Ethylene biosynthesis and cell-wall digestion in citrus peel

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Title:
Ethylene biosynthesis and cell-wall digestion in citrus peel
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Citrus peel
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xii, 118 leaves : ill. ; 28 cm.
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English
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Baldwin, Elizabeth A., 1952-
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Subjects / Keywords:
Ethylene   ( lcsh )
Plant cell walls   ( lcsh )
Horticultural Science thesis Ph. D
Dissertations, Academic -- Horticultural Science -- UF
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

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

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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oclc - 14238982
notis - ACZ3381
sobekcm - AA00004852_00001
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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 FULFILL.IEliT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY




UNIVERSITY OF FLORIDA


1986

















ACKNOWLEDGEMENTS


There are many friends, acquaintances, 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

research 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 ...................................... ii

LIST OF TABLES ........................................ vii

LIST OF FIGURES. ...................................... viii

ABSTRACT .............................................. xi

CHAPTER

I INTRODUCTION ................................. 1

II LITERATURE REVIEW ............................ 4

Introduction ................................. 4
Primary Cell Walls of Plants................. 4
Plant Cell-Wall Structure ................ 5
Plant Cell-Wall Function ................. 9
Plant Cell-Wall Lysing Enzymes ........... 12
Ethylene .................................... 15
Ethylene Biosynthesis .................... 15
Ethylene and Defense...................... 17
Ethylene and Cell Walls .................. 19
Ethylene and Citrus ...................... 19

III CELL WALL DEPOLYMERKASE ENZYMES AND ETHYLENE
PRODUCTION IN CITRUS PEEL .................... 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











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











CHAPTER Page

VI SUIVMARY ...................................... 94

LITERATURE CITED ...................................... 100

BIOGRAPHICAL SKETCH ................................... 117
















LIST OF TABLES

Table Page

4.1 Green Valencia oranges, harvested 9/10/85
or 9/30/85, were treated with 6 x 20 pl
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


vii
















LIST OF FIGURES

Figure Page

3.1 Green Valencia oranges, harvested 10/30/84,
were treated with 6 x 10 p1 injections of
fungal cell-wall lysing enzymes in............... 28

3.2 Green Navel oranges, harvested 9/7/83, were
treated with 6 x 10 p1 injections of o.1%
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 ul 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 ul 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 ul 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 p1 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 p1 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 p1 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


3.11 Coomassie blue-stained 11% polyacrylimide
gels of pectolyase and the
Geotrichum candidum PG ......................... 43

3.12 Transmission micrograph of orange peel
tissue treated with 10 p1 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 p1 of 0.1% pectolyase
in 0.1 M phosphate buffer...................... 45

3.14 Transmission micrograph of orange peel
tissue treated with 10 ul 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 p1 injections of
sugar-containing fractions ...... ............... 62

4.4 Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 p1 injections of
sugar-containing fractions...................... 64

4.5 Regreened Valencia oranges, harvested
5/6/85, were treated with 6 x 20 p1
injections of a 20% solution ................... 65

4.6 Green Valencia oranges, harvested 7/16/85,
were treated with 6 x 20 p1 injections of
various albedo pectin-pectolyase ............... 68

4.7 Green Valencia oranges, harvested 9/10/85,
were treated with 6 x 20 p1 injections of
sugar-containing fractions ...................... 69

4.8 Green Valencia oranges, harvested 9/30/85,
were treated with 6 x 20 p1 injections of
sugar-containing fractions ..................... 71










Figure Page

4.9 Regreened Valencia oranges, harvested
5/6/85, were treated with 6 x 20 pi
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 11% 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 ul of 0.1'
pectolyase in 0.1 M acetate buffer ............. 91

















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











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.


xii
















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 B 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







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 (48, 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.











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







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 B-1,4-linked glucose










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 2-3 and S-4 linked

glycopyranosyl residues. These are commonly called 3-

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











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 3-4-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

extension, arabinogalactan proteins, rhamnogalactan I and II,

xyloglucan, arabinoglucan, and glucuronoarabinoxylans (108,

111).











Angiosperm primary cell walls also contain many

different glycoproteins. One common and important such

glycoprotein is the hydroxyproline-rich extension 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%-10% 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). Nalate

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











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











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- x-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 a-4-

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).











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

uptake and incorporation of [ C]leucine 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











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 control over flowering in this species

(111).



Plant Cell u.all 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-B

-1,4- or B-1,3-glucanases have been found in cell walls of

plants (82). These enzymes degrade S-1,4- cellulose as well

as the hemicellulose polymers xyloglucan and 3-1,4-xylan

(23, 76, 81, 83). The role of -1,3-glucanase is curious as

there appears to be no 3-l,3-glucans in plant cell walls

(41, 70). Glycosidases, which are enzymes that hydrolyze











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). B-1,4-Xylanase, a-

arabinosidase and B-xylosidase have been reported to

breakdown arabinoxylan (55, 88) and a B-1,4-mannase may be

responsible for the breakdown of mannose-rich lettuce

endosperm (74).

6-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 B-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











terminal a-D(1,4) 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-(1,4) 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










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










14
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 CH3S 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 regulatory 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).











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.,phytuberin and phytuberol, than slices not subjected to

ethrel treatment (75). Ethrel also induced production of a










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). U.ounding

and/or ethylene induced wound resistance to cellulase in oat

leaves (67).

Accumulation of hydroxyproline-rich glycoprotein IHRGP)

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











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 chloroplasts to

convert to chromoplasts (156).


















CHAPTER III

CELL-WALL DEPOLYMERASE ENZYMES AND ETHYLE;;E 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











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 ul 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











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 (PG) was obtained from Charles

Barmore (CREC, Univ. of Fla., IFAS, Lake Alfred Fl.) 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 alginicc acid











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 MI 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 0.03% 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 ?i NaCI 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 iM phosphate buffer, pH 6.0, or the same

buffer alone. Ethylene measurements were made four hours

after treatment following which the injection sites were









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 0.1% 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 Fl.) 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 conditions to an












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 0.1%

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 11% 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, 0.1% 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











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 3% 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























0 100.





S80-
go


Hours After Treatment


Figure 3.1. Green Valencia oranges, harvested 10/30/84,
were treated with 6 x 10 p1 injections of
fungal cell-wall lysing enzymes in 0.1 M
acetate buffer pH 5.0. A) 0.1% pectolyase and
boiled pectolyase. B) G. candidum PG and
boiled P.G. All points are means of 3
replications +/- S.E.











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. 3.1B) 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, 3.1B, 3.2A, and 3.2B). Although

ethylene levels then decreased by 24 hour (Fig. 3.1A, 3.1B,

3.2A, and 3.2B) in some treatments ethylene production was
























S200



i 100
I1


" 0
u


Hours After Treatment


Figure 3.2. Green Navel oranges, harvested 9/7/83, were
treated with 6 x 10 pl injections cell-wall
lysing enzymes in 0.1 M phosphate buffer pH
6.0. A) 0.1% pectolyase and boiled pectolyase.
B) 0.1% drislase and boiled drislase. All
points are means of 3 replications +/- S.E.






















0

\ 400-

U.


" 300-
V



200



100




0














Figure 3.3.


Hours After Treatment


Green Navel oranges, harvested 8/15/83, were
treated with 6 x 10 pk 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.











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,




















I-



600-



4 800-




200-



3.0 3.6 4.2 46 5.0 5.0 5.6 6 66 7.0

pH of Pectolyase Treatment Solutions














Figure 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 buffer at
different pH's, and boiled pectolyase in same
buffer at pH 5. All bars are means of 3
replications +/- S.E.
















































Hours After Treatment


Figure 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 buffer pH
6.0. A) 0.1% pectolyase and boiled pectolyase.
B) 0.1% pectinmethylesterase, and boiled
pectinmethylesterase (PME). Enzyme solutions
had been desalted on lyphogel. All points are
means of 3 reps +/- S.E.





















o=
N 7.0-



6.0


.2t 5.0--
=


N 4.0-
U
U

3-0

C 2.0--


1.0--














Figure 3.6.


SV_) ) U)

C0
A. .


Green Valencia oranges, harvested 8/23/84, were
treated with 6 x 10 ul 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.











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


















1200-


1000-


800-


600-


400-


Hours After Treatment










Figure 3.7. Green Valencia oranges, harvested 9/30/85, were
treated with 6 x 20 p1 injections of 30 ppm CHI
+ 0.1% 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.


PECTOLYASE + CHI


*-- Boiled Pectolyase
A- 30 ppm CHI
a--w 30ppm CHI +
Pectolyase
o-a H20 + Pectolyase


1 -























120-








60-
40-




20"



3 8 24
Hours After Treatment




Figure 3.8. Yellow-green Valencia oranges, harvested
1/8/85, were treated with 6 x 10 pi 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.











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-Wall 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 pi
injections were administered/fruit with 3 fruit
replications +/- S.E.






41




4 Hours After Treatment

So.






F, F2 F, Fu Fu Fu Fu
SO----------------
8 leN After Treatment

45


40-


3S-


|30- j
s

_:s- -













F, F, F, 2 F, Fs F F Fu F
50.






















30. 24 HNws After Treatment


25-

S20-



o0-







F, F2 F, F, F, F. Fu F" Fn Fu.









A B C D E F G














-l9









I. -* -



Figure 3.10. Coomassie blue-stained 11% 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









A B C

'- -


U

i

II



I
I)C
|I


Figure 3.11. Coomassie blue-stained 11% polyacrylimide gels
of pectolyase and the Geotrichum candidum PG


A) standards
B) pectolyase
C) Geotrichum candidum P.G.


M























































Figure 3.12. 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. ML=middle Lamellae at 26,OOOX.























4, L































Figure 3.13. Transmission micrograph of orange peel tissue
treated with 10 pl 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,OOOX.











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 TElI 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,































CW






















Figure 3.14. Transmission micrograph of orange peel tissue
treated with 10 p1 of 0.1 M phosphate buffer pH
6, 24 hours after treatment. CW=cell wall at
34,000X.












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).











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











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 pl, 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.











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 >I 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.










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 retentate was

suspended in methanol at approximately 1:1 (v/v) and

evaporated to dryness. This procedure 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 pM 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











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










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 NaCI. 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 NaCI 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










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.










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.













350-


300. H nya. .a.mmwrcial
Citrus Pectin
o-o Boiled Hyd. Pectin
A-& Unhyd. Pectin
*-- Pectolyase
250- 0-a Boiled Pectolyase



S200-



v 150-



100-



50




4 8 24
HOURS AFTER TREATMENT






Figure 4.1. Yellow Valencia oranges, harvested 2/26/85,
were treated with 6 x 20 pi injections of crude
orange pectin hydrolysate, boiled crude
hydrolysate, unhydrolyzed pectin, 0.1%
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.


PARTIALLY HYDROLYZED PECTIN
and
PECTO LYASE

1 _J i --I










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#1ll-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 pl injections of
pooled fractions containing crude pectin
hydrolysate material that had been passed
through a G-25 Sephadex column washed with 0.1
M NaCI. Ethylene measurements were taken at 4
and 24 hours after treatment. All points are
means of 3 replications +/-S.E.
















































> C4 '0


TREATMENT


> TREATMENT

TREATMENT


A G-25 FRACTIONS -4 HRS.
and
PECTOLYASE

VVoid
*1-20 Fraction
P Pectolyasr
BP Boiled
Pc tolyase
NS Non sugar
Fraction


ISO
QC


~ ~----


I


n--,-f4















S20- -40

18- 0

S1.6- -32
IIA

oa' 1.2- -24 C

i. "
VOIDI


S0.8- -16 !A
TR 0}
JTi MONO
0.6- 4 DI

0.4"

0.2-


24 32 40 48 56 64

P-2 FRACTION NUMBERS



Figure 4.3. Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 pl 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.











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%













Z.U0- Moo-oi yT 40 -

1.8- 0

% 1.6- .32 ^

" 1.4-
-,U "I 1

S1.2- 24'
In /

- 0.8- 4 1 "" 16 !
z 9
I 0.6- >-

0.4- VOID -8

0 2 PENTA TETRA T


18 26 34 42 50 58 66 74
P-2 FRACTION NUMBERS





Figure 4.4. Yellow Valencia oranges, harvested 4/16/85,
were treated with 6 x 20 pl 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.

















S32
N


S24

i.
16


u 8



















Figure 4.5.


HOURS AFTER TREATMENT


Regreened Valencia oranges, harvested 5/6/85,
were treated with 6 x 20 p1 injections of a 20%
solution of polygalacturonase in 0.1 M acetate
buffer pH 5.0, incubated with 0.2% or 0.4%
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.










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 pl injections of various
albedo pectin-pectolyase digest mixtures. 2.5
g of extracted albedo pectin was incubated with
0.4% 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.














A PECTOLYASE DIGESTION OF ALBEDO PECTIN

4 HOURS


-. Enzyme Treated
Pectin
i Boiled Enzyme
Treated Pectin
P Pc tolyose
BP= Boiled Pectolyase


S600-



S500-



' 400-



300-



200-



100-













800



700,



600-



500-



400-



-C 300-



200-



100-


Treatments


Treatments





















1.6
t 1.4
4<- 1.2-


< 0.8-

4 0.6'


P-2 FRACTION NUMBERS


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.











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 5% solution of chitin (w/v) in acetate buffer induced

a small amount of ethylene production at 8 and 24 hours


















0.9- -
0.8- .32 I
SnN
S 7 o0.7-
VMS
'A 0.6- ,24
05 0.4- --

U 0.4- \16
S+0.3- tH -'-U-- 9

0.2 I8I

0.1

0.0-V I
26 34 42 50 58 66 74 82 90
P-4 FRACTION NUMBER





Figure 4.8. Green Valencia oranges, harvested 9/30/85, were
treated with 6 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
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.














Table 4.1.


Green Valencia oranges, harvested 9/10/85 or
9/30/85, were treated with 6 x 20 ul injections
of sugar containing fractions resulting from
gel filtration of pooled ethylene-inducing
albedo pectin-pectolyase 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, Su7.(uq)/Fru. nl C2H,/fruit/2 hr nl C H,/10u2 Su?.
2 4lM l- 2, 4


7.3

0.6
5.0


4.4
5.4
0.6
3.3


8 Hrs after Treatment


0.6
6.4
0.0
0.4


0.4
3.2
0.0
0.3


24-27
23-31
32-35
39-46


67.2
73.2
27.9
10.8


27-34
43-51
52-62
63-67


14.7
17.1
33.0
14.7














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


-7
ARABINOSE 2x10_7
GALACTOSE 7x10


Trace amts: xylose, mannose, and unk.sug.


ARABINOSE
XYLOSE
GALACTOSE


-7
2x10-7
3x10 -
9x10-7


Trace amts: mannose and unk. sug.


ARABINOSE
GALACTOSE


2x10-7
2x10 -l


Trace amts: xylose and unk. sug.


43-51


4x10-6


52-62


5.0%
18.0%


6x10-6


63-67


4.0%
5.0%
16.0%


2x10-6


12.0%
0.01%














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 MOLE% of AS



28-31 7x10-6 ARABINOSE 6x10-7 8.8%
RHAMNOSE 6x10 0.9%
XYLOSE 2x10 7 1.4%
GALACTOSE 3x10- 4.4%

Trace amts unk. sug.


32-35 3x10-6 ARABINOSE 2x10-7 6.2%
XYLOSE 2x10- 6.2%
GALACTOSE 1x10- 3.1%

Trace amts unk. sug.


39-46 3x10-6 ARABINOSE 2x10-8 0.5%
XYLOSE ixl10- 0.4%










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






































HOURS AFTER TREATMENT


Figure 4.9. Regreened Valencia oranges, harvested 5/6/85,
were treated with 6 x 20 pi injections of a 5%
chitin solution (w/v), in 0.1 M acetate buffer
pH 5.6, incubated with a 0.5% solution of
chitinase (w/v) or boiled chitinase in same
buffer for 4 hours, or 0.5% Chitinase alone
(A). Other fruit were injected with a 0.5%
solution of chitin in same buffer, or buffer
alone (B). Ethylene measurements were made at
4, 8, and 24 hours after treatment and all
points are means of 3 replications +/- S.E.










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 placental (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










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










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.









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 2%

osmium tetraoxide. Sections were then dehydrated in an

alcohol series, critical point dried in 100% 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 pi cell-wall lysing enzyme solution containing

0.1% 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










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 mls

of deionized water. The resulting solution was loaded onto

4Z stacking and 11% 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.










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 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



































Cu


Cl













Figure 5.1. 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.










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


























^-^ *4


Cu


Cl















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.






















































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.