.
The relationship between pericarp and whole fruit firmness
was investigated. Tomato genotypes varying in intrinsic
firmness were examined to determine the quantitative relation
ships between polygalacturonase (PG) activity, autolysis,
firmness, and other ripening parameters including rate and
intensity of ripening. Firmness, respiration, and ethylene
production were monitored in the immature-green throughout
ripening. Polygalacturonase activity was measured by direct
assay of salt-extractable wall protein or by monitoring the
release of pectins from isolated, enzymically active wall.
Pectin content, hemicellulose fractionation, ethanol-insoluble
solids, and calcium were determined in mealy and non-mealy
fruit.
v


Whole fruit firmness was not highly correlated with
firmness of inner and outer pericarp (r = -0.139 and -0.582,
respectively). Mealiness was not correlated with firmness.
Calcium and pectin content were significantly higher in mealy
fruit than in less-mealy fruit (25 to 104% and 12 to 44%,
respectively). There were no significant differences in
ethanol-insoluble-solids and % dry weight between the two
fruit types. In all fruit, polygalacturonase activity was
highly correlated with pericarp softening, but only moderately
correlated with softening of whole fruit (r = 0.920 and 0.757,
respectively). Polygalacturonase activity was positively
correlated with cell-wall autolytic activity in pink (r =
0.969) and red (r = 0.900) fruit. Firmer genotypes exhibited
lower rates of respiration and ethylene production during
ripening. Polygalacturonase activity in isolates prepared
from fruit at the climacteric peak was positively correlated
with ethylene production and respiration, and negatively
correlated with days to ripen (r = 0.929, 0.805, and -0.791,
respectively).
The data indicate 1) whole fruit firmness is not indica
tive of pericarp firmness; 2) mealiness and firmness are
separate metabolic events; 3) changes in firmness of pericarp
are due to the activity of PG. Furthermore, the data are
consistent with the hypothesis that pectin fragments released
by PG contribute to the production of autocatalytic (System
II) ethylene.
vi


CHAPTER 1
INTRODUCTION AND REVIEW OF LITERATURE
Introduction
Tomato (Lvcooersicon esculentum Mill.) fruit is the
primary vegetable crop produced in Florida. Three hundred
forty million kgs were marketed in the 1986-1987 season with
a wholesale F.O.B. value of over 408 million dollars (Florida
Tomato Committee, 1987). Seventy percent of the crop is sold
to be consumed fresh, with the balance used by the processing
industry. Terminal markets may be roadside, local, distant,
or export. Firm fruit are needed for shipping and most
handling. The highly mechanized bulk fast food market
requires firm fruit for slicing. On the other hand, softer,
fully ripe fruit are desired by the roadside market and for
processing. Due to these diverse marketing requirements, past
and current breeding efforts have been aimed at developing
tomato varieties with handling characteristics suitable to a
wide range of shipping, storage, and marketing conditions,
while attempting to meet consumers' demands for high quality
fruit (Hamson, 1952b; Rushing and Huber, 1983).
Firmness has long been recognized as an important
physical attribute of tomato fruit to be shipped successfully
(Hamson, 1952a, 1952b). Past workers have undertaken studies
to identify mechanical parameters of specific cultivars in


2
order to characterize and predict handling qualities of
certain breeding lines (Hall and Augustine, 1981; Hamson,
1952b; Rushing and Huber, 1983) These attempts have been
largely successful and are a tribute to past breeding and
selection programs. Today, there are tomato varieties
available that offer increased firmness over cultivars
available in the past (Rushing and Huber, 1983). However,
excessive firmness has often been associated with 'mealiness,'
an undesirable texture trait (John Scott, Gulf Coast Research
and Education Center, Bradenton, FI, personal communication).
The biochemical and physiological bases of firmness have been
studied in the past and are of great current interest (e.g.,
Haller and Harding, 1937; Shafshak and Winsor, 1964; Smith et
al., 1988). On the other hand, investigations into the
chemical basis of mealiness and its relationship to ripening
and firmness have received little attention. Recent inves
tigations have focused on the relationship of firmness, cell-
wall degradation, and ripening (Baldwin and Pressey, 1988;
Brecht and Huber, 1988; DellaPenna et al., 1987; Smith et al.,
1988) .
Tomato fruit are classified as climacteric. That is,
during ripening they exhibit a rise and peak in respiration
and autocatalytic ethylene production (Abeles, 1973; Biale,
1960). The onset of ripening and increased carbon dioxide
and ethylene production are temporally correlated with many
ripening phenomena among which are de novo mRNA accumulation
(Lee et al., 1987) and protein synthesis (Bartley et al.,


3
1982; Grierson and Tucker, 1983? Grierson et al., 1985; Tucker
and Grierson, 1982; Tucker et al., 1980), increases in the
activity of certain enzymes (Bartley, 1974; Bartley et al.,
1982; Brady et al., 1983; Buescher and Tigchelaar, 1975;
Buescher et al., 1976? Crookes and Grierson, 1983; Hobson,
1964, 1965; Knegt et al., 1988; Poovaiah and Nukaya, 1979;
Sawamura et al., 1978; Tucker and Grierson, 1982; Tucker et
al., 1980), fruit softening (Ahmed and Labavitch, 1980;
Bartley, 1974; Bartley et al., 1982; Brady et al., 1983;
Buescher and Tigchelaar, 1975; Buescher et al., 1976; Hobson,
1965; Knee, 1973; Yamaki et al., 1979), changes in acid and/or
sugar composition (Ahmed and Labavitch, 1980; Bartley, 1974?
Bartley et al., 1982; Gross, 1984; Gross and Sams, 1984; Gross
and Wallner, 1979; Huber, 1984; Knee, 1973; Yamaki et al.,
1979), and carotenoid synthesis (Tucker and Grierson, 1982).
Another characteristic of climacteric species is that fruit
harvested after a specific point in development (physiological
maturity), but before the climacteric rise, can be unripe, yet
will continue the normal ripening process even though detached
from the plant (Biale, 1960). The time it takes for fruit to
reach physiological maturity, the nature of the climacteric,
and length and characteristics of the ripening process are
characteristic for individual cultivars (Biale and Young,
1981) As an aid in marketing and to assure uniform prac
tices, the USDA has classified tomatoes into ripeness categor
ies according to their external color (U.S. Dept, of Agri.,
1975) Fruit in each category is perceived by wholesalers and


4
retailers as having certain handling characteristics, with
greener fruit being firmer and able to withstand longer
periods in shipping channels without breakdown. The earliest
stage at which tomato fruit can be harvested and still ripen
satisfactorily is designated the 'mature-green' stage. Fruit
in this classification are physiologically mature but show no
external signs of ripeness. Internally, ripening has been
initiated, but changes in quality attributes such as softening
are minimal at this point (Rick, 1978) Fruit of this class
are used for shipping to the most distant markets, for
specific types of processing, and generally can withstand the
rigors of marketing with the least damage. Another advantage
of harvesting at the mature-green stage is that they can be
allowed to ripen under controlled conditions. With the
manipulation of temperature and ethylene, it is common
practice for entire lots of tomatoes to be ripened uniformly
to meet particular market slots and demands (Ethygen Catalytic
Generators, 1988). As a rule, as tomatoes ripen and move
through the ripeness categories of mature-green, to breaker,
pink, and finally red, they soften (Brady et al., 1985;
Gertman and Fuchs, 1974; Rushing and Huber, 1983). It is
believed that changes in the structure of the plant cell wall
result in softening. The cell wall by its composition and
location is the most important structural element of the plant
cell (Bartnicki-Garcia, 1984). A primary component in this
softening is the enzymic hydrolysis of certain wall polymers
(Huber, 1983b).


5
Cell Wall Structure and Composition
Albersheim's group (Albersheim, 1978; Keegstra et al.,
1973) has proposed a model of cell-wall structure based on
their work with cell wall from suspension-cultured sycamore
cells. A diagramatic representation of this model is shown
in figure 1-1. The primary cell wall can be divided into two
general structural components (microfibrils and matrix) and
four general chemical components (cellulose, hemicellulose,
pectin, and protein).
Microfibrils
Cellulose molecules consist of long unbranched chains of
a 1-4 glycosidic linked D-glucopyranose residues (8000
residues chain'1, average). These molecules overlap, have
their long axis' arranged parallel to each other, and are
grouped together into unit structures called microfibrils
(Frey-Wyssling, 1969). A cross section of a microfibril would
cut across roughly 150 cellulose chains. The core of the
microfibril is composed of cellulose molecules organized into
a three-dimensional crystalline lattice held together by
hydrogen bonds. This core is in turn surrounded by other
cellulose molecules running parallel to the core, but not
organized into a crystalline structure. This area is called
the paracrystalline region or cortex (Mark, 1967; Wilson,
1964). Included in the cortex are non-cellulosic polysac
charides and glycoprotein.


6
Matrix
The microfibrils are embedded in a matrix which is
analogous to steel reinforcing rods being embedded in concrete
(Mark, 1967). The primary component of the matrix is polysac
charide which can be subdivided into pectins (Aspinall, 1980;
Worth, 1967) and hemicelluloses (Whistler and Richards, 1970)
classified on the basis of their solubility in water/chelator
or alkali, respectively.
Hemicelluloses were originally thought to be precursors
of cellulose, but they now are known to be made up of polysac
charides classified according to their monosaccharide com
ponents (Bauer et al., 1973). There is considerable diversity
among the hemicelluloses, and entities in this group are
related only by their solubility (Towle and Whistler, 1973).
Albersheim (1978) has found that the only hemicellulose
present in primary cell walls of suspension-cultured sycamore
cells is a xyloglucan, which also appears to be the case for
primary walls of all dicotyledons.
Pectin in higher plants is comprised mostly of polyuronic
acid (unbranched chains of /3 1-4 linked galacturonic acid
residues). The major identified configuration consists of a
zig-zag arrangement of a variable number of galacturonic acid
residues separated from one another by a rhamnogalactose
trisaccharide. The trisaccharide appears to be the site of
a glycosidic link to other pectin components, specifically the
arabinogalactans. These are thought to be the 'connecting
bridge' between the pectins and the hemicelluloses. In


7
Albersheim's model (1978), the cellulose microfibrils are
coated by the hemicellulose xyloglucans, attached by hydrogen
bonding. The xyloglucan is glycosidically linked to a pectic
arabinogalactan. Further, this arabinogalactan is in turn
glycosidically linked to the pectic rhamnogalacturonan.
Proteins can be either structural or enzymic in nature.
Protein isolates prepared from isolated cell wall have been
shown to contain a variety of distinct enzymes including
exopolygalacturonase and /?-galactosidase in apple (Pvrus malus
L.) fruit (Bartley, 1974, 1977, 1978), a- and 0-galactosidas-
es and glucosidases, a-mannosidase, a-arabinosidase, ¡3-
xylosidase, pectin esterase (sic), and polygalacturonase in
pear (P. communis L.) fruit (Ahmed and Labavitch, 1980;
Bartley et al., 1982); /?-galactosidase, /3-l,3- and exo-/3-l,4-
glucanase, Cx-cellulase, pectinmethylesterase (PME), and
endopolygalacturonase (endoPG) in tomato fruit (Buescher and
Tigchelaar, 1975; Gross and Wallner, 1979; Huber and Lee,
1988; Kivilaan et al., 1961; Poovaiah and Nukaya, 1979;
Sobotka and Stelzig, 1974; Strand et al., 1976; Themmen et
al., 1982; Tucker et al., 1980; Wallner and Bloom, 1977;
Wallner and Walker, 1975).
Lee et al. (1967) proposed that cell walls of corn (Zea
mays L.). coleoptiles contained hydrolytic as well as syn
thetic enzymes. A decrease in weight of isolated wall in
buffer was thought to be due to autolysis of a non-cellulosic
glucan component with concurrent release of arabinose and
xylose. Huber and Nevins (1981) determined that this


8
autolysis was the result of glucanases hydrolyzing hemicell-
ulose glucans. Other cell-wall associated proteins include
the polygalacturonases (PG), which play an important role in
cell-wall degradation and associated fruit softening in
numerous fruit types (Crookes and Grierson, 1983; Hobson,
1964; Wallner and Walker, 1975).
Fruit Softening
The major structural component of the cell is the cell
wall (Bartnicki-Garcia, 1984) It is obvious that the
modification of this structure should play a major role in
changes or differences in fruit texture. Huber (1983b)
reviewed the role of cell-wall hydrolases in fruit softening.
Cellulases (individual enzymes or complexes) have been
identified in avocado (Hatfield and Nevins, 1986; Pesis et
al., 1978), peach (Hinton and Pressey, 1974), pear (Yamaki et
al., 1979), strawberry (Fragaria ananassa Duch.) (Barnes and
Patchett, 1976), and tomato fruit (Buescher and Tigchelaar,
1975; Hall, 1963, 1964; Huber, 1985; Pharr and Dickinson,
1973; Poovaiah and Nukaya, 1979; Sobotka and Stelzig, 1974;
Sobotka and Watada, 1971; Wallner and Walker, 1975). Although
reports of cellulase complexes apparently capable of complete
ly degrading native cellulose have been characterized in
tomato fruit (Sobotka and Stelzig, 1974), there is no evidence
for extensive degradation of this polymer in vivo in any
fruit. Cellulose degradation has been shown to be minimal in
apple (Nelmes and Preston, 1968), avocado (Hatfield and


9
Nevins, 1986), pear (Jermyn and Isherwood, 1956), and peaches
(Sterling, 1961) However, Cx-cellulase does possibly play a
role in the terminal stages of the degradation of the locular
gel in tomato fruit (Huber, 1985).
Few studies have been undertaken to identify changes
occurring in the alkali-soluble (hemicellulose) components of
the cell wall during ripening and softening of fruit. Huber
(1983a) reported an increase in smaller-molecular-weight and
a decrease in larger-molecular-weight hemicelluloses in ripe
tomato fruit as compared with mature-green fruit. This shift
in polymer molecular-weight was coincident with pectin
degradation, but was shown to be an independent phenomenon
(Huber, 1983a) The basis for this modification of the
hemicellulose fraction is unknown. As mentioned by Huber
(1983a), there could be a precursor-product relationship
and/or separate synthesis of modified, smaller polymers as
ripening progresses. Similar results were reported in a study
on strawberry fruit (Huber, 1984). On a mole-% basis, sugar
composition of the hemicelluloses showed little change during
ripening from small-green through red. However, as with
tomato fruit, there was a loss of large-molecular-weight
polymers and a gain in smaller-molecular-weight polymers over
the same developmental period.


10
Models of Softening in Fruits
Pear Fruit
Jermyn and Isherwood (1956) reported on changes in the
cell wall of pear during ripening. There is a loss of
arabinan, galactan, and total hemicellulose. Total polysac
charides fall, with a slight decrease in cell wall on a per
fruit basis. Yamaki et al. (1979) reported similar data for
the Japanese pear. They found a decrease in arabinose and
galactose content and concluded that these sugars were lost
from the hemicellulose fraction, possibly due to increased
arabanase and /3-galactosidase activity (Yamaki and Matsuda,
1979). Cellulose decreased, while total pectin and water
soluble pectin (WSP) increased. It was concluded that the WSP
increase was due to the activity of PG (Yamaki and Matsuda,
1977), and the decrease in cellulose due to the activity of
exocellulase (Yamaki and Kakiuchi, 1979) However,
Labavitch's group (Ahmed and Labavitch, 1980b) failed to
identify cellulase and arabanase activities in 'Bartlett'
pear.
Ahmed and Labavitch confirmed the substantial loss of
arabinose and soluble pectin from ripening pear (1980a).
However, the most rapid loss of these polysaccharides does not
occur until after substantial fruit softening. They suggested
that the initial loss of firmness was due to the metabolism
of cell-wall components which did not affect polyuronide
solubility. Treatment of unripe tissue with a purified PG


11
preparation solubilized a pectic arabinan similar to that
which was solubilized in vivo. The large MW of the arabinan
released indicated that extensive degradation was not neces
sary to release the polymer. Based on these studies, they
concluded that PG was the only enzyme participating in cell-
wall metabolism in ripening pear fruit. Bartley et al. (1982)
agreed that PG was the major determinant in pear softening and
furthermore reported a coordinated degradation of pectin
polymers with the appearance of endoPG.
Apple Fruit
Although also a pome, apple-fruit cortical softening
appears to be distinctly different than that which occurs in
pear. Knee (1973) demonstrated that apple fruit exhibit an
increase in soluble polyuronide, a decrease in hemicellulose
correlated with loss of wall glucan, and no change in cel
lulose during postharvest ripening. Bartley (1974) described
a 0-galactosidase in ripening apples, the activity of which
preceded the increase in soluble polyuronides. However,
whether this enzyme contributed to polyuronide solubilization
was unclear. Later (1977), Bartley proposed that in the
absence of changes in other components of the cell wall
(Bartley, 1976) hydrolysis of the galactan (probably through
the activity of /3-galactosidase) in cell wall is responsible
for the loss of firmness in apple. Lidster et al. (1985) were
able to retain firmness in 'McIntosh' and 'Gravenstein' apples
in store at 20C after vacuum infusion of a partially purified


12
/3-galactosidase inhibitor. These results indicate that /3-
galactosidase can contribute markedly to loss of firmness in
apple fruit.
Knee (1974) described a two-stage breakdown of the cell
wall during apple ripening, similar to that suggested by
Bartley (1974). In the first stage, changes in firmness are
minimal while there is a decrease in wall galactan. The
galactose decrease was speculated to originate from a polygal-
acturonan. In the second stage, there is a marked accelera
tion in softening and an increase in soluble polyuronide,
thought to be bound to a hydroxyproline protein in the middle
lamella. Endopolygalacturonase activity was not evident in
apple, an observation supported by the fact that the MW of the
liberated polyuronide was very high. However, Bartley (1977)
identified an exoPG in apple fruit. Still, there is no
plausable explanation of how this enzyme contributes to wall
degradation.
Knee (1978) studied properties of polygalacturonate and
cell cohesion in apple fruit and suggested that cohesion
depended on the degree of esterification of the polygalac
turonate. He suggested that free carboxyl groups maintained
cell cohesion through cooperative binding of calcium ions.
Strawberry Fruit
A different mechanism was proposed for softening in
strawberry fruit (Barnes and Patchett, 1976). In these fruit,
PE increases during fruit development to the early red stage


13
and decreases thereafter, while PG and polymethylgalacturonase
activities are not observed at any point in development. C-
type cellulytic activity increases as fruit senesce. The
authors suggested that loss of firmness in strawberry was not
due to degradation of pectic material, but (in agreement with
Neal, 1965) rather to an increase in pectic methylation,
thereby eliminating calcium cross-links, leading to a scission
of the middle lamella.
Knee et al. (1977) determined that cell expansion in
strawberry receptacles continued through the period that the
fruit turned red and softened. There is no increase in total
polysaccharide during ripening, whereas the middle lamella
swells and 70% of the polyuronide cell wall becomes soluble,
with a concurrent loss of arabinose and galactose. The
authors concluded that polysaccharide synthesis failed to keep
pace with expansion and wall maintenance requirements. This
causes the wall to weaken and allows cellular contents to
hydrate the middle lamella. Huber (1984) agreed that total
polysaccharides may remain constant (or decrease on a fruit
FW basis) but provided evidence which showed that polyuronide
increased on a per fruit basis during ripening. Based on
neutral sugar analysis of polyuronides throughout ripening,
it appears that the increase in polyuronide solubility is due
to the synthesis of new, more-soluble polymers. In support
of previous work, no evidence of PG-mediated cell-wall
degradation was found.


14
Tomato Fruit
Tomato fruit softening appears to be more related to the
system found in pear than in apple or strawberry fruit.
Hobson (1964, 1965) first suggested that the firmness of
tomato fruit was related predominately to the activity of PG.
Softer genotypes show increased PG activity (Hobson, 1964).
In addition, pericarp from areas exhibiting the physiological
disorder 'blotchy ripening' (Picha, 1987) fail to soften as
ripening procedes. Hobson (1964) reported that this disor
dered tissue is lower in PG activity than the surrounding
'normal' areas. Hobson (1965) confirmed his earlier work by
measuring firmness of tomato fruit at various stages of
ripeness and correlating the measurements with PG activity.
There was a high positive correlation (r = 0.952) between
softness (whole fruit compression) and PG activity. This
study was substantiated by Buescher and Tigchelaar (1975) and
Buescher et al. (1976) who reported similar results working
with 'Rutgers' and tomato ripening mutants.
Wallner and Walker (1975) reported on the presence of
/3-glycosidases in ripening tomato fruit. As it was under
stood at this time that arabinans and galactans were major
structural components of primary plant cell walls (Talmadge
et al., 1973), Wallner and Walker (1975) speculated that these
glycosidases possibly function to hydrolyze these polymers and
aid PG in degrading the cell wall (Wallner and Walker, 1975) .
Wallner and Bloom (1977) described degradation of tomato cell
walls in vitro and in situ. They reported less wall galactose


15
loss in situ, leading to their suggestion that PG is only
partially responsible for the wall hydrolysis which accom
panies fruit softening. Sawamura et al. (1978) agreed with
these findings. They exposed tomato fruit to exogenous
ethylene. Polygalacturonase activity increased, but only
after an initial increase in WSP, leading to further specula
tion that other enzymes were involved in softening. However,
these studies did not follow changes in firmness. Gross and
Wallner (1979) confirmed that galactose as well as arabinose
are lost from the cell wall during ripening. This loss
appeared to be separate from polyuronide solubilization.
However, they suggested that the effect of the decline in
these sugars on fruit firmness was minimal. Themmen et al.
(1982) exposed cell wall from normal and mutant tomato fruit
to protein extracts and purified PG from ripe fruit and agreed
that PG was the major enzyme responsible for cell-wall
degradation. However, they did point out that other enzymes
may contribute to softening in vivo.
Huber (1983a) followed the changes in molecular weights
of polyuronides during ripening in tomato fruit and confirmed
that extensive degradation occurred as evidenced by the
appearance of lower-molecular-weight polymers. This degrada
tion corresponded with the trend of PG activity. Ultrastruc
ture studies of ripening tomato fruit (Crookes and Grierson,
1983) showed a dissolution of the middle lamella and eventual
disruption of the primary cell wall. These changes were
correlated with the appearance of PG. Application of PG


16
isolates to unripe fruit tissue brought about identical
ultrastructural changes. Other workers have continued to
confirm that PG activity and loss of polyuronides from the
cell wall during ripening are the primary agents responsible
for softening in tomato fruit (Brady et al., 1985; Gross,
1984; Huber and Lee, 1986, 1988; Pressey, 1986; Rushing and
Huber, 1984); however, there have been some suggestions that
this relationship is only a general one (Brady et al., 1983,
1985) .
Most researchers agree that the major cell wall event
occurring during tomato softening is solubilization of
polyuronide (Brady et al., 1985; Buescher and Tigchelaar,
1975; Buescher et al., 1986; Crookes and Grierson, 1983; Gross
and Wallner, 1979; Hobson, 1964, 1965; Huber, 1983a; Huber and
Lee, 1986, 1988; Pressey and Avants, 1971; Sawamura et al.,
1978; Themmen et al., 1982; Wallner and Bloom, 1977; Wallner
and Walker, 1975). It is recognized that there is a general
inverse relationship between PG activity and fruit firmness
within cultivars as fruit ripen (and soften) and between
cultivars varying in intrinsic firmness. Of those studied,
firmer tomato cultivars had less PG (Brady et al., 1983;
Buescher and Tigchelaar, 1975; Buescher et al., 1976; Hobson,
1964; Malis-Arad et al., 1983; Poovaiah and Nukaya, 1979;
Tucker et al., 1980). In addition, Rushing and Huber (1984)
developed an in vitro system for indirectly quantifying the
levels of PG activity by measuring the autolytic activity of


17
enzymically active cell wall. Wall from firmer fruit release
less soluble polyuronide than does wall from softer fruit.
One might anticipate a high relationship between PG
levels and firmness rather than the general trends previously
reported (Brady et al., 1983, 1985). However, these inves
tigators determined firmness based on compression of whole
fruit. Hall (1987) has emphasized the importance of measuring
tissue (pericarp) firmness rather than whole-fruit firmness
when investigating the relationship between enzyme levels and
changes in texture. When measuring whole fruit firmness, the
relationship between enzymes and texture may be confounded by
the contribution to fruit texture of parameters which have no
relationship to the enzyme in question. Such parameters could
include the thickness of the pericarp and radial fruit-walls,
the amount and composition of the locular material, the
internal morphology of the fruit, the presence of columnella,
the point of measurement of compression (whether taken over
a locule or at a radial wall intersection), and the overall
water status of the fruit (Shafshak and Winsor, 1964). By
measuring only tissue firmness, the effects of most of these
confounding influences can be either elimnated or greatly
reduced.
Calcium Content of Pericarp and Fruit Firmness
Calcium is a divalent ion. Due to its double positive
charge, it readily associates with pectin, forming calcium
pectate. The stereochemistry of the pectin chain may allow
for intra- or inter-polymeric binding (Rendleman, 1978). It


18
has been proposed that this cooperative binding may strengthen
the chains by holding hydrolyzed segments together through the
ionic bonds, or alternately making the polymers less vul
nerable to attack due to stereochemic interference. The
degree of binding would depend on the amount of calcium
available and the degree to which the polymers were esteri-
fied. In this light, PME may play a role in fruit softening
by regulating the number of binding sites available for
calcium.
If calcium does regulate PG activity, it would appear that
the concentration or distribution of this cation would change
during fruit softening, or be correlated with firmness among
cultivars of varying firmness. Investigations in this area
have yielded conflicting data. Poovaiah (1979), and Suwwan
and Poovaiah (1978) reported a decrease in total calcium as
fruit ripened and softened. Fruit were harvested at selected
stages of development and assayed. This study was supported
by Rigney and Wills (1981) who showed a shift in calcium
partitioning during ripening of on-vine-developed tomato
fruit. Total calcium increased as fruit developed, to a
maximum level at 80% of full development (incipient color)
then decreased as fruit ripened. Concurrently, bound calcium
(that calcium thought to be associated with pectin) decreased
and soluble calcium increased as a percent of total calcium.
However, Brady et al. (1985), using similar vine-attached
fruit, reported no change in calcium during ripening, whether
measured as total or acid-extractable. Ferguson et al. (1980)


19
pointed out that calcium extraction techniques are often
unreliable in determining calcium partitioning. Any method
which uses tissue homogenization and water extractions will
be influenced by other ionic components of the cytosolic
fluids. For instance, citric acid, which is often present at
high levels in fruits, readily complexes with calcium.
However, in an artificial system, Ferguson et al. (1980) were
able to recover over 90% of the calcium in calcium pectate
using an 80% acetic acid extraction. In order to overcome the
problems inherent in any extraction procedures, Burns and
Pressey (1987) measured calcium in the cell wall-middle
lamella (CW-ML) of tomato fruit using energy-dispersive X-ray
microanalysis. They reported that as fruit ripen and soften
on the vine, calcium in the CW-ML increases. The hypothesis
offered to explain this phenomenon was that calcium served to
hold the hydrolyzed polymers together, allowing for some
pericarp softening, but preventing cell-wall degradation until
the later stages of ripening and senescence.
Calcium infiltration has proved effective at delaying
flesh softening in apple fruit (Glenn and Poovaiah, 1987? Sams
and Conway, 1984). In addition, a deficiency in calcium in
tomato fruit has been linked to 'blossom end rot' (Geraldson,
1957; Gerard and Hipp, 1968; Spurr, 1959). In this disorder,
tissues in the area of the stylar end soften, become water
soaked, and are subject to invasions by pathogens.


20
Polygalacturonase Activity and Ripening in Tomato Fruit
Polygalacturonase as a Ripening Initiator
The most striking characteristic of tomatoes is that they
undergo a change in color (internal and external) during
ripening (Rick, 1978) As an aid in shipping and handling,
the United States Department of Agriculture has developed a
visual aid based on the external color of tomatoes which is
used in describing the 'ripeness' of the fruit (U. S. Dept.
Agri., 1975). This aid has become useful to researchers as
a reference for stages of development during ripening.
Typical commercial cultivars of tomatoes move through the
color classifications (from mature-green to red) in around 12
days (Rick, 1978). Another feature of developing tomato fruit
is that they soften dramatically as they ripen (Garrett et
al., 1960; Rushing and Huber, 1983; Shafshak and Winsor,
1964). As mentioned previously, PG activity is implicated as
the major cause of softening in tomato fruit.
Fruit with the physiological disorder known as 'blotchy
ripening' ( Picha, 1987) have areas which do not synthesize
lycopene and which fail to soften. Hobson (1964) first
demonstrated that these areas were lacking in PG. This was
the first recorded instance of PG being associated with
abnormal ripening. Buescher and Tigchelaar (1975) did a
comparative investigation of the tomato ripening mutant rin
and the normal cultivar Rutgers. Rin does not soften appreci
ably, fails to develop lycopene (Robinson and Tomes, 1968) and


21
lacks the normal climacteric of tomato fruit (Herner and Sink,
1973). Buescher and Tigchelaar (1975) concluded that the
failure of rin to soften was due to the lack of PG activity.
No inhibitors of PG activity were detected. The authors,
along with Hobson (1964) speculated that the lack of PG
activity could be a primary or secondary agent responsible for
the failure of the fruit to ripen.
Attempts have been made to overcome the ripening inhibi
tion expressed in rin and other mutants. Since ethylene
levels are low in these mutants, application of exogenous
ethylene (or its analogs) has been used in an effort to induce
ripening. Propylene caused an initial increase in respiration
in rin, but endogenous ethylene production was not initiated
(McGlasson et al., 1975). However, development of a slight
yellow color was somewhat advanced over control rin. Mizrahi
et al. (1975) demonstrated that attached rin fruit develop
lycopene if exposed to 130 ppm ethylene, although only at
levels a third of that in normal fruit. This response is the
only report of ripening related changes in rin due to exo
genously applied ethylene.
Tigchelaar et al. (1978) reviewed genetic regulation of
ripening in tomato fruit, based on studies of the ripening
mutants. They reported that exogenous ethylene does not
stimulate PG activity in rin. Based on this and evidence that
rin exhibits normal levels of preclimacteric ethylene produc
tion, yet fails to ripen when exposed to higher concentrations
of ethylene, the authors proposed that the 'ripening event'


22
which was suppressed in rin and nor mutants was PG activity.
In their model, PG hydrolyzes cell-wall pectin, resulting in
the release of cell wall-bound enzymes. They speculated that
these enzymes could contribute to various ripening phenomena
such as ethylene synthesis, carotenoid synthesis, and flavor
and volatile development. Studies by Hobson et al. (1983)
supported this hypothesis by demonstrating that tomato PG
released protein from tomato cell wall preparations. In
addition, an earlier investigation by Strand et al. (1976)
showed that the action of fungal PG released cell wall-bound
proteins.
Suwwan and Poovaiah (1978) determined that bound calcium
levels in attached rin were high and continued to increase
with age compared to normal attached tomato fruit, which
showed a drop in bound calcium. Poovaiah (1979) agreed that
low PG activity in rin was responsible for the failure of the
fruit to ripen; furthermore, this low activity was due to the
influence of calcium functioning in maintaining cell-wall
structure and membrane integrity. A subsequent study
(Poovaiah and Nakaya, 1979) determined that PG (not cellulase)
was inhibited in rin. Autocatalytic ethylene production and
the respiratory climacteric do not occur in the absence of PG
and in normal fruit PG is detected prior to the respiratory
climacteric. Based on these results, the authors suggested
that the failure of rin to ripen was a consequence of the lack
of active PG.


23
Other investigators did not support the idea that PG
initiated ripening. Sawamura et al. (1978) reported that the
rise in ethylene production preceded an increase in water-
soluble pectins and PG activity in tomato fruit. They
concluded that ethylene initiated ripening in tomato and that
PG was a secondary ripening response. Salveit and McFeeters
(1980) demonstrated that PG activity increased in immature
cucumber fruit in response to exogenously applied ethylene.
Polygalacturonase activity was not detectable until after the
initial burst in ethylene production.
Hobson (1980) sought to introduce the non-ripening mutant
genes, through backcrossing, into normal tomato lines. Except
for the heterozygote (rin rin) all progeny had lower PG
activity, prolonged shelf life, and were firmer. The mutant
fruit also showed reduced levels of phosphofructokinase and
NADP+-malic enzyme, offering an explanation for the weak
climacteric in the mutants.
Grierson and Tucker (1983) followed ethylene production
and PG synthesis in normal tomato fruit. Enhanced ethylene
production occurred prior to the appearance of PG. Further
more, fruit held in containers with the ethylene absorber
mercuric perchlorate remained green and failed to produce PG
during the same time period as control fruit. The authors
suggested that ethylene was in part required for the synthesis
of PG. Maunders et al. (1987) supported the role of PG as a
secondary ripening agent by demonstrating that ethylene
stimulates the accumulation of mRNAs in tomato, one of which


24
yielded in vitro translation products similar in molecular
weight to products obtained from known PG mRNA.
Brecht (1987) demonstrated that the initial increase in
System II (McMurchie et al., 1972) ethylene synthesis during
the early stages of ripening occurs in placental tissues.
This tissue has low, if any, detectable PG (Wallner and
Walker, 1975), yet is temporally advanced over pericarp tissue
in terms of cell-wall changes (Huber and Lee, 1986) and
lycopene accumulation.
Polygalacturonase and System II Ethylene
Recent studies have suggested that PG may contribute to
the production of System II ethylene in pericarp tissue.
VanderMolen et al. (1983) reported on the induction of
vascular-plugging gels in castor bean and banana by extracts
of Fusarium cultures. Polygalacturonase was isolated from the
pathogen-free extract. Data show that PG alone is able to
stimulate the production of the gels, possibly through the
solubilization of wall components. However, PG was shown to
stimulate ethylene production in castor bean leaves. Applica
tion of exogenous ethylene to leaves produced the occluding
gels. These data indicate that PG of fungal origin elicits
ethylene production which, in turn, causes production of the
gels. Roby et al. (1985, 1986) treated melon leaves with an
elicitor isolated from melon leaf cell walls. This envoked
the synthesis of ethylene and cell-wall hydroxyproline-rich
glycoprotein.


25
Baldwin and Pressey (1988) vacuum-infiltrated PG and PME
into unripe normal and mutant tomato fruit and found that PG
had only a transitory effect on ethylene production in normal
fruit, but that in the mutant lines ethylene was maintained
at high levels. Pectinmethylesterase administered alone has
no effect. They suggested that possibly low and as yet
undetectable levels of PG prior to the onset of System II
ethylene in green fruit promote ethylene production to levels
that reach a physiologically active threshhold, which in turn
would cause an increase in PG levels. These higher levels
would induce even more ethylene, which would be System II
ethylene. Kim et al. (1987) reported that infiltration of
galactose into pre-ripe tomato fruit, at levels present in
ripening fruit due to degradation of wall galactan polymers,
enhanced ethylene biosynthesis. Thus, it might be that a
variety of products of wall origin may contribute to enhanced
ethylene biosynthesis.
Brecht and Huber (1988) obtained pectin fragments from
autolytically active cell wall and vacuum infiltrated them
into pre-ripe tomato fruit. Ethylene biosynthesis was
enhanced, possibly due to an elicitor effect of the infil
trated material. The response was specific in that only
certain size classes of fragments were effective. The authors
advanced the hypothesis that PG promotes System II ethylene
production.


26
Fruit Mealiness
Mealiness is a general term used to describe a dry,
grainy, coarse texture in fruits. It is an important cellular
feature affecting a number of horticultural commodities. In
apples, fruit that become soft concurrently exhibit mealy
symptoms and the terms are often used interchangeably (Fisher,
1942? Haller and Harding, 1937). Peaches which have been
stored continuously at chilling temperatures become mealy upon
rewarming (Ben-Arie et al., 1970? Buescher and Furmanski,
1978). There is currently no literature available on meali
ness in tomato fruit. However, tomato fruit which are firm
are thought to be mealy, although there are cultivars which
appear to be soft and yet have a grainy, coarse internal
appearance (John W. Scott, Gulf Coast Research and Education
Center, Bradenton, FI, personal communication).
Mealiness Determinations
For some time it has been recognized that mealiness can
reduce consumer acceptance of fruits (Haller and Harding,
1937? Harding and Haller, 1932? Liu and King, 1978). There
exist many pre- and postharvest programs aimed at detecting,
evaluating, and reducing this internal textural parameter?
however, there has to date been no objective means available
to directly quantify this cellular trait. Direct determina
tion is often made by subjective sensory evalaution (Clark and
Rao, 1977? Finney, 1971? Finney et al., 1978? Liu and King,
1978? Looney, 1975a, 1975b). Although sensory panels are


27
discriminating and able to distinguish mealy tissue, they are
limited in accuracy and precision, and do not provide a
reproducible numerical distinction.
Based on the observation that soft apple tissue is mealy
(Fisher, 1942; Haller and Harding, 1937), most tests for
mealiness determination in apple have incorporated resistance
to puncture (Liu and King, 1978; Mason et al. 1975; Riley and
Kolattukudy, 1976). There are reports of using sonic reson
ance, which produces a "stiffness coefficient" (Finney et al.,
1978). In addition to sensory evaluations and puncture
(pressure) tests, bulk compression (Clark and Rao, 1977) has
been used to determine 'wooliness' (mealiness) in peach
tissue. Since mealy fruit appear dry and cakey, Buescher and
Furmanski (1978) have used % expressible juice to guantify
mealiness in peach fruit.
Physiological Aspects of Mealiness
Meheriuk and Porritt (1971) reported on the effect of
high (2%) C02 on mealiness of apples in storage. Apples under
C02 were firmer than those in air. Looney (1975b) confirmed
these findings. A sensory panel found that 'McIntosh' apples
which had been stored under C02 were crisper and firmer than
those which had been stored in air. Meheriuk et al. found
f
that either prestorage (1977b) or during-storage (1977a) C02
treatments of apples resulted in less softening in storage as
compared to control fruit; however, the high concentration of
C02 required for the prestorage treatment damaged 'Golden


28
Delicious' apples beyond consumer acceptance (Meheriuk et al.,
1977b). Unrath (1972) demonstrated that preharvest applica
tion of 2-chloroethylphosphonic acid (ethephon) increased
softness of apple flesh at harvest. This was later confirmed
by Looney (1975a). Miller (1975) looked at the effect of
ethephon on apples in storage. Treated 'McIntosh' apples were
softer after 1 week in storage, but effects as compared to
controls were minimal after 3 months. Hammett (1976) reported
similar but improved findings with ethephon treatment of
'Starkrimson Delicious' apples. These results all indicate
that apples become mealy in storage due to the action of
ethylene. Indeed, Lougheed et al. (1973) were able to
demonstrate that ethylene removal from storage atmospheres
allowed apples to remain firm.
Bramlage et al. (1974) demonstrated that calcium has an
effect on firmness. Apples which had received up to 700 ppm
Ca were firmer than control fruit after 7 months storage. A
postharvest dip of calcium chloride solution was shown to be
effective in reducing softness in 'McIntosh' apples after 4
months (Mason et al., 1975). The inclusion of a thickener
(for retention of the dip solution) gave greater results.
This same treatment was repeated by Mason (1976) with the same
results. Riley and Kolattukudy (1976) confirmed the effects
of calcium on apple firmness by demonstrating that prestorage
dips of any of several calcium-containing solutions effective
ly reduced poststorage softness in 'Golden Delicious' apples.
Glenn and Poovaiah (1987) have recently determined the mode


29
of action of calcium treatments on delaying softening in apple
fruit. Dipping apples in calcium solutions prior to storage
prevents or delays the mealy condition by preventing the
dissolution of the CW-ML. Fruit which received calcium
treatments were judged to be less mealy and had less breakdown
and dissolution of the CW-ML as evidenced by scanning and
transmission electron microscopy. There were fewer cell-to-
cell contacts and less middle lamellar material in non-treated
fruit.
Peach fruit are harvested prior to the initiation of
ripening. They are stored in this unripe state at low
temperatures for periods ranging from several weeks up to
several months (Buescher and Furmanski, 1978; Harding and
Haller, 193 2) At the end of the storage period, they are
returned to ambient temperature and are allowed to ripen.
However, unripe peaches are chilling sensitive (Harding and
Haller, 1932, 1934). A symptom of this chilling injury is
mealiness of the pericarp (wooliness), which becomes evident
upon rewarming (Haller and Harding, 1939). In order to
overcome expression of this symptom, the fruit are intermit
tently warmed during the second week of storage (Anderson and
Penney, 1975; Ben-Arie et al., 1970). Buescher and Furmanski
(1978) determined that reduced levels of pectinesterase and
PG were associated with reduced juiciness and mealiness in
peaches. The authors proposed that intermittent warming
prevents the development of mealiness by protecting the


30
ability of the tissue to produce adequate levels of these
enzymes during post-storage ripening.
While mealiness as a textural trait in tomato warrants
attention in breeding programs (John W. Scott, Gulf Coast
Research and Education Center, Bradenton, FI, personal
communication), there is no documented evidence of the
relationship between mealiness and development (on the plant
or during storage) or its underlying biochemical causes.
However, it is often thought that firmer fruit tend to be
mealier than softer fruit.


31
Rhamnogalacturonan
Xyloglucan
Cellulose
Arabinogalactan
Figure 1-1. Model of primary cell wall components of suspen
sion-cultured sycamore cells as proposed by Albersheim. The
model is not intended to be drawn to scale, but only repre
sents relationships. After Albersheim, 1978.


CHAPTER 2
PREAMBLE
Discussion
Changes in texture are a function of PG activity and are
related to the ripening process. In this research I want to
address the very broad area of the relationship between
texture modifications, PG activity, and ripening and to some
extent how calcium influences their interactions. Several
questions come to mind. What is the character of texture in
tomatoes? Is mealiness related to firmness? If not, what is
the biochemical basis of mealiness? What role does PG play
in mealiness and in ripening in general? Do softer fruit
ripen more quickly than firmer fruit? Does PG have more of
a role in ripening than just texture modification? And, how
does calcium influence and/or regulate these processes?
Polygalacturonase Activity and Texture
Polygalacturonase activity is an accepted major component
of tomato fruit softening; however, correlations between this
enzyme and changes in firmness have ranged from quite high to
only moderate. All previous investigators have extracted PG
from specific tissues, yet have measured firmness by compres
sion of whole fruit. In this methodology, effects of the
enzyme are dilluted and confounded. Furthermore, most
32


33
researchers in this area report firmness as absolute compres
sion. This introduces another error. Cultivars, especially
non-improved lines, vary in size. A compression of 10 mm on
a 25 mm diameter fruit is not comparable to the same compres
sion on a 150 mm diameter fruit. Using % compression would
minimize errors of this type and allow for more precise
comparisons between cultivars.
In apple and watermelon, mealy fruit are soft. In the
previous discussion, it was seen that treatments which
diminish the effects of ethylene delay or reduce mealiness in
apple. However, in peach, mealy fruit are excessively dry,
but not soft, and there has been no evidence linking ethylene
activity to mealiness in peach fruit. On the contrary,
mealiness is due to a failure of PG to adequately degrade
pectin after storage upon rewarming. It is completely unknown
at this time which system (if either) most closely represents
the one existing in tomato fruit.
Because a knife blade tends to pass between cell walls
of mealy fruit and through the cell walls of non-mealy tissue,
the cell wall in mealy tissue is probably stronger than that
in normal tissue, and/or the middle lamella in mealy tissue
is weaker than that in non-mealy tissue. As an alternative
hypothesis to lower PG activity causing mealiness, possibly
an increase in the hemicellulose component coupled with a
higher PG activity could result in mealy fruit.


34
Polygalacturonase Activity and Ripening
In comparing the amounts of C02 evolved by fruits of
different species, it appears that those fruits which have the
highest rates of respiration during ripening require less time
to ripen. In addition, bananas, which evolve copius amounts
of ethylene and C02, have high levels of PG. Avocados also
ripen quickly and have high levels of PG, yet less than
bananas. Tomatoes are intermediate in rate (days to ripen)
and intensity (amount of ethylene and C02 evolved at during
ripening), and have less PG than avocado. Recent preliminary
observations have indicated that those tomato cultivars which
evolve the most ethylene, take less time to ripen (once
initiated) yet application of excessive amounts of exogenous
ethylene does not increase this rate. Lastly, apples take
much longer to ripen, and lack endoPG, having only exoPG. It
appears intuitive that the level of PG in a ripening fruit
directly affects the rate and intensity of ripening in that
fruit. While there are cases of fruits which evolve moderate
amounts of ethylene and C02 yet produce no PG (muskmelon),
there are no known or reported instances of fruits which have
active PG and do not evolve moderate amounts of ethylene.
Calcium
Bound calcium has been reported to decrease, increase,
or remain the same during tomato fruit ripening and softening.
There have been two major flaws with these investigations.
First, there are recognized problems with fractionating


35
calcium. Second, these investigators harvested fruit at
selected stages of development and attempted to correlate the
amount of bound and free calcium in the pericarp to the
fruit's point of development. Any changes in calcium could
have been influenced by the dynamic flux of calcium into or
out of the fruit while attached to the plant. Investigations
which attempt to look at changes in the state and amount of
calcium in relationship to changes in development during
ripening need to analyze detached fruit harvested un-ripe.
Objectives of this Study
Three major related hypotheses are proposed.
1. Texture is inherently linked to ripening in tomato fruit,
but in addition, its primary modifying agent (PG activ
ity) is directly related to ripening regulation through
the biological activity of cell wall-released pectin
fragments.
2. Mealiness in tomato fruit results from a failure of the
cell wall to rupture under mechanical force. The binding
forces between cells are greater than that within
individual cell walls.
3. Mealiness is related to solubility of hemicellulose in
conjunction with differences in pectin solubilization.
In order to address these theories, this work will
attempt to:
1. Characterize the texture of selected tomato cultivars
from "immature" through "red" ripeness classifications;


36
2. Evaluate alternative methods of determining firmness in
tomato fruit with a view to contrasting whole fruit and
tissue firmness;
3. Develop an objective method of determining mealiness?
4. Identify those anatomical characteristics that define
mealiness;
5. Determine the relationship between firmness and meali
ness ;
6. Follow changes in respiration and autocatalytic ethyl
ene production through the climacteric and relate these
parameters to PG activity and texture;
7. Characterize changes associated with the cell wall
including changes in hemicellulose and pectin polymers;
8. Relate cell-wall autolysis to texture;
9. Determine the role of calcium in modifying texture.


CHAPTER 3
FIRMNESS AND MEALINESS: ATTRIBUTES OF
TEXTURE IN TOMATO FRUIT AND THEIR MEASUREMENT
Introduction
The Incidence of Mealiness and Firmness
Texture is an important quality parameter in many
horticultural commodities. In fruit, texture can be sub
divided into two basic components: firmness and mealiness.
Apple fruit. In apple fruit the terms are often used
interchangeably, since soft apple flesh is typically mealy
(Fisher, 1942; Liu and King, 1978). Data from subjective
evaluations which have determined fruit flesh to be mealy have
been highly correlated with objective tests for firmness as
determined using the Magness-Taylor and Effe-gi devices
(Finney, 1971; Finney et al., 1978; Liu and King, 1978). In
one investigation where the author did attempt to separately
analyze mealiness and firmness (Finney, 1971), mealiness was
found to be highly correlated (r = 0.93 to 0.96) with soft
ness, as determined by sensory evaluation. Scanning electron
micrographs of the cut surface of apple fruit show that mealy
and soft fruit have less middle lamellar material between
cells, resulting in fewer cell to cell contact points (Glenn
and Poovaiah, 1987).
37


38
Peach fruit. Mealiness and firmness appear to be
independent aspects of texture in peach fruit. Clark and Rao
(1977) demonstrated that sensory panelists are able to
discriminate between maturity stages of peaches based upon
"elasticity", "hardness", and "wetness" (prob > F = 0.0001 in
each case), but not by "graniness" (prob > F = 0.8699).
Wooliness in peach fruit is described as a "dry and mealy"
condition (Buescher and Furmanski, 1978). It is a chilling
injury symptom which becomes evident upon rewarming. Harding
and Haller (1932) demonstrated that peaches stored at high
temperatures (25 to 30C) soften and are juicy. Those that are
stored at lower temperatures (2 to 4C) and then allowed to
ripen at 25C are less juicy, display a chilling injury symptom
called "breakdown". We can infer from these reports that in
peaches, mealiness, breakdown, and wooliness are terms
describing the same disorder, mealiness is independent of
firmness, and mealy fruit are dry. The only similarities in
mealiness between apple and peach fruit appears to be that the
texture is dry and coarse.
Tomato fruit. There have been numerous studies involv
ing firmness measurements of tomato. Hamson extensively
reviewed literature on firmness and the underlying causes in
tomato (1952a) and contrasted firmness in several varieties
(1952b). Hall and Augustine (1981) evaluated three tomato
cultivars during extended storage. Rushing and Huber (1983)
reported on efforts by breeders to improve firmness of tomato,
detailing progress in this area. However, mealiness, as a


39
separate texture trait, has received little attention in
breeding programs (John W. Scott, Gulf Coast Research and
Education Center, Bradenton, FI, personal communication). A
preliminary study using sensory evaluation indicated that
mealiness does not appear to be related to firmness in tomato
fruit. The firmest and softest cultivars studied were the
first and second mealiest, respectively. Furthermore, within
each cultivar, it appeared that mealiness does not increase
during ripening and softening of the fruit.
Texture Methodology
Although there exist no objective tests for measuring
mealiness, there are numerous devices and techniques for
determining firmness in fruit. Hamson (1952b) first described
a device which objectively measured firmness in tomato fruit
by simulating squeezing by hand. This device has become known
as the Cornell Pressure Tester (Garrett et al., 1960; Kattan,
1957). Kattan (1957) attempted to improve on this methodology
by constructing a multi-point compression device named the
Firm-o-meter. This device was later commercially manufactured
by Agricultural Specialty Company (Hyattsville, Maryland) and
is known as the Asco Firmness Meter (Garrett et al., 1960).
Correlations between these two devices (Cornell Pressure
Tester and the Firm-o-meter) in one study were r = 0.923 to
0.947 (Kattan, 1957). Other devices based on the single point
compression test have been constructed (Shafshak and Winsor,
1964) and have offered some improvement in ease of use. Gull


40
(1987) has modified the Cornell device to greatly facilitate
its operation. Compression devices have also been constructed
to measure firmness in cherry and prune (Verner, 1930),
strawberry (Bouyoucos and Marshall, 1951), and peach (Clark
and Rao, 1977).
The Magness-Taylor pressure tester (1925) has been widely
adopted in determining the firmness of apples, peaches, and
pears (Bouyoucos and Marshall, 1951). This spring-driven
device measures the amount of force required to drive a
plunger a given distance into a fruit. In addition to the
Magness-Taylor device and its derivatives, which are destruc
tive, sonic resonance has been employed as a nondestructive
test for determining texture in apples (Finney, 1971; Finney
et al., 1978).
Universal testing devices for measuring rheological
properties, such as the Instron Testing Device, measure the
force required to shear, compress, or pull (tension) samples
prepared from a variety of sources (Corey and Schlimme, 1988;
Hall, 1987; Mohsenin and Gohlich, 1962). These methods are
precise and accurate and they lend themselves to testing a
number of horticultural products; however, they are costly,
destructive, and may not correlate well with consumer evalua
tions .
Summary
Mealiness and firmness may or may not be related in
tomato fruit, although a preliminary study indicated they are


41
not. There are examples in other fruits where mealiness is
associated with softening (apple) and where it is not (peach).
Much of the public and many breeders feel excessively firm
tomato fruit tend to be mealy. Any study investigating
texture must first address the relationship between firmness
and mealiness. In order to do so, objective techniques for
determining each component must be evaluated. The purpose of
this study is to characterize the texture of several tomato
cultivars. In order to do this, a variety of devices to
measure firmness will be evaluated and the collected data
correlated with the results from sensory panel evaluations.
Second, mealiness in tomato fruit will be determined by
sensory panel and an attempt be made to develop an objective
test for determining mealiness. Last, the relationship
between firmness and mealiness will be investigated.
Materials and Methods
Subjective and Objective Measurement of Tomato Fruit Firmness
Plant material. Initially, seven tomato cultivars were
grown in the Spring of 1986 at the Gulf Coast Research and
Education Center, University of Florida, Bradenton, FI.
Transplants were set in a completely randomized block design
in the field March of 1986, 6 plants per block, 3 blocks per
cultivar. Harvests were initiated in June.
Fruit (50) from each block were harvested green at a size
characteristic of fully ripe fruit of each cultivar. The
pedicel and calyx were removed to prevent injury during


42
transport. Fruit were shaded and transported in an air-
conditioned vehicle directly to the laboratories at
Gainesville, arriving within 3.5 hours.
Upon arrival, fruit were lightly rinsed in tap water to
remove field debris, dipped in 1% hypochlorite, rerinsed, and
allowed to air-dry. Decayed, abnormal, or damaged fruit were
discarded. Fruit were regraded for uniformity, within
cultivars, of shape and size. Fruit were placed on trays,
stem-end down, in storage rooms at 20C and allowed to ripen
to selected stages of development. These stages were deter
mined visually by comparison of external fruit color to a
United States Dept. Agri. visual aid (U.S. Dept. Agri. Visual
Aid TM-L-1, The John Henry Company, P.0. Box 17099, Lansing,
Michigan). To insure the exclusion of immature fruit, those
fruit that had not begun to ripen (develop incipient color)
within 2 weeks of harvest were discarded.
Additional fruit were harvested green, rinsed, and graded
as above, and evaluated for firmness with 12 hours of harvest.
Immature-green fruit were separated from mature-green fruit
after firmness determination by cutting through the equatorial
plane and observing the cut surface. Seed coats and locular
gel have not fully developed in immature-green fruit (Brecht,
1987; Kader and Morris, 1976).
Eleven, 9, 14, and 18 cultivars were transplanted into
the field in the Fall of 1986,. Spring and Fall of 1987, and
the Spring of 1988. Planting and harvesting schedules are
shown in table 3-1. Fruit handling procedures in all cases
were as outlined above.


43
Subjective texture determinations. Sensory evaluations
were conducted by panelists in the Fall of 1986 and the Spring
of 1987. The panels were managed under the guidelines of
Heintz and Kader (1983). Each panel was comprised of 11
individuals, 9 of whom were on both panels. In the fall
evaluation, panelists were not trained other than to be given
instructions on filling out the evaluation form, which is
reproduced in figure 3-1. In the spring test, panelists were
familiar with the range of texture to be expected (having
participated in the previous evaluation). Furthermore, they
received instruction by being provided examples of what a non-
mealy and severely mealy fruit were prior to beginning the
examinations.
Fifty uniform red-ripe fruit were placed on trays, stem-
end down, in front of the panelists. Each tray contained one
cultivar. Three fruit per cultivar were selected by the
panelists at random. Prior to cutting the tomatoes, firmness
of whole fruit was judged by hand. With a utility knife,
panelists halved the fruit through the equatorial plane and
observed the cut surface. Afterwards, firmness, mealiness,
and mouthfeel were ascertained upon mastication. Each
panelist scored 3 separate fruit of each of the 9 (spring) and
11 (fall) cultivars. Individual scores of each fruit were
recorded as determined using a 5-point scale for mealiness and
firmness (figure 3-1). In another test, members were asked
to rank cultivars by mealiness without regard to individual
scores.


44
Objective firmness determinations. Initially, in the
Spring of 1986, 10 red-ripe fruit of each cultivar from each
block were examined for firmness objectively as follows. A
Cornell Pressure Tester (Hamson, 1952b) as modified by Gull
(1987), was used. This device will hereafter be referred to
as the IFAS (Institute of Food and Agricultural Science)
Firmness Device (figure 3-2). Measurements were taken on
whole fruit at the blossom end and at 3 equidistant points
along the equatorial plane. No effort was made to identify
locular areas or radial walls, since in some cultivars these
areas are not apparent from the exterior. Initial height was
determined and a 1 kg weight was applied to the fruit at these
points. Millimeters of deformation (deflection or compres
sion) were noted at 5 seconds. Firmness values were expressed
as % deformation of whole fruit. Lower values reflect firmer
fruit.
Following the compression test, the same fruit were
peeled by hand with a paring knife, taking care to remove the
least amount of tissue as possible. Two areas, 180 degrees
apart along the equatorial plane, were selected at random and
tested for firmness using a Magness-Taylor puncture device
(Magness and Taylor, 1925). Data were the force required (in
grams) to drive a 7.9 mm diameter convex tip into the flesh.
Higher values reflect firmer tissue.
The locular, placental, and columnella material, includ
ing the blossom and stem scar tissues, of the above fruit were
removed and discarded. The remaining pericarp (fruit wall)


45
tissue of each individual fruit was further divided into outer
and inner (radial) pericarp by carefully excising the radial
walls intact. This left the pericarp of each fruit in 4
pieces: 2 shells of outer pericarp and 2 hemispheres of inner
radial wall. The pericarp types of each fruit were weighed
separately and placed in a Kramer shear cell attached to an
Instron Universal Testing Device (model #1132, Instron Corp.,
100 Royall St., Canton, Mass.) (figure 3-3). The Instron was
equipped with a 500 kg mechanical load cell. Crosshead speed
was 20 cm min'1. The device was set to drive the shear plates
completely through the cell. Peak heights were recorded and
data were expressed as Newtons (GFW pericarp)'1. Higher values
reflect firmer tissue.
In subsequent tests, firmness was determined at the
immature-green (IM), mature-green (MG), breaker (B) pink (P) ,
and red (R) stages of development, using the IFAS and Instron
devices.
Objective Mealiness Determination
Plant material. Tomato fruit were harvested and handled
as previously described. Initial evaluations were carried out
on 5 cultivars (Rutgers, Sunny, FL-7136, Suncoast, and Flora-
Dade) and later expanded to include most of the cultivars
grown each season. For subjective determinations, of
mealiness, all cultivars were evaluated in the Fall of 1986
and the Spring of 1987.


46
Sensory evaluations. The sensory evaluations were
conducted as previously described.
Determination of tissue tonicity. Discs were cut from
the outer pericarp of ripe fruit using a 5-mm cork borer. The
peel was excised from each disc and enough endodermal material
removed to bring the disc thickness to 3 mm. The discs were
incubated for 12 hours at room temperature in 50 ml of a range
of Sorbitol concentrations from 0.2 through 1.0 M to determine
which one concentration would most closely approximate an
isotonic solution for a number of tomato cultivars. Using 5
cultivars, 10 discs were excised from 3 fruit per cultivar per
Sorbitol concentration. Isotonic strength was determined by
measuring change in resistance of the bathing solution using
a conductance bridge (Radiometer CDM, The London Co., 811
Sharon Dr. Cleveland, Ohio), and comparing this against
visual observation of plasmolysis and rupture via light
microscopy.
Cell separation and count determination. Ten discs
(total) were recovered from 3 to 4 fruit as outlined above and
placed in 50 ml of isotonic Sorbitol (0.6 M, as determined
above) in 125-ml erlenmeyer flasks (3 flasks per cultivar).
Each flask was then sealed with parafilm and shaken (120 rpm)
at room temperature using a gyrotory shaker. At the end of
1 hour, 10 ml aliquots of the bathing solution were drawn from
each flask and pipetted into a 25-mm petri plate. Total cell
counts of each sample were determined using a light microscope
(Nikon Diaphot Type 108).


47
Scanning electron microscopy of mealv tissue. Ripe,
mealy and non-mealy tomato fruit were cut through the equa
torial plane and a slice of tissue 3 mm thick was excised from
the surface using a utility knife. These sections were freeze
dried for 1 week at -62C and 80 mtorr (Virtis Freezemobile II,
model 10-MR-TR, The Virtis Co., Gardiner, NY), then immediate
ly transferred to a desiccator. After 2 days, samples were
excised from the outer equatorial pericarp region of the dried
sections with a scapel, gold coated using an IB-2 Ion Coater
(Eiko Engineering, Perkin Elmer, Rockville, MD) and viewed at
50X with an Hitachi S-450 Scanning Electron Microscope
(Hitachi Inc., Tokyo, Japan).
Results and Discussion
Objective and Subjective Firmness of Tomato Fruit
Objective measurements. Firmness of ripe fruit in the
Spring of 1986 as determined by the IFAS device, Instron
device, and Magnus-Taylor is shown in table 3-2. Each
technique was able to discriminate between cultivars, and with
precision. However, ranking of the cultivars by firmness
differed according to technique employed. Correlations among
objective measurement techniques are shown in table 3-3.
Whole fruit firmness as determined by the IFAS device was
poorly correlated with the Magness-Taylor device (r = -0.572) .
This is not surprising since the Magness-Taylor device, in
measuring resistance of the outer pericarp to puncture, is
influenced by the contribution of the underlying tissue.


48
Unlike apples, peaches, and pears, tomatoes are not uniform
throughout their interior. Locular areas containing a jelly
and water-like consistency are surrounded by firmer ovary
wall. In addition to the IFAS device, the Magness-Taylor was
not correlated highly with the Instron inner wall (r = 0.301)
or outer wall (r = 0.435) determinations. The Instron wall
measurements are of a single tissue type (ovary wall), whereas
the Magness-Taylor device measures a composite parameter, as
pointed out. These data indicate that the Magness-Taylor
device is unsuitable for use in determining firmness of fruits
that are not uniform throughout, such as the tomato.
Like the puncture test, the IFAS device measures a
composite type of firmness. Resistance to compression is
likely influenced by inner wall morphology and texture of the
locular material. Correlations between the Instron and IFAS
devices were low. Correlations coefficients were -0.139 and
-0.582 for inner and outer wall, respectively.
The Instron measurements of inner and outer pericarp wall
were highly correlated (r = 0.834). This is not surprising
since these measurements were taken on the same tissue type.
The inner wall was softer than the outer wall (table 3-2) ,
reflecting the observed pattern of ripening in tomato fruit.
That is, interior radial walls ripen before outer walls and
are developmentally advanced over outer walls (Brecht, 1987).
Hall (1987) found identical patterns when he examined outer
and radial walls of 'Walter', 'Flora-Dade', and 'MH-1' tomato
fruit using an Instron device equipped with a penetrating
probe.


49
Ripening related changes in firmness of selected culti-
vars are graphically illustrated in figures 3-4 and 3-5. For
3 of the cultivars, firmness of whole fruit (as determined by
IFAS device) was not significantly different until the breaker
stage (figure 3-4). Thereafter, the cultivars segregated into
very soft ('Rutgers') and midrange firmness ('Flora-Dade' and
'Sunny') groupings. 'Sunny' is considered 'ideal firm' by the
tomato industry (John W. Scott, Gulf Coast Research and
Education Center, Bradenton, FI, personal communication). A
fourth cultivar (Fl-7136) consistently increased in softness
from the immature-green through the red stage. However, this
cultivar was the firmest of those tested, and the overall rate
of change was the least. 'Flora-Dade' and 'Fl-7136' were not
significantly different at the pink stage, yet varied drama
tically at the red stage. These data (figure 3-4) illustrate
the point that firmness of whole fruit up to and including the
breaker stage, and even in one instance through the pink
stage, is not indicative of firmness at the red-ripe stage of
development. Figure 3-5 shows firmness of fruit pericarp (as
determined by Instron device) at selected stages of develop
ment as fruit ripen. Data are expressed in this figure as the
reciprocal of Newtons (GFW pericarp)'1 for easy comparison with
data in figure 3-4. For all 4 cultivars, differences and
changes in firmness were not significant until the pink stage.
At this point, rate of change in firmness of 'Rutgers'
exceeded the rate of softening of the other 3 cultivars which,


50
although had begun to change, did not differ significantly
from one another. However, at the red stage, the 4 cultivars
had segregated into 3 categories similar to the whole fruit
firmness (fig 3-4). Unlike whole fruit firmness, tissue
firmness was not indicative of final firmness until after the
pink stage.
Subjective texture evaluation. Table 3-4 shows firmness
of 9 cultivars in the Spring of 1987 as determined by the IFAS
device and sensory panel. There was a very high correlation
between blossom-end compression and eguatorial compression (r
= 0.946). It appears that the blossom-end measurement can
serve as an indication of whole fruit firmness without sacri
ficing accuracy. In this test, blossom-end firmness was
higher than equatorial firmness, reflecting the absence of
softer underlying locular material through the polar axis.
Sensory panel determinations were equally correlated with the
IFAS blossom-end measurement (r = -0.877) and equatorial (r
= -0.857) determinations (table 3-4), confirming either
objective technique From the Duncan's Multiple Range Test,
it appears that sensory panelists were not as discriminating
as the objective measurements. Trends in the fall sensory
evaluations were similar to the spring tests (data not shown).


51
Mealiness Determinations
Preliminary studies have shown that in mealy tomato
fruit, cells appear to easily separate one from another under
slight pressure. Glenn and Poovaiah (1987) have shown that
in mealy apple tisse, cell to cell contacts are reduced.
Scanning electron micrographs of the cut surface of mealy
cortical tissue showed little rupture of cells as compared to
non-mealy tissue. Overripe watermelon, whose pericarp appears
mealy (Donald J. Huber, Vegetable Crops Dept., Univ. of
Florida, Gainesville, FL, personal communication), show a
similar breakdown of the middle lamella (Elkashif and Huber,
1988). It was surmised that the tensile strength between
adjacent cells in mealy tissue is less than that within the
cell walls. This results in cells separating rather than
rupturing when sheared; the contents are not released, thus
the characteristic dry, coarse, grainy appearance of the cut
surface. Figure 3-6 shows the cut surface of non-mealy,
mealy, and extremely mealy tomato fruit. With these assump
tions in mind, the possibility of determining mealiness by
direct measurement was investigated, based on the propensity
of the cells to separate under slight mechanical force.
Tissue tonicity. Six hundred millimolar (0.60 M)
Sorbitol was the concentration which most closely matched an
isotonic solution for the cultivars tested (table 3-5). Light
microscopy confirmed that there was only slight plasmolysis
at this concentration and little evidence of cell rupture.


52
Cell separation and count determination. Figure 3-7
shows examples of randomly chosen viewing areas of the
aliquots of the disc bathing solution as seen through the
light microscope at 4X. The fields correspond to aliquots
from slightly mealy (figure 3-7A), mealy (figure 3-7B), and
very mealy (figure 3-7C) fruit. Table 3-6 lists subjective
mealiness scores of 11 cultivars in the Fall of 1986. Also
included are objective determinations (cell counts) of 5
cultivars. Fruit judged not mealy to slightly mealy had
average cell counts of 76 to 101, whereas fruit judged to be
mealy and extremely mealy had average cell counts of 374 and
512. Table 3-7 lists and compares subjective and objective
mealiness scores of 8 cultivars in the Spring of 1987. In
every instance, mealy fruit (determined by sensory evaluation)
had greater average cell counts than less mealy fruit.
Average cell counts of 57 to 116, 160, 244 to 371, and 480
corresponded to slightly mealy, mealy, very mealy, and
extremely mealy, respectively. When sensory panelists were
asked to rank tomato fruit according to mealiness, there was
a 1 to 1 correlation between the sensory panel determinations
and cell count method (table 3-8).
Scanning electron microscopy. Scanning electron micro
graphs of the surfaces of tomato tissue which had been sliced
with a utility knife are shown in figure 3-8. Cells of very
mealy tissue (figure 3-8A) had less points of cohesion than
did cells of mealy tissue (figure 3-8B), which in turn had
less than cells from non-mealy tissue (figure 3-8C). The


53
blade appeared to have gone between cells in mealy tissue
(figure 3-8A) rather than through them as in non-mealy tissue
(figure 3-8C). Cells of moderately mealy tissue had a
combination of ruptured and whole cells (figure 3-8B). This
phenomenon in tomato is similar to that reported to occur in
apples (Glenn and Poovaiah, 1987), and substantiates the
hypothesis regarding the physical manisfestation of the mealy
characteristic.
Mealiness as a function of firmness. Results of the
objective mealiness determinations for the Fall of 1986 are
contrasted with the objective IFAS firmness determinations in
table 3-9. The overall correlation across all cultivars
between mealiness and firmness was r = 0.089. The highest
correlation between these textural traits occurred within the
cultivar FL-7155 in the Spring of 1987 (r = 0.451). A
scattergram of mealiness as a function of firmness is depicted
in figure 3-9. These data overwhelmingly indicate that there
is no relationship between firmness and mealiness in tomato
fruit.


54
Table 3-1. Planting and harvest dates of tomato cultivars,
conducted at the
Bradenton, FI.
Gulf Coast
Research and
Education
Center,
GROWING SEASON
SEEDED
TRANSPLANT
SET IN
FIELD
fifWEST
SPRING 1986
JAN 2 6
FEB 09
MAR 07
JUN 06
FALL 1986
JUL 28
AUG 05
AUG 28
NOV 13
SPRING 1987
JAN 31
FEB 17
MAR 16
JUN 18
FALL 1987
JUL 16
JUL 2 6
AUG 26
NOV 23
SPRING 1988
JAN 08
JAN 18
FEB 22
MAY 26


Table 3-2. Objective firmness determinations of ripe tomato
fruit harvested in the Spring of 1986. Methodology is as
outlined in the text. Each number is the mean of 30 fruit,
followed by the standard error.
INSTRON2
CULTIVAR
IFASy
MAGNESS
TAYLORx
INNER
WALL
OUTER
WALL
Flora-Dade
11+0.083
10.10.065
2.99+0.096
4.63+0.112
MR-1
12+0.334
7.5+0.118
1.97+0.082
3.40+0.156
Fl-7136
5+0.042
8.8+0.118
2.29+0.063
4.76+0.132
Suncoast
90.196
8.0+0.063
2.03+0.036
3.35+0.072
Sunny
100.170
7.7+0.056
3.02+0.051
4.68+0.125
Walter
16+0.171
7.0+0.043
1.85+0.031
2.70+0.055
Marglobe
14+0.290
6.1+0.078
2.71+0.134
4.30+0.130
2Newtons (GFW pericarp)'1. Larger values reflect firmer fruit
Y% compression. Larger values reflect softer fruit.
xgrams X 100. Larger values reflect firmer fruit.


56
Table 3-3. Correlations among objective firmness measurement
techniques, between means of firmness of ripe tomato fruit
harvested in the Spring of 1986.
I FAS
MAGNESS
TAYLOR
INNER
WALL
INSTRON
OUTER
WALL
I FAS

-0.572Z
-0.139
-0.582
M-T
-0.572

0.301
0.435
INSTRON
INNER
-0.139
0.301

0.835
OUTER
-0.582
0.435
0.834

ZA11 values are significant at the 0.0001 level.


57
Table 3-4. Firmness of ripe tomato fruit harvested in the
spring of 1987, as determined by sensory panel and IFAS
device. Smaller IFAS values are firmer. Larger sensory panel
values are firmer.
CULTIVAR
BLOSSOM
IFAS
END
DEVICE2
EQUATORIAL1
SENSORY PANELX
Angora-Hairy
14au
25a
2.2a
Rutgers
lib
16b
3.0b
Angora
10b
16b
2.3a
FI MH-1
8c
14c
2.8b
Sunny
6d
14c
3.0b
Flora-Dade
5de
12d
3.7d
Suncoast
4e
9e
3.3c
FL-7136
3 f
It
3.9d
FL-7155
3 f
8e
4.2d
z% compression, means of 10 fruit
y3 measurements per fruit
xmeans of 33 fruit
wmeans within columns followed by the same letter are not
significantly different at the 0.05 level


58
Table 3-5. Effect of molarity of Sorbitol bathing solution
on electrolyte leakage from tomato pericarp discs. Data are
means (to nearest %) of three determinations of % change in
conductivity of solutions after 12 hours of incubation at room
temperature.
MOLARITY
CULTIVAR
0.20
0.40
0.60
0.80
1.00
ANGORA
124
74
30
52
69
RUTGERS
71
42
28
22
94
FL-7136
112
71
37
21
23
SUNNY
80
49
33
39
51


59
Table 3-6. Mealiness of ripe tomato cultivars as determined
by sensory panel and objective cell count, Fall 1986.
MEALINESS2
CULTIVAR PANEL1 CELL COUNT*
2 33-A
2.2b
RUTGERS
1.9a
76a
MARGLOBE
1.3a
SUNNY
1.4a
101b
SUNCOAST
2.8c
374c
FL-7136
3.7d
512d
FL-MH1
1.9a
FL-7155
3.2d
FLORA-DADE
1.9a
97a
WALTER
2.5c
7136 X (NC-8276 X 7065)
2.7c
zmeans in columns followed by the same letter are not signifi
cantly different at the 0.05 level.
Yrange of 1 (not mealy) to 5 (extremely mealy)
xmeans of 3 determinations


60
Table 3-7. Mealiness of ripe tomato cultivars as determined
by sensory panel and cell count, Spring 1987.
CULTIVAR
PANEL1
MEALINESS*
CELL COUNTX
SUNNY
2.3a
57a
RUTGERS X SUNCOAST
2.7b
116b
RUTGERS
2 la
67a
RUTGERS X ANGORA
3.3c
160c
ANGORA
4.6ef
371e
SUNCOAST
4.3d
253d
FL-7136
4.5de
244d
FL-7136 X SUNCOAST
4.8 f
480f
zmeans within columns followed by the same letter are not sig
nificantly different at the 0.05 level
Yrange of 1 (not mealy) to 5 (extremely mealy)
xmeans of 3 determinations


61
Table 3-8. Ranking of ripe tomato cultivars by mealiness as
determined by sensory evaluation and cell count.
CULTIVAR
PANEL*
MEALINESS
CELL COUNT1
RUTGERS
1
65
SUNNY
2
132
FLORA-DADE
2
171
FL-7136
4
262
ANGORA
5
344
zleast (1) to most (5) mealy
Ymeans of 3 determinations


62
Table 3-9. Objective
mealiness (cell count)
Fall of 1986.
firmness (IFAS
of ripe tomato
device) and objective
fruit harvested in the
CULTIVAR
IFASZ
CELL C0UNTy
SUNNY
6.5
57
RUTGERS X SUNCOAST
7.2
116
RUTGERS
10.5
67
RUTGERS X ANGORA
13.2
160
ANGORA
12.9
371
SUNCOAST
5.1
253
FL-7136
4.9
244
FL-7136 X SUNCOAST
4.7
480
z% deformation. Larger numbers reflect softer fruit.
Ymeans of 3 determinations. Larger numbers reflect mealier
tissue.


TOMATO TEXTURE EVALUATION
CULTIVAR CODE
NAME DATE
Please rate the following texture parameters on a 1 to 5
scale. Use any criteria or technique to determine the
ratings. Choose three (3) fruit PER CULTIVAR. Evaluate one
(1) fruit per form. You will have completed a total of
forms.
FIRMNESS
Very Soft
Slightly
Soft
Firm
Very Firm
Hard
1
FIRMNESS -
2
CENTER
3
4
5
Very Soft
Slightly
Soft
Firm
Very Firm
Hard
1
2
3
4
5
MEALINESS
None
Slightly Mealy
Mealy
Very Mealy
Severe
1
2
3
4
5
Figure 3-1. Form used to record sensory evaluation of tomato
texture.


64
Figure 3-2. Cornell Firmness Tester as modified by Gull. A
fruit is placed under the 1 kg weight and the handle is
lowered until contact is made with the fruit. The sliding
inclined plane under the depth gauge is used to zero the
instrument. The handle is then released and compression is
noted at 5 seconds.


65
Figure 3-3. Instron Universal Testing Device fitted with a
Kramer Shear Cell. The machine is set to drive the plates
completely through the sample box. A 500 kg mechanical load
cell transfers the force into electrical energy which is sent
to a strip recorder.


66
FIRMNESS OF WHOLE
TOMATO FRUIT
STAGE OF DEVELOPMENT
Figure 3-4. Whole fruit firmness, as determined by IFAS
device, of tomato fruit at selected stages of development.
IM, MG, B, P, and R are immature-green, mature-green, breaker,
pink, and red fruit, respectively. Bars are SE of 30 fruit.
Higher values reflect softer fruit.


67
FIRMNESS OF TOMATO PERICARP
CL
cr
<
o
CL
UJ
CL
o

30 n
10-
0
i 1 1 r
IM MG B P
STAGE OF DEVELOPMENT
R
Figure 3-5. Firmness of tomato pericarp, as determined by
Instron device, from tomato fruit at selected stages of
development. IM, MG, B, P, and R are immature-green, mature-
green, breaker, pink, and red, respectively. Data is ex
pressed as the reciprocal of Newtons GFW pericarp*1. Bars are
SE of 10 measurements.


68
Figure 3-6. Examples of, from top to bottom, non-mealy,
mealy, and extremely mealy ripe tomato fruit.


69
Figure 3-7. Typical field views through the light microscope
at 4X of aliquots taken from the bathing solution containing
discs from A) non-mealy, B) mealy, and C) very mealy tomato
pericarp.


70
Figure 3-8. Scanning electron micrographs of the cut surfaces
of A) very mealy, B) mealy, and C) non-mealy tomato fruit.
Bars are 500 microns.


CELL COUNT
71
FIRMNESS VS MEALINESS IN
RED TOMATO FRUIT
500
400
300
200
100
0-j | | | |
0 100 200 300 400
FIRMNESS (NEWTONS PER G PERICARP)
Figure 3-9. Mealiness (cell count) as a function of pericarp
firmness as determined by Instron device. Overall correlation
between mealiness and firmness was r = 0.089.7


CHAPTER 4
POLYGALACTURONASE ACTIVITY, RESPIRATION, AND ETHYLENE
PRODUCTION IN RIPENING TOMATO FRUIT
Introduction
The levels of PG activity in tomato fruit show a general
inverse correlation with whole fruit firmness (Brady et al.,
1985; Hobson, 1965). Firmer genotypes tend to have less PG
activity. Within a cultivar, the softening which occurs
during ripening is accompanied by increases in the levels of
PG. The ripening mutant rin. which fails to ripen and soften
normally, has been reported to lack this enzyme (Buescher et
al., 1976; Hobson, 1980; Tucker and Grierson, 1982), although
this claim has been recently disputed by DellaPenna et al.
(1987). Some tomato genotypes are prone to the disorder
'blotchy ripening', in which affected areas of the pericarp
fail to ripen; these regions have been shown to have a greatly
reduced level of PG (Hobson, 1965) .
On the basis of studies with rin and other tomato
ripening mutants, Tigchelaar et al. (1978) proposed that PG
functioned to initiate a number of ripening parameters,
including an increase in ethylene production, by releasing
specific but unidentified enzymes from the cell wall. The
idea that PG functioned to initiate ripening did not receive
support from subsequent studies showing that the initial
72


73
increase in ethylene synthesis occurred prior to the ap
pearance of PG transcripts (Grierson, 1985; Grierson and
Tucker, 1983; Maunders et al., 1987). However, these studies
were based on ethylene production in whole fruit, whereas PG
levels were assessed in isolates of pericarp tissue. Brecht
(1987) demonstrated that the initial increase in System II
(McMurchie et al., 1972) ethylene synthesis during the early
stages of ripening occurs in the locular gel which, while not
containing PG, is temporally advanced over pericarp tissue in
terms of cell-wall changes (Huber and Lee, 1986) and lycopene
accumulation. Therefore, the absence of PG in the locular gel
argues against an initiative role of the enzyme in ripening.
Recent studies have provided evidence that PG may
contribute to the production of System II ethylene in pericarp
tissue. Brecht and Huber (1988) demonstrated that vacuum
infiltration of pectin fragments generated from autolytically
active cell wall from ripe tomato fruit into pre-ripe tomato
fruit enhanced ethylene biosynthesis, possibly due to an
'elicitor' effect. The response was specific in that only
certain size classes were effective. That PG is not asso
ciated with ethylene production in locular gel does not weaken
the tenability of the idea of PG-enhanced System II ethylene.
Kim et al. (1987) demonstrated that infiltration of galactose
into pre-ripe tomato fruit, at levels present in ripening
fruit due to degradation of wall galactan polymers, enhanced
ethylene biosynthesis. Thus it is possible that a variety of
products of wall origin may contribute to enhanced ethylene


74
biosynthesis. Numerous studies, primarily those directed at
the biochemistry of host-pathogen interactions (Chappell et
al., 1984; Roby et al., 1985, 1986; VanderMolen et al., 1983),
have demonstrated the capacity of pectin and other carbohy
drate fragments to elicit a variety of responses, including
ethylene synthesis.
If PG serves in a regulatory manner in fruit ripening,
there would presumably be a high correlation between PG
activity and ethylene production during ripening. Similarly,
one might anticipate a high relationship between PG levels and
firmness rather than the general trends previously reported
(Brady et al., 1985; Hobson, 1965). However, these invest
igators measured firmness of whole fruit firmness when
investigating the relationship between enzyme (PG) levels and
changes in texture. When measuring whole fruit firmness, the
relationship between enzymes and texture may be confounded by
the contribution to fruit texture of the locular material and
internal morphology of the fruit.
In this study, the relationship between whole fruit and
tissue (pericarp) texture and how these parameters are
correlated with PG levels were examined. Additionally, four
genotypes differing in firmness were examined to ascertain
whether a relationship exists between PG levels and autocata-
lytic (System II) ethylene production.


75
Materials and Methods
Plant Material
Eleven tomato genotypes were grown at the University of
Florida, Gulf Coast Research Center, Bradenton, FL. Fruit
were harvested at selected developmental stages, determined
by comparing external fruit color with a United States Dept.
Agri. visual aid (United States Dept. Agri. Visual Aid TM-L-
1, The John Henry Company, P.0. Box 17099, Lansing, MI). On
the basis of initial firmness evalautions of all genotypes,
fruit from 4 selected genotypes (Rutgers, Sunny, Flora-Dade,
and FL-7136) were harvested green and at their typical full-
red size. Fruit were rinsed with tap water to remove field
debris, dipped in 1% hypochlorite, and rinsed with deionized
water. After air drying, the fruit were held in storage rooms
and ripened at 23C, or used immediately. Fruit that had not
begun to ripen within 1 week were eliminated. At desired
stages of development, fruit were sectioned through the
equatorial plane and each half quartered by cutting through
the polar (stem-blossom) axis. The blossom and stem scars and
locular and placental material were removed and discarded.
Fruit pieces were randomized within genotypes and 1 kg of
material was stored in high-density polyethylene bags at -30C.
Additional fruit were utilized for firmness measurements as
described below.


76
Firmness Measurements
Fifty fruit per cultivar were harvested at the red-ripe
stage and evaluated for whole fruit firmness using a Cornell
Firmness Device (Hamson, 1952) as modified by Gull (1987).
Measurements were taken at 3 equidistant points along the
equatorial plane. A 1 kg weight was applied and deflection
in millimeters noted at 5 seconds. Data were expressed using
the equation
Percent Deformation = (d X h'1) X 100
where d = deflection after 5 seconds and h = original height
(diameter at point of applied force). For pericarp firmness
determinations, discs were cut from the outer pericarp
equatorial wall using a 15-mm cork borer. The discs were
trimmed to a thickness of 5 mm by excising the peel and
exocarp, then compressed to 3 mm through the short axis.
Compression was accomplished with a 5-cm diameter plate
attached to an Instron Universal Testing Instrument (Model
#1132, Instron Corporation, 100 Royall Street, Canton, MA)
equipped with a 50-kg mechanical load cell. Crosshead speed
was 20 cm min1. Peak heights over the course of travel were
recorded and data were expressed as the reciprocal of Newtons
disc1. Out of the 11 genotypes screened, Rutgers, Sunny,
Flora-Dade, and a non-released line, FL-7136 were selected as
representative of soft, 2 mid-range, and very firm fruit,
respectively.


77
Respiration and Ethvlene Determinations
Respiration was determined by placing individual fruit
(10 per cultivar) in 500-ml glass containers with lids fitted
with rubber septa. Samples of 0.5 ml were drawn from the
container atmosphere after 30 min and analyzed for C02 using
a Fisher gas-partitioner (model #1200, Fisher Scientific
Company, 711 Forbes Avenue, Pittsburg, Penn.). Column and
injection port temperatures were 60 and 87C, respectively.
Data were expressed as ml C02 [(kg FW) hour]'1. Ethylene
production was determined by sampling as above, analyzed with
a Photovac model 10A10 (Photovac Inc., Unit 2, 134 Doncaster
Avenue, Thornhill, Ontario), and expressed as ul ethylene [(kg
FW) h]'1. Both ethylene and C02 production were monitored
throughout ripening at 24-hour intervals.
Preparation of Enzvmicallv-active Cell Walls
Enzymically-active cell walls were isolated from pink and
red fruit pericarp tissue and release of uronic acids deter
mined as described by Rushing and Huber (1984) with the
exception that the incubation temperature was 30C.
Extraction and Assay of Polygalacturonase
Polygalacturonase was extracted by homogenizing 40 g of
partially thawed pericarp in 40 ml of cold (5C) 100 mM Na-
acetate, 2.4 M NaCl, pH 6.5, in a Sorvall Omni-mixer in ice
for 2 min at full speed. The homgenate was held on ice for
1 hour, filtered through 2 layers of cheesecloth, and the


78
filtrate centrifuged at 39200 g (JA-20 rotor) for 20 min at
5C. The pellet was discarded and the supernatant brought to
75% saturation with solid ammonium sulphate and held on ice
for 2 hours. The suspension was centrifuged at 39200 g for
25 min at 5C and the supernatant discarded. The pellet was
resuspended in 2 ml of the extraction buffer and centrifuged
at 490 g for 15 min. A 1 ml aliquot of the supernatant was
desalted on a Sephadex G-25 column (1.6 x 15 cm) equilibrated
with 50 mM Na-acetate, 150 mM NaCl, pH 4.5. Fractions of 2
ml were collected at a flow rate of 50 ml hour1. Presence of
protein in each fraction was determined visually after
addition of 100 ul of Coomasie reagent (Bradford, 1976) to an
equal volume of aliquots of each fraction. Fractions which
contained protein (elution volume 20 through 40 ml) were
combined. Polygalacturonase activity was determined using a
reaction mixture containing 100 ul of the desalted protein
preparation and 400 ul of ethanol-purified polygalacturonic
acid (2 mg ml'1) in 30 mM Na-acetate, 150 mM NaCl, 0.01%
Thimerosal, pH 4.5. Enzyme activity after 1 hour at 34C was
determined by the reductometric method of Milner and Avigad
(1967). Protein was determined by the method of Bradford
(1976) using bovine serum albumin as a standard. Activity of
PG was expressed as umoles galacturonic acid equivalents [ (GFW
pericarp) hour]'1.


Results and Discussion
Over 3 growing seasons, 'Rutgers' fruit were the softest,
'Sunny' and 'Flora-Dade' were intermediate in firmness and
'Fl-7136' were the firmest of the 4 genotypes tested (figures
4-1 and 4-2). All fruit softened as they ripened; however,
differences in firmness among all genotypes were not consis
tent or continuously significant over the course of ripening
when determined by whole fruit firmness (figure 4-1).
Initiation of softening varied according to genotype. In
whole fruit, softening was not readily apparent until the pink
stage of development in 'FL-7136', the breaker stage in
'Sunny' and 'Flora-Dade', and the mature-green stage in
'Rutgers'. In addition, the rates of change in firmness
varied within and among genotypes. The change in firmness of
1 midrange ('Flora-Dade') and the firm genotype ('FL-7136')
were nearly linear once softening had been initiated. In the
other midrange ('Sunny') and the softest genotype ('Rutgers'),
the pattern appeared sigmoidal. In contrast to whole fruit
firmness, differences in pericarp firmness were discrete past
the breaker stage and rates of change nearly consistent
throughout ripening (figure 4-2) Softening initiation
occurred past the breaker stage of development in all geno
types. Firmness at the early developmental stages (mature-
green and breaker) was not indicative of firmness at the later
stages of development as measured by whole fruit (figure 4-1)
or tissue firmness (figure 4-2). Absolute differences between
genotypes and the rate of change in firmness within genotypes


80
varied according to measurement technique. Since outer
pericarp firmness remained unchanged up through the breaker
stage (figure 4-2), the firmness changes occurring in whole
fruit prior to the breaker stage (figure 4-1) may be inter
preted as the contribution of changes occurring in internal
tissues. The locular gel, for example, undergoes significant
textural changes during the transition from the immature-green
to the mature-green stage (Huber and Lee, 1986). Other
factors possibly contributing to whole fruit firmness include
carpel morphology, thickness of outer fruit wall, and overall
water status of all fruit tissues. In this study, overall
correlation between whole fruit firmness and tissue firmness
was 0.668. Although the general order of firmness was
conserved between techniques, at the pink and red-ripe stage
differences in firmness as depicted by the two measurement
techniques was dramatic (figures 4-1 and 4-2).
Rushing and Huber (1984) reported on the use of enzymic-
ally active cell wall as a system for studying tomato fruit
softening. They demonstrated that cell wall autolysis
increases with ripening, reflecting progressively higher
levels of endogenous PG. Consistent with this relationship
between autolysis and tissue PG levels, autolysis (table 4-1)
was also related to tissue firmness (figure 4-2) among the 4
genotypes examined here. Genotypes with greater autolytic
activity had softer pericarp tissue. Autolysis in wall
prepared from the 4 genotypes was highly correlated with
levels of tissue PG in pink (r = 0.969) and red (r = 0.900)


81
pericarp tissue (table 4-1). Similarly, PG activity was
highly correlated with pericarp tissue firmness (r = 0.920).
Previous workers (Brady et al., 1984; 1985; Hobson, 1965,
1981), measuring whole fruit, found only general trends in
relating PG activity to firmness. Consistent with these
reports, PG was only moderately correlated with firmness of
whole red-ripe fruit (r = 0.757). In a recent study, Smith
et al. (1988) were able to reduce active PG levels in tomato
fruit to 10% of that in control fruit using antisense RNA
inhibition. Yet, they reported no concurrent decrease in
softness as measured by compression of whole fruit. As
previously mentioned, changes in whole fruit firmness are not
a good indication of changes in tissue firmness. When
relating these changes to metabolic activity in pericarp
alone, they are indirect at best. Smith et al. (1988) point
out underlying causes of changes in texture are complex. In
this study, the lowest levels of PG were found in the very
firm genotype 'Fl-7136' (table 4-1). These levels (1/4 of
that found in the softest genotype 'Rutgers') were apparently
insufficient to soften the tissue of 'Fl-7136 over the course
of normal ripening (figure 4-2). However, on a whole fruit
basis (compression), softening of this genotype did indeed
occur (figure 4-1). Those genotypes which characteristically
have a large locule to pericarp ratio or vary in size within
the genotype, would show even greater disparity between PG
levels and whole fruit firmness, making comparisons between
genotypes even more difficult.


82
Ethylene production showed a pattern similar to firmness
in that differences between genotypes were not significant
until after the breaker stage (figure 4-3). Maximum ethylene
production occurred at the pink stage, at which time the
genotypes were concurrently segregated into firm, inter
mediate, and soft categories. In agreement with Poovaiah and
Nukaya (1979), firmer fruit had lower levels of respiration
at the climacteric peak (figure 4-4). Polygalacturonase
activity was highly correlated (r = 0.929) with peak ethylene
production (table 4-2). This is supportive evidence that
these 2 processes are related. Brecht and Huber (1988)
reported that the onset of ripening in green tomato fruit was
advanced by infiltration of pectin fragments generated by
autolytically-active cell wall from ripe tomato pericarp.
Initiation of ripening in relation to controls was indicated
by advanced autocatalytic ethylene production and lycopene
synthesis. This work was supported by Baldwin and Pressey
(1988) who reported that tomato fruit vacuum infiltrated with
purified tomato PG displayed elevated levels of ethylene
production over control fruit. In the present study, in
addition to greater autocatalytic ethylene production,
genotypes with higher PG had greater levels of respiration and
required less days to ripen (table 4-2, r = 0.805 and -0.791,
respectively). Kagan-Zur and Mizrahi (1987) found that
tetraploid tomato fruit had greater levels of PG, climacteric
ethylene production, and required less days to reach the
climacteric maximum than did diploid fruit of the same


83
isogenic line with similar pre-climacteric levels of ethylene
production. They pointed out that if ethylene is the trigger
and coordinator of ripening processes, then fruits of the same
genetic background with identical pre-climacteric rates of
ethylene production should have similar ripening responses.
However, DellaPenna et al. (1987) reported finding low
levels of PG in the non-ripening mutant rin, which has been
reported to show no enhanced ethylene production during
development. This appears to indicate that PG activity and
ethylene production are not coupled through the activity of
PG-generated elicitors. However, the presence of PG mRNA or
its product may not, in rin. be coordinated with production
or activity of 1-aminocyclopropane-l-carboxylic acid (ACC)
synthase or ethylene-forming enzyme (EFE), thereby rendering
the tissue incapable of enhanced ethylene production in
response to PG.
Numerous studies have provided evidence for a relation
ship between stress and ethylene (Lee et al., 1987; Roby et
al., 1985, 1986; Romani et al., 1968; VanderMolen et al.,
1983). Romani (1984) suggests that ethylene produced during
fruit ripening and senescence is in response to stress imposed
by the genetically programmed demise of various cellular
entities, including membranes and cell walls. It is his view
that ethylene serves as a ripening augmentor or accelerator,
but is not the primary causative agent. Other factors, such
as tissue sensitivity to ethylene, determine the capacity of
a fruit to ripen. In climacteric fruit, one of the ripening


84
parameters is the production of System II ethylene. In
addition to autocatalytic ethylene production, climacteric
fruit show a burst of respiration during senescence. Romani
(1984) proposed that this increase in respiration occurs as
a response to ripening stresses and is a homeostatic response
of the mitochondrion.
These studies demonstrate that there is a highly sig
nificant relationship between PG levels and pericarp tissue
firmness and, more interestingly, between PG levels and System
II ethylene. Whether the latter relationship is a direct one
is yet unknown. The extensive evidence of pectin-fragment-
mediated increases in ethylene production in host-pathogen
interactions (Roby et al., 1985, 1986; VanderMolen et al.,
1983? West et al., 1984) argues in favor of a direct relation
ship. The data are consistent with this theory and indicate
a strong dose-response regulatory relationship between PG
activity and rate and intensity of ripening.


85
Table 4-1. Polygalacturonase activity [umoles galacturonic
acid equivalents (GFW pericarp) hour'1] and autolysis [pectin
released as (ug galacturonic acid equivalents) (mg cell DW)'1,
after 4 hours at 30C] in pink and red tomato fruit. Data in
columns followed by the same letter are not significantly
different at the 0.01 level.
STAGE OF DEVELOPMENT
PINK RED
CULTIVAR
PG
ACTIVITY
PECTIN
RELEASED
PG
ACTIVITY
PECTIN
RELEASED
RUTGERS
3.57a
76.0a
4.46a
105.6a
FLORA-DADE
1.86b
62.9b
2.43b
96.8b
SUNNY
1.59b
55.3c
2.02b
73.7c
FL-7136
0.95c
46.4d
1.22c
62.7d
r =
0.967
r = 0.
900


86
Table 4-2. Polygalacturonase activity [umole galacturonic
acid equivalents (GFW pericarp) hour'1] of pink tomato fruit
and rate (days to ripen) and intensity [uL ethylene and ml C02
(kg FW) hour'1 at climacteric peak] of ripening in tomato
fruit. Data in columns followed by the same letter are not
significantly different at the 0.01 level.
CULTIVAR
PG
ACTIVITY
RATE OF
RIPENING
ETHYLENE
PRODUCTION
RESPIRATION
RUTGERS
3.56a
5.0a
6.82a
31.0a
FLORA-DADE
1.86b
6.4b
4.31b
21.5b
SUNNY
1.59b
6.6b
4.03b
28.0c
FL-7136
0.95c
12.4c
0.65c
17.5d


87
FIRMNESS OF WHOLE TOMATO FRUIT
STAGE OF DEVELOPMENT
Figure 4-1. Whole fruit firmness, as determined by modified
Cornell device, of tomato fruit at selected stages of develop
ment. IM, MG, B, P, and R are immature-green, mature-green,
breaker, pink, and red, respectively. Bars are SE of 30
fruit. Higher values reflect softer fruit.


NEWTONS
88
FIRMNESS OF TOMATO PERICARP DISCS
STAGE OF DEVELOPMENT
Figure 4-2. Firmness (resistance to compression) of pericarp
discs, as determined by Instron device, from tomato fruit as
selected stages of development. MG, B, P, and R are mature-
green, breaker, pink, and red, respectively. Bars are SE of
30 discs. Data are expressed as the reciprocal of Newtons
disc'1. Higher values reflect softer fruit.


89
ETHYLENE PRODUCTION OF TOMATO FRUIT
STAGE OF DEVELOPMENT
Figure 4-3. Ethylene production of tomato fruit at selected
stages of development at 23C. MG, B, T, P, LR, and R are
mature-green, breaker, turning, pink, light red, and red,
respectively. Bars are SE of 24 fruit.


90
C02 PRODUCTION OF TOMATO FRUIT
STAGE OF DEVELOPMENT
Figure 4-4. Respiration of tomato fruit at selected stages
of development as measured by carbon dioxide evolution at 23C.
MG, B, T, P, LR, and R are mature-green, breaker, turning,
pink, light red, and red, respectively. Bars are SE of 24
fruit.


CHAPTER 5
HEMICELLULOSE MODIFICATIONS, POLYURONIDE CONTENT,
AND CALCIUM CONCENTRATION IN MEALY TOMATO FRUIT
Introduction
Little is known regarding the biochemical basis of
mealiness in horticultural crops. In apple (Malus domestica
Borkh.) fruit, exogenously applied calcium has been shown to
modify mealiness in cortical tissue by reducing the loss of
the cell wall-middle (CW-ML) (Glenn and Poovaiah, 1987). It
remains unknown, however, if changes in endogenous calcium
concentrations in apple lead to mealiness in vivo. Ben-Arie
and Laves (1971) demonstrated that mealiness ('wooliness') in
peach (Prunus prsica L.) was due to a decreased solubiliza
tion of pectic substances. Buescher and Furmanski (1978)
proposed that the basis for this was the failure of the tissue
to develop adequate pectinesterase and polygalacturonase (PG)
activity. From these limited studies, two very different
forms of mealiness can be described. In apple, mealiness is
associated with softening and loss of the CW-ML, and in peach,
a failure to solubilize the pectins of the CW-ML.
Scanning electron micrographs of the cut surface of mealy
apple tissue (Glenn and Poovaiah, 1987) reveal discreet,
intact cells which are not ruptured. Non-mealy tissue cells
are cut open and apparently release their contents. Because
91


92
of this, the mealy tissue appears 'dry' and the non-mealy
tissue appears 'wet'. Preliminary investigations (Ahrens,
unpublished) involving scanning electron micrographs of mealy
and non-mealy tomato (Lvcopersicon esculentum Mill.) tissue
showed similar results. However, initial studies have
revealed that, unlike apple fruit, in tomato fruit mealiness
and firmness (and concommitant PG activity) are not associated
(Ahrens, unpublished).
Changes in cell-wall components other than polyuronides
in relation to the development of mealiness have not been
investigated. Modifications in the cellulose fraction of the
cell wall could conceivably lead to the mealiness trait.
Although Cx-cellulase activity has been demonstrated in tomato
(Buescher and Tigchelaar, 1075' Hall, 1963, 1964; Pharr and
Dickinson, 1973; Poovaiah and Nukaya, 1979; Sobotka and
Stelzig, 1974; Sobotka and Watada, 1971) and avocado (Persea
americana Mill.) (Hatfield and Nevins, 1986; Pesis et al.,
1978) fruit, no evidence exists for the in vivo degradation
of native crystalline cellulose. It appears unlikely that
modifications of this cell-wall fraction are responsible for
the development of mealiness.
Hemicelluloses have been reported to be modified during
ripening in tomato (Huber, 1983a) and strawberry (Fragaria X
ananassa Duch.) (Huber, 1984). Since these changes were
monitored throughout ripening in single cultivars, no infer
ence can be drawn regarding mealiness and these modifications.
It is conceivable that relative differences in modification


93
of this cell-wall fraction are responsible for the mealiness
trait in tomato fruit.
The purpose of this study was to examine several tomato
cultivars known to vary in mealiness and relate differences
in their cell wall composition to differences in the expres
sion of this textural trait.
Materials and Methods
Plant Material
Four tomato genotypes varying in intrinsic mealiness were
grown at the Gulf Coast Research and Education Center,
Bradenton, Florida, in the Fall of 1987. Fruit were harvested
green, the calyxes and pedicels removed, and transported in
an air-conditioned vehicle to Gainesville, arriving within 3.5
hours. Tomatoes were then lightly rinsed in tap water to
remove field debris, dipped in 1% hypochlorite, and rerinsed
in tap water before air drying at ambient temperature. Fruit
were graded by removing misshapen, damaged, and under- and
oversized fruit, placed on trays stem-end down, and allowed
to ripen at 2 0C. When ripe, fruit were halved through the
equatorial plane and each half was quartered through the polar
(stem-blossom) axis. All locular material was removed and
discarded, as well as the stem and blossom scar material, and
the columnella. The remaining pericarp was stored at -30C.
To guard against inclusion of immature-green fruit in the
experiments, fruit in which ripening had failed to commence
by 1 week were discarded.


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