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Ethylene biosynthesis and cell-wall digestion in citrus peel

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Ethylene biosynthesis and cell-wall digestion in citrus peel
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Citrus peel
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Baldwin, Elizabeth A., 1952-
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English
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xii, 118 leaves : ill. ; 28 cm.

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Cell walls ( jstor )
Cells ( jstor )
Colors ( jstor )
Enzymes ( jstor )
Ethylene production ( jstor )
Pathogens ( jstor )
Peels ( jstor )
pH ( jstor )
Ripening ( jstor )
Sugars ( jstor )
Dissertations, Academic -- Horticultural Science -- UF
Ethylene ( lcsh )
Horticultural Science thesis Ph. D
Plant cell walls ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1986.
Bibliography:
Bibliography: leaves 100-116.
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Typescript.
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Vita.
Statement of Responsibility:
by Elizabeth A. Baldwin.

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Full Text


ETHYLENE BIOSYNTHESIS AND
CELL-WALL DIGESTION IN
CITRUS PEEL
BY
ELIZABETH A. BALDWIN
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
1986


ACKNOWLEDGEMENTS
There are many friends, aquaintances, and co-workers in
Gainesville and at the University of Florida to whom I am
very grateful for their support, moral or technical, in the
reseach and writing of this manuscript. It would be
impossible to name everyone, but I think most of these
people know who they are. A few, to whom I am especially
grateful, however, need mentioning:
To my husband, Mike, for his patience,
love, babysitting and computer skills. I especially
appreciate the fact that his goals and career were put on
hold in order for me to pursue mine.
To Hilton Biggs to whom I can only say that it has
been a long and elightening relationship (from my point of
view) for I feel privileged to have experienced.
To Dixie Biggs, who has helped me in so many ways.
Were it not for her friendship, creative input, and
technical support, this manuscript would not now exist in
completed form.
To Don Huber, for his invaluable help, advice,
laboratory facilities, and equipment. I appreciate the


time and effort he invested on my research problem.
To John Munson, for always being ready to help with
laboratory equipment, advice, and for his sense of humor
when needed.
To Cadance Lowell, for her help and instruction on the
electron microscope which was essential for some of the
work presented here.
To Charles Barmore, for his brief service on my
committee, and more importantly, for his enzyme material,
ideas, and support from which evolved some of the
accomplishments presented in this work.
To Karen Koch, for her friendship, enthusiasm,
advice and use of equipment.
To Richard Smith, for his instruction in plant
physiology which provided the groundwork for this or any
other physiological research I may attempt in my career.
To Chesley Hall, for his participation on both my
masters and doctoral committees and for use of
laboratory equipment.
iii


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS i i
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT xi
CHAPTER
I INTRODUCTION
1
II
III
LITERATURE REVIEW
4
Introduction
Primary Cell Walls of Plants
Plant Cell-Wall Structure
Plant Cell-Wall Function
Plant Cell-Wall Lysing Enzymes
Ethylene
Ethylene Biosynthesis
Ethylene and Defense
Ethylene and Cell Walls
Ethylene and Citrus
CELL WALL DEPOLYMERASE ENZYMES AND ETHYLENE
PRODUCTION IN CITRUS PEEL
9
12
15
15
17
19
19
21
Introduction 21
Materials and Methods 22
Fruit Injection Method and Ethylene
Determination 22
Affinity Chromotography 23
ACC Determination in Pectolyase treated
Fruit 24
Cycloheximide Application 25
PG Inhibitor 25
Treatment of Callus Tissue 25
Electrophoresis 26
Transmission Electron Microscopy 26
IV


CHAPTER Page
Results 27
Effect of Cell-Wall Lysing Enzymes on
Ethylene Production 27
Effect of Pectolyase on the Ethylene
Pathway and Cell-walls 32
PG Inhibitor and Pectolyase-Induced
Ethylene Production 36
SDS-Gel Profiles of Cell-Wall Lysing
Enzymes 39
Transmission Electron Microscopy of
Digested Cell Walls 39
Conclusion 46
IV PRODUCTS OF CELL-WALL DEGRADATION ELICIT
ETHYLENE IN CITRUS 49
Introduction 49
Materials and Methods 51
Fruit Injection Method and Ethylene
Determination 51
Preparation of Crude Pectin Hydrolysate. 53
Preparation of Carbohydrate-Enzyme
Digest Treatments 53
Gel Filtration of Pectin Materials 55
Analysis of Neutral Sugars 56
Results 57
Acid Hydrolyzed Pectin 57
Pectin-Digestion End-Products 63
Analysis of Neutral Sugar Content 70
Effect of Chitin on Ethylene Production. 70
Conclusion 75
V STRUCTURAL AND BIOCHEMICAL DEFENSE
MECHANISMS IN CITRUS 78
Introduction 78
Materials and Methods 80
Harvesting of fruit 80
Investigation of Citrus Peel Structure.. 80
Orange Peel X-ray Diffraction Analysis.. 81
Electrophoresis of Citrus Peel Proteins. 81
Ethylene Determination 83
Results 83
Orange Peel Structure 83
Orange Peel Mineral Analysis 85
Effect of Pectolyase on Orange Peel
Protein Profiles 85
Pectolyase and Orange Peel Filtrate
Induce Ethylene 89
Conclusion 92
v


CHAPTER
paRe
VI SUMMARY 94
LITERATURE CITED 100
BIOGRAPHICAL SKETCH 117
VL


LIST OF TABLES
Table Page
or 9/30/85, were treated with 6 x 20 pi
injections of sugar-containing 72
4.2 Neutral and acid sugar analysis was made on
the albedo pectin-pectolyase end product
material that had been fractionated 73
4.3 Neutral and acid sugar analysis was made on
the albedo pectin-pectolyase digest material
that had been fractionated on a Bio-gel 74
vi i


LIST OF FIGURES
Figure Page
3.1 Green Valencia oranges, harvested 10/30/84,
were treated with 6 x 10 ¡a 1 injections of
fungal cell-wall lysing enzymes in 28
3.2 Green Navel oranges, harvested 9/7/83, were
treated with 6 x 10 |jl injections of o.l?
cell-wall lysing enzymes in 0.1 M phosphate.... 30
3.3 Greel Navel oranges, harvested 8/15/83,
were treated with 6 x 10 ¡al injections of
0.1?, 0.05?, or 0.01!? pectolyase 31
3.4 Green Valencia oranges, harvested 8/7/85,
were treated with 6 x 20 pil injections of
0.1? pectolyase in 0.1 M citrate-phosphate 33
3.5 Green Valencia oranges, harvested 7/30/84,
were treated with 6 x 10 pi injections of
cell-wall lysing enzymes in 0.1 M phosphate.... 34
3.6 Green Valencia oranges, harvested 8/23/84,
were treated with 6 x 10 pi injections of
either 0.1? desalted pectolyase in 0.1 M 35
3.7 Green Valencia oranges, harvested 9/30/85,
were treated with 6 x 20 pi injections of 30
ppm CHI + 0.1? pectolyase in 0.1 M acetate 37
3.8 Yellow-green Valencia oranges, harvested
1/8/85, were treated with 6 x 10 pi of 0.1?
pectolyase, boiled pectolyase, and 38
3.9 Yellow Valencia oranges, harvested 2/27/85,
were treated with protein-containing
fractions #1-6 and 10, and control fractions... 41
3.10 Coomassie blue-stained 11? polyacrylimide
gels of different enzyme preparations used
in protoplast isolation 42
viii


Figure Page
3.11Coomassie blue-stained 11% polyacrylimide
gels of pectolyase and the
Geotrichum candidum PG 43
3.12 Transmission micrograph of orange peel
tissue treated with 10 pil 0.1 % pectolyase
in 0.1 M phosphate buffer pH 6, 24 hours 44
3.13 Transmission micrograph of orange peel
tissue treated with 10 pi of 0.1% pectolyase
in 0.1 M phosphate buffer 45
3.14 Transmission micrograph of orange peel
tissue treated with 10 pi of 0.1 M phosphate
buffer pH 6, 24 hours after 47
4.1 Yellow Valencia oranges, harvested 2/26/85,
were treated with 6 x 20 pi injections of
crude orange pectin hdrolysate, 58
4.2 Yellow Valencia oranges, harvested 3/19/85,
were treated with 6 x 20 pi injections of
pooled fractions containing 61
4.3 Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 pi injections of
sugar-containing fractions 62
4.4 Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 pi injections of
sugar-containing fractions 64
4.5 Regreened Valencia oranges, harvested
5/6/85, were treated with 6 x 20 pi
injections of a 20% solution 65
4.6 Green Valencia oranges, harvested 7/16/85,
were treated with 6 x 20 pi injections of
various albedo pectin-pectolyase 68
4.7 Green Valencia oranges, harvested 9/10/85,
were treated with 6 x 20 pi injections of
sugar-containing fractions 69
4.8 Green Valencia oranges, harvested 9/30/85,
were treated with 6 x 20 pi injections of
sugar-containing fractions 71
IX


Figure Page
4.9 Regreened Valencia oranges, harvested
5/6/85, were treated with 6 x 20 ptl
injections of a 5% chitin 76
5.1 Green Valencia oranges, were harvested,
10/30/84, and the peel was cut in 30
micron sections and viewed 84
5.2 Green Valencia were harvested 10/30/84
and the peel was cut in 30 micron sections
and viewed under the light 86
5.3 Green Valencia oranges were harvested
10/30/84 and the peel was fixed, dehydrated
crital point dried, and gold 87
5.4 X-Ray diffraction analysis of dehydrated
orange peel show peaks for sulphur,
chlorine, potassium, and calcium 88
5.5 Coumassie blue-stained 112 polyacrylimide
gels of Valencia orange peel proteins from
fruit treated with boiled pectolyase 90
5.6 Green Valencia oranges, harvested 7/16/85,
were treated with 6 x 20 pil of 0.12
pectolyase in 0.1 M acetate buffer 91
x


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ETHYLENE BIOSYNTHESIS AND
CELL-WALL DIGESTION
IN CITRUS PEEL
By
Elizabeth A. Baldwin
May 1986
Chairman: Robert Hilton Biggs
Major Department: Horticultural Science (Fruit Crops)
Increased ethylene production is often associated with
pathogens that cause cell wall dissolution. Cell wall
lysing enzymes are also associated with ethylene
production, softening during ripening of certain fruits, or
cell wall lysis during abscission of plant parts. Control
over of such events through the manipulation of ethylene
would be of benefit to the horticultural industry.
Ethylene was produced by orange peel in response to
injections of solutions containing pectolyase (obtained from
Aspergillus japonicus) as well as some other commercial cell
wall lysing enzyme mixtures. These cell wall lysing enzyme
mixtures showed a complex protein profile when analyzed by
xi


SDS-PAGE. Polygalacturonase (PG) isolated from citrus peel
that had been infected by the citrus sour rot fungus,
Geotrichum candidum, and a similar enzyme isolated from the
pectolyase mixture, caused ethylene to be produced when
injected into orange peel tissue. Neither pectolyase nor
fungal PG induced ethylene production when applied to orange
callus tissue. Transmission electron microscopy revealed
middle lamellae dissolution and SDS gels showed differences
in protein profiles in orange peel treated with pectolyase
when compared to controls.
Sugar fragments are known to induce phytoalexin
production in different plant tissues. Pectic fragments,
released from citrus pectin by acid hydrolysis or pectolyase
digestion, induced ethylene production when injected into
citrus peel. Oligosaccharides of around nine to ten sugar
units were found to be the most potent elicitors of
ethylene synthesis, although a range of fragment sizes
induced more ethylene than the summed ethylene production of
individual fragments. This suggests synergism between the
individual fragments in terms of ethylene production.
Such elicitor fragments would be useful for manipulation of
ethylene to promote such events as degreening of citrus,
abscission of citrus fruits, and possibly resistance against
pathogen invasion.
xi i


CHAPTER I
INTRODUCTION
Of all the major classes of biological molecules,
polysaccharides have been the least studied. Proteins and
nucleic acids have been intensely researched, and some of
their structures deciphered. With the knowledge of
structure came the understanding of function such as
catalytic activity or encoding ability. With the exception
of glycoproteins and lectins, polysaccharidies are still
thought to be relatively unimportant except as protective
coatings for cells, energy sources, or energy storage
substrates. Part of the problem is the difficulty of
carbohydrate chemistry. Good analytical methods for
determination of carbohydrate structure were lacking until
recently (111). Even so, carbohydrates are far more
chemically complex and more difficult to decipher than
polypeptides and nucleic acids. This is because of the many
ways in which they can be linked and that their structures
can be branched (6). Furthermore, sugars can attach in
either a or 3 configuration of either five or six-membered
rings. Polysaccharides can also be modified by the addition
of other non-sugar substituents and can bind cations such as
1


2
calcium. The possibilities for linkages and configurations
for the one-hundred known natural sugars is staggering. The
question is: why such complexity? This has recently been
explained in part by research that has shown that
carbohydrates have diverse roles aside from the obvious
structural and energy storage functions. Sugar polymers are
now thought to be involved in signaling events such as
pathogen invasion (43, 117) as well as promoting hormonal
changes and affecting development (40, 111), or acting as
toxins (160). Cell recognition of such signals is little
understood, but study of the structures of such molecules
has lead to a greater understanding of their functions.
The plant cell wall, like the polysaccharides of which
it is made, has also been much ignored in plant physiology.
Other than interest in cell-wall expansion during growth,
and the passive roles of protection, structure, and turgor
maintenance, little importance has been assigned to it. The
idea that cell walls may play a more direct regulatory role
in cellular events is new and, as yet, not well accepted.
Lately cell walls have been shown to contain many types of
proteins, some of whose functions remain obscure (41, 42,
90, 111). The regulation and mechanisms for cell wall
growth, softening, changes during abscission, or signalling
activity are little understood. Further studies on this
complex "inverted organelle" are required to fully
understand all the functional aspects.


3
Ethylene and auxin are two plant hormones that seem to
be closely associated with cell wall activity. This is
interesting as these two hormones are mutually regulatory
(25). Ethylene is known to have important roles in
ripening, growth, abscission, defense, and auxin transport
(25), while the role of auxin in growth, abscission, and
regulation of the ethylene biosynthetic pathway is well
known (16, 25). The connection between auxin and cell wall
growth has become more obvious over the years, but the
involvement of ethylene in cell wall changes is more
obscure. It is not clear whether the endogenous cell wall
digesting enzymes involved in ripening or abscission are
regulated by ethylene, but they are certainly associated
with ethylene production (68, 93, 106, 121). On the other
hand, ethylene production is reported to be induced by
fungal cell wall digestion enzymes in various plant species,
as well as by pathogen invasion in general (40, 54, 93,
148). Further elucidating the relationship between ethylene
and cell wall changes has important consequences for the
areas of postharvest physiology and host-pathogen relations.
This knowledge could lead to new ways of manipulating
ripening, abscission, and plant defense responses which
could prove to be of great benefit to the horticultural
field.


CHAPTER II
LITERATURE REVIEW
Introduction
This review of the literature is an attempt to cover
various aspects of plant research that pertain to the data
presented in this dissertation. Recently the plant primary
cell wall structure has been intensely studied by Albersheim
and co-workers (6, 111) and many of its components and
interconnections have been identified which will be
summarized here. Evidence that has accumulated documenting
various cell wall functions, other than that of structure,
will also be presented. Cell wall lysing enzymes,
especially those that degrade pectic substances in nature,
will be reviewed in terms of their mode of action, substrate
preference, and roles in cell wall digestion. Finally,
ethylene biosynthesis, and subsequent hormonal role in
ripening, abscission, growth, and plant defense will be
analyzed and discussed.
Primary Cell Walls of Plants
Cell walls are an essential structural component of the
majority of eukaryotes and almost all prokaryotes. Most
4


5
likely, primitive prokaryotes evolved cell walls through
natural selection for reinforced plasma membranes. Probably
the advantage to such coated cells was that they were able
to operate at higher turgor and at higher metabolic rates,
enabling them to grow faster (21). This selective advantage
has populated this planet with many walled organisms.
Plant Cell Wall Structure
The structural components of all cell walls are 90!?
polysaccharide and 10? protein, of which most of the latter
are components of glycoproteins. The ability to determine
the structures of complex carbohydrates has only recently
become possible through improved techniques involving gas
chromotography, mass spectrometry, fast atom-bombardment
mass spectrometry, liquid chromotrgraphy, x-ray diffraction,
electron microscopy, and nuclear magnetic resonance
spectroscopy (111,143). Cellulose constitutes 20?-30? of
primary cell walls and is a polymer of B-4-linked glucose.
The other polysaccharides of cell walls are complex in
structure and, for the most part, undefined (111).
Cellulose chains are aggregated into microfibrils which are
laid down perpendicular to the axis of cell growth (89).
Hemicellulose is that non-cellulosic fraction of the
cell wall whose polysaccharides can be extracted with alkali
(43). Xyloglucans, present in cell walls of plants,
especially dicots, are constructed of a 8-1,4-linked glucose


6
backbone with side chains composed of xylose. Occasionally,
galactose, fucose or arabinose residues are found linked to
xylose of some xyloglucans (23,111). Some or most of the
xyloglucan is hydrogen bonded to cellulose (23).
Xylans, the major hemicellulose in the primary cell
walls of monocots, have a backbone of B-4-linked xylose and
also hydrogen bond to cellulose. Like xyloglucans, xylans
have various side chains attached to them, usually
containing arabinose (111). Monocot cell walls also contain
polysaccharides containing a mixture on B~3 and g-4 linked
glycopyranosyl residues. These are commonly called g-
glucans and so far have been found to be linear (111).
The pectic components of cell walls are
homogalaturonans or rhamnogalacturonans of which the latter
is divided into two groups (rhamnogalacturonans I and II).
Homogalacturonans are a-4-linked galacturonic acid units
which tend to be insoluble. They may be covalently linked
to other wall polymers (89) and have been found in dicot and
monocot tissues. These polymers tend to form insoluble gels
in the presence of calcium, suggesting that they may play a
role in the structure of cell walls (113). The linkages,
ring form, configurations, and degree of polymerization of
homogalacturonans are, for the most part, unknown (116,
143) .
Another pectic polysaccharide that was isolated from
several dicots and one monocot is rhamnogalacturonan I. It


7
has an alternating 2-linked rhamnose and 4-linked
galacturonic acid backbone (109), of which about half of the
rhamnosyl residues are branched. The side chains are varied
in nature and contain galactose, arabinose, and small
amounts of fucose (109, 110). The other rhamnogalacturonan,
that has been found in both dicots and monocots, is called
rhamnogalacturonan II. This polymer also contains a high
proportion of rhamnosyl residues as does rhamnogalacturonan
I, but with different linkages (111). Rhamnogalacturonan
II, from suspension cultured sycamore cells, contains some
unusual sugars including a methylated fucose, a methylated
xylose, and a new sugar called aceric acid (111). Since
this polysaccharide can be solubilized by endo-a-1,4-
polygalacturonase, it would appear to be covalently linked
in the primary cell wall through a series of 2,4-linked
galacturonic acid residues (143).
Pure arabinan polymers are also found in cell walls
containing 5-linked a-L-arabinofuranosyl residues as well as
polymers that consist almost totally of 8-4-linked
galactans, or mixtures of 6-linked and 4-linked galactose
units (111). Arabinogalactans in cell walls consist of two
types that differ in the linkage of the galactose and
arabinose residues. The neutral sugars arabinose and
galactose are found in many cell wall polymers such as
extensin, arabinogalactan proteins, rhamnogalactan I and II,
xyloglucan, arabinoglucan, and glucuronoarabinoxylans (108,
111) .


8
Angiosperm primary cell walls also contain many
different glycoproteins. One common and important such
glycoprotein is the hydroxyproline-rich extensin molecule.
Through peroxidase-catalyzed internal isodityrosine cross-
linkage, it forms a three-dimensional network around
cellulose microfibrils (44, 45). Extensin is 50^
carbohydrate, of which most is arabinose and galactose
residues. The protein portion is rich in hydroxyproline,
serine, lysine, tyrosine and isodityrosine (141).
Arabinogalactan proteins are water soluble and are found
extracellularly as well as in the cytoplasm. They are
acidic and the protein portion, which makes up 27-l07o of the
molecule, is rich in hydroxyproline, serine, alanine, and
glycine. The carbohydrate portions of these molecules are
of relatively high degrees of polymerization and contain
galactose, arabinose, rhamnose, mannose, galactoarabinose,
and glucoarabinose. Their biological role is unclear (111).
Cell walls of higher plants also contain various
enzymes, some of which are ionically bound in the wall and
all of which so far studied are glycoproteins (111). Malate
dehydrogenases, peroxidases, phosphatases, and proteases
have been reported. In addition, many glycosyl hydrolases,
transferases, endoglycanases, pectinases and pectinesterases
have been documented in cell walls (90, 123, 125, 111).
Plant cell walls also posess enzymes that are capable of
degrading walls of invading fungi (41, 42) and other


9
proteins capable of inhibiting polysaccharide-degrading
enzymes secreted by fungal pathogens (2, 17).
Plant Cell Wall Function
The primary cell wall dictates the growth and
morphology of plant cells. In addition to the obvious
structural importance, cell wall components have been
postulated to have functions such as control over genetic
expression in plants. This hypothesis arose partly from the
complexity of cell wall polysaccharides and also because a
fungal wall oligosaccharide of branched 3-glucans was shown
to exert control on the plant genetic level (48). Fungal
cell walls have been shown to elicit phytoalexins, lignan
synthesis, and protease inhibitor activity in plants (65,
91, 154). Specific fragments of plant cell walls were shown
to control various physiological responses in plants (116,
137, 160). These reports gave rise to the view that complex
carbohydrates can be regulatory molecules. It has been
shown that a xyloglucan fragment in nanomolar concentrations
inhibited 2,4-D-stimulated (2,4-dichlorophenoxy-ace tic acid )
elongation in etiolated pea epicotyls (111). The fragment
was obtained from suspension-cultured sycamore cells treated
with endo-1,4-glucanase. Fragments of plant cell wall
homogalacturonan have been shown to elicit phytoalexin
production in plants (35, 116, 155, 154). Such elicitors
induce receptive plant cells to synthesize the mRNA's and


10
enzymes responsible for phytoalexin synthesis, but the
manner of such specific gene activation is as yet not
completely understood.
Homogalacturonan elicitor fragments have been produced
from cell walls of different plant systems in two different
ways. One method involved partial acid hydrolysis of
soybean cell walls and citrus pectin. A D-
dodecagalacturonide fraction of the resulting material
contained the greatest elicitor activity with some activity
present in oligosaccharides from 10-13 residues long (116).
Another elicitor of phytoalexins was identified after
incubation of various substrates with an endo-cx-1,4-
polygalacturonic acid lyase (PGA lyase) or endo
polygalacturonase (PG.) (35, 51). The elicitor active
fragments produced by such treatments were shown to be an a -
4-linked dodecagalacturonide, for the lyase, and an de
linked tridecagalacturonide, for the PG, when assayed on
soybean or castor bean, respectively.
Mechanical injury, such as is found with microbes and
insects, will induce systemic synthesis of plant proteins
that inhibit microbial and insect proteinases (153). The
signal that induces this response in tissues distant from
the injury was shown to be fragments of pectic and chitosan
polysaccharides in suspension cultured sycamore cells (137),
tomato leaves, pea pods, and castor beans (154).


11
Hypersensitive cell death is a common response of
plants to microbial and viral invasion (24, 111). It is
hypothesized that cell death slows down the pathogen
invasion allowing time for induction of other plant defense
responses. Partial acid hydrolysis of suspension-cultured
sycamore cells produced toxic fragments which inhibited
14
uptake and incorporation of [ CJleucine into protein by
the cultured cells (160). In this study, the measure of
protein synthesis was equated to a measure of cell vitality.
The fragments were thought to be pectic in nature, as small
as trisaccharides (111), and may explain the observed
toxicity of pectic enzymes (78). Plasmolysis of cells was
found to protect tissues from the toxicity of both the
pectic enzymes and their fragment products (22). Cells
isolated for protoplasmic fusion or for tissue culture are
sometimes stimulated to produce ethylene by the enzymatic
mixtures used to digest their cells (11). CelluLysin and
macerase have been reported to induce ethylene production in
tobacco and pear suspension cells, respectively (40, 73,
147) and ethylene was observed to be produced by oranges in
response to both fungal enzymes and partially digested
citrus pectin (99). Castor bean explants produced ethylene
when exposed to heterogeneous enzymes or purified fungal
P.G. (152) and fungal cell walls elicited ethylene and
phytoalexin production in soybean (117). Ethylene is also
often associated with the hypersensitive necrotic response


12
to pathogen invasion (24, 30, 54, 158) and with the injury
due to insect or mechanical wounding (63, 64) all of which
may involve cell wall lysing enzymes and/or cell wall
fragments as well as eventual loss of cell vitality.
Whether cell wall fragments induce cell senescence or loss
of vitality via ethylene is unknown.
Partial acid-hydrolysis of suspension-cultured sycamore
cell walls produced a pectic fragment that both inhibited
flowering and promoted vegetative growth of fronds in
duckweed (Lemna gibba) This indicates that the fragments
had some regulatory contol over flowering in this species
(111) .
Plant Cell Wall Lysing Enzymes
There are many cell wall digesting enzymes that are
involved in ripening, growth, abscission, and pathogen
invasion of plant cells. Some of these enzymes digest
cellulose and others pectin or other polymers. Some remove
sugars only from the ends of polymers, some attack internal
bonds, while still others remove side chains. Endo- and exo~6
-1,4- or 6~1,3-glucanases have been found in cell walls of
plants (82). These enzymes degrade 6-1,4- cellulose as well
as the nemicellulose polymers xyloglucan and 6-1,4-xylan
(23, 76, 81, 83). The role of 6-1,3-glucanase is curious as
there appears to be no g-1,3-glucans in plant cell walls
(41, 70). Glycosidases, which are enzymes that hydrolyze


13'
oligosacchardes to monomers, are also active in plant
tissues. Cellulase and 1,3-glucanase have been associated
with growing tissues and autohydrolysis of cell walls (50,
70, 83). Cellulases are also reported to be involved in
locular formation in tomato ripening (81) as well as
ripening of papaya (118) and peach (77). 3~1,4-Xylanase, a-
arabinosidase and g-xylosidase have been reported to
breakdown arabinoxylan (55, 38) and a B_1,4-mannase may be
responsible for the breakdown of mannose-rich lettuce
endosperm (74).
B-Galactosidase is an enzyme that has been shown to be
associated with ripening in apple (19), tomato (123), and
pear (5). A similar enzyme showed high activity in growing
pea epicotyl cells and appeared to be involved with the
process of autolysis. a-Arabinase and exo-glucanase also
showed some activity is this system although less than that
of the 8-galactosidase (100).
Endo-polygalacturonase catalyzes the hydrolytic
cleavage of -1,4-bonds between non-esterified galacturonic
acid residues releasing oligomeric products.
Polygalacturonases are often thought to be responsible for
cell wall softening during ripening (20, 47) and abscission
(71, 131, 138). These enzymes prefer high molecular weight D
galacturonans and the rate of splitting the glycosidic bonds
decreases with the shortening of the substrate chain. Exo-D
galacturonases catalyze the hydrolytic cleavage of the


14
terminal a-D(l,4) bonds starting at the non-reducing end of
the galacturonan chains, releasing D-galactopyranuronic acid
as a product (130). Polymethylgalacturonases are enzymes
capable of degrading highly esterified D-galacturonans by
endo-action pattern and are ineffective toward de-esterified
substrates. Generally, D-galacturonases have their pH
optimum in a weakly acidic region between pH 4.0 and 6.5
(130). Polygalacturonases are also produced by many
pathogenic fungi and some bacteria (18, 51). Lyases
catalyze the cleavage of -D-(l,4) glycosidic bonds of
esterified and non-esterified D-galacturonans by the 8-
elimination mechanism giving rise to a double bond at the
non-reducing end. Endopectate lysases characteristically
have high pH optimums, such as 8.0-9.5, and a requirement
for calcium ions. Exopectate lyases cleave bonds starting
at the reducing end and prefer univalent cations and an
alkaline pH (130). Pectate lyases are produced by bacteria
and fugaria (96). Pectin lyases preferentially split the
highly esterified D-galacturonans producing esterified
unsaturated oligo-D-galacturonans. These enzymes have a pH
optimum of 5.1-6.6, are activated by calcium, and are of
fungal origin (130).
Pectinesterases attack polymethyl esters of D-
galacturonans. Enzymatic de-esterification of methyl esters
of pectin proceeds linearly along the chain of the molecule
resulting in free carboxyl groups. This enzyme apparently


15
is subject to end product inhibition and therefore does not
completely de-esterify pectin. Plant and some microbial
pectinesterases have the pH optimum in the range of pH 7.0-
9.0 although most microbial pectinesterases tend to prefer
neutral to acid pH (130). These enzymes can be found in all
plants and can be bound to cell walls. They may be involved
in growth as well as ripening (122). Pectinesterases are
also found in many plant pathogens of fungal and bacterial
nature (130). The action of these enzymes can sometimes
enhance the activity of plant or microbial
polygalacturonases and pectin lyases (118, 124, 130). This
would explain their association with ripening (118, 126).
Ethylene
Ethylene is a plant hormone that can have a dramatic
effect on ripening (37, 47, 58, 121, 139,), abscission (38,
71, 131, 138), breaking of dormancy (57), senescence (92,
159), sex expression (103), and flowering (25). This
hormone is unique in that it is a simple hydrocarbon gas
(103) and is therefore easily transported in plant tissues
(25). Other effects of ethylene are subtle and are modified
by interaction with other hormones especially auxin (1, 25,
36, 57, 103).
Ethylene Biosynthesis
Methionine was proposed by Lieberman and Mapson (105)
to be a precursor of ethylene which was later confirmed


16
using C-labeled methionine (104). The methyl-thio group
of this amino acid is cycled back through several
intermediates to reform methionine (4). In this scheme,
methionine is converted to S-adenosylmethionine (SAM) and
the CH^S group is released from SAM as methylthioadenosine
(MTA) which is hydrolyzed to methylthioribose (MTR). MTR is
then incorporated into a 2-aminobutyrate moiety which is
condensed into methionine through a series of as yet
unidentified steps (166). Meanwhile, the 3 and 4 carbons
from SAM split off to form 1-aminocyclopropane-l-carboxylic
acid (ACC) which is then converted to ethylene (3). ACC can
also be converted to N-malonyl ACC which appears to be an
inactive dead end-product and therefore represents a
mechanism for regulation of ethylene biosynthesis (79). An
important step in the regulation of ethylene biosynthesis is
the conversion of SAM to ACC by the enzyme ACC synthase.
This enzyme requires pyridoxyl phosphate for maximal
activity and is inhibited by inhibitors of pyridoxyl
phosphase enzymes such as aminovinylglycine (AVG) and
aminooxyacetic acid (AOA) (3, 32, 163, 165). Auxin
stimulates ethylene production at this step
(162). Another important reulatory point in ethylene
production is the conversion of ACC to ethylene which
requires oxygen (3), membrane integrity (103), and is
sensitive to free radical scavengers (13).


17
Ethylene and Defense
Ethylene Is involved in plant-pathogen relationships in
many ways. An attack by pathogens is a stress that often
leads to ethylene production (54, 64, 93). It has been
suggested that ethylene may be a signal for the plant to
activate biochemical defenses against potential pathogens
(30). This could be the hormonal function of stress
ethylene in that physical or chemical wounding provides
preferred entry sites for many pathogens (30).
Phytoalexins are compounds that accumulate in certain
plants in response to infection which generally hinder
pathogen growth (24). Their accumulation can be elicited by
true infection by living pathogens or by biotic factors such
as fungal enzymes, or plant and fungal cell wall fragments
(35, 51, 91, 154). In a few cases ethylene has been found
to act as an elicitor of phytoalexins. Wounded peas
produced pistatin in response to ethylene treatment (14).
Accumulation of glyceollin in soybean in response to a
fungal glucan elicitor was determined to some extent by
ethylene (94). An earlier report however claimed that
ethylene was not a messenger in the induction of this
particular phytoalexin (117). Potato slices treated with
ethrel (2-chloroethylphosphonic acid) and then innoculated
with a fungal pathogen accumulated more of the phytoalexins,
i.e.,phytuber in and pnytuberol, than slices not subjected to
ethrel treatment (75). Ethrel also induced production of a


18
phytoalexin compound in tobacco leaves (151). On the other
hand, the toxin, coronatine, produced by Pseudomonas
syringae elicited ethylene in bean leaf discs and caused
chlorosis whereas an unrelated pseudomonad phytotoxin, which
also causes chlorosis, did not stimulate ethylene production
(66) .
Ethylene has been reported to influence activities of
various enzymes. Pectinmethylesterase activity was
decreased in vitro with ethylene treatments (69) although
this was not supported by later studies. Ethylene induced
chitinase activity in bean and melon (31, 149). Wounding
and/or ethylene induced wound resistance to cellulase in oat
leaves (67).
Accumulation of hydroxyproline-rich glycoprotein (HRGP)
in muskmellon cell walls was shown to be induced by a
pathogenic fungus (59). This glycoprotein was later found
to be involved in the plant defense reaction to this
pathogen, imparting some resistance to invasion. The
increase in the HRGP was mediated through ethylene (60) and
inhibition of ethylene synthesis by AVG decreased both
ethylene and HRGP levels (150). A cell wall elicitor from
the pathogen was shown to cause inhibition of protein
synthesis after 18 hours, while promoting both ethylene and
HRGP production earlier in elicitor treated melon seedlings.
Ethylene may be involved in HRGP elicitation as ACC, the
precursor to ethylene which triggered HRGP synthesis


19
to the same extent as the fungal elicitor mentioned above
(134).
Ethylene and Cell Walls
Ethylene has often been associated with cell wall
lysing enzymes in cases of ripening (118, 121, 135, 139),
abscission (38, 71, 138), and pathogen invasion of plant
tissue (54, 64, 93). As discussed in the section on cell
wall function, recent evidence has shown that fungal cell
wall lysing enzymes can induce ethylene production. Various
naturally occurring carbohydrates stimulated ethylene
production in tobacco leaf discs and galactose promoted
ethylene evolution and ripening in tomato fruit (72, 112,
119). Ethylene has also been shown to be associated with
cell wall changes in terms of growth of rice and other
plants (85, 87, 98, 114, 127, 140).
Ethylene and Citrus
Citrus is a non-climacteric fruit, and produces only
low amounts of ethylene under normal conditions (164),
although small amounts of ethylene are produced in all
stages of development (128). Citrus fruit, however, can be
induced to produce relatively high levels of ethylene by
chemical or mechanical wounding and this in turn promotes
abscission (27, 63, 86, 164). The fact that citrus peel is
capable of producing substantial amounts of ethylene was


20
shown with peel disc explants (62, 63) and with whole fruits
on trees sprayed with abscission chemicals (27, 49, 97).
This is especially true of the albedo portion of citrus peel
(63, 64, 84). Yang and coworkers have shown citrus ethylene
production to be autoinhibitory in grapefruit flavedo (133)
and autocatalytic in orange leaves (132). Ethylene can
promote degreening in citrus fruit, causing c'nloroplasts to
convert to chromoplasts (156).


CHAPTER III
CELL-WALL DEPOLYMERASE ENZYMES AND ETHYLENE PRODUCTION
Introduction
Pathogen invasion of higher plant tissue usually
promotes ethylene production (30, 54) and often is
associated with cell wall lysing enzymes (143, 152, 142).
Similarly, there is a rise in ethylene production that
accompanies the activity of endogenous cell wall softening
enzymes during ripening of certain fruits (28, 47, 121, 139,
155) and cell-wall separation during abscission (131, 138).
In each case ethylene is associated with cell-wall degrading
enzymes of pathogen or plant origin. Furthermore, single
cells, isolated for protoplasmic fusion or for tissue
culture, are stimulated to produce ethylene by the enzymatic
mixtures used to separate the cells (11). Cell wall
digesting enzyme mixtures have also been shown to produce
ethylene in tobacco leaf discs as well as in other tissues
(10, 12, 39)
Little is known about the mechanism by which ethylene
is produced during the hypersensitive response in host
pathogen interactions. In order to further investigate this
21


22
process, cell-wall digesting enzymes, isolated from citrus
pathogens, and some commercial cell-wall lysing enzyme
mixtures were studied as to their effects on citrus cell
wall structure and ethylene production.
Similarities between the initiation of ethylene
production during pathogen induced hypersensivity and
abscission may also extend to its production during
ripening. In all cases ethylene is associated with cell
wall hydrolysis. Cell-wall lysing enzymes or the resulting
cell wall fragments may be general elicitors of ethylene
biosynthesis.
Materials and Methods
Fruit Injection Method and Ethylene Determination
Navel or Valencia oranges (Citrus sinensis (L.) Osb.)
were harvested at various times of the year with 4 cm stems.
Fruit stems were stripped of leaves and put in 20 ml aqua
pics (self-sealing tubes which maintain water around the cut
end of plant or flower stems). The fruit were stored in
portable coolers for transport to the laboratory.
Enzyme solutions of 10 or 20 pil containing 0.1% ,0.05%,
or 0.01% solutions (w/v) of commercial cell-wall lysing
enzyme mixtures in 10 mis phosphate, acetate, or citrate
buffer at various pH values were injected into orange peel


23
just under the flavedo at six locations around the equator
of the fruit using serum syringes with 25 gauge needles.
Some enzyme solutions were boiled and then cooled before
injection, while others were desalted on lyphogel (Gelman
Instrument Co.). Fruits, with stems in water, were then
placed in glass jars which were periodically capped for
ethylene determinations. Ethylene samples were taken at the
end of one or two hours by syringe and analyzed on a Hewlett
Packard flame ionization G.C. model #5706 A equipped with an
activated alumina column. Carrier gas flow rate was adjusted
to give a sharp ethylene peak that eluted at 0.4 minute
retention time.
Commercial cell-wall lysing enzyme mixtures used in
these experiments were cellulysin (Calbiochem), macerozyme
(Yakult Biochemicals, Japan), cellulase (Worthington),
pectolyase (Seichin Pharmaceutical, Japan and Sigma),
pectinmethylesterase (Sigma), drislase (Plenum Scientific),
and pectinase (Sigma).
Affinity Chromatography
Polygalacturonase (PC) was obtained from Charles
Barmore (CREC, Univ. of Fla., IFAS, Lake Alfred FI.) who
extracted it by the following procedure. Commercial orange
juice, innoculated with Geotrichum candidum or Diplodia
natalensis, was centrifuged and then 140 ml of supernatant
were loaded onto an alginate affinity column (alginic acid


24
cross-linked with epichlorohydrin, 1.6 x 5.5 cm). The
protein was eluted with acetate buffer (0.5 M at pH 5
containing 0.5 M NaCl). One milliliter fractions were
collected with the protein eluting in fraction #2-10 after
the void volume. All steps were carried out at 4C. The
protein recovered was assayed for PG activity by measuring
the release of reducing groups from polygalacturonic acid.
The glucose equivalents were determined by the procedure of
Nelson (115 ) .
In a similar manner as for G. candidum and D.
natalensis PG's, 200 ml of a 0.032 solution of pectolyase
(w/v) in acetate buffer (0.5 M at pH 5.0) was loaded onto an
alginate affinity column and 1 ml fractions were collected.
A protein was eluted with 0.5 M NaCl in fractions #1-6 and
10, similar to the elution of the previous fungal PG's.
Protein-containing fractions were assayed for ethylene
activity by injection of fraction material into the peel of
Valencia oranges, six 20 1 injections per fruit. Three
fractions that did not contain protein were also injected
into fruit for controls.
ACC Determination in Pectolyase-Treated Fruit
Valencia oranges were treated with 0.12 desalted
pectolyase in 0.1 M phosphate buffer, pH 6.0, or the same
buffer alone. Ethylene measurements were made four hours
after treatment following which the injection sites were


25
excised from the fruit with a 1 cm diameter cork borer. The
peel discs were then ground in ethanol and ACC was extracted
by the method of Lizada and Yang (107).
Cycloheximide Application
Valencia oranges were injected with 20 1 of 30 ppm
cycloheximide (CHI) or deionized water. One hour later the
same fruit were reinjected in the same areas with 0.11 mg
pectolyase solution in 0.1 M acetate buffer pH 5.0.
Ethylene measurements were made at 4, 8, and 24 hours after
the pectolyase treatment. Some fruits were injected with 30
ppm CHI alone or with boiled pectolyase.
PG Inhibitor
This protein was obtained from Charles Barmore (CREC,
Univ. of Florida, IFAS, Lake Alfred FI.) who extracted it
from Valencia orange albedo tissue, the white spongy portion
of the peel (17). The protein was purified by sephadex G-
100 column chromotography and inhibition of PG was assayed
by the liberation of reducing groups from polygalacturonic
acid as described above. This inhibitor was applied with
0.1^ pectolyase solution in 10 ml 0.1 M phosphate buffer, pH
5.0.
Treatment of Callus Tissue
Approximately 5 mg fresh weight of embryogenic Hamlin
orange callus was transferred under sterile conditons to an


26
agar nutrient media in 20 ml scintillation vials and allowed
to grow for a few days. The callus produced a wound ethylene
upon transfer which subsided after 1-2 days. After the
ethylene had subsided the callus was treated with 10 ul of
treatment solution, administered by auto-pipet with a
sterile tip. The treatment solutions included desalted Q.1%
pectolyase, cellulysin, or pectinase in 0.1 M phosphate
buffer pH 6.0, a boiled control for each enzyme treatment,
and phosphate buffer alone.
Electrophoresis
The commercial enzyme mixtures and the proteins isolated
from the fungal organims G. candidum and D. natalensis were
fractionated on 4^ stacking and 111 running SDS
polyacrylimide tube gels. These were run at a constant
current of 2 mA/gel for three hours after which they were
fixed and stained with coomassie blue.
Transmission Electron Microscopy
Green Valencia oranges, harvested 7/18/84, were treated
with injections of pectolyase, 0.11 in 0.1 M phosphate
buffer pH 6.0, or buffer alone. Fruit were then incubated
at room temperature for 24 hours during which the pectolyase-
treated group produced ethylene over and above the buffer
controls. After 24 hours the injection sites


27
were explanted in 1 mm squares and soaked in 0.075 M half
strength Karnovski fixative for 2 hours followed by 1 %
osmium tetraoxide for 2 hours. The tissue was then
dehydrated in an alchohol-acetone series and embedded in
Spur's resin (95). Thin sections were cut, applied to
forumvar coated copper grids and post-stained with lead and
uranyl acetate. Other sections on forumvar coated nickel
grids were treated for 25 minutes with a solution of 1%
periodate, washed in deionized water, and floated on a
solution of 37o methanamine, 5% silver nitrate, and 5% sodium
tetraborate for 40 minutes at 60C. The silver ions are
reduced to metalic silver by the aldehyde groups formed from
periodate oxidation of free hydroxyl groups on adjacent
carbon atoms, appearing as small black dots on micrographs.
This results in a fairly specific stain for polysaccharides
(95). These sections were later post-stained with lead and
uranyl acetate.
Results
Effect of Cell-Wall Lysing Enzymes on Ethylene Production
The commercial cell-wall lysing mixtures usually have a
high salt content which in itself caused the orange peel to
produce some wound ethylene (Fig. 3.1A). For this reason
desalting techniques or boiled enzyme controls were used to


28
Green Valencia oranges, harvested 10/30/84,
were treated with 6 x 10 (jl injections of
fungal cell-wall lysing enzymes in 0.1 M
acetate buffer pH 5.0. A) 0.1l pectolyase and
boiled pectolyase. B) G. candidum PG and
boiled P.G. All points are means of 3
replications +/- S.E.
Figure 3.1.


29
distinguish between ethylene produced in response to a
protein and wound ethylene due to salts or the possible
presence of other non-protein contaminants such as fungal
cell walls. Pectolyase stimulated the most ethylene above
boiled control levels in Valencia oranges 6 hours after
treatment (Fig. 3.1A). In Navel oranges the boiled enzyme
control also produced significant amounts of ethylene at 6
hours probably due to higher sensitivity of these oranges to
salts (Fig. 3.2A). Drislase (Fig. 3.2B) and the G. candidum
PG (Fig. 3.IB) also produced significantly more ethylene
than their boiled controls at 24 hours for drislase and 6
and 24 hours for the PG. However, pectolyase produced at
least 10 to 40 times more ethylene than either commercial
drislase or the purified fungal PG. A three-fold dilution
of pectolyase had little effect on ethylene production at 8
hours but produced slightly decreased ethylene levels at 24
hours (Fig. 3.3). This indicates that these concentrations
of pectolyase saturated the ethylene response to cell wall
dissolution at 8 hours, but that the diluted pectolyase
treatments became less than that necessary for a saturated
response at 24 hours. The cells in response to middle
lamellae dissolution by fungal enzymes, produced increasing
levels of ethylene which peaked around 6-8 hours after
treatment (Fig. 3.1A, 3.IB, 3.2A, and 3.2B). Although
ethylene levels then decreased by 24 hour (Fig. 3.1A, 3.IB,
3.2A, and 3.2B) in some treatments ethylene production was


ni C2H / Fruit / Hour
30
Hours After Treatment
Figure 3.2. Green Navel oranges, harvested 9/7/83, were
treated with 6 x 10 ¡al injections cell-wall
lysing enzymes in 0.1 M phosphate buffer pH
6.0. A) 0.1l pectolyase and boiled pectolyase.
B) 0.1Z drislase and boiled drislase. All
points are means of 3 replications +/- S.E.


ni C2H4 / Fruit / Hour
31
Figure 3.3. Green Navel oranges, harvested 8/15/83, were
treated with 6 x 10 ¡al injections of 0.1%,
0.05%, or 0.01% mg pectolyase in 0.1 M
phosphate buffer pH 6, and phosphate buffer
alone. All points are means of 3 replications
+/- S.E.


32
found to undergo a second rise by 48 hours after treatment
(Fig. 3.2A and 3.2B). This was associated with fruit
abscission, yellowing and general senesence, often occurring
in control fruit as well as treated. The optimum pH for
pectolyase-induced ethylene activity appeared to be around
pH 5.0 (Fig. 3.4) which is the optimum pH range reported for
several fungal PG's and a bacterial pectin lyase (17, 18,
51, 101, 157).
Cellulysin, macerozyme, cellulase, pectinase, and the
PG from D. natalensis did not produce ethylene levels above
that of their boiled or buffer controls at the
concentrations tested (data not shown). Desalted
pectinmethylesterase (PME) did produce small amounts of
ethylene above a buffer control (Fig. 3.5B), although much
less than that produced by pectolyase (Fig. 3.5A). This may
be due to changes in free calcium levels and pH as a result
of pectin desterification, to a low salt content still
present in the enzyme treatment solution, or to
contamination by other enzymes, possibly cell wall lysing in
nature.
Effect of Pectolyase on the Ethylene Pathway and Cell Walls
Pectolyase stimulated an increase in ACC levels (Fig.
3.6), a precursor to ethylene (161), as well as ethylene
indicating that it stimulates the ethylene pathway at some
point before ACC synthesis, possibly at ACC synthase. CHI,


pH of Pectolyase Treatment Solutions
Figure 3.4. Green Valencia oranges, harvested 8/7/85, were
treated with 6 x 20 |al injections of O.lt
pectolyase in 0.1 M citrate-phosphate buffer at
different pH's, and boiled pectolyase in same
buffer at pH 5. All bars are means of 3
replications +/- S.E.


34
igure 3.5. Green Valencia oranges, harvested 7/30/34, were
treated with 6 x 10 |jl injections of cell-wall
lysing enzymes in 0.1 M phosphate buffer pH
6.0. A) 0.17o pectolyase and boiled pectolyase.
3) 0.17o pectinmethylesterase, and boiled
pectinmethylesterase (PME). Enzyme solutions
had been desalted on lyphogel. All points are
means of 3 reps + /- S.E.


nMole ACC / 6 Injection Sites/Fruit
35
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
L.
O)
k-
O)
O)

O)

**-
re
H
re
3
3
CO
"o
CO
o
+*
CJ
V
OJ
Q)
O.
Q.
Figure 3.6. Green Valencia oranges, harvested 8/23/84, were
treated with 6 x 10 ial injections of either
0.1% desalted pectolyase, in 0.1 M phosphate
buffer pH 6, or 0.1 M phosphate buffer alone.
Ethylene production was determined and peel
injection sites were analyzed for ACC content
4 hours after treatment. Bars are means of 3
replications +/- S.E.


36
a reported protein inhibitor (43, 46, 61), by contrast,
decreased pectolyase-induced ethylene production by over one-
half at 4 hours after treatment. This inhibition was less
effective as the tissue recovered from the wound ethylene
response at 8 and 24 hours (Fig. 3.7). Also demonstrated
here is the difference between immature and mature fruit
response to pectolyase. In previous experiments with mature
fruit, the pectolyase-induced ethylene production peaked at
around 6-8 hours after treatment with the enzymes. In this
case with immature fruit, pectolyase-induced ethylene
production peaks at or before 4 hours after treatment and
the levels of ethylene are somewhat higher than those levels
observed for mature fruit. In effect, the immature fruit
respond more rapidly to pectolyase treatment, the magnitude
of the response is greater, and the recover is faster than
that for mature fruit.
PG Inhibitor and Pectolyase-induced Ethylene Production
Since the PG from G. candidum induced ethylene
production, a hypothesis was formulated that an ethylene
promoting protein in the pectolyase preparation might be a
pectin-digesting enzyme. This was tested by mixing the PG.
inhibitor with pectolyase. The inhibitor did significantly
decrease the amount of ethylene produced by the fruit in
response to pectolyase at 8 hours after treatment (Fig.
3.8), although not below the level of the boiled control. A


37
Hours After Treatment
Figure 3.7. Green Valencia oranges, harvested 9/30/85, were
treated with 6 x 20 |jl injections of 30 ppm CHI
+ 0.11 pectolyase in 0.1 M acetate buffer pH 5,
D.I. water + 1 mg pectolyase/10 ml same buffer,
30 ppm CHI alone, or boiled pectolyase. All
points are means of 3 replications + /- S.E.


ni C.H. / Fruit / Hour
38
Figure 3.8. Yellow-green Valencia oranges, harvested
1/8/85, were treated with 6 x 10 ul of 0.1?
pectolyase, boiled pectolyase, and pectolyase +
a PG inhibitor in 0.1 M phosphate buffer pH 6.
All points are means of 3 replications +/- S.E.


39
protein then was isolated by affinity chromotography from
pectolyase which caused increased levels of ethylene to be
produced compared to controls (Fig. 3.9), but not within the
range of pectolyase. However, the activity and the
concentration of this protein and the fungal PG's, isolated
by affinity chromotography, may have been too low for an
ethylene response such as was found with pectolyase. On the
other hand, there may be some other protein(s) in the
commercial mix which are not cell wall-lysing enzymes or
were not eluted from the affinity column. These may be
causing some of the ethylene production or may be necessary
to enhance the activity of the pectin-digesting enzyme.
SDS Gel Profiles of Cell-Mall Lysing Enzymes
SDS polyacrylimide gel electrophoresis shows complex
protein profiles for the different enzyme mixtures (Fig.
3.10). The ethylene-inducing PG isolated from G. candidum
did, however, correspond to a similar molecular weight
protein of around 60,000 D in pectolyase (Fig. 3.11).
However it is not known which of these bands in pectolyase
are responsible to inducing ethylene production.
Transmission Electron Microscopy of Digested Cell Walls
The TEM micrographs of desalted pectolyase treated
orange peel show middle lamellae digestion (Fig. 3.12 and
3.13) whereas buffer-treated tissues show no signs of cell


Figure 3.9. Yellow Valencia oranges, harvested 2/27/85,
were treated with protein-containing fractions
#1-6, and 10, and control fractions #13, 21,
and 24 which did not contain protein, from an
alginate affinity column. Six x 20 |ul
injections were administered/fruit with 3 fruit
replications +/- S.E.


41


42
A B C D E F G
II ;|i
Figure 3.10. Coomassie blue-stained 111 polyacrylimide gels
of different enzyme preparations used in
protoplast isolation.
A)standards: bovine albumin, 66,000 D; egg
allbumin,
45,000 D; glyceraldehyde-3-P-dehydrogenase,
36,000 D;
carbonic anhydrase, 29,000 D;
trypsinogen,24,000 D;
trypsin inhibitor, 20,000 D; a-lactalbumin,
14,000 D.
B) cellulase
C) PME
D) pectolyase
E) cellulysin
F) drislase
G) pectinase


Figure 3.13. Transmission micrograph of orange peel tissue
treated with 10 pi of 0.1 % pectolyase in 0.1 M
phosphate buffer pH 6, 24 hours after
treatment. These sections were later treated
with periodate and then a silvermethanamine
solution tor polysaccharide staining.
ML=middle lamellae at 90,000X.


44
Figure 3.12. Transmission micrograph of orange peel tissue
treated with 10 pil of 0.1Z pectolyase in 0.1 M
phosphate buffer pH 6, 24 hours after
treatment. ML=middle Lamellae at 26,000X.


45
Figure 3.13. Transmission micrograph of orange peel tissue
treated with 10 |ul of 0.1 % pectolyase in 0.1 M
phosphate buffer pH 6, 24 hours after
treatment. These sections were later treated
with periodate and then a silvermethanamine
solution for polysaccharide staining.
ML=middle lamellae at 90,000X.


46
wall breakdown (Fig. 3.14). This type of digestion would be
expected from a pectin-digesting enzyme such as a PG.
Callus tissue, treated with pectolyase, did not produce any
ethylene above boiled control levels although TEM studies
showed middle lamellae digestion (data not shown). This is
interesting because the callus produced wound ethylene upon
transfer into the experimental vials, showing that the cells
were capable of a wound ethylene response. It may be that
these cells are not able to perceive signals of cell wall
breakdown. The silver stained pectolyase-treated sections
of citrus peel show the digested area clearly as there is
little polysaccharide silver staining along the middle
lamellae area (Fig. 13).
Conclusion
Pectolyase appears to induce ACC synthesis and
subsequent ethylene production due to a pectin degrading
enzyme either directly or indirectly through some cell-wall
fragment product. Protein synthesis seems to be necessary
for optimal ethylene production induced by pectolyase.
Fungal PG' s and bacterial pectin lyases have been reported
to be elicitors of phytoalexins in plant tissues (101, 102)
often via their plant cell wall oligomeric products (35, 52,
53, 116). In addition, fungal cell walls have been
implicated as elicitors of an ethylene signal that was
linked to phytoalexin production in soybean (117). Finally,


47
Figure 3.14. Transmission micrograph of orange peel tissue
treated with 10 ul of 0.1 M phosphate buffer pH
6, 24 hours after treatment. CW=cell wall at
34,000X.


48
ethylene has been shown to have a diverse role in plant
defense responses ranging from effecting wound-induced
resistance to cellulase in oat leaves (68) to increased
chitinase activity in bean (31) and melon (149), to
hydroxyproline-rich glycoprotein accumulation in diseased
plants (60, 150), and phytoalexin accumulation in tobacco
(151), potato (75), and other plants (14). It is possible
that ethylene has a role in the initiation of a defense
response in citrus.


CHAPTER IV
PRODUCTS OF CELL-WALL DEGRADATION ELICIT ETHYLENE IN CTRUS
Introduction
Cell wall changes and ethylene production are highly
correlated in many physiological events such as mechanical
or chemical wounding (62, 63, 146) pathogen invasion (54,
64, 93), cell wall softening in ripening of certain fruits
(5, 26, 106, 118, 121), abscission (71, 93, 129, 131, 138),
and growth (98, 114, 127, 140). A cause and effect
relationship between physiological cell wall changes and
ethylene evolution has not been established. It may be that
they are unrelated products of a common stimulus or
interdependent events. Albersheim and others have shown
that the primary structures of the cell-wall polymers are
exceedingly complex (22, 89, 143). This enormous structural
complexity lead to the postulation that the cell-wall
polysaccharides may have regulatory functions in plant cells
via genomic expression (15). Such carbohydrates may control
various physiological responses of plants such as rate of
cell growth, time of flowering, activation mechanisms for
resistance to potential pathogens (112), and perhaps
ripening (80).
49


50
Single cells isolated for protoplasmic fusion or for
tissue culture are sometimes stimulated to produce ehylene
by the enzymatic mixtures used to separate the cells (11).
Cellulysin and macerase have been reported to induce
ethylene in tobacco and pear suspension cells respectively
(40, 73, 147). Preliminary studies were also conducted on
fruit ethylene production in response to partially digested
citrus pectin and fungal enzymes (99). Castor bean explants
produced ethylene when exposed to a mixture of enzymes or
purified polygalacturonase (PG), isolated from the fungus
Fusarum oxysporum (152). Fungal cell walls were shown to
elicit ethylene and phytoalexins in soybean (117). Cell
wall digesting enzymes and the resulting fungal or plant
cell wall fragments may regulate ethylene synthesis in
certain tissues. Regulation of ethylene by cell wall
digestion products would be a possible mechanism by which
plant cells could recognize pathogen invasion. Ethylene has
been shown to stimulate certain plant defense reactions such
as activation of chitinase (31, 149), wound-induced
resistance to cellulase (67, 68), and induction of
phytoalexins (9, 75, 151). In melons, both fungal and plant
cell wall elicitors induced synthesis of ethylene which in
turn elicited hydroxyproline-rich glycoprotein (HRGP).
Ethylene has been implicated in the accumulation of HRGP in
healthy (60) and infected plants (150). Finally,
carbohydrates, such as galactose, sucrose, and lactose, have


51
been shown to stimulate ethylene production in tobacco (112,
119) and galactose promoted ethylene evolution as well as
ripening in tomatoes (72).
This study is an investigation of the mechanism by
which certain enzyme mixtures induce ethylene production in
citrus peel. It was found that such enzymatic action on
cell walls may elicit ethylene indirectly via oligomeric
products.
Materials and Methods
Fruit Injection Method and Ethylene Determination
Valencia oranges (Citrus sinensis (L.) Osb.) were
harvested at various times of the year with 4 cm stems.
Fruit stems were stripped of leaves and put in 20 ml aqua
pics (self-sealing tubes which maintain water around the cut
end of plant or flower stems). The fruit were stored in
portable coolers for transport to the laboratory.
Treatment solutions of 20 |jl, containing carbohydrate,
enzyme, or carbohydrate plus enzyme solutions, were injected
into orange peel just under the flavedo at six locations
around the equator of the fruit. Some of these treatment
solutions were boiled and then cooled before injection.
Fruits, with stems in water, were then placed in glass jars
which were periodically capped for ethylene determinations.


52
Ethylene samples were taken at the end of two hours by
syringe and analyzed on a Hewlett Packard flame ionization
G.C., model #5706 A equipped with an activated alumina
column. Carrier gas flow rate was adjusted to give a sharp
ethylene peak that eluted at 0.4 minute retention time.
The carbohydrate solutions used to inject orange peel
contained either partially hydrolyzed commercial orange
pectin in deionized water (polygalacturonic acid methyl
ester from citrus fruits, grade I, Sigma), different size
oligomers of gel filtrated partially hydrolyzed pectin in 1
mM NaCl, sodium polypectate (polygalacturonic acid sodium
salt, a commercial preparation of de-esterified pectin,
Sigma), sodium polypectate digested for different time
periods by 0.2% or 0.4% pectolyase (w/v) in 0.1 M acetate
buffer pH 5.0, albedo pectin isolated from freshly harvested
Valencia oranges digested for different time periods by 0.2%
or 0.4% pectolyase in 0.2 M acetate buffer pH 5.0, pectin
oligomers obtained from an albedo pectin-pectolyase digest
via gel filtration in 30 mM acetate buffer pH 5.0 with 5 mM
disodium EDTA, a 5% chitin solution (Sigma) in 0.1 M acetate
buffer pH 5.6, chitin digested for 4 hours with a 0.05%
solution of chitinase (Sigma) in same buffer, 0.1 or 0.2 M
acetate buffer pH 5.0, 30 mM same buffer with 5 mM disodium
EDTA, 0.1% pectolyase (w/v) in 0.1 M same buffer, and boiled
pectolyase.


53
Preparation of Crude Pectin Hydrolysate
Five grams of commercial orange pectin were partially
hydrolyzed in 2 N trifluoracetic acid by the method of
Nothnagel et al. (116) at a concentration of 10 mg pectin
per ml acid. Suspensions were refluxed in an 85C water
bath for 4 hours, then cooled and passed through a Whatman
GF/A glass microfiber filter. The resulting retntate was
suspended in methanol at approximately 1:1 (v/v) and
evaporated to dryness. This proceedure was repeated a total
of four times. The final dried solids were suspended in
deionized water (10 ml water per initial gram of pectin) and
titrated to pH 7.0 with 5 M imidizole base. The resulting
suspension was cleared of particulates by filtration through
a Whatman GF/A millipore type HA 0.45 piM filter. The final
filtrate was the crude "pectin hydrolysate" used to inject
the fruit. Some of this material was boiled for five
minutes and cooled before injection into fruit.
"Unhydrolyzed pectin" consisted of 5 g of commercial orange
pectin to which imidizole base had been added in the same
amount that was used to neutralize the hydrolyzed pectin.
The resulting suspension was then titrated to pH 7.0 by
trifluoroacetic acid and injected into fruit.
Preparation of Carbohydrate-Enzyme Digest Treatments
One milliliter of a 20^ solution of Polygalacturonic
acid in 0.1 M acetate buffer pH 5.0 was incubated at 30C


54
with 1 ml of a 0.2% or 0.4% solution of pectolyase or boiled
pectolyase (w/v) in 0.2 M same buffer. All incubation
mixtures were then boiled at the end of either one or two
hours incubation time to stop all enzyme activity. The
mixtures were filtered through a Whatman #1 filter and the
resulting filtrate was used to inject fruit.
In a similar experiment pectin was extracted from
albedo tissue of Valencia oranges and incubated with
pectolyase. Two grams of chopped albedo tissue were ground
for five minutes with a Tekmar homogenizer in 10 ml cold
acetone and vacuum filtered through a Whatman #1 filter.
The solids were then re-homogenized and filtered again.
This step was repeated for a total of three times after
which the acetone was evaporated from the resulting powder.
The powder was stirred overnight in 40 mM acetate
buffer pH 5.0, containing 1 mM disodium EDTA at room
temperature. This suspension was then centrifuged at 27,000
g-max for 15 minutes and the supernatant was discarded. Two
and one-half grams of pellet material were homogenized with
30 ml of a 0.2% or 0.4% (w/v) pectolyase or boiled
pectolyase in 0.2 M acetate buffer pH 5 and incubated for
0.5, 1, 1.5, or 3 hours at 30C. At the end of the
appropriate incubation period, all mixtures were boiled and
filtered as described above and injected into fruit.
A 5% chitin (Sigma) solution (w/v) in 0.1 M acetate
buffer, pH 5.6 was incubated with 1 ml of a solution


55
containing 0.5!? chitinase (w/v) or boiLed chitinase (Sigma)
in same buffer for 4 hours. At the end of the incubation
period all mixtures were boiled to stop enzymic activity and
injected into fruit. Some fruit were also injected with a
5% chitin solution in 0.1 M acetate buffer pH 5.6 or buffer
alone .
Gel Filtration of Pectin Materials
The 5 g of partially acid-hydrolyzed pectin material,
which had been taken up in 50 ml of deionized water, was
concentrated to 7 ml by rotary evaporation. This material
was then passed through a 40 x 1.5 cm G-25 Sephadex column
eluted with 0.1 M NaCl. The void and 2 ml fractions of
included material were collected and injected into fruit (6
injections/fruit of 20 pi each). Every two fractions of the
included volume were pooled before injection.
The fractions most active in eliciting ethylene
production in fruit from the G-25 Sephadex column and pectin-
enzyme digest solutions, were each applied to a 46 x 1.5 cm
Bio-gel P-2 or Bio-gel P-4 column. The columns were washed
with 0.1 M NaCl or 30 mM sodium acetate buffer with 5 mM
disodium EDTA and 1 ml fractions were collected. These
fractions were tested colormetrically for total sugar
content by the phenol-sulfuric acid method (56) or for acid
sugar content by the Blumenkranz-Asboe-Hansen methods (29).
In some cases every 3 fractions, of those found to contain


56
sugar, were pooled and injected into fruit (6
injections/fruit of 20 |ul each) to assay for ethylene
inducing activity. In other cases, the void and individual
sugar peaks were pooled before injection. The P-2 column
had been calibrated with galacturonic acid, cellibiose,
raffinose and stachiose to determine the elution profile of
up to 4 sugar units.
Analysis of Neutral Sugars
Pooled sugar fractions from the P-2 and P-4 column
material were analyzed for neutral sugar content as
described by Albersheim et al. (7). Fraction material,
containing 0.3-1.0 mg of total acid sugar (29), was
hydrolyzed to aldoses and reduced to alditols by
trifluoroacetic acid and sodium borohydride, respectively.
The sugars were then acetylated by acetic anhydride using
pyridine as a catalyst. The acetylated sugars were analyzed
on a Hewlett Packard G.C. model #5710 A with a Spelco 23-40
column. Rhamnose, xylose, arabinose, mannose, galactose,
and inositol standards were used to determine retention
times. Inositol also served as the internal standard.


57
Results
Acid Hydrolyzed Pectin
The partially acid hydrolyzed pectin material
dramatically induced ethylene production over and above that
produced by the 0.1% pectolyase (w/v) treatment by 24 hours
after injection (Fig. 4.1). The pattern of ethylene
induction was different from that of pectolyase in that the
cells did not respond as quickly in terms of ethylene
production and did not show signs of decreasing ethylene
production by 24 hours after treatment. It may be that
fragments generated in the peel by pectolyase were more
localized, less concentrated and therefore metabolized more
quickly than the hydrolyzed pectin material which was
injected into the fruit. Also the concentration of
fragments generated by pectolyase may have been less than
those in the hydrolyzed pectin solutions. Boiling the
hydrolyzed pectin material caused a slight decrease in its
ability to elicit ethylene. This may have been due to
further hydrolysis of the fragment population or to
denaturation of possible ethylene-inducing proteins
contained in the pectin material. The unhydrolyzed pectin
produced very little ethylene, less than that of the boiled
pectolyase control. Pectolyase produced more ethylene than
the hydrolyzed pectin material only at 4 hours, and more
ethylene than the boiled control at all cime periods.


58
Figure 4.1. Yellow Valencia oranges, harvested 2/26/85,
were treated with 6 x 20 |al injections of crude
orange pectin hydrolysate, boiled crude
hydrolysate, unnydrolyzed pectin, Q.1%
pectolyase (w/v) in 0.1 M acetate buffer pH 5.0
and boiled pectolyase. Ethylene measurements
were taken at 4, 8, and 24 hours after
treatment. All points are means of 3
replications +/- S.E.


59
Gel filtration of the partially acid-hydrolyzed pectin
on a G-25 Sephadex column produced some fractions that
showed greater ethylene-inducing capacity than others at 4
and 24 hours after treatment (Fig. 4.2A and B). Some of the
fractions produced almost as much ethylene as pectolyase at
4 hours (Fig. 4.2A) and much more ethylene at 24 hours (Fig.
4.2B). Interestingly, fractions that induced the most
ethylene at 4 hours were surpassed in ethylene elicition
capacity by other fractions, which contained smaller sugar
fragments, at 24 hours. The most active ethylene-inducing
fraction at 4 hours (F#7-8) and the most active ethylene-
inducing fraction at 24 hours (F#ll-12) were then each
chromatographed on a Bio-gel P-2 column. The resulting
fractions were analyzed colorimetrically for acid sugar
content. Every three fractions from sugar peaks were pooled
before injection into fruit, including some non-sugar
control fractions. These pooled fractions were injected
into fruit and ethylene production was determined 4 hours
after injection. Certain fractions induced more ethylene
synthesis than others, although none produced as much as the
original crude pectin hydrolysate or G-25 materials. The
Biogel P-2 fractions, #32-34, of the G-25 7 and 8 fraction
material, correspond roughly to a nona or decasaccaride and
produced the most ethylene, yet contained a relatively low
amount of sugar (Fig. 4.3). The P-2 fractions, #44-46, of
the G-25 11 and 12 fraction material, correspond to a


Figure 4.2. Yellow Valencia oranges, harvested 3/19/85,
were treated with 6 x 20 ¡ul injections of
pooled fractions containing crude pectin
hydrolysate material that had been passed
through a G-25 Sephadex column washed with 0.1
M NaCl. Ethylene measurements were taken at 4
and 24 hours after treatment. All points are
means of 3 replications +/-S.E.


/ FRUIF / 2 HRS
51
TREATMENT


62
Figure 4.3. Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 ¡al injections of sugar-
containing fractions resulting from gel
filtration of the G-25 Sephadex fractions 7 and
8 on a Bio-gel P-2 column washed with 0.1 M
NaCl. The fractions were tested for total
sugar content by the phenol-sulfuric acid
assay. Every 3 fractions, of those found to
contain sugar, as well as 3 non-sugar control
fractions were pooled before injection into
fruit. Ethylene measurements were made at 4
hours after treatment and all ethylene
measurements are means of 3 replications +/-
S.E.


63
pentasaccharide and also produced slightly more ethylene
than the non-sugar control, yet contained a relatively low
amount of sugar (Fig. 4.4). The large mono, disaccharide
peaks in comparison induced less ethylene than the non-sugar
control fractions, indicating that ethylene production was
not determined by total sugar concentration or osmotic
effects .
Pectin-Digestion End Products
Both pectolyase and certain pectolyase-pectin digestion
mixtures induced ethylene production in citrus peel. The
digestion mixtures had been boiled before injection into the
fruit so that their ethylene-inducing activity was not due
to active enzymes, but to the enzyme digestion products and
salts. The amount of enzyme present and the length of the
digestion period altered the ethylene-inducing activity of
these mixtures. A 0.2% solution of pectolyase (w/v)
incubated with polygalacturonic acid (PGA) for 2 hours
induced more ethylene production at 4 and 8 hours after
treatment than either its boiled control or a 0.4^ solution
of pectolyase incubated with PGA for one hour (Fig. 4.5).
The latter solution induced ethylene levels above its boiled
control only at 24 hours.
Pectolyase digestion of albedo pectin showed the most
dramatic inducement of ethylene production dependent upon
amount of enzyme and length of digestion period. A 0.4^


64
I
2.0-
1.8-
>
<
v 16-1
tfi 1-01
^ CD
1.4-1
E -
3 03 1.2-
u-<
3 1.0-1
(/)
0.8-
0.6-
0.4
0.2
o
z
Ui
x
.a.
VOID
A
18
26
ACID HYDROLYSIS OF PECTIN
MONO-OI
/
Y
I,
34 42 50 58
P-2 FRACTION NUMBERS
-32
-24
9
f

o
PENT* T"RA TR) / i
i
34 42 50 58 66
-8
74
Figure 4.4. Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 ;ul injections of sugar-
containing fractions resulting from gel
filtration of the G-25 Sephadex fractions 11
and 12 on a Bio-gel P-2 column washed with 0.1
M NaCl. Every 3 fractions, of those found to
contain sugar as well as 3 non-sugar control
fractions, were pooled before injection into
fruit. 3 non-sugar fractions were also pooled
for a control. Ethylene measurements were made
at 4 hours after treatment and all ethylene
measurements are means of 3 replications +/-
S.E.
nl C2H4/ FRUIT / 2 HRS.


65
Figure 4.5. Regreened Valencia oranges, harvested 5/6/85,
were treated with 6 x 20 (jl injections of a 201
solution of polygalacturonase in 0.1 M acetate
buffer pH 5.0, incubated with 0.22 or 0.42
pectolyase (w/v) or boiled pectolyase in same
buffer for 1 and 2 hours respectively.
Ethylene measurements were taken at 4, 8, and
24 hours after treatment and all points are
means of 3 replications + /- S.E.


66
pectolyase solution (w/v) induced large amounts of ethylene
4 hours after treatment when incubated with albedo pectin
for 1 hour, over double that of the boiled control (Fig.
4.6A). The same solution, however, failed to induce much
ethylene above boiled controls when incubated with albedo
pectin for either 0.5, or 1.5 hours. Similarly a 0.2%
solution of pectolyase induced more ethylene when incubated
with albedo pectin for 1 or 2 hours than at 3 hours at both
time periods tested (Fig. 4.6A and B). All incubation
periods, in this case, resulted in ethylene production above
that of the corresponding boiled controls. A 0.1% pectolyase
solution (w/v) alone induced more ethylene than any of the
enzyme-pectin digest mixtures.
The most active ethylene-inducing enzyme-pectin digest
mixtures (0.4% pectolyase, incubated with 2.5 g albedo
pectin for 1 hr and 0.2% pectolyase, incubated with 2.5 g
albedo pectin for 1 and 2 hours) were pooled and
chromatographed on a Bio-gel P-2 column. The resulting void
and sugar peaks were individually pooled and aliquots
injected into fruit. The void (F#24-27) and a shoulder off
the void (F#28 32 ) corresponding to around 9-10
galacturonic acid units, appeared to contain the most
ethylene-inducing capacity (Fig. 4.7), although ethylene
levels were relatively low when compared to the enzyme-
pectin digest mixtures (Fig. 4.6). Salts, present in the
pectolyase enzyme mixture, co-eluted with the


Figure 4.6. Green Valencia oranges, harvested 7/16/85, were
treated with 6 x 20 jul injections of various
albedo pectin-pectolyase digest mixtures. 2.5
g of extracted albedo pectin was incubated with
0.4Z or 0.2Z pectolyase (w/v) or boiled
pectolyase in 0.2 M acetate buffer pH 5, for
0.5, 1.0, 1.5, 2.0, or 3.0 hours. Ethylene
measurements were made at 4 (A) or 8 (B) hours
after treatment and all bars are means of 3
replications +/- S.E.


O o
900*'
PECTOLYASE DIGESTION OF ALBEDO PECTIN
4 HOURS
Traf ment


69
Figure 4.7. Green Valencia oranges, harvested 9/10/85, were
treated with 6 x 20 pi injections of sugar-
containing fractions resulting from gel
filtration of pooled albedo pectin-pectolyase
digest mixtures (albedo pectin digested by 0.4^
pectolyase for 1 hour and by 0.2% pectolyase or
1 and 2 hours) on a Bio-gel P-2 column washed
with 30 mM acetate buffer pH 5.0 with 5 mM
disodium EDTA. The void, individual sugar
peaks, and 3 non-sugar fraction controls were
pooled before injection into fruit. Ethylene
measurements were made at 4 hours after
injection and all ethylene measurements are
means of 3 replications +/- S.E.


70
monosaccharides and are probably contributing to the
ethylene production observed for this small peak. Since the
ethylene inducing sugar peak was not well separated from the
void, the same material was chromatographed on a Bio-gel P-4
column. The resulting sugar profile was tested for ethylene
production as described before. Again, one sugar peak (F #
43-51) induced slightly more ethylene than the rest (Fig.
4.8). Ethylene production, expressed per gram of sugar
injected into fruit for sugar peaks from both columns, shows
more clearly that certain P-2 and P-4 fractions elicited
more ethylene than others (Table 4.1).
Analysis of Neutral Sugar Content
Analysis of neutral sugar content for both the Bio-gel
P-2 and P-4 column material (Table 4.2 and 4.3) showed that
rhamnose, arabinose, xylose, and galactose were detected
with arabinose, galactose, and xylose occurring in the
highest amounts. The fractions which showed the most
ethylene-inducing activity contained relatively high amounts
of arabinose and galactose, small amounts of rhamnose and
xylose, and trace amounts of mannose and another unknown
sugar.
Effect of Chitin on Ethylene Production
A 57c solution of chitin (w/v) in acetate buffer induced
a small amount of ethylene production at 8 and 24 hours


ACID SUGAR ASSAY
ABS. 520
71
Figure 4.8. Green Valencia oranges, harvested 9/30/85, were
treated with 6 20 ¡al injections of sugar-
containing fractions resulting .from gel
filtration of pooled albedo pectin-pectolyase
digest mixtures (albedo pectin digested by 0.4^
pectolyase for 1 hour and by 0.2% pectolyase
for 1 and 2 hours) on a Bio-gel P-4 column
washed with 30 mM acetate buffer Ph 5.0 with 5
mM disodium EDTA. The void, individual sugar
peaks and 3 non-sugar fraction controls were
pooled before injection into fruit. Ethylene
measurements were made at 4 hours after
treatment and all ethylene measurements are
means of 3 replications +/- S.E.
nl C,H4 / FRUIT/2 HRS.


72
Tabla 4.1. Green Valencia oranges, harvested 9/10/85 or
9/30/85, were treated with 6 x 20 i_il injections
of sugar containing fractions resulting from
gel filtration of pooled ethylene-inducing
albedo pec tin-pec tolyase digest mixtures on Bio
gel P-2 or P-4 columns. Ethylene measurements
were made at 4 and 8 hours after treatment for
tne P-2 and P-4 column materials respectively.
The resulting ethylene levels in this table
reflect only the ethylene produced above
control levels.
Treatment
4 Hrs after Treatment
P-2 F# Sue.(ug)/Fru.
nl C.-jH^/fruit/2 hr nl CoH^/10ug Sug.
24-27
67.2
23-31
73.2
32-35
27.9
39-46
10.8
7.8
11.6
0.6
5.0
4.4
5.
0.
3.
P-4
£7
f TT
8 Hrs
27-34
14.7
43-51
17.1
52-62
33.0
63-67
14.7
0.6
6.4
0.0
0.4
0.4
3.2
0.0
0.3
after Treatment
U> O'


73
Table 4.2. Neutral and acid sugar analysis was made on the
albedo pectin-pectolyase digest material that
had been fractionated on a Bio-gel P-2 column.
Fractions comprising sugar peaks were pooled
for analysis of sugar content.
P-4 F# MOLE ACID SUG. MOLE NEUTRAL SUG. NS MOLE? of AS
43-51
4x10 6 ARABINOSE
2x10 l
5.0?
GALACTOSE
7x10
18.0?
Trace amts: xylose,
mannose, and
unk.sug.
52-62
6xl0_6 ARABINOSE
XYLOSE
GALACTOSE
2x10
3x10 7
9x10 7
4.0?
5.0?
16.0?
Trace amts: mannose
and unk. sug.
63-67
2xl0'6 ARABINOSE
GALACTOSE
2xl0-10
2x10 1U
12.0?
0.01?
Trace amts: xylose and unk.
sug.


74
Table 4.3. Neutral and acid sugar analysis was made on the
albedo pectin-pectolyase digest material that
had been fractionated on a Bio-gel P-4 column.
Fractions comprising sugar peaks were pooled
for analysis of sugar content.
P-2 F#
MOLE ACID SUGAR.
MOLE NEUTRAL SUG.
NS MOLEZ of AS
28-31
7xlO_6
ARABINOSE 6xl0_^
RHAMNOSE 6x10 5
8.82
0.92
XYLOSE 2x10 '
1.42
GALACTOSE 3x10
4.42
Trace amts unk. sug.
32-35 3x10 6 ARABINOSE 2xl0_^ 6.22
XYLOSE 2x10 6.22
GALACTOSE 1x10 3.12
Trace amts unk. sug.
39-46
3x10 6 ARABINOSE 2xlO_5?
XYLOSE 1x10 s
0.52
0.4 2


75
after injection that was slightly higher than a buffer
control (Fig. 4.9B). Digestion of the chitin with chitinase
for 4 hours, however, increased ethylene production over
four times that of the boiled control at 8 hours after
treatment (Fig. 4.9A). The chitinase itself showed some
ethylene-inducing activity, probably due to salts present in
the enzyme preparation.
Conclusion
Products of cell wall digestion, whether by acid or
enzymes, were capable of eliciting ethylene production in
citrus peel of intact citrus fruit. Data indicate that more
material is needed of specific fragment size or size range
to induce the level of ethylene such as is observed for
pectolyase. The response to pectolyase, however, may not be
due solely to cell-wall lysing enzymes. Salts in the
pectolyase mixture also induce some wound ethylene, and
lysis of cell walls could promote leakage of toxic materials
from oil glands in citrus peel which may promote ethylene
production in neighboring cells. Since activity is lost as
the sugar materials are further separated into their
corresponding fragment sizes, it is possible that certain
oligomers act synergistically in elicitation of ethylene
production. Albersheim and others have noted that both
fungal and plant cell wall fragments elicit phytoalexin
production in several plant species (91, 116, 154). It has


ni C2H4 / FRUIT / 2 HRS.
76
Regre
ened
Valencia
orang
es, harves
t
ed 5/6/85,
were
treat
ed with
6 x 20
(u inject
i
ons of a 5%
chiti
n sol
ution (w
/v), i
n 0.1 M ac
e
tate buffer
pH 5.
6, incubated
with a
0.5Z solut
ion of
chiti
nase
(w/v) or
boile
d chitinas
e
in same
buf f e
r for
4 hours
, or 0
.5Z Chitin
a
se alone
(A) .
Othe
r fruit
were i
njected wi
th a 0.57o
solut
ion o
f chitin
in sa
me buffer,
or buffer
alone
(B) .
Ethyle
ne mea
surements
were made at
4, 8,
and
24 hours
after
treatment
and all
point
s are
means of 3 re
plications
+/- S.E.


77
also been suggested that fungal and plant cell wall
fragments may act synergistically in elicition of
phytoalexin production in soybeans (48, 159). It would be
interesting to investigate whether these oligomeric products
of cell wall digestion induce synthesis of new mRNA or
protein, such as ACC synthase, that result in increased
ethylene production. The ethylene, in turn, promotes
chlorophyll breakdown (59) and abscission (71, 120) in
citrus fruits. If this also involves synthesis in new mRNA
and protein then a complex model emerges for plant detection
and response to fungal invasion. Similar processes may
occur in cases of ripening and abscission.


CHAPTER V
STRUCTURAL AND BIOCHEMICAL DEFENSE MECHANISMS IN CITRUS
Introduction
A Citrus fruit is considered to be a berry of unusual
structure (8). The thick peel includes exo- and mesocarp
while the pulp originates from proliferations of the
endocarp. The fruit has a superior ovary (8, 136) which
consists of 10-13 carpels and the ovules are formed in two
rows in each locule on marginal and central placentai (136).
The exocarp, or flavedo, which is the colored portion of the
peel, contains chloroplasts or chromoplasts. Chloroplasts,
that are present in the early stages of fruit development,
later transform into chromoplasts due to the disappearance
of chlorophyll and the unmasking and synthesis of
caroteniods. Thus the green fruits turn yellow to orange at
maturity. These changes are temperature and ethylene
related and can be reversed by chromoplasts reconversion to
chloroplasts in a process called regreening (144, 145). The
flavedo also contains oil glands formed by special cells
that produce terpenes and oils, which then later lyse to
form the oil gland cavities These cells are rich in
78


79
protoplasm and oils and eventually undergo disintegration of
their cell walls. The parenchymous cell adjacent to the
resulting cavity are thick walled and resist the lysigenous
process (33, 34). Flavedo epidermal cells produce cutin and
waxes which protect their outer surface and tangential walls
by a layer of wax and cutin (8, 136). This outer epidermal
layer also contains actinocytic type stomates (136).
The mesocarp or white colored albedo portion of the
orange peel consists of colorless cells which are typically
multi-armed, parenchymous and highly vacuolated. The tissue
contains large air spaces imparting to it a spongy nature.
The albedo is less specialized than the flavedo and although
some cells contain leucoplasts, it mainly serves as a
storage parenchyma. Some cells have been observed to
contain starch in young fruit but this is rarely found in
mature tissue (8, 136). The endocarp portion of the citrus
fruit is the most complex, giving rise to the juice sacs
which fill the locules entirely at fruit maturity (136).
Pathogens must penetrate citrus peel to invade the
fruit. The waxy cuticle and toxic terpenes and oils of the
flavedo offer a formidable defense to most pathogens.
Invasion of citrus fruits, therefore, usually occurs through
the stylar or button (stem) ends, or through a wounded
portion of the peel (18). The albedo, although much more
vulnerable to pathogen invasion if exposed, has been found
to contain a polygalacturonase (PG) inhibitor (17). This


80
inhibitor may be a form of defense against pathogens such as
Diplodia natalensis, a fungal pathogen of citrus that uses a
PG enzyme to lyse through host cell walls. The albedo is
also capable of producing much ethylene when wounded (64,
84). This may be another form of defense in that it can
lead to fruit abscission (27). This study describes the
structural features of citrus peel as they relate to
possible defense mechanisms and responses to cell-wall lysis
which typically occur during pathogen invasion of fruit.
Materials and Methods
Harvesting of Fruit
Valencia oranges (Citrus sinensis L. Osb.) were
harvested at various times of the year with 4 cm stems.
Fruit stems were stripped of leaves and put in 20 ml aqua
pics (self-sealing tubes which maintain water around the
ends of cut plant or flower stems). The fruit were stored
in portable coolers for transport to the laboratory.
Investigation of Citrus Peel Structure
Light Microscopy. Fresh Valencia orange peel was
sectioned longitudinally at 30 microns with a vibratome.
These sections were placed in water under a coverslip and
photographed in phase contrast and under ultraviolet
illumination.


81
Scanning Electron Microscopy. Valencia orange peel was
2
cut into 1 mm sections and fixed in 2% gluteraldehyde in
0.1 M cacdyllate buffer for 2 hours and then overnight in 2Z
osmium tetraoxide. Sections were then dehydrated in an
alchohol series, critical point dried in 100Z ethanol, and
coated with a thin layer of gold for observation under the
scanning electron microscope.
Orange Peel X-ray diffraction Analysis
2
Valencia orange peel sections were cut into 1 mm
sections, dehydrated for 48 hours over dry-rite and observed
uncoated with the scanning electron microscope for
characteristic X-rays of elements contained in the peel.
Electophoresis of Citrus Peel Proteins
A 20 |jl cell-wall lysing enzyme solution containing
0.1Z pectolyase (w/v) in 0.1 M acetate buffer pH 5.0, was
injected into Valencia orange peel just under the flavedo at
six locations around the equator of the fruit using serum
syringes with 25 gauge needles. Some of the enzyme
solutions were boiled and then cooled before injection.
There were 8 fruit replications for each of the treatments.
After four hours, injection sites of four fruit from
each treatment were excised with a 7 mm cork borer. The 72
discs resulting from each treatment were stored for 24 hours
in liquid nitrogen. Discs were then thawed and ground for 5


82
minutes with a Tekmar homogenizer in 10 ml cold acetone and
then vacuum filtered through a Whatman #1 filter.
Homogination and filtration were repeated three times on
remaining solids after which the acetone was evaporated.
The resulting filtrate was collected, lypholized and stored
at 4 C. Equal amounts of the powder (850 mg) from each
treatment were ground for 30 minutes with the Tekmar
homogenizer at low speed in 10 ml cold 0.05 M tris-glycine
buffer pH 6.6. This mixture was then centrifuged at 27,000
g-max for 15 minutes after which the pellet was discarded.
The supernatant was filtered through glass wool and combined
with cold acetone to make up an 80Z solution and put in the
freezer for 18 hours for maximal protein precipitation.
Precipitate was then centrifuged at 27,000 g-max for 15
minutes and the supernatant was discarded. The pellet was
drained and dissolved in a buffer that consisted of 1.5 g
tris, 20 mis glycerol, 4 g sodium dodecalsulfate (SDS), 10
ml 2-mercaptoethanol and 0.002 g bromophenol blue in 100 mis
of deionized water. The resulting solution was loaded onto
4X stacking and 111 running tube gels. These were run at a
constant current of 2 mA per gel for three hours after which
they were fixed with coomassie blue. The same procedure was
repeated for the remaining 4 fruit from each treatment at 8
hours after injection.


83
Ethylene Determination
Valencia oranges were treated with solutions of 0.1!?
pectolyase (w/v) in 0.1 M acetate buffer pH 5.0, boiled
pectolyase, or orange peel filtrate obtained by grinding
orange peel in acetone as described above. The treatments
consisted of 6 x 20 pi 1 injections of these solutions into
the peel by serum syringes with 25 gauge needles. The
fruits, with stems in water, were then placed in glass jars
which were capped periodically for two hours to determine
ethylene content at 4, 8, and 24 hours after treatment.
Ethylene samples were taken by syringe and analyzed on a
Hewlett Packard flame ionization G.C. model #5706 A equipped
with an activated alumina column. Carrier gas flow rate was
adjusted to give a sharp ethylene peak that eluted at 0.4
minute retention time.
Results
Orange Peel Structure
Citrus peel structure is most clearly observed when
illuminated with ultraviolet light. Sections from a green
orange show fluorescing cuticle on the outer surface and
along the tangential walls of flavedo epidermal cells and
faintly fluorescing chloroplasts in the cells of the flavedo
layer (Fig. 5.1). The same tissue when viewed in phase


84
Green Valencia oranges were harvested 10/30/84,
and the peel was cut in 30 micron sections and
viewed under the light microscope in fresh
condition under fluorescent light. Fluorescing
chloroplasts (Cl), and cuticle (Cu) are visible
in this flavedo section at 640X.
Figure 5.1.


85
contrast, reveals chloroplast organelles within cells and a
stomata in the epidermal layer (Fig. 5.2). A scanning
electron micrograph clearly shows the transition from the
comparatively dense flavedo cells to the albedo with its
abundant air spaces (Fig. 5.3). An oil gland is also
clearly defined as a cavity bordered by closely packed
thick-walled cells. The bordering cells are seen at the
back of the cavity.
Orange Peel Mineral Analysis
Orange peel that had been dehydrated and observed
uncoated under the scanning electron microscope for X-ray
microanalysis displayed an interesting spectrum of elements
(Fig. 5.4). The peel is shown here to contain significant
amounts of sulphur, chlorine, potassium, and calcium. The
other peaks in the spectrum are aluminum and silica and are
X-rays coming from equipment metals. X-ray diffraction
analysis reveals elements in a specimen with atomic numbers
above that of sodium in amounts above trace levels, although
quantification is not possible by this method. Of these
elements, calcium has been shown to have a stimulating
effect on ethylene synthesis (25) and is required for
maximal activity of pectin lyase enzymes (130)
Effect of Pectolyase on Orange Peel Protein Profiles
The gels from pectolyase-treated orange peel show an
increase in some proteins compared to those from the boiled


86
Green Valencia oranges were harvested 10/30/84,
and the peel was cut in 30 micron sections and
viewed under the light microscope in phase
contrast. Chloroplasts (Cl) are visible within
flavedo cells and one stomata (S) is evident in
the epideral layer at 580X.


87
A
F
OG
Figure 5.3. Green Valencia were harvested 10/30/84, and the
peel was fixed, dehydrated, crital point dried,
and gold coated for observation under the
scanning electron microscope. An oil gland
(OG) is visible in the flavedo (F) tissue and
large intercellular spaces are visible in the
albedo (A) tissue.


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hfl 3-ZS-ll
e'hylenebiosynthOObald



ETHYLENE BIOSYNTHESIS AND
CELL-WALL DIGESTION IN
CITRUS PEEL
BY
ELIZABETH A. BALDWIN
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
1986

ACKNOWLEDGEMENTS
There are many friends, aquaintances, and co-workers in
Gainesville and at the University of Florida to whom I am
very grateful for their support, moral or technical, in the
reseach and writing of this manuscript. It would be
impossible to name everyone, but I think most of these
people know who they are. A few, to whom I am especially
grateful, however, need mentioning:
To my husband, Mike, for his patience,
love, babysitting and computer skills. I especially
appreciate the fact that his goals and career were put on
hold in order for me to pursue mine.
To Hilton Biggs to whom I can only say that it has
been a long and elightening relationship (from my point of
view) for I feel privileged to have experienced.
To Dixie Biggs, who has helped me in so many ways.
Were it not for her friendship, creative input, and
technical support, this manuscript would not now exist in
completed form.
To Don Huber, for his invaluable help, advice,
laboratory facilities, and equipment. I appreciate the

time and effort he invested on my research problem.
To John Munson, for always being ready to help with
laboratory equipment, advice, and for his sense of humor
when needed.
To Cadance Lowell, for her help and instruction on the
electron microscope which was essential for some of the
work presented here.
To Charles Barmore, for his brief service on my
committee, and more importantly, for his enzyme material,
ideas, and support from which evolved some of the
accomplishments presented in this work.
To Karen Koch, for her friendship, enthusiasm,
advice and use of equipment.
To Richard Smith, for his instruction in plant
physiology which provided the groundwork for this or any
other physiological research I may attempt in my career.
To Chesley Hall, for his participation on both my
masters and doctoral committees and for use of
laboratory equipment.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS i i
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT xi
CHAPTER
I INTRODUCTION
1
II
III
LITERATURE REVIEW
4
Introduction
Primary Cell Walls of Plants
Plant Cell-Wall Structure
Plant Cell-Wall Function
Plant Cell-Wall Lysing Enzymes
Ethylene
Ethylene Biosynthesis
Ethylene and Defense
Ethylene and Cell Walls
Ethylene and Citrus
CELL WALL DEPOLYMERASE ENZYMES AND ETHYLENE
PRODUCTION IN CITRUS PEEL
9
12
15
15
17
19
19
21
Introduction 21
Materials and Methods 22
Fruit Injection Method and Ethylene
Determination 22
Affinity Chromotography 23
ACC Determination in Pectolyase treated
Fruit ' 24
Cycloheximide Application 25
PG Inhibitor 25
Treatment of Callus Tissue 25
Electrophoresis 26
Transmission Electron Microscopy 26
IV

CHAPTER Page
Results 27
Effect of Cell-Wall Lysing Enzymes on
Ethylene Production 27
Effect of Pectolyase on the Ethylene
Pathway and Cell-walls 32
PG Inhibitor and Pectolyase-Induced
Ethylene Production 36
SDS-Gel Profiles of Cell-Wall Lysing
Enzymes 39
Transmission Electron Microscopy of
Digested Cell Walls 39
Conclusion 46
IV PRODUCTS OF CELL-WALL DEGRADATION ELICIT
ETHYLENE IN CITRUS 49
Introduction 49
Materials and Methods 51
Fruit Injection Method and Ethylene
Determination 51
Preparation of Crude Pectin Hydrolysate. 53
Preparation of Carbohydrate-Enzyme
Digest Treatments 53
Gel Filtration of Pectin Materials 55
Analysis of Neutral Sugars 56
Results 57
Acid Hydrolyzed Pectin 57
Pectin-Digestion End-Products 63
Analysis of Neutral Sugar Content 70
Effect of Chitin on Ethylene Production. 70
Conclusion 75
V STRUCTURAL AND BIOCHEMICAL DEFENSE
MECHANISMS IN CITRUS 78
Introduction 78
Materials and Methods 80
Harvesting of fruit 80
Investigation of Citrus Peel Structure.. 80
Orange Peel X-ray Diffraction Analysis.. 81
Electrophoresis of Citrus Peel Proteins. 81
Ethylene Determination 83
Results 83
Orange Peel Structure 83
Orange Peel Mineral Analysis 85
Effect of Pectolyase on Orange Peel
Protein Profiles 85
Pectolyase and Orange Peel Filtrate
Induce Ethylene 89
Conclusion 92
v

CHAPTER
paRe
VI SUMMARY 94
LITERATURE CITED 100
BIOGRAPHICAL SKETCH 117
vi

LIST OF TABLES
Table Page
or 9/30/85, were treated with 6 x 20 pi
injections of sugar-containing 72
4.2 Neutral and acid sugar analysis was made on
the albedo pectin-pectolyase end product
material that had been fractionated 73
4.3 Neutral and acid sugar analysis was made on
the albedo pectin-pectolyase digest material
that had been fractionated on a Bio-gel 74
vi i

LIST OF FIGURES
Figure Page
3.1 Green Valencia oranges, harvested 10/30/84,
were treated with 6 x 10 ¡a 1 injections of
fungal cell-wall lysing enzymes in 28
3.2 Green Navel oranges, harvested 9/7/83, were
treated with 6 x 10 |jl injections of o.l%
cell-wall lysing enzymes in 0.1 M phosphate.... 30
3.3 Greel Navel oranges, harvested 8/15/83,
were treated with 6 x 10 pi injections of
0.17o, 0.05%, or 0.01!? pectolyase 31
3.4 Green Valencia oranges, harvested 8/7/85,
were treated with 6 x 20 pi injections of
0.1% pectolyase in 0.1 M citrate-phosphate 33
3.5 Green Valencia oranges, harvested 7/30/84,
were treated with 6 x 10 pi injections of
cell-wall lysing enzymes in 0.1 M phosphate.... 34
3.6 Green Valencia oranges, harvested 8/23/84,
were treated with 6 x 10 pi injections of
either 0.1% desalted pectolyase in 0.1 M 35
3.7 Green Valencia oranges, harvested 9/30/85,
were treated with 6 x 20 pi injections of 30
ppm CHI + 0.1% pectolyase in 0.1 M acetate 37
3.8 Yellow-green Valencia oranges, harvested
1/8/85, were treated with 6 x 10 pi of 0.1%
pectolyase, boiled pectolyase, and 38
3.9 Yellow Valencia oranges, harvested 2/27/85,
were treated with protein-containing
fractions #1-6 and 10, and control fractions... 41
3.10 Coomassie blue-stained 11% polyacrylimide
gels of different enzyme preparations used
in protoplast isolation 42
viii

Figure Page
3.11Coomassie blue-stained 11% polyacrylimide
gels of pectolyase and the
Geotrichum candidum PG 43
3.12 Transmission micrograph of orange peel
tissue treated with 10 pi 0.1 % pectolyase
in 0.1 M phosphate buffer pH 6, 24 hours 44
3.13 Transmission micrograph of orange peel
tissue treated with 10 pi of 0.1 X pectolyase
in 0.1 M phosphate buffer 45
3.14 Transmission micrograph of orange peel
tissue treated with 10 |jl of 0.1 M phosphate
buffer pH 6, 24 hours after 47
4.1 Yellow Valencia oranges, harvested 2/26/85,
were treated with 6 x 20 |jl injections of
crude orange pectin hdrolysate, 58
4.2 Yellow Valencia oranges, harvested 3/19/85,
were treated with 6 x 20 pi injections of
pooled fractions containing 61
4.3 Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 pi injections of
sugar-containing fractions 62
4.4 Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 pi injections of
sugar-containing fractions 64
4.5 Regreened Valencia oranges, harvested
5/6/85, were treated with 6 x 20 pi
injections of a 20i solution 65
4.6 Green Valencia oranges, harvested 7/16/85,
were treated with 6 x 20 pi injections of
various albedo pectin-pectolyase 68
4.7 Green Valencia oranges, harvested 9/10/85,
were treated with 6 x 20 pi injections of
sugar-containing fractions 69
4.8 Green Valencia oranges, harvested 9/30/85,
were treated with 6 x 20 pi injections of
sugar-containing fractions 71
IX

Figure Page
4.9 Regreened Valencia oranges, harvested
5/6/85, were treated with 6 x 20 ptl
injections of a 5% chitin 76
5.1 Green Valencia oranges, were harvested,
10/30/84, and the peel was cut in 30
micron sections and viewed 84
5.2 Green Valencia were harvested 10/30/84
and the peel was cut in 30 micron sections
and viewed under the light 86
5.3 Green Valencia oranges were harvested
10/30/84 and the peel was fixed, dehydrated
crital point dried, and gold 87
5.4 X-Ray diffraction analysis of dehydrated
orange peel show peaks for sulphur,
chlorine, potassium, and calcium 88
5.5 Coumassie blue-stained 112 polyacrylimide
gels of Valencia orange peel proteins from
fruit treated with boiled pectolyase 90
5.6 Green Valencia oranges, harvested 7/16/85,
were treated with 6 x 20 |jl of 0.12
pectolyase in 0.1 M acetate buffer 91
x

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ETHYLENE BIOSYNTHESIS AND
CELL-WALL DIGESTION
IN CITRUS PEEL
By
Elizabeth A. Baldwin
May 1986
Chairman: Robert Hilton Biggs
Major Department: Horticultural Science (Fruit Crops)
Increased ethylene production is often associated with
pathogens that cause cell wall dissolution. Cell wall
lysing enzymes are also associated with ethylene
production, softening during ripening of certain fruits, or
cell wall lysis during abscission of plant parts. Control
over of such events through the manipulation of ethylene
would be of benefit to the horticultural industry.
Ethylene was produced by orange peel in response to
injections of solutions containing pectolyase (obtained from
Aspergillus japonicus) as well as some other commercial cell
wall lysing enzyme mixtures. These cell wall lysing enzyme
mixtures showed a complex protein profile when analyzed by
xi

SDS-PAGE. Polygalacturonase (PG) , isolated from citrus peel
that had been infected by the citrus sour rot fungus,
Geotrichum candidum, and a similar enzyme isolated from the
pectolyase mixture, caused ethylene to be produced when
injected into orange peel tissue. Neither pectolyase nor
fungal PG induced ethylene production when applied to orange
callus tissue. Transmission electron microscopy revealed
middle lamellae dissolution and SDS gels showed differences
in protein profiles in orange peel treated with pectolyase
when compared to controls.
Sugar fragments are known to induce phytoalexin
production in different plant tissues. Pectic fragments,
released from citrus pectin by acid hydrolysis or pectolyase
digestion, induced ethylene production when injected into
citrus peel. Oligosaccharides of around nine to ten sugar
units were found to be the most potent elicitors of
ethylene synthesis, although a range of fragment sizes
induced more ethylene than the summed ethylene production of
individual fragments. This suggests synergism between the
individual fragments in terms of ethylene production.
Such elicitor fragments would be useful for manipulation of
ethylene to promote such events as degreening of citrus,
abscission of citrus fruits, and possibly resistance against
pathogen invasion.
xi i

CHAPTER I
INTRODUCTION
Of all the major classes of biological molecules,
polysaccharides have been the least studied. Proteins and
nucleic acids have been intensely researched, and some of
their structures deciphered. With the knowledge of
structure came the understanding of function such as
catalytic activity or encoding ability. With the exception
of glycoproteins and lectins, polysaccharidies are still
thought to be relatively unimportant except as protective
coatings for cells, energy sources, or energy storage
substrates. Part of the problem is the difficulty of
carbohydrate chemistry. Good analytical methods for
determination of carbohydrate structure were lacking until
recently (111). Even so, carbohydrates are far more
chemically complex and more difficult to decipher than
polypeptides and nucleic acids. This is because of the many
ways in which they can be linked and that their structures
can be branched (6). Furthermore, sugars can attach in
either a or 3 configuration of either five or six-membered
rings. Polysaccharides can also be modified by the addition
of other non-sugar substituents and can bind cations such as
1

2
calcium. The possibilities for linkages and configurations
for the one-hundred known natural sugars is staggering. The
question is: why such complexity? This has recently been
explained in part by research that has shown that
carbohydrates have diverse roles aside from the obvious
structural and energy storage functions. Sugar polymers are
now thought to be involved in signaling events such as
pathogen invasion (43, 117) as well as promoting hormonal
changes and affecting development (40, 111), or acting as
toxins (160). Cell recognition of such signals is little
understood, but study of the structures of such molecules
has lead to a greater understanding of their functions.
The plant cell wall, like the polysaccharides of which
it is made, has also been much ignored in plant physiology.
Other than interest in cell-wall expansion during growth,
and the passive roles of protection, structure, and turgor
maintenance, little importance has been assigned to it. The
idea that cell walls may play a more direct regulatory role
in cellular events is new and, as yet, not well accepted.
Lately cell walls have been shown to contain many types of
proteins, some of whose functions remain obscure (41, 42,
90, 111). The regulation and mechanisms for cell wall
growth, softening, changes during abscission, or signalling
activity are little understood. Further studies on this
complex "inverted organelle" are required to fully
understand all the functional aspects.

3
Ethylene and auxin are two plant hormones that seem to
be closely associated with cell wall activity. This is
interesting as these two hormones are mutually regulatory
(25). Ethylene is known to have important roles in
ripening, growth, abscission, defense, and auxin transport
(25), while the role of auxin in growth, abscission, and
regulation of the ethylene biosynthetic pathway is well
known (16, 25). The connection between auxin and cell wall
growth has become more obvious over the years, but the
involvement of ethylene in cell wall changes is more
obscure. It is not clear whether the endogenous cell wall
digesting enzymes involved in ripening or abscission are
regulated by ethylene, but they are certainly associated
with ethylene production (68, 93, 106, 121). On the other
hand, ethylene production is reported to be induced by
fungal cell wall digestion enzymes in various plant species,
as well as by pathogen invasion in general (40, 54, 93,
148). Further elucidating the relationship between ethylene
and cell wall changes has important consequences for the
areas of postharvest physiology and host-pathogen relations.
This knowledge could lead to new ways of manipulating
ripening, abscission, and plant defense responses which
could prove to be of great benefit to the horticultural
field.

CHAPTER II
LITERATURE REVIEW
Introduction
This review of the literature is an attempt to cover
various aspects of plant research that pertain to the data
presented in this dissertation. Recently the plant primary
cell wall structure has been intensely studied by Albersheim
and co-workers (6, 111) and many of its components and
interconnections have been identified which will be
summarized here. Evidence that has accumulated documenting
various cell wall functions, other than that of structure,
will also be presented. Cell wall lysing enzymes,
especially those that degrade pectic substances in nature,
will be reviewed in terms of their mode of action, substrate
preference, and roles in cell wall digestion. Finally,
ethylene biosynthesis, and subsequent hormonal role in
ripening, abscission, growth, and plant defense will be
analyzed and discussed.
Primary Cell Walls of Plants
Cell walls are an essential structural component of the
majority of eukaryotes and almost all prokaryotes. Most
4

5
likely, primitive prokaryotes evolved cell walls through
natural selection for reinforced plasma membranes. Probably
the advantage to such coated cells was that they were able
to operate at higher turgor and at higher metabolic rates,
enabling them to grow faster (21). This selective advantage
has populated this planet with many walled organisms.
Plant Cell Wall Structure
The structural components of all cell walls are 90!?
polysaccharide and 10? protein, of which most of the latter
are components of glycoproteins. The ability to determine
the structures of complex carbohydrates has only recently
become possible through improved techniques involving gas
chromotography, mass spectrometry, fast atom-bombardment
mass spectrometry, liquid chromotrgraphy, x-ray diffraction,
electron microscopy, and nuclear magnetic resonance
spectroscopy (111,143). Cellulose constitutes 20?-30? of
primary cell walls and is a polymer of B-4-linked glucose.
The other polysaccharides of cell walls are complex in
structure and, for the most part, undefined (111).
Cellulose chains are aggregated into microfibrils which are
laid down perpendicular to the axis of cell growth (89).
Hemicellulose is that non-cellulosic fraction of the
cell wall whose polysaccharides can be extracted with alkali
(43). Xyloglucans, present in cell walls of plants,
especially dicots, are constructed of a 8-1,4-linked glucose

6
backbone with side chains composed of xylose. Occasionally,
galactose, fucose or arabinose residues are found linked to
xylose of some xyloglucans (23,111). Some or most of the
xyloglucan is hydrogen bonded to cellulose (23).
Xylans, the major hemicellulose in the primary cell
walls of monocots, have a backbone of B-4-linked xylose and
also hydrogen bond to cellulose. Like xyloglucans, xylans
have various side chains attached to them, usually
containing arabinose (111). Monocot cell walls also contain
polysaccharides containing a mixture on g-3 and g-4 linked
glycopyranosyl residues. These are commonly called g-
glucans and so far have been found to be linear (111).
The pectic components of cell walls are
homogalaturonans or rhamnogalacturonans of which the latter
is divided into two groups (rhamnogalacturonans I and II).
Homogalacturonans are a-4-linked galacturonic acid units
which tend to be insoluble. They may be covalently linked
to other wall polymers (89) and have been found in dicot and
monocot tissues. These polymers tend to form insoluble gels
in the presence of calcium, suggesting that they may play a
role in the structure of cell walls (113). The linkages,
ring form, configurations, and degree of polymerization of
homogalacturonans are, for the most part, unknown (116,
143) .
Another pectic polysaccharide that was isolated from
several dicots and one monocot is rhamnogalacturonan I. It

7
has an alternating 2-linked rhamnose and 4-linked
galacturonic acid backbone (109), of which about half of the
rhamnosyl residues are branched. The side chains are varied
in nature and contain galactose, arabinose, and small
amounts of fucose (109, 110). The other rhamnogalacturonan,
that has been found in both dicots and monocots, is called
rhamnogalacturonan II. This polymer also contains a high
proportion of rhamnosyl residues as does rhamnogalacturonan
I, but with different linkages (111). Rhamnogalacturonan
II, from suspension cultured sycamore cells, contains some
unusual sugars including a methylated fucose, a methylated
xylose, and a new sugar called aceric acid (111). Since
this polysaccharide can be solubilized by endo-a-1,4-
polygalacturonase, it would appear to be covalently linked
in the primary cell wall through a series of 2,4-linked
galacturonic acid residues (143).
Pure arabinan polymers are also found in cell walls
containing 5-linked a-L-arabinofuranosyl residues as well as
polymers that consist almost totally of 8-4-linked
galactans, or mixtures of 6-linked and 4-linked galactose
units (111). Arabinogalactans in cell walls consist of two
types that differ in the linkage of the galactose and
arabinose residues. The neutral sugars arabinose and
galactose are found in many cell wall polymers such as
extensin, arabinogalactan proteins, rhamnogalactan I and II,
xyloglucan, arabinoglucan, and glucuronoarabinoxylans (108,
111) .

8
Angiosperm primary cell walls also contain many
different glycoproteins. One common and important such
glycoprotein is the hydroxyproline-rich extensin molecule.
Through peroxidase-catalyzed internal isodityrosine cross-
linkage, it forms a three-dimensional network around
cellulose microfibrils (44, 45). Extensin is 50^
carbohydrate, of which most is arabinose and galactose
residues. The protein portion is rich in hydroxyproline,
serine, lysine, tyrosine and isodityrosine (141).
Arabinogalactan proteins are water soluble and are found
extracellularly as well as in the cytoplasm. They are
acidic and the protein portion, which makes up 2%-lOZ of the
molecule, is rich in hydroxyproline, serine, alanine, and
glycine. The carbohydrate portions of these molecules are
of relatively high degrees of polymerization and contain
galactose, arabinose, rhamnose, mannose, galactoarabinose,
and glucoarabinose. Their biological role is unclear (111).
Cell walls of higher plants also contain various
enzymes, some of which are ionically bound in the wall and
all of which so far studied are glycoproteins (111). Malate
dehydrogenases, peroxidases, phosphatases, and proteases
have been reported. In addition, many glycosyl hydrolases,
transferases, endoglycanases, pectinases and pectinesterases
have been documented in cell walls (90, 123, 125, 111).
Plant cell walls also posess enzymes that are capable of
degrading walls of invading fungi (41, 42) and other

9
proteins capable of inhibiting polysaccharide-degrading
enzymes secreted by fungal pathogens (2, 17).
Plant Cell Wall Function
The primary cell wall dictates the growth and
morphology of plant cells. In addition to the obvious
structural importance, cell wall components have been
postulated to have functions such as control over genetic
expression in plants. This hypothesis arose partly from the
complexity of cell wall polysaccharides , and also because a
fungal wall oligosaccharide of branched g-glucans was shown
to exert control on the plant genetic level (48). Fungal
cell walls have been shown to elicit phytoalexins, lignan
synthesis, and protease inhibitor activity in plants (65,
91, 154). Specific fragments of plant cell walls were shown
to control various physiological responses in plants (116,
137, 160). These reports gave rise to the view that complex
carbohydrates can be regulatory molecules. It has been
shown that a xyloglucan fragment in nanomolar concentrations
inhibited 2,4-D-stimulated (2,4-dichlorophenoxy-acetic acid )
elongation in etiolated pea epicotyls (111). The fragment
was obtained from suspension-cultured sycamore cells treated
with endo-1,4-glucanase. Fragments of plant cell wall
homogalacturonan have been shown to elicit phytoalexin
production in plants (35, 116, 155, 154). Such elicitors
induce receptive plant cells to synthesize the mRNA's and

10
enzymes responsible for phytoalexin synthesis, but the
manner of such specific gene activation is as yet not
completely understood.
Homogalacturonan elicitor fragments have been produced
from cell walls of different plant systems in two different
ways. One method involved partial acid hydrolysis of
soybean cell walls and citrus pectin. A D-
dodecagalacturonide fraction of the resulting material
contained the greatest elicitor activity with some activity
present in oligosaccharides from 10-13 residues long (116).
Another elicitor of phytoalexins was identified after
incubation of various substrates with an endo-a-1,4-
polygalacturonic acid lyase (PGA lyase) or endo
polygalacturonase (PG.) (35, 51). The elicitor active
fragments produced by such treatments were shown to be an a -
4-linked dodecagalacturonide, for the lyase, and an de¬
linked tridecagalacturonide, for the PG, when assayed on
soybean or castor bean, respectively.
Mechanical injury, such as is found with microbes and
insects, will induce systemic synthesis of plant proteins
that inhibit microbial and insect proteinases (153). The
signal that induces this response in tissues distant from
the injury was shown to be fragments of pectic and chitosan
polysaccharides in suspension cultured sycamore cells (137),
tomato leaves, pea pods, and castor beans (154).

11
Hypersensitive cell death is a common response of
plants to microbial and viral invasion (24, 111). It is
hypothesized that cell death slows down the pathogen
invasion allowing time for induction of other plant defense
responses. Partial acid hydrolysis of suspension-cultured
sycamore cells produced toxic fragments which inhibited
14
uptake and incorporation of [ CJleucine into protein by
the cultured cells (160). In this study, the measure of
protein synthesis was equated to a measure of cell vitality.
The fragments were thought to be pectic in nature, as small
as trisaccharides (111), and may explain the observed
toxicity of pectic enzymes (78). Plasmolysis of cells was
found to protect tissues from the toxicity of both the
pectic enzymes and their fragment products (22). Cells
isolated for protoplasmic fusion or for tissue culture are
sometimes stimulated to produce ethylene by the enzymatic
mixtures used to digest their cells (11). CelluLysin and
macerase have been reported to induce ethylene production in
tobacco and pear suspension cells, respectively (40, 73,
147) and ethylene was observed to be produced by oranges in
response to both fungal enzymes and partially digested
citrus pectin (99). Castor bean explants produced ethylene
when exposed to heterogeneous enzymes or purified fungal
P.G. (152) and fungal cell walls elicited ethylene and
phytoalexin production in soybean (117). Ethylene is also
often associated with the hypersensitive necrotic response

12
to pathogen invasion (24, 30, 54, 158) and with the injury
due to insect or mechanical wounding (63, 64) all of which
may involve cell wall lysing enzymes and/or cell wall
fragments as well as eventual loss of cell vitality.
Whether cell wall fragments induce cell senescence or loss
of vitality via ethylene is unknown.
Partial acid-hydrolysis of suspension-cultured sycamore
cell walls produced a pectic fragment that both inhibited
flowering and promoted vegetative growth of fronds in
duckweed (Lemna gibba) . This indicates that the fragments
had some regulatory contol over flowering in this species
(111) .
Plant Cell Wall Lysing Enzymes
There are many cell wall digesting enzymes that are
involved in ripening, growth, abscission, and pathogen
invasion of plant cells. Some of these enzymes digest
cellulose and others pectin or other polymers. Some remove
sugars only from the ends of polymers, some attack internal
bonds, while still others remove side chains. Endo- and exo-6
-1,4- or 6~1,3-glucanases have been found in cell walls of
plants (82). These enzymes degrade 6-1,4- cellulose as well
as the nemicellulose polymers xyloglucan and 6-1,4-xylan
(23, 76, 81, 83). The role of 6-1,3-glucanase is curious as
there appears to be no g-1,3-glucans in plant cell walls
(41, 70). Glycosidases, which are enzymes that hydrolyze

13'
oligosacchardes to monomers, are also active in plant
tissues. Cellulase and 1,3-glucanase have been associated
with growing tissues and autohydrolysis of cell walls (50,
70, 83). Cellulases are also reported to be involved in
locular formation in tomato ripening (81) as well as
ripening of papaya (118) and peach (77). 3-1,4-Xylanase, a-
arabinosidase and g-xylosidase have been reported to
breakdown arabinoxylan (55, 38) and a g-1,4-mannase may be
responsible for the breakdown of mannose-rich lettuce
endosperm (74).
B-Galactosidase is an enzyme that has been shown to be
associated with ripening in apple (19), tomato (123), and
pear (5). A similar enzyme showed high activity in growing
pea epicotyl cells and appeared to be involved with the
process of autolysis. a-Arabinase and exo-glucanase also
showed some activity is this system although less than that
of the 8-galactosidase (100).
Endo-polygalacturonase catalyzes the hydrolytic
cleavage of -1,4-bonds between non-esterified galacturonic
acid residues releasing oligomeric products.
Polygalacturonases are often thought to be responsible for
cell wall softening during ripening (20, 47) and abscission
(71, 131, 138). These enzymes prefer high molecular weight D
galacturonans and the rate of splitting the glycosidic bonds
decreases with the shortening of the substrate chain. Exo-D
galacturonases catalyze the hydrolytic cleavage of the

14
terminal a-D(l,4) bonds starting at the non-reducing end of
the galacturonan chains, releasing D-galactopyranuronic acid
as a product (130). Polymethylgalacturonases are enzymes
capable of degrading highly esterified D-galacturonans by
endo-action pattern and are ineffective toward de-esterified
substrates. Generally, D-galacturonases have their pH
optimum in a weakly acidic region between pH 4.0 and 6.5
(130). Polygalacturonases are also produced by many
pathogenic fungi and some bacteria (18, 51). Lyases
catalyze the cleavage of -D-(l,4) glycosidic bonds of
esterified and non-esterified D-galacturonans by the 6-
elimination mechanism giving rise to a double bond at the
non-reducing end. Endopectate lysases characteristically
have high pH optimums, such as 8.0-9.5, and a requirement
for calcium ions. Exopectate lyases cleave bonds starting
at the reducing end and prefer univalent cations and an
alkaline pH (130). Pectate lyases are produced by bacteria
and fugaria (96). Pectin lyases preferentially split the
highly esterified D-galacturonans producing esterified
unsaturated oligo-D-galacturonans. These enzymes have a pH
optimum of 5.1-6.6, are activated by calcium, and are of
fungal origin (130).
Pectinesterases attack polymethyl esters of D-
galacturonans. Enzymatic de-esterification of methyl esters
of pectin proceeds linearly along the chain of the molecule
resulting in free carboxyl groups. This enzyme apparently

15
is subject to end product inhibition and therefore does not
completely de-esterify pectin. Plant and some microbial
pectinesterases have the pH optimum in the range of pH 7.0-
9.0 although most microbial pectinesterases tend to prefer
neutral to acid pH (130). These enzymes can be found in all
plants and can be bound to cell walls. They may be involved
in growth as well as ripening (122). Pectinesterases are
also found in many plant pathogens of fungal and bacterial
nature (130). The action of these enzymes can sometimes
enhance the activity of plant or microbial
polygalacturonases and pectin lyases (118, 124, 130). This
would explain their association with ripening (118, 126).
Ethylene
Ethylene is a plant hormone that can have a dramatic
effect on ripening (37, 47, 58, 121, 139,), abscission (38,
71, 131, 138), breaking of dormancy (57), senescence (92,
159), sex expression (103), and flowering (25). This
hormone is unique in that it is a simple hydrocarbon gas
(103) and is therefore easily transported in plant tissues
(25). Other effects of ethylene are subtle and are modified
by interaction with other hormones especially auxin (1, 25,
36, 57, 103).
Ethylene Biosynthesis
Methionine was proposed by Lieberman and Mapson (105)
to be a precursor of ethylene which was later confirmed

16
using C-labeled methionine (104). The methyl-thio group
of this amino acid is cycled back through several
intermediates to reform methionine (4). In this scheme,
methionine is converted to S-adenosylmethionine (SAM) and
the CH^S group is released from SAM as methylthioadenosine
(MTA) which is hydrolyzed to methylthioribose (MTR). MTR is
then incorporated into a 2-aminobutyrate moiety which is
condensed into methionine through a series of as yet
unidentified steps (166). Meanwhile, the 3 and 4 carbons
from SAM split off to form 1-aminocyclopropane-l-carboxylic
acid (ACC) which is then converted to ethylene (3). ACC can
also be converted to N-malonyl ACC which appears to be an
inactive dead end-product and therefore represents a
mechanism for regulation of ethylene biosynthesis (79). An
important step in the regulation of ethylene biosynthesis is
the conversion of SAM to ACC by the enzyme ACC synthase.
This enzyme requires pyridoxyl phosphate for maximal
activity and is inhibited by inhibitors of pyridoxyl
phosphase enzymes such as aminovinylglycine (AVG) and
aminooxyacetic acid (AOA) (3, 32, 163, 165). Auxin
stimulates ethylene production at this step
(162). Another important reulatory point in ethylene
production is the conversion of ACC to ethylene which
requires oxygen (3), membrane integrity (103), and is
sensitive to free radical scavengers (13).

17
Ethylene and Defense
Ethylene Is involved in plant-pathogen relationships in
many ways. An attack by pathogens is a stress that often
leads to ethylene production (54, 64, 93). It has been
suggested that ethylene may be a signal for the plant to
activate biochemical defenses against potential pathogens
(30). This could be the hormonal function of stress
ethylene in that physical or chemical wounding provides
preferred entry sites for many pathogens (30).
Phytoalexins are compounds that accumulate in certain
plants in response to infection which generally hinder
pathogen growth (24). Their accumulation can be elicited by
true infection by living pathogens or by biotic factors such
as fungal enzymes, or plant and fungal cell wall fragments
(35, 51, 91, 154). In a few cases ethylene has been found
to act as an elicitor of phytoalexins. Wounded peas
produced pistatin in response to ethylene treatment (14).
Accumulation of glyceollin in soybean in response to a
fungal glucan elicitor was determined to some extent by
ethylene (94). An earlier report however claimed that
ethylene was not a messenger in the induction of this
particular phytoalexin (117). Potato slices treated with
ethrel (2-chloroethylphosphonic acid) and then innoculated
with a fungal pathogen accumulated more of the phytoalexins,
i.e.,phytuber in and pnytuberol, than slices not subjected to
ethrel treatment (75). Ethrel also induced production of a

18
phytoalexin compound in tobacco leaves (151). On the other
hand, the toxin, coronatine, produced by Pseudomonas
syringae elicited ethylene in bean leaf discs and caused
chlorosis whereas an unrelated pseudomonad phytotoxin, which
also causes chlorosis, did not stimulate ethylene production
(66) .
Ethylene has been reported to influence activities of
various enzymes. Pectinmethylesterase activity was
decreased in vitro with ethylene treatments (69) although
this was not supported by later studies. Ethylene induced
chitinase activity in bean and melon (31, 149). Wounding
and/or ethylene induced wound resistance to cellulase in oat
leaves (67).
Accumulation of hydroxyproline-rich glycoprotein (HRGP)
in muskmellon cell walls was shown to be induced by a
pathogenic fungus (59). This glycoprotein was later found
to be involved in the plant defense reaction to this
pathogen, imparting some resistance to invasion. The
increase in the HRGP was mediated through ethylene (60) and
inhibition of ethylene synthesis by AVG decreased both
ethylene and HRGP levels (150). A cell wall elicitor from
the pathogen was shown to cause inhibition of protein
synthesis after 18 hours, while promoting both ethylene and
HRGP production earlier in elicitor treated melon seedlings.
Ethylene may be involved in HRGP elicitation as ACC, the
precursor to ethylene which triggered HRGP synthesis

19
to the same extent as the fungal elicitor mentioned above
(134).
Ethylene and Cell Walls
Ethylene has often been associated with cell wall
lysing enzymes in cases of ripening (118, 121, 135, 139),
abscission (38, 71, 138), and pathogen invasion of plant
tissue (54, 64, 93). As discussed in the section on cell
wall function, recent evidence has shown that fungal cell
wall lysing enzymes can induce ethylene production. Various
naturally occurring carbohydrates stimulated ethylene
production in tobacco leaf discs and galactose promoted
ethylene evolution and ripening in tomato fruit (72, 112,
119). Ethylene has also been shown to be associated with
cell wall changes in terms of growth of rice and other
plants (85, 87, 98, 114, 127, 140).
Ethylene and Citrus
Citrus is a non-climacteric fruit, and produces only
low amounts of ethylene under normal conditions (164),
although small amounts of ethylene are produced in all
stages of development (128). Citrus fruit, however, can be
induced to produce relatively high levels of ethylene by
chemical or mechanical wounding and this in turn promotes
abscission (27, 63, 86, 164). The fact that citrus peel is
capable of producing substantial amounts of ethylene was

20
shown with peel disc explants (62, 63) and with whole fruits
on trees sprayed with abscission chemicals (27, 49, 97).
This is especially true of the albedo portion of citrus peel
(63, 64, 84). Yang and coworkers have shown citrus ethylene
production to be autoinhibitory in grapefruit flavedo (133)
and autocatalytic in orange leaves (132). Ethylene can
promote degreening in citrus fruit, causing c'nloroplasts to
convert to chromoplasts (156).

CHAPTER III
CELL-WALL DEPOLYMERASE ENZYMES AND ETHYLENE PRODUCTION
Introduction
Pathogen invasion of higher plant tissue usually
promotes ethylene production (30, 54) and often is
associated with cell wall lysing enzymes (143, 152, 142).
Similarly, there is a rise in ethylene production that
accompanies the activity of endogenous cell wall softening
enzymes during ripening of certain fruits (28, 47, 121, 139,
155) and cell-wall separation during abscission (131, 138).
In each case ethylene is associated with cell-wall degrading
enzymes of pathogen or plant origin. Furthermore, single
cells, isolated for protoplasmic fusion or for tissue
culture, are stimulated to produce ethylene by the enzymatic
mixtures used to separate the cells (11). Cell wall
digesting enzyme mixtures have also been shown to produce
ethylene in tobacco leaf discs as well as in other tissues
(10, 12, 39)
Little is known about the mechanism by which ethylene
is produced during the hypersensitive response in host
pathogen interactions. In order to further investigate this
21

22
process, cell-wall digesting enzymes, isolated from citrus
pathogens, and some commercial cell-wall lysing enzyme
mixtures were studied as to their effects on citrus cell
wall structure and ethylene production.
Similarities between the initiation of ethylene
production during pathogen induced hypersensivity and
abscission may also extend to its production during
ripening. In all cases ethylene is associated with cell
wall hydrolysis. Cell-wall lysing enzymes or the resulting
cell wall fragments may be general elicitors of ethylene
biosynthesis.
Materials and Methods
Fruit Injection Method and Ethylene Determination
Navel or Valencia oranges (Citrus sinensis (L.) Osb.)
were harvested at various times of the year with 4 cm stems.
Fruit stems were stripped of leaves and put in 20 ml aqua
pics (self-sealing tubes which maintain water around the cut
end of plant or flower stems). The fruit were stored in
portable coolers for transport to the laboratory.
Enzyme solutions of 10 or 20 pil containing 0.1% ,0.05%,
or 0.01% solutions (w/v) of commercial cell-wall lysing
enzyme mixtures in 10 mis phosphate, acetate, or citrate
buffer at various pH values were injected into orange peel

23
just under the flavedo at six locations around the equator
of the fruit using serum syringes with 25 gauge needles.
Some enzyme solutions were boiled and then cooled before
injection, while others were desalted on lyphogel (Gelman
Instrument Co.). Fruits, with stems in water, were then
placed in glass jars which were periodically capped for
ethylene determinations. Ethylene samples were taken at the
end of one or two hours by syringe and analyzed on a Hewlett
Packard flame ionization G.C. model #5706 A equipped with an
activated alumina column. Carrier gas flow rate was adjusted
to give a sharp ethylene peak that eluted at 0.4 minute
retention time.
Commercial cell-wall lysing enzyme mixtures used in
these experiments were cellulysin (Calbiochem), macerozyme
(Yakult Biochemicals, Japan), cellulase (Worthington),
pectolyase (Seichin Pharmaceutical, Japan and Sigma),
pectinmethylesterase (Sigma), drislase (Plenum Scientific),
and pectinase (Sigma).
Affinity Chromatography
Polygalacturonase (PC) was obtained from Charles
Barmore (CREC, Univ. of Fla., IFAS, Lake Alfred FI.) who
extracted it by the following procedure. Commercial orange
juice, innoculated with Geotrichum candidum or Diplodia
natalensis, was centrifuged and then 140 ml of supernatant
were loaded onto an alginate affinity column (alginic acid

24
cross-linked with epichlorohydrin, 1.6 x 5.5 cm). The
protein was eluted with acetate buffer (0.5 M at pH 5
containing 0.5 M NaCl). One milliliter fractions were
collected with the protein eluting in fraction #2-10 after
the void volume. All steps were carried out at 4°C. The
protein recovered was assayed for PG activity by measuring
the release of reducing groups from polygalacturonic acid.
The glucose equivalents were determined by the procedure of
Nelson (115 ) .
In a similar manner as for G. candidum and D.
natalensis PG' s, 200 ml of a 0.03!? solution of pectolyase
(w/v) in acetate buffer (0.5 M at pH 5.0) was loaded onto an
alginate affinity column and 1 ml fractions were collected.
A protein was eluted with 0.5 M NaCl in fractions #1-6 and
10, similar to the elution of the previous fungal PG's.
Protein-containing fractions were assayed for ethylene
activity by injection of fraction material into the peel of
Valencia oranges, six 20 1 injections per fruit. Three
fractions that did not contain protein were also injected
into fruit for controls.
ACC Determination in Pectolyase-Treated Fruit
Valencia oranges were treated with 0.1 % desalted
pectolyase in 0.1 M phosphate buffer, pH 6.0, or the same
buffer alone. Ethylene measurements were made four hours
after treatment following which the injection sites were

25
excised from the fruit with a 1 cm diameter cork borer. The
peel discs were then ground in ethanol and ACC was extracted
by the method of Lizada and Yang (107).
Cycloheximide Application
Valencia oranges were injected with 20 1 of 30 ppm
cycloheximide (CHI) or deionized water. One hour later the
same fruit were reinjected in the same areas with 0.11 mg
pectolyase solution in 0.1 M acetate buffer pH 5.0.
Ethylene measurements were made at 4, 8, and 24 hours after
the pectolyase treatment. Some fruits were injected with 30
ppm CHI alone or with boiled pectolyase.
PG Inhibitor
This protein was obtained from Charles Barmore (CREC,
Univ. of Florida, IFAS, Lake Alfred FI.) who extracted it
from Valencia orange albedo tissue, the white spongy portion
of the peel (17). The protein was purified by sephadex G-
100 column chromotography and inhibition of PG was assayed
by the liberation of reducing groups from polygalacturonic
acid as described above. This inhibitor was applied with
0.1Z pectolyase solution in 10 ml 0.1 M phosphate buffer, pH
5.0.
Treatment of Callus Tissue
Approximately 5 mg fresh weight of embryogenic Hamlin
orange callus was transferred under sterile conditons to an

26
agar nutrient media in 20 ml scintillation vials and allowed
to grow for a few days. The callus produced a wound ethylene
upon transfer which subsided after 1-2 days. After the
ethylene had subsided the callus was treated with 10 ul of
treatment solution, administered by auto-pipet with a
sterile tip. The treatment solutions included desalted Q.1%
pectolyase, cellulysin, or pectinase in 0.1 M phosphate
buffer pH 6.0, a boiled control for each enzyme treatment,
and phosphate buffer alone.
Electrophoresis
The commercial enzyme mixtures and the proteins isolated
from the fungal organims G. candidum and D. natalensis were
fractionated on 470 stacking and 111 running SDS
polyacrylimide tube gels. These were run at a constant
current of 2 mA/gel for three hours after which they were
fixed and stained with coomassie blue.
Transmission Electron Microscopy
Green Valencia oranges, harvested 7/18/84, were treated
with injections of pectolyase, 0.11 in 0.1 M phosphate
buffer pH 6.0, or buffer alone. Fruit were then incubated
at room temperature for 24 hours during which the pectolyase-
treated group produced ethylene over and above the buffer
controls. After 24 hours the injection sites

27
were explanted in 1 mm squares and soaked in 0.075 M half¬
strength Karnovski fixative for 2 hours followed by 1 %
osmium tetraoxide for 2 hours. The tissue was then
dehydrated in an alchohol-acetone series and embedded in
Spur's resin (95). Thin sections were cut, applied to
forumvar coated copper grids and post-stained with lead and
uranyl acetate. Other sections on forumvar coated nickel
grids were treated for 25 minutes with a solution of 1%
periodate, washed in deionized water, and floated on a
solution of 3l methanamine, 5% silver nitrate, and 5% sodium
tetraborate for 40 minutes at 60°C. The silver ions are
reduced to metalic silver by the aldehyde groups formed from
periodate oxidation of free hydroxyl groups on adjacent
carbon atoms, appearing as small black dots on micrographs.
This results in a fairly specific stain for polysaccharides
(95). These sections were later post-stained with lead and
uranyl acetate.
Results
Effect of Cell-Wall Lysing Enzymes on Ethylene Production
The commercial cell-wall lysing mixtures usually have a
high salt content which in itself caused the orange peel to
produce some wound ethylene (Fig. 3.1A). For this reason
desalting techniques or boiled enzyme controls were used to

28
Green Valencia oranges, harvested 10/30/84,
were treated with 6 x 10 (jl injections of
fungal cell-wall lysing enzymes in 0.1 M
acetate buffer pH 5.0. A) 0.1l pectolyase and
boiled pectolyase. B) G. candidum PG and
boiled P.G. All points are means of 3
replications +/- S.E.
Figure 3.1.

29
distinguish between ethylene produced in response to a
protein and wound ethylene due to salts or the possible
presence of other non-protein contaminants such as fungal
cell walls. Pectolyase stimulated the most ethylene above
boiled control levels in Valencia oranges 6 hours after
treatment (Fig. 3.1A). In Navel oranges the boiled enzyme
control also produced significant amounts of ethylene at 6
hours probably due to higher sensitivity of these oranges to
salts (Fig. 3.2A). Drislase (Fig. 3.2B) and the G. candidum
PG (Fig. 3.IB) also produced significantly more ethylene
than their boiled controls at 24 hours for drislase and 6
and 24 hours for the PG. However, pectolyase produced at
least 10 to 40 times more ethylene than either commercial
drislase or the purified fungal PG. A three-fold dilution
of pectolyase had little effect on ethylene production at 8
hours but produced slightly decreased ethylene levels at 24
hours (Fig. 3.3). This indicates that these concentrations
of pectolyase saturated the ethylene response to cell wall
dissolution at 8 hours, but that the diluted pectolyase
treatments became less than that necessary for a saturated
response at 24 hours. The cells in response to middle
lamellae dissolution by fungal enzymes, produced increasing
levels of ethylene which peaked around 6-8 hours after
treatment (Fig. 3.1A, 3.IB, 3.2A, and 3.2B). Although
ethylene levels then decreased by 24 hour (Fig. 3.1A, 3.IB,
3.2A, and 3.2B) in some treatments ethylene production was

ni C2H« / Fruit / Hour
30
Hours After Treatment
Figure 3.2. Green Navel oranges, harvested 9/7/83, were
treated with 6 x 10 ¡jl injections cell-wall
lysing enzymes in 0.1 M phosphate buffer pH
6.0. A) 0.1l pectolyase and boiled pectolyase.
B) 0.11 drislase and boiled drislase. All
points are means of 3 replications +/- S.E.

ni C2H4 / Fruit / Hour
31
Figure 3.3. Green Navel oranges, harvested 8/15/83, were
treated with 6 x 10 ¡al injections of 0.1%,
0.05%, or 0.01% mg pectolyase in 0.1 M
phosphate buffer pH 6, and phosphate buffer
alone. All points are means of 3 replications
+/- S.E.

32
found to undergo a second rise by 48 hours after treatment
(Fig. 3.2A and 3.2B). This was associated with fruit
abscission, yellowing and general senesence, often occurring
in control fruit as well as treated. The optimum pH for
pectolyase-induced ethylene activity appeared to be around
pH 5.0 (Fig. 3.4) which is the optimum pH range reported for
several fungal PG's and a bacterial pectin lyase (17, 18,
51, 101, 157).
Cellulysin, macerozyme, cellulase, pectinase, and the
PG from D. natalensis did not produce ethylene levels above
that of their boiled or buffer controls at the
concentrations tested (data not shown). Desalted
pectinmethylesterase (PME) did produce small amounts of
ethylene above a buffer control (Fig. 3.5B), although much
less than that produced by pectolyase (Fig. 3.5A). This may
be due to changes in free calcium levels and pH as a result
of pectin desterification, to a low salt content still
present in the enzyme treatment solution, or to
contamination by other enzymes, possibly cell wall lysing in
nature.
Effect of Pectolyase on the Ethylene Pathway and Cell Walls
Pectolyase stimulated an increase in ACC levels (Fig.
3.6), a precursor to ethylene (161), as well as ethylene
indicating that it stimulates the ethylene pathway at some
point before ACC synthesis, possibly at ACC synthase. CHI,

pH of Pectolyase Treatment Solutions
Figure 3.4. Green Valencia oranges, harvested 8/7/85, were
treated with 6 x 20 |jl injections of 0.1%
pectolyase in 0.1 M citrate-phosphate buffer at
different pH's, and boiled pectolyase in same
buffer at pH 5. All bars are means of 3
replications +/- S.E.

34
igure 3.5. Green Valencia oranges, harvested 7/30/34, were
treated with 6 x 10 |jl injections of cell-wall
lysing enzymes in 0.1 M phosphate buffer pH
6.0. A) 0.17o pectolyase and boiled pectolyase.
3) 0.110 pectinmethylesterase, and boiled
pectinmethylesterase (PME). Enzyme solutions
had been desalted on lyphogel. All points are
means of 3 reps + /- S.E.

nMole ACC / 6 Injection Sites/Fruit
35
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
L.
O)
O)
O)
0)

**-
re
H—
re
3
3
CO
"o
00
o
CJ
u
OJ
Oí
Q.
Q.
Figure 3.6. Green Valencia oranges, harvested 8/23/84, were
treated with 6 x 10 ial injections of either
0.1% desalted pectolyase, in 0.1 M phosphate
buffer pH 6, or 0.1 M phosphate buffer alone.
Ethylene production was determined and peel
injection sites were analyzed for ACC content
4 hours after treatment. Bars are means of 3
replications +/- S.E.

36
a reported protein inhibitor (43, 46, 61), by contrast,
decreased pectolyase-induced ethylene production by over one-
half at 4 hours after treatment. This inhibition was less
effective as the tissue recovered from the wound ethylene
response at 8 and 24 hours (Fig. 3.7). Also demonstrated
here is the difference between immature and mature fruit
response to pectolyase. In previous experiments with mature
fruit, the pectolyase-induced ethylene production peaked at
around 6-8 hours after treatment with the enzymes. In this
case with immature fruit, pectolyase-induced ethylene
production peaks at or before 4 hours after treatment and
the levels of ethylene are somewhat higher than those levels
observed for mature fruit. In effect, the immature fruit
respond more rapidly to pectolyase treatment, the magnitude
of the response is greater, and the recover is faster than
that for mature fruit.
PG Inhibitor and Pectolyase-induced Ethylene Production
Since the PG from G. candidum induced ethylene
production, a hypothesis was formulated that an ethylene
promoting protein in the pectolyase preparation might be a
pectin-digesting enzyme. This was tested by mixing the PG.
inhibitor with pectolyase. The inhibitor did significantly
decrease the amount of ethylene produced by the fruit in
response to pectolyase at 8 hours after treatment (Fig.
3.8), although not below the level of the boiled control. A

37
Hours After Treatment
Figure 3.7. Green Valencia oranges, harvested 9/30/85, were
treated with 6 x 20 |jl injections of 30 ppm CHI
+ 0.11 pectolyase in 0.1 M acetate buffer pH 5,
D.I. water + 1 mg pectolyase/10 ml same buffer,
30 ppm CHI alone, or boiled pectolyase. All
points are means of 3 replications +/- S.E.

ni C.H. / Fruit / Hour
38
Figure 3.8. Yellow-green Valencia oranges, harvested
1/8/85, were treated with 6 x 10 ul of 0.1%
pectolyase, boiled pectolyase, and pectolyase +
a PG inhibitor in 0.1 M phosphate buffer pH 6.
All points are means of 3 replications +/- S.E.

39
protein then was isolated by affinity chromotography from
pectolyase which caused increased levels of ethylene to be
produced compared to controls (Fig. 3.9), but not within the
range of pectolyase. However, the activity and the
concentration of this protein and the fungal PG's, isolated
by affinity chromotography, may have been too low for an
ethylene response such as was found with pectolyase. On the
other hand, there may be some other protein(s) in the
commercial mix which are not cell wall-lysing enzymes or
were not eluted from the affinity column. These may be
causing some of the ethylene production or may be necessary
to enhance the activity of the pectin-digesting enzyme.
SDS Gel Profiles of Cell-Mall Lysing Enzymes
SDS polyacrylimide gel electrophoresis shows complex
protein profiles for the different enzyme mixtures (Fig.
3.10). The ethylene-inducing PG isolated from G. candidum
did, however, correspond to a similar molecular weight
protein of around 60,000 D in pectolyase (Fig. 3.11).
However it is not known which of these bands in pectolyase
are responsible to inducing ethylene production.
Transmission Electron Microscopy of Digested Cell Walls
The TEM micrographs of desalted pectolyase treated
orange peel show middle lamellae digestion (Fig. 3.12 and
3.13) whereas buffer-treated tissues show no signs of cell

Figure 3.9. Yellow Valencia oranges, harvested 2/27/85,
were treated with protein-containing fractions
#1-6, and 10, and control fractions #13, 21,
and 24 which did not contain protein, from an
alginate affinity column. Six x 20 |ul
injections were administered/fruit with 3 fruit
replications +/- S.E.

41

42
A B C D E F G
Figure 3.10. Coomassie blue-stained 111 polyacrylimide gels
of different enzyme preparations used in
protoplast isolation.
A)standards: bovine albumin, 66,000 D; egg
allbumin,
45,000 D; glyceraldehyde-3-P-dehydrogenase,
36,000 D;
carbonic anhydrase, 29,000 D;
trypsinogen,24,000 D;
trypsin inhibitor, 20,000 D; a-lactalbumin,
14,000 D.
B) cellulase
C) PME
D) pectolyase
E) cellulysin
F) drislase
G) pectinase

43
ABC
Figure 3.11. Coomassie blue-stained 11l polyacrylimide gels
of pectolyase and the Geotrichum candidum PG
A) standards
B) pectolyase
C) Geotrichum candidum P.G.

44
Figure 3.12. Transmission micrograph of orange peel tissue
treated with 10 pil of 0.1% pectolyase in 0.1 M
phosphate buffer pH 6, 24 hours after
treatment. ML=middle Lamellae at 26,000X.


44
Figure 3.12. Transmission micrograph of orange peel tissue
treated with 10 pil of 0.1Z pectolyase in 0.1 M
phosphate buffer pH 6, 24 hours after
treatment. ML=middle Lamellae at 26,000X.

45
Figure 3.13. Transmission micrograph of orange peel tissue
treated with 10 |ul of 0.1 % pectolyase in 0.1 M
phosphate buffer pH 6, 24 hours after
treatment. These sections were later treated
with periodate and then a silvermethanamine
solution for polysaccharide staining.
ML=middle lamellae at 90,000X.

46
wall breakdown (Fig. 3.14). This type of digestion would be
expected from a pectin-digesting enzyme such as a PG.
Callus tissue, treated with pectolyase, did not produce any
ethylene above boiled control levels although TEM studies
showed middle lamellae digestion (data not shown). This is
interesting because the callus produced wound ethylene upon
transfer into the experimental vials, showing that the cells
were capable of a wound ethylene response. It may be that
these cells are not able to perceive signals of cell wall
breakdown. The silver stained pectolyase-treated sections
of citrus peel show the digested area clearly as there is
little polysaccharide silver staining along the middle
lamellae area (Fig. 13).
Conclusion
Pectolyase appears to induce ACC synthesis and
subsequent ethylene production due to a pectin degrading
enzyme either directly or indirectly through some cell-wall
fragment product. Protein synthesis seems to be necessary
for optimal ethylene production induced by pectolyase.
Fungal PG' s and bacterial pectin lyases have been reported
to be elicitors of phytoalexins in plant tissues (101, 102)
often via their plant cell wall oligomeric products (35, 52,
53, 116). In addition, fungal cell walls have been
implicated as elicitors of an ethylene signal that was
linked to phytoalexin production in soybean (117). Finally,

47
Figure 3.14. Transmission micrograph of orange peel tissue
treated with 10 ul of 0.1 M phosphate buffer pH
6, 24 hours after treatment. CW=cell wall at
34,000X.

48
ethylene has been shown to have a diverse role in plant
defense responses ranging from effecting wound-induced
resistance to cellulase in oat leaves (68) to increased
chitinase activity in bean (31) and melon (149), to
hydroxyproline-rich glycoprotein accumulation in diseased
plants (60, 150), and phytoalexin accumulation in tobacco
(151), potato (75), and other plants (14). It is possible
that ethylene has a role in the initiation of a defense
response in citrus.

CHAPTER IV
PRODUCTS OF CELL-WALL DEGRADATION ELICIT ETHYLENE IN CTRUS
Introduction
Cell wall changes and ethylene production are highly
correlated in many physiological events such as mechanical
or chemical wounding (62, 63, 146) pathogen invasion (54,
64, 93), cell wall softening in ripening of certain fruits
(5, 26, 106, 118, 121), abscission (71, 93, 129, 131, 138),
and growth (98, 114, 127, 140). A cause and effect
relationship between physiological cell wall changes and
ethylene evolution has not been established. It may be that
they are unrelated products of a common stimulus or
interdependent events. Albersheim and others have shown
that the primary structures of the cell-wall polymers are
exceedingly complex (22, 89, 143). This enormous structural
complexity lead to the postulation that the cell-wall
polysaccharides may have regulatory functions in plant cells
via genomic expression (15). Such carbohydrates may control
various physiological responses of plants such as rate of
cell growth, time of flowering, activation mechanisms for
resistance to potential pathogens (112), and perhaps
ripening (80).
49

50
Single cells isolated for protoplasmic fusion or for
tissue culture are sometimes stimulated to produce ehylene
by the enzymatic mixtures used to separate the cells (11).
Cellulysin and macerase have been reported to induce
ethylene in tobacco and pear suspension cells respectively
(40, 73, 147). Preliminary studies were also conducted on
fruit ethylene production in response to partially digested
citrus pectin and fungal enzymes (99). Castor bean explants
produced ethylene when exposed to a mixture of enzymes or
purified polygalacturonase (PG), isolated from the fungus
Fusarum oxysporum (152). Fungal cell walls were shown to
elicit ethylene and phytoalexins in soybean (117). Cell
wall digesting enzymes and the resulting fungal or plant
cell wall fragments may regulate ethylene synthesis in
certain tissues. Regulation of ethylene by cell wall
digestion products would be a possible mechanism by which
plant cells could recognize pathogen invasion. Ethylene has
been shown to stimulate certain plant defense reactions such
as activation of chitinase (31, 149), wound-induced
resistance to cellulase (67, 68), and induction of
phytoalexins (9, 75, 151). In melons, both fungal and plant
cell wall elicitors induced synthesis of ethylene which in
turn elicited hydroxyproline-rich glycoprotein (HRGP).
Ethylene has been implicated in the accumulation of HRGP in
healthy (60) and infected plants (150). Finally,
carbohydrates, such as galactose, sucrose, and lactose, have

51
been shown to stimulate ethylene production in tobacco (112,
119) and galactose promoted ethylene evolution as well as
ripening in tomatoes (72).
This study is an investigation of the mechanism by
which certain enzyme mixtures induce ethylene production in
citrus peel. It was found that such enzymatic action on
cell walls may elicit ethylene indirectly via oligomeric
products.
Materials and Methods
Fruit Injection Method and Ethylene Determination
Valencia oranges (Citrus sinensis (L.) Osb.) were
harvested at various times of the year with 4 cm stems.
Fruit stems were stripped of leaves and put in 20 ml aqua
pics (self-sealing tubes which maintain water around the cut
end of plant or flower stems). The fruit were stored in
portable coolers for transport to the laboratory.
Treatment solutions of 20 |jl, containing carbohydrate,
enzyme, or carbohydrate plus enzyme solutions, were injected
into orange peel just under the flavedo at six locations
around the equator of the fruit. Some of these treatment
solutions were boiled and then cooled before injection.
Fruits, with stems in water, were then placed in glass jars
which were periodically capped for ethylene determinations.

52
Ethylene samples were taken at the end of two hours by
syringe and analyzed on a Hewlett Packard flame ionization
G.C., model #5706 A equipped with an activated alumina
column. Carrier gas flow rate was adjusted to give a sharp
ethylene peak that eluted at 0.4 minute retention time.
The carbohydrate solutions used to inject orange peel
contained either partially hydrolyzed commercial orange
pectin in deionized water (polygalacturonic acid methyl
ester from citrus fruits, grade I, Sigma), different size
oligomers of gel filtrated partially hydrolyzed pectin in 1
mM NaCl, sodium polypectate (polygalacturonic acid sodium
salt, a commercial preparation of de-esterified pectin,
Sigma), sodium polypectate digested for different time
periods by 0.2% or 0.4% pectolyase (w/v) in 0.1 M acetate
buffer pH 5.0, albedo pectin isolated from freshly harvested
Valencia oranges digested for different time periods by 0.2%
or 0.4% pectolyase in 0.2 M acetate buffer pH 5.0, pectin
oligomers obtained from an albedo pectin-pectolyase digest
via gel filtration in 30 mM acetate buffer pH 5.0 with 5 mM
disodium EDTA, a 5% chitin solution (Sigma) in 0.1 M acetate
buffer pH 5.6, chitin digested for 4 hours with a 0.05%
solution of chitinase (Sigma) in same buffer, 0.1 or 0.2 M
acetate buffer pH 5.0, 30 mM same buffer with 5 mM disodium
EDTA, 0.1% pectolyase (w/v) in 0.1 M same buffer, and boiled
pectolyase.

53
Preparation of Crude Pectin Hydrolysate
Five grams of commercial orange pectin were partially
hydrolyzed in 2 N trifluoracetic acid by the method of
Nothnagel et al. (116) at a concentration of 10 mg pectin
per ml acid. Suspensions were refluxed in an 85°C water
bath for 4 hours, then cooled and passed through a Whatman
GF/A glass microfiber filter. The resulting reténtate was
suspended in methanol at approximately 1:1 (v/v) and
evaporated to dryness. This proceedure was repeated a total
of four times. The final dried solids were suspended in
deionized water (10 ml water per initial gram of pectin) and
titrated to pH 7.0 with 5 M imidizole base. The resulting
suspension was cleared of particulates by filtration through
a Whatman GF/A millipore type HA 0.45 piM filter. The final
filtrate was the crude "pectin hydrolysate" used to inject
the fruit. Some of this material was boiled for five
minutes and cooled before injection into fruit.
"Unhydrolyzed pectin" consisted of 5 g of commercial orange
pectin to which imidizole base had been added in the same
amount that was used to neutralize the hydrolyzed pectin.
The resulting suspension was then titrated to pH 7.0 by
trifluoroacetic acid and injected into fruit.
Preparation of Carbohydrate-Enzyme Digest Treatments
One milliliter of a 20^ solution of Polygalacturonic
acid in 0.1 M acetate buffer pH 5.0 was incubated at 30°C

54
with 1 ml of a 0.2^ or 0.47a solution of pectolyase or boiled
pectolyase (w/v) in 0.2 M same buffer. All incubation
mixtures were then boiled at the end of either one or two
hours incubation time to stop all enzyme activity. The
mixtures were filtered through a Whatman #1 filter and the
resulting filtrate was used to inject fruit.
In a similar experiment pectin was extracted from
albedo tissue of Valencia oranges and incubated with
pectolyase. Two grams of chopped albedo tissue were ground
for five minutes with a Tekmar homogenizer in 10 ml cold
acetone and vacuum filtered through a Whatman #1 filter.
The solids were then re-homogenized and filtered again.
This step was repeated for a total of three times after
which the acetone was evaporated from the resulting powder.
The powder was stirred overnight in 40 mM acetate
buffer pH 5.0, containing 1 mM disodium EDTA at room
temperature. This suspension was then centrifuged at 27,000
g-max for 15 minutes and the supernatant was discarded. Two
and one-half grams of pellet material were homogenized with
30 ml of a 0.2^ or 0.41 (w/v) pectolyase or boiled
pectolyase in 0.2 M acetate buffer pH 5 and incubated for
0.5, 1, 1.5, or 3 hours at 30°C. At the end of the
appropriate incubation period, all mixtures were boiled and
filtered as described above and injected into fruit.
A 57c chitin (Sigma) solution (w/v) in 0.1 M acetate
buffer, pH 5.6 was incubated with 1 ml of a solution

55
containing 0.5!? chitinase (w/v) or boiLed chitinase (Sigma)
in same buffer for 4 hours. At the end of the incubation
period all mixtures were boiled to stop enzymic activity and
injected into fruit. Some fruit were also injected with a
5% chitin solution in 0.1 M acetate buffer pH 5.6 or buffer
alone .
Gel Filtration of Pectin Materials
The 5 g of partially acid-hydrolyzed pectin material,
which had been taken up in 50 ml of deionized water, was
concentrated to 7 ml by rotary evaporation. This material
was then passed through a 40 x 1.5 cm G-25 Sephadex column
eluted with 0.1 M NaCl. The void and 2 ml fractions of
included material were collected and injected into fruit (6
injections/fruit of 20 pi each). Every two fractions of the
included volume were pooled before injection.
The fractions most active in eliciting ethylene
production in fruit from the G-25 Sephadex column and pectin-
enzyme digest solutions, were each applied to a 46 x 1.5 cm
Bio-gel P-2 or Bio-gel P-4 column. The columns were washed
with 0.1 M NaCl or 30 mM sodium acetate buffer with 5 mM
disodium EDTA and 1 ml fractions were collected. These
fractions were tested colormetrically for total sugar
content by the phenol-sulfuric acid method (56) or for acid
sugar content by the Blumenkranz-Asboe-Hansen methods (29).
In some cases every 3 fractions, of those found to contain

56
sugar, were pooled and injected into fruit (6
injections/fruit of 20 |ul each) to assay for ethylene
inducing activity. In other cases, the void and individual
sugar peaks were pooled before injection. The P-2 column
had been calibrated with galacturonic acid, cellibiose,
raffinose and stachiose to determine the elution profile of
up to 4 sugar units.
Analysis of Neutral Sugars
Pooled sugar fractions from the P-2 and P-4 column
material were analyzed for neutral sugar content as
described by Albersheim et al. (7). Fraction material,
containing 0.3-1.0 mg of total acid sugar (29), was
hydrolyzed to aldoses and reduced to alditols by
trifluoroacetic acid and sodium borohydride, respectively.
The sugars were then acetylated by acetic anhydride using
pyridine as a catalyst. The acetylated sugars were analyzed
on a Hewlett Packard G.C. model #5710 A with a Spelco 23-40
column. Rhamnose, xylose, arabinose, mannose, galactose,
and inositol standards were used to determine retention
times. Inositol also served as the internal standard.

57
Results
Acid Hydrolyzed Pectin
The partially acid hydrolyzed pectin material
dramatically induced ethylene production over and above that
produced by the 0.1% pectolyase (w/v) treatment by 24 hours
after injection (Fig. 4.1). The pattern of ethylene
induction was different from that of pectolyase in that the
cells did not respond as quickly in terms of ethylene
production and did not show signs of decreasing ethylene
production by 24 hours after treatment. It may be that
fragments generated in the peel by pectolyase were more
localized, less concentrated and therefore metabolized more
quickly than the hydrolyzed pectin material which was
injected into the fruit. Also the concentration of
fragments generated by pectolyase may have been less than
those in the hydrolyzed pectin solutions. Boiling the
hydrolyzed pectin material caused a slight decrease in its
ability to elicit ethylene. This may have been due to
further hydrolysis of the fragment population or to
denaturation of possible ethylene-inducing proteins
contained in the pectin material. The unhydrolyzed pectin
produced very little ethylene, less than that of the boiled
pectolyase control. Pectolyase produced more ethylene than
the hydrolyzed pectin material only at 4 hours, and more
ethylene than the boiled control at all cime periods.

58
Figure 4.1. Yellow Valencia oranges, harvested 2/26/85,
were treated with 6 x 20 |al injections of crude
orange pectin hydrolysate, boiled crude
hydrolysate, unnydrolyzed pectin, 0.1Z
pectolyase (w/v) in 0.1 M acetate buffer pH 5.0
and boiled pectolyase. Ethylene measurements
were taken at 4, 8, and 24 hours after
treatment. All points are means of 3
replications +/- S.E.

59
Gel filtration of the partially acid-hydrolyzed pectin
on a G-25 Sephadex column produced some fractions that
showed greater ethylene-inducing capacity than others at 4
and 24 hours after treatment (Fig. 4.2A and B). Some of the
fractions produced almost as much ethylene as pectolyase at
4 hours (Fig. 4.2A) and much more ethylene at 24 hours (Fig.
4.2B). Interestingly, fractions that induced the most
ethylene at 4 hours were surpassed in ethylene elicition
capacity by other fractions, which contained smaller sugar
fragments, at 24 hours. The most active ethylene-inducing
fraction at 4 hours (F#7-8) and the most active ethylene-
inducing fraction at 24 hours (F#ll-12) were then each
chromatographed on a Bio-gel P-2 column. The resulting
fractions were analyzed colorimetrically for acid sugar
content. Every three fractions from sugar peaks were pooled
before injection into fruit, including some non-sugar
control fractions. These pooled fractions were injected
into fruit and ethylene production was determined 4 hours
after injection. Certain fractions induced more ethylene
synthesis than others, although none produced as much as the
original crude pectin hydrolysate or G-25 materials. The
Biogel P-2 fractions, #32-34, of the G-25 7 and 8 fraction
material, correspond roughly to a nona or decasaccaride and
produced the most ethylene, yet contained a relatively low
amount of sugar (Fig. 4.3). The P-2 fractions, #44-46, of
the G-25 11 and 12 fraction material, correspond to a

Figure 4.2. Yellow Valencia oranges, harvested 3/19/85,
were treated with 6 x 20 |il injections of
pooled fractions containing crude pectin
hydrolysate material that had been passed
through a G-25 Sephadex column washed with 0.1
M NaCl. Ethylene measurements were taken at 4
and 24 hours after treatment. All points are
means of 3 replications +/-S.E.

/ FRUIF / 2 HRS
51
TREATMENT

62
Figure 4.3. YeLLow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 ¡al injections of sugar-
containing fractions resulting from gel
filtration of the G-25 Sephadex fractions 7 and
8 on a Bio-gel P-2 column washed with 0.1 M
NaCl. The fractions were tested for total
sugar content by the phenol-sulfuric acid
assay. Every 3 fractions, of those found to
contain sugar, as well as 3 non-sugar control
fractions were pooled before injection into
fruit. Ethylene measurements were made at 4
hours after treatment and all ethylene
measurements are means of 3 replications +/-
S.E.

63
pentasaccharide and also produced slightly more ethylene
than the non-sugar control, yet contained a relatively low
amount of sugar (Fig. 4.4). The large mono, disaccharide
peaks in comparison induced less ethylene than the non-sugar
control fractions, indicating that ethylene production was
not determined by total sugar concentration or osmotic
effects .
Pectin-Digestion End Products
Both pectolyase and certain pectolyase-pectin digestion
mixtures induced ethylene production in citrus peel. The
digestion - mixtures had been boiled before injection into the
fruit so that their ethylene-inducing activity was not due
to active enzymes, but to the enzyme digestion products and
salts. The amount of enzyme present and the length of the
digestion period altered the ethylene-inducing activity of
these mixtures. A 0.2% solution of pectolyase (w/v)
incubated with polygalacturonic acid (PGA) for 2 hours
induced more ethylene production at 4 and 8 hours after
treatment than either its boiled control or a 0.4^ solution
of pectolyase incubated with PGA for one hour (Fig. 4.5).
The latter solution induced ethylene levels above its boiled
control only at 24 hours.
Pectolyase digestion of albedo pectin showed the most
dramatic inducement of ethylene production dependent upon
amount of enzyme and length of digestion period. A 0.4^

64
I
2.0-
1.8-
>
<
ví 16-1
tfi 1-01
^ CD
1.4-1
E ,
3 03 1.2-
u-<
3 1.0-1
(/)
0.8-
0.6-
0.4
0.2
o
z
Ui
x
.a.
VOID
i
18
26
ACID HYDROLYSIS OF PECTIN
MONO-DI —
/
Y
I,
34 42 50 58
P-2 FRACTION NUMBERS
-32
-24
9
♦
ó
o
PENTA T"RA TRI / 1
34 42 50 58 66
-8
74
Figure 4.4. Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 ;ul injections of sugar-
containing fractions resulting from gel
filtration of the G-25 Sephadex fractions 11
and 12 on a Bio-gel P-2 column washed with 0.1
M NaCl. Every 3 fractions, of those found to
contain sugar as well as 3 non-sugar control
fractions, were pooled before injection into
fruit. 3 non-sugar fractions were also pooled
for a control. Ethylene measurements were made
at 4 hours after treatment and all ethylene
measurements are means of 3 replications +/-
S.E.
nl C2H4/ FRUIT / 2 HRS.

65
Figure 4.5. Regreened Valencia oranges, harvested 5/6/85,
were treated with 6 x 20 (jl injections of a 201
solution of polygalacturonase in 0.1 M acetate
buffer pH 5.0, incubated with 0.22 or 0.42
pectolyase (w/v) or boiled pectolyase in same
buffer for 1 and 2 hours respectively.
Ethylene measurements were taken at 4, 8, and
24 hours after treatment and all points are
means of 3 replications + /- S.E.

66
pectolyase solution (w/v) induced large amounts of ethylene
4 hours after treatment when incubated with albedo pectin
for 1 hour, over double that of the boiled control (Fig.
4.6A). The same solution, however, failed to induce much
ethylene above boiled controls when incubated with albedo
pectin for either 0.5, or 1.5 hours. Similarly a 0.2%
solution of pectolyase induced more ethylene when incubated
with albedo pectin for 1 or 2 hours than at 3 hours at both
time periods tested (Fig. 4.6A and B). All incubation
periods, in this case, resulted in ethylene production above
that of the corresponding boiled controls. A 0.1% pectolyase
solution (w/v) alone induced more ethylene than any of the
enzyme-pectin digest mixtures.
The most active ethylene-inducing enzyme-pectin digest
mixtures (0.4% pectolyase, incubated with 2.5 g albedo
pectin for 1 hr and 0.2% pectolyase, incubated with 2.5 g
albedo pectin for 1 and 2 hours) were pooled and
chromatographed on a Bio-gel P-2 column. The resulting void
and sugar peaks were individually pooled and aliquots
injected into fruit. The void (F#24-27) and a shoulder off
the void (F#28 — 32), corresponding to around 9-10
galacturonic acid units, appeared to contain the most
ethylene-inducing capacity (Fig. 4.7), although ethylene
levels were relatively low when compared to the enzyme-
pectin digest mixtures (Fig. 4.6). Salts, present in the
pectolyase enzyme mixture, co-eluted with the

Figure 4.6. Green Valencia oranges, harvested 7/16/85, were
treated with 6 x 20 jul injections of various
albedo pectin-pectolyase digest mixtures. 2.5
g of extracted albedo pectin was incubated with
0.4Z or 0.2% pectolyase (w/v) or boiled
pectolyase in 0.2 M acetate buffer pH 5, for
0.5, 1.0, 1.5, 2.0, or 3.0 hours. Ethylene
measurements were made at 4 (A) or 8 (B) hours
after treatment and all bars are means of 3
replications +/- S.E.

O o
900*'
PECTOLYASE DIGESTION OF ALBEDO PECTIN
4 HOURS
Tr«af mentí

69
Figure 4.7. Green Valencia oranges, harvested 9/10/85, were
treated with 6 x 20 pi injections of sugar-
containing fractions resulting from gel
filtration of pooled albedo pectin-pectolyase
digest mixtures (albedo pectin digested by 0.4^
pectolyase for 1 hour and by 0.2% pectolyase or
1 and 2 hours) on a Bio-gel P-2 column washed
with 30 mM acetate buffer pH 5.0 with 5 mM
disodium EDTA. The void, individual sugar
peaks, and 3 non-sugar fraction controls were
pooled before injection into fruit. Ethylene
measurements were made at 4 hours after
injection and all ethylene measurements are
means of 3 replications +/- S.E.

70
monosaccharides and are probably contributing to the
ethylene production observed for this small peak. Since the
ethylene inducing sugar peak was not well separated from the
void, the same material was chromatographed on a Bio-gel P-4
column. The resulting sugar profile was tested for ethylene
production as described before. Again, one sugar peak (F #
43-51) induced slightly more ethylene than the rest (Fig.
4.8). Ethylene production, expressed per gram of sugar
injected into fruit for sugar peaks from both columns, shows
more clearly that certain P-2 and P-4 fractions elicited
more ethylene than others (Table 4.1).
Analysis of Neutral Sugar Content
Analysis of neutral sugar content for both the Bio-gel
P-2 and P-4 column material (Table 4.2 and 4.3) showed that
rhamnose, arabinose, xylose, and galactose were detected
with arabinose, galactose, and xylose occurring in the
highest amounts. The fractions which showed the most
ethylene-inducing activity contained relatively high amounts
of arabinose and galactose, small amounts of rhamnose and
xylose, and trace amounts of mannose and another unknown
sugar.
Effect of Chitin on Ethylene Production
A 57o solution of chitin (w/v) in acetate buffer induced
a small amount of ethylene production at 8 and 24 hours

ACID SUGAR ASSAY
ABS. 520
71
Figure 4.8. Green Valencia oranges, harvested 9/30/85, were
treated with 6 20 ¡al injections of sugar-
containing fractions resulting .from gel
filtration of pooled albedo pectin-pectolyase
digest mixtures (albedo pectin digested by 0.4^
pectolyase for 1 hour and by 0.2% pectolyase
for 1 and 2 hours) on a Bio-gel P-4 column
washed with 30 mM acetate buffer Ph 5.0 with 5
mM disodium EDTA. The void, individual sugar
peaks and 3 non-sugar fraction controls were
pooled before injection into fruit. Ethylene
measurements were made at 4 hours after
treatment and all ethylene measurements are
means of 3 replications +/- S.E.
nl C,H4 / FRUIT/2 HRS.

72
Table 4.1. Green Valencia oranges, harvested 9/10/85 or
9/30/85, were treated with 6 x 20 i_il injections
of sugar containing fractions resulting from
gel filtration of pooled ethylene-inducing
albedo pec tin-pec tolyase digest mixtures on Bio¬
gel P-2 or P-4 columns. Ethylene measurements
were made at 4 and 8 hours after treatment for
tne P-2 and P-4 column materials respectively.
The resulting ethylene levels in this table
reflect only the ethylene produced above
control levels.
Treatment
4 Hrs after Treatment
P-2 F# Sue.(ug)/Fru.
nl C0H/(/fruit/2 hr nl CoH^/10u-g Sug.
24-27
67.2
23-31
73.2
32-35
27.9
39-46
10.8
7.8
11.6
0.6
5.0
4.4
5.
0.
3.
P-4
£7 'L
f TT
8 Hrs
27-34
14.7
43-51
17.1
52-62
33.0
63-67
14.7
0.6
6.4
0.0
0.4
0.4
3.2
0.0
0.3
after Treatment
U> O' 4>

73
Table 4.2. Neutral and acid sugar analysis was made on the
albedo pectin-pectolyase digest material that
had been fractionated on a Bio-gel P-2 column.
Fractions comprising sugar peaks were pooled
for analysis of sugar content.
P-4 F# MOLE ACID SUG. MOLE NEUTRAL SUG. NS MOLE? of AS
43-51
4x10 6 ARABINOSE
2x10 l
5.0?
GALACTOSE
7x10 ‘
18.0?
Trace amts: xylose,
mannose, and
unk.sug.
52-62
6xl0_6 ARABINOSE
XYLOSE
GALACTOSE
2x10~7
3x10 7
9x10 7
4.0?
5.0?
16.0?
Trace amts: mannose
and unk. sug.
63-67
2xl0'6 ARABINOSE
GALACTOSE
2xl0-10
2x10 1U
12.0?
0.01?
Trace amts: xylose and unk.
sug.

74
Table 4.3. Neutral and acid sugar analysis was made on the
albedo pectin-pectolyase digest material that
had been fractionated on a Bio-gel P-4 column.
Fractions comprising sugar peaks were pooled
for analysis of sugar content.
P-2 F#
MOLE ACID SUGAR.
MOLE NEUTRAL SUG.
NS MOLEZ of AS
28-31
7xlO_6
ARABINOSE 6xl0_^
RHAMNOSE 6x10 5
8.82
0.92
XYLOSE 2x10 '
1.42
GALACTOSE 3x10
4.42
Trace amts unk. sug.
32-35 3x10 6 ARABINOSE 2xl0_^ 6.22
XYLOSE 2x10 ' 6.22
GALACTOSE 1x10 3.12
Trace amts unk. sug.
39-46
3x10 6 ARABINOSE 2xlO_5?
XYLOSE 1x10 s
0.52
0.4 2

75
after injection that was slightly higher than a buffer
control (Fig. 4.9B). Digestion of the chitin with chitinase
for 4 hours, however, increased ethylene production over
four times that of the boiled control at 8 hours after
treatment (Fig. 4.9A). The chitinase itself showed some
ethylene-inducing activity, probably due to salts present in
the enzyme preparation.
Conclusion
Products of cell wall digestion, whether by acid or
enzymes, were capable of eliciting ethylene production in
citrus peel of intact citrus fruit. Data indicate that more
material is needed of specific fragment size or size range
to induce the level of ethylene such as is observed for
pectolyase. The response to pectolyase, however, may not be
due solely to cell-wall lysing enzymes. Salts in the
pectolyase mixture also induce some wound ethylene, and
lysis of cell walls could promote leakage of toxic materials
from oil glands in citrus peel which may promote ethylene
production in neighboring cells. Since activity is lost as
the sugar materials are further separated into their
corresponding fragment sizes, it is possible that certain
oligomers act synergistically in elicitation of ethylene
production. Albersheim and others have noted that both
fungal and plant cell wall fragments elicit phytoalexin
production in several plant species (91, 116, 154). It has

ni C2H4 / FRUIT / 2 HRS.
76
Regre
ened
Valencia
orang
es, harves
t
ed 5/6/85,
were
treat
ed with
6 x 20
(uí inject
i
ons of a 5Z
chiti
n sol
ution (w
/v), i
n 0.1 M ac
e
tate buffer
pH 5.
6, incubated
with a
0.5Z solut
ion of
chiti
nase
(w/v) or
boile
d chitinas
e
in same
buf f e
r for
4 hours
, or 0
.5Z Chitin
a
se alone
(A) .
Othe
r fruit
were i
njected wi
th a 0.57o
solut
ion o
f chitin
in sa
me buffer,
or buffer
alone
(B) .
Ethyle
ne mea
surements
were made at
4, 8,
and
24 hours
after
treatment
and all
point
s are
means of 3 re
plications
+/- S.E.

77
also been suggested that fungal and plant cell wall
fragments may act synergistically in elicition of
phytoalexin production in soybeans (48, 159). It would be
interesting to investigate whether these oligomeric products
of cell wall digestion induce synthesis of new mRNA or
protein, such as ACC synthase, that result in increased
ethylene production. The ethylene, in turn, promotes
chlorophyll breakdown (59) and abscission (71, 120) in
citrus fruits. If this also involves synthesis in new mRNA
and protein then a complex model emerges for plant detection
and response to fungal invasion. Similar processes may
occur in cases of ripening and abscission.

CHAPTER V
STRUCTURAL AND BIOCHEMICAL DEFENSE MECHANISMS IN CITRUS
Introduction
A Citrus fruit is considered to be a berry of unusual
structure (8). The thick peel includes exo- and mesocarp
while the pulp originates from proliferations of the
endocarp. The fruit has a superior ovary (8, 136) which
consists of 10-13 carpels and the ovules are formed in two
rows in each locule on marginal and central placentai (136).
The exocarp, or flavedo, which is the colored portion of the
peel, contains chloroplasts or chromoplasts. Chloroplasts,
that are present in the early stages of fruit development,
later transform into chromoplasts due to the disappearance
of chlorophyll and the unmasking and synthesis of
caroteniods. Thus the green fruits turn yellow to orange at
maturity. These changes are temperature and ethylene
related and can be reversed by chromoplasts reconversion to
chloroplasts in a process called regreen'ing (144, 145). The
flavedo also contains oil glands formed by special cells
that produce terpenes and oils, which then later lyse to
form the oil gland cavities These cells are rich in
78

79
protoplasm and oils and eventually undergo disintegration of
their cell walls. The parenchymous cell adjacent to the
resulting cavity are thick walled and resist the lysigenous
process (33, 34). Flavedo epidermal cells produce cutin and
waxes which protect their outer surface and tangential walls
by a layer of wax and cutin (8, 136). This outer epidermal
layer also contains actinocytic type stomates (136).
The mesocarp or white colored albedo portion of the
orange peel consists of colorless cells which are typically
multi-armed, parenchymous and highly vacuolated. The tissue
contains large air spaces imparting to it a spongy nature.
The albedo is less specialized than the flavedo and although
some cells contain leucoplasts, it mainly serves as a
storage parenchyma. Some cells have been observed to
contain starch in young fruit but this is rarely found in
mature tissue (8, 136). The endocarp portion of the citrus
fruit is the most complex, giving rise to the juice sacs
which fill the locules entirely at fruit maturity (136).
Pathogens must penetrate citrus peel to invade the
fruit. The waxy cuticle and toxic terpenes and oils of the
flavedo offer a formidable defense to most pathogens.
Invasion of citrus fruits, therefore, usually occurs through
the stylar or button (stem) ends, or through a wounded
portion of the peel (18). The albedo, although much more
vulnerable to pathogen invasion if exposed, has been found
to contain a polygalacturonase (PG) inhibitor (17). This

80
inhibitor may be a form of defense against pathogens such as
Diplodia natalensis, a fungal pathogen of citrus that uses a
PG enzyme to lyse through host cell walls. The albedo is
also capable of producing much ethylene when wounded (64,
84). This may be another form of defense in that it can
lead to fruit abscission (27). This study describes the
structural features of citrus peel as they relate to
possible defense mechanisms and responses to cell-wall lysis
which typically occur during pathogen invasion of fruit.
Materials and Methods
Harvesting of Fruit
Valencia oranges (Citrus sinensis L. Osb.) were
harvested at various times of the year with 4 cm stems.
Fruit stems were stripped of leaves and put in 20 ml aqua
pics (self-sealing tubes which maintain water around the
ends of cut plant or flower stems). The fruit were stored
in portable coolers for transport to the laboratory.
Investigation of Citrus Peel Structure
Light Microscopy. Fresh Valencia orange peel was
sectioned longitudinally at 30 microns with a vibratome.
These sections were placed in water under a coverslip and
photographed in phase contrast and under ultraviolet
illumination.

81
Scanning Electron Microscopy. Valencia orange peel was
2
cut into 1 mm sections and fixed in 2% gluteraldehyde in
0.1 M cacdyllate buffer for 2 hours and then overnight in 2Z
osmium tetraoxide. Sections were then dehydrated in an
alchohol series, critical point dried in 100Z ethanol, and
coated with a thin layer of gold for observation under the
scanning electron microscope.
Orange Peel X-ray diffraction Analysis
2
Valencia orange peel sections were cut into 1 mm
sections, dehydrated for 48 hours over dry-rite and observed
uncoated with the scanning electron microscope for
characteristic X-rays of elements contained in the peel.
Electophoresis of Citrus Peel Proteins
A 20 ¡ul cell-wall lysing enzyme solution containing
0.1Z pectolyase (w/v) in 0.1 M acetate buffer pH 5.0, was
injected into Valencia orange peel just under the flavedo at
six locations around the equator of the fruit using serum
syringes with 25 gauge needles. Some of the enzyme
solutions were boiled and then cooled before injection.
There were 8 fruit replications for each of the treatments.
After four hours, injection sites of four fruit from
each treatment were excised with a 7 mm cork borer. The 72
discs resulting from each treatment were stored for 24 hours
in liquid nitrogen. Discs were then thawed and ground for 5

82
minutes with a Tekmar homogenizer in 10 ml cold acetone and
then vacuum filtered through a Whatman #1 filter.
Homogination and filtration were repeated three times on
remaining solids after which the acetone was evaporated.
The resulting filtrate was collected, lypholized and stored
at 4° C. Equal amounts of the powder (850 mg) from each
treatment were ground for 30 minutes with the Tekmar
homogenizer at low speed in 10 ml cold 0.05 M tris-glycine
buffer pH 6.6. This mixture was then centrifuged at 27,000
g-max for 15 minutes after which the pellet was discarded.
The supernatant was filtered through glass wool and combined
with cold acetone to make up an 80Z solution and put in the
freezer for 18 hours for maximal protein precipitation.
Precipitate was then centrifuged at 27,000 g-max for 15
minutes and the supernatant was discarded. The pellet was
drained and dissolved in a buffer that consisted of 1.5 g
tris, 20 mis glycerol, 4 g sodium dodecalsulfate (SDS), 10
ml 2-mercaptoethanol and 0.002 g bromophenol blue in 100 mis
of deionized water. The resulting solution was loaded onto
4X stacking and 11Z running tube gels. These were run at a
constant current of 2 mA per gel for three hours after which
they were fixed with coomassie blue. The same procedure was
repeated for the remaining 4 fruit from each treatment at 8
hours after injection.

83
Ethylene Determination
Valencia oranges were treated with solutions of 0.1!?
pectolyase (w/v) in 0.1 M acetate buffer pH 5.0, boiled
pectolyase, or orange peel filtrate obtained by grinding
orange peel in acetone as described above. The treatments
consisted of 6 x 20 pi 1 injections of these solutions into
the peel by serum syringes with 25 gauge needles. The
fruits, with stems in water, were then placed in glass jars
which were capped periodically for two hours to determine
ethylene content at 4, 8, and 24 hours after treatment.
Ethylene samples were taken by syringe and analyzed on a
Hewlett Packard flame ionization G.C. model #5706 A equipped
with an activated alumina column. Carrier gas flow rate was
adjusted to give a sharp ethylene peak that eluted at 0.4
minute retention time.
Results
Orange Peel Structure
Citrus peel structure is most clearly observed when
illuminated with ultraviolet light. Sections from a green
orange show fluorescing cuticle on the outer surface and
along the tangential walls of flavedo epidermal cells and
faintly fluorescing chloroplasts in the cells of the flavedo
layer (Fig. 5.1). The same tissue when viewed in phase

84
Green Valencia oranges were harvested 10/30/84,
and the peel was cut in 30 micron sections and
viewed under the light microscope in fresh
condition under fluorescent light. Fluorescing
chloroplasts (Cl), and cuticle (Cu) are visible
in this flavedo section at 640X.
Figure 5.1.

85
contrast, reveals chloroplast organelles within cells and a
stomata in the epidermal layer (Fig. 5.2). A scanning
electron micrograph clearly shows the transition from the
comparatively dense flavedo cells to the albedo with its
abundant air spaces (Fig. 5.3). An oil gland is also
clearly defined as a cavity bordered by closely packed
thick-walled cells. The bordering cells are seen at the
back of the cavity.
Orange Peel Mineral Analysis
Orange peel that had been dehydrated and observed
uncoated under the scanning electron microscope for X-ray
microanalysis displayed an interesting spectrum of elements
(Fig. 5.4). The peel is shown here to contain significant
amounts of sulphur, chlorine, potassium, and calcium. The
other peaks in the spectrum are aluminum and silica and are
X-rays coming from equipment metals. X-ray diffraction
analysis reveals elements in a specimen with atomic numbers
above that of sodium in amounts above trace levels, although
quantification is not possible by this method. Of these
elements, calcium has been shown to have a stimulating
effect on ethylene synthesis (25) and is required for
maximal activity of pectin lyase enzymes (130)
Effect of Pectolyase on Orange Peel Protein Profiles
The gels from pectolyase-treated orange peel show an
increase in some proteins compared to those from the boiled

86
Figure 5.2. Green Valencia oranges were harvested 10/30/84,
and the peel was cut in 30 micron sections and
viewed under the light microscope in phase
contrast. Chloroplasts (Cl) are visible within
flavedo cells and one stomata (S) is evident in
the epideral layer at 580X.

87
A
F
OG
Figure 5.3. Green Valencia were harvested 10/30/84, and the
peel was fixed, dehydrated, crital point dried,
and gold coated for observation under the
scanning electron microscope. An oil gland
(OG) is visible in the flavedo (F) tissue and
large intercellular spaces are visible in the
albedo (A) tissue.

88
Figure 5.4. X-ray diffraction analysis of dehydrated orange
peel show peaks for sulphur, chlorine,
potassium, and calcium. Silica and aluminum
peaks are due to the presence to these metals
in the scanning microscope equipment.

89
pectolyase treated peel. This is evident due to a darkening
of some low molecular weight bands of around 15,000 and
18,000 d (Fig. 5.5). Other protein bands of around 67,000,
66,000, and 53,000 d and again in the 28,000-31,000 d range,
present in boiled pectolyase treated tissue, are very faint
or not evident in the pectolyase treated peel. There are
also differences between the bands found in the boiled
pectolyase gels at 4 hours after treatment and those at 8
hours after treatment with all the lower molecular weight
bands becoming very faint in the 8 hour tissue. These
proteins in particular may be stress related as all
treatments resulted in some ethylene production (data not
shown) and ethylene is thought to be associated with stress
(25, 63).
Pectolyase and Orange Peel Filtrate Induce Ethylene
Pectolyase induced ethylene production over and above
the boiled pectolyase controls (Fig. 5.6). The response to
pectolyase may be additive, however. Cells could be induced
to produce ethylene by the cell-wall lysing enzymes
themselves or cell-wall digestion products. A similar
response has been shown with tobacco treated with cellulysin
(10, 39), and pear suspension cells treated with macerase
(147). Furthermore, wound ethylene can be elicited by salts
in the pectolyase mixture included to stabilize the enzymes.
This is probably the reason for the small amount of ethylene

90
A B C D E
Figure 5.5. Coomassie blue-
stained 11% polyacrylimide gels of Valencia
orange peel proteins from fruit treated with
boiled pectolyase and pectolyase at different
times after treatment.
A) standards: bovine albumin, 66,000 d;
egg albumin, 45,000 d; glyceraldehyde-3-
P-dehydrogenase, 36,000 d; carbonic
anhydrase, 29,000 d; trypsinogen, 24,000
d: trypsin inhibitor, 20,000 d;
lactalbumin, 14,000 d.
B) Boiled pectolyase-treated orange peel
analyzed at 4 hours after treatment.
C) Pectolyase-treated orange peel analyzed
at 4 hours after treatment.
D) Boiled pectolyase-treated orange peel
analyzed at 8 hours after treatment.
E) Pectolyase-treated orange peel analyzed
at 8 hours after treatment.

91
4 8 24
Hours After Treatment
Figure 5.6. Green Valencia oranges, harvested 7/16/85, were
treated with 6 x 20 pi of 0.12 pectolyase in
0.1 M acetate buffer pH 5.0, boiled pectolyase,
buffer alone, and cell filtrate from Valencia
orange peel dried down and then taken up in
same buffer. All points are means of 3
replications +/- S.E.

92
produced in response to boiled pectolyase. Finally, the
lysis of cell walls around cells and oil glands could cause
membrane leakage, and/or leakage of terpenes and oils from
oil glands which could cause neighboring cells to produce
ethylene. The fact that the filtrate from ground peel
tissue caused substantial ethylene production (Fig. 5.6)
when injected into the peel (although less than that of
pectolyase) is evidence for this last point.
Conclusion
Damage to citrus peel has been shown to induce peel
ethylene production, whether it be due to abscission
chemicals or pathogens (49, 62, 63). The latter also occurs
in different plant tissues (54, 64, 93), but the stimulus
for ethylene production in citrus may be partly due to the
contact by neighboring cells with materials leaked from
damaged cells or oil glands. Also sugar fragments produced
during lysis of cell walls by pathogenic enzymes, could
serve as signals for ethylene production in neighboring
cells as well as for other possible cellular defense
mechanisms. Such a process would likely involve mRNA and
protein synthesis resulting in in altered protein profiles.
This hypothesis is consistant with results obtained when
pectolyase was used to mimic pathogen invasion.
This type of signalling has been observed before. It
has been shown that fungal cell-wall lysing enzymes and

93
their cell-wall fragment products induced plant defense
responses such as the synthesis of phytoalexins and lignan,
and an increase in protease inhibitor activity in various
plant species. This, in turn, involved synthesis of enzymes
necessary for production of these compounds (65, 91, 154).
In addition, fungal cell walls have been shown to elicit
both ethylene and phytoalexins in soybean (117). In the
case of citrus, however, ethylene and the PG inhibitor (17)
are the only defense mechanisms stimulated by pathogens so
far discovered.

CHAPTER VI
SUMMARY
Certain cell-wall lysing enzymes can induce ethylene
production in citrus peel. Data indicating a possible
mechanism by which these enzymes induce ethylene is
summarized here, as well as the associated structural and
biochemical attributes of citrus peel. Implications of this
work for plant physiology and the horticultural industry are
discussed and areas for future research are suggested.
Of the different types of cell-wall lysing enzymes
tested, the commercial mixture, pectolyase, and a PC from
the citrus pathogen Geotrichum candidum were found to induce
ethylene production when injected into citrus peel.
Pectolyase stimulated the most dramatic ethylene release at
concentrations tested. Commercial cell-wall lysing enzyme
mixtures were shown by SDS polyacrylimide gel
electrophoresis to contain many different proteins. Salts
were also present for enzyme stabilization and these
appeared to induce low levels of wound ethylene evident when
boiled controls were compared to desalted mixtures. The
injection process itself produced little or no ethylene
94

95
response. It is not known at this point which protein(s) in
pectoLyase were responsible for inducing citrus peel cells
to produce ethylene, but evidence suggests that it is a
pectin-digesting enzyme. Data from previous chapters show
that a PG from G. candidum induces ethylene, that a PG
inhibitor partially inhibits pectolyase-induced ethylene
synthesis in citrus peel and that the optimal pH for
pectolyase-induced ethylene production is the same as that
reported for most fungal PG.'s. Also, transmission
micrographs of pectolyase-treated citrus peel show middle
lamellae digestion indicating the presence of a pectin¬
degrading enzyme such as PG. It is possible that a PG
and/or a pectin lyase could be responsible for the middle
lamellae digestion and ethylene production since pectolyase
could contain either or both of these enzymes. Fungal and
bacterial pectin lyases and PG's have been reported to be
elicitors of phytoalexins in plant tissues (35, 101, 102),
and other commercial cell-wall lysing enzymes have been
shown to induce ethylene production in tobacco (10, 11,
147) .
The ethylene biosynthesic pathway was next examined to
determine at which point its induction by pectolyase
occured. Pectolyase induced ACC synthesis indicating that
it probably had some effect on ACC synthase activity or
synthesis. Partial inhibition of pectolyase-induced
ethylene production by cycloheximide suggested that protein

96
synthesis was necessary for the maximal ethylene response.
Whether this is necessarily the de novo synthesis of ACC
synthase requires more research. It was discovered through
gel electrophoresis that pectolyase caused citrus peel to
synthesize more of certain proteins and less of others when
compared to boiled pectolyase treated tissue. Again this
would seem to suggest that de novo synthesis of some
proteins is a response to pectolyase.
Effects of cell-wall lysing enzymes themselves on
ethylene production in citrus peel were then compared to
those of cell-wall digestion products such as pectic
polysaccharides. Commercial citrus pectin, partially
hydrolyzed by trifluoracetic acid, and albedo cell walls,
partially digested by pectolyase, were both capable of
inducing ethylene production equal to and above that
produced by a 0.1% pectolyase solution. Separation of these
sugar oligomers showed that polymers of certain size ranges
stimulated more ethylene production. The most potent
elicitor appeared to be an oligosaccharide of around 10
galacturonic acid units, the same size range reported for
elicitors of some phytoalexins (111). Chemical analysis of
the neutral sugars associated with the fraction examined
here indicated that they differed from those that elicit
phytoalexins, however, maximum stimulation of ethylene
release at 4 hours was also stimulated by a larger fragment
than was that at 24 hours after treatment. Further studies

97
are necessary to determine the significance of this. In
general, a range of fragment sizes induced more ethylene
than the summed ethylene production of individual fragments,
suggesting synergism between the individual fragments in
terms of ethylene production. It has been suggested that
fungal and plant cell wall fragments act synergistically in
elicition of phytoalexin production in soybeans (48, 159).
Oligomeric products of pectin-degrading enzymes have been
reported to be elicitors of phytoalexins in plants (35, 53,
116). Ethylene has been shown to be induced by cell-wall
lysing enzyme products in pear suspension cells (147).
Chitin also induced some ethylene production when injected
into citrus peel especially if digested by chitinase before
injection. This is not surprising in view of the fact that
fungal cell wall fragments elicited ethylene and
phytoalexins in soybean (117).
The structure of citrus peel suggests that cell-wall
lysis in the area of an oil gland could lead to leakage of
the glandular contents which might induce neighboring cells
to produce ethylene. Cell-wall lysis may cause cellular
membranes to become leaky due to possible changes in turgor
pressure, extracellular free ion levels and extracellular
pH. Such leakage of cellular contents may also induce
ethylene synthesis in neighboring cells as indicated by
ethylene production after injection of filtrate from ground
peel tissue induced substantial ethylene production when

98
injected into the peel. The filtrate contained cellular and
glandular materials. The response to pectolyase therefore
could be a triple response: ethylene produced due to salt
stress, ethylene produced due to cell-wall lysis, and
ethylene produced due to leakage of cellular and glandular
materials .
Release of ethylene reported here and elsewhere in
response to products of cell wall lysis suggests that cell-
wall fragments may be universal signals for ethylene
synthesis. The type or size of ethylene-inducing fragments
could conceivably vary from species to species. Some plants
may respond more to fungal cell-wall fragments and others to
plant cell-wall fragments or to both. Albersheim and others
are proponents of the theory that cell-wall fragments are
mediators of diverse plant responses (111). Since ethylene
has been shown to have a diverse role in plant defense
mechanims (30), it would be logical for plant cells to
recognize a signal for pathogen invasion which could turn on
ethylene production and/or the production of other defense
chemicals such as phytoalexins, lignin and protein synthesis
inhibitors. Cell-wall fragments, which are produced by
pathogenic cell-wall lysing enzymes, would be logical
candidates for such a role.
Ethylene is also associated with endogenous cell-wall
lysing enzymes during ripening (28, 47, 121), abscission
(131, 138), and growth (98, 114, 127). If similar

99
mechanisms are operating in response to endogenous cell-wall
lysing enzymes, new possibilities may exist for manipulation
of ripening, abscission and perhaps even growth. This area
needs to be investigated in order to determine whether
ethylene turns on synthesis of endogenous cell-wall lysing
enzymes or enhances their activity, or whether these enzymes
turn on ethylene production. It could be that any or all of
these things are happening in some sort of autocatalytic
operation in climacteric fruit, or in abscission zones.

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87 .
i

BIOGRAPHICAL SKETCH
Elizabeth Amory Baldwin was born in New York, N.Y., in
1952 and was raised in and around the New York City area.
After finishing highschool in 1970, she worked for Ralph
Nader and participated in the research and writing of the
book Old Age, the Last Segregation. In 1971 she left to
travel for a year throughout Europe, the middle east, India,
and Shrilanka. During her travels, she worked in a home for
brain damaged children, on a rice paddy farm in Shrilanka,
and on a kibbutz in Israel.
In 1972, she entered Fordham University in New York
City switching later to Hunter College of the City
University of New York in 1973. She completed a Bachelor of
Arts degree with honors in 1976 in anthropology. After
working in several positions from 1975-1977, she became co¬
owner and manager of a fruit farm in Jamaica W.I., living
and working there for several years. Having cultivated an
interest in horticulture, she entered Middle Tennessee State
University in 1979, receiving a Bachelor of Science degree
with honors in plant and soil science in 1980.
In 1981 Elizabeth entered a graduate program at the
University of Florida under the direction of Dr. Robert
117

118
Hilton Biggs, receiving her Master of Science degree in
1982. From there she continued, under the guidance of Dr.
Biggs, to work on her Ph.D. and will receive her degree in
May 1986. During her time with the Fruit Crops Department,
she served as vice-president of the Fruit Crops Graduate
Students (1984-1985), was awarded the Hughes Foundation
award in 1984 and the Harry Spyke Memorial Scholarship in
1985.
Elizabeth is the wife of Michael P. Timpe and mother of
2 year old son, Ulrich. Upon completion of her graduate
studies she plans to move to Athens, Georgia, where she will
be working for the USDA investigating the biochemistry of
ripening in the laboratory of Dr. Russel Pressey.

I certify that
opinion it conforms
presentation and is
a dissertation for
I certify that
opinion it conforms
presentation and is
a dissertation for
I certify that
opinion it conforms
presentation and is
a dissertation for
I certify tha t
opinion it conforms
presentation and is
a dissertation for
I certify that
opinion it conforms
presentation and is
a dissertation for
I have read this study and that in my
to acceptable standards of scholarly
fully adequate, in scope and quality,
the degree of Doctor of Philosophy.
a
LC.
\
/Robert H. Biggs /^¿hairnai
Professor of Fruit Crops
as
I have read this study and that in my
to acceptable standards of scholarly
fully adequate, in scope and quality, as
the degree of Doctor of Philosophy.
I have read this study and that in my
to acceptable standards of scholarly
fully adequate, in scope and quality,
the degree of Doctor of Philosophy.
\r'
Cs
\ \
v\
Donald J .- ,Huber
Assistant Professor oj
Vegetable Crops
I have read this study and that in my
to acceptable standards of scholarly
fully adequate, in scope and quality, as
the degree of Doctor of Philosophy.
"Chesley D. Hall
Professor of Ve
Crops
geta ble
I have read this study and that in my
to acceptable standards of scholarly
fully adequate, in scope and quality,
the degree of Doctor of Philosophy.
-L-
S m i t h
R i c n a r d
Proressor of botany
3. S

This dissertation was submitted to the Graduate Faculty of
the College of Education and to the Graduate School and was
accepted as partial
degree of Doctor of
fulfillment of the requirements for the
Philosophy.
May 1986
ef. c
Deary,/ College of ¿Education
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08554 1406


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