• TABLE OF CONTENTS
HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Literature review
 Materials and methods
 Results
 Discussion
 Summary
 Bibliography
 Biographical sketch
 Copyright






Title: Physiological and ultrastructural studies of oat membranes treated with Helminthosporium victoriae toxin
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Title: Physiological and ultrastructural studies of oat membranes treated with Helminthosporium victoriae toxin
Physical Description: Book
Language: English
Creator: Gracen, Vernon Edward
Publisher: Vernon Edward Gracen
Publication Date: 1970
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Bibliographic ID: UF00089979
Volume ID: VID00001
Source Institution: University of Florida
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Resource Identifier: alephbibnum - 000428531
oclc - 37752836

Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
    List of Tables
        Page v
        Page vi
    List of Figures
        Page vii
    Abstract
        Page viii
        Page ix
    Introduction
        Page 1
        Page 2
    Literature review
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Materials and methods
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
    Results
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
    Discussion
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
    Summary
        Page 81
        Page 82
        Page 83
    Bibliography
        Page 84
        Page 85
        Page 86
        Page 87
        Page 88
    Biographical sketch
        Page 89
        Page 90
    Copyright
        Copyright
Full Text










PHYSIOLOGICAL AND ULTRASTRUCTURAL STUDIES

OF OAT MEMBRANES TREATED WITH

Helminthosporium victoria TOXIN











By
VERNON EDWARD GRACEN, JR.


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











UNIVERSITY OF FLORIDA
1970


































Dedicated to my
wife, Sharon













ACKNOWLEDGMENTS


The author would like to express his sincere appreciation

to Dr. S. H. West for serving as Chairman of the Supervisory

Committee and for guidance during the course of this

research.

The author is especially grateful to Dr. H. H. Luke

for his help, guidance, and advice during the course of

this study. Dr. A. T. Wallace is also thanked for his

inspiration and assistance which were instrumental in

the development of this study.

Dr. R. C. Smith and Dr. R. G. Stanley are thanked

for serving on the Supervisory Committee and for their

helpful criticism and advice. The author is also grateful

to Dr. H. C. Aldrich who provided the training and laboratory

facilities that made the electron microscopic work possible.

The financial support supplied by the Department of

Agronomy in the form of an NDEA fellowship and graduate

assistantship is gratefully acknowledged.

The author is deeply appreciative of the understanding

and help provided by his wife during the course of this

work.


iii


















TABLE OF CONTENTS


Page


ACKNOWLEDGMENTS . .


LIST OF TABLES . .


LIST OF FIGURES . .


ABSTRACT . . . .


INTRODUCTION .. .


LITERATURE REVIEW. .


MATERIALS AND METHODS.


RESULTS. . . . .


DISCUSSION . . .


SUMMARY . . . .


BIBLIOGRAPHY . . .


BIOGRAPHICAL SKETCH. .


iii


S . . . . . . ii


. . . . . . . . iii
* C C C C C C C C C C VJ.1


. C C C C C


. C C C C C


S. . .. . . . . 17


. . . . . . . . 26








~


~


. . . . . . . . 89














LIST OF TABLES


Table Page

1 COMPARISON OF THE RELATIVE TOXICITY OF CULTURE
FILTRATE AND PARTIALLY PURIFIED CULTURE FILTRATE .27

2 FINAL 86Rb CONTENT OF RESISTANT AND SUSCEPTIBLE
OAT ROOT TISSUE TREATED WITH VICTORIN. . . . 32

3 INFLUENCE OF DURATION OF VICTORIN PRETREATMENT
ON FINAL 8Rb CONTENT IN SUSCEPTIBLE ROOT TISSUE . 34

4 FINAL 86Rb CONTENT IN SUSCEPTIBLE OAT ROOTS
TREATED WITH VICTORIN SOLUTIONS CONTAINING VARIOUS
CALCIUM CONCENTRATIONS . . . . . . . 35

5 RETENTION OF 86Rb AFTER VICTORIN POST-TREATMENT
IN SUSCEPTIBLE OAT ROOTS PREVIOUSLY EXPOSED TO
VARIOUS CALCIUM CONCENTRATIONS . . .. . . 38

6 FINAL CONTENT OF 86Rb IN RESISTANT AND SUSCEPTIBLE
OAT ROOTS AFTER EDTA TREATMENT . . . . . 39

7 FINAL CONTENT OF 86Rb IN RESISTANT ROOTS
PRETREATED WITH EDTA, VICTORIN, AND EDTA PLUS
VICTORIN. . . . . . . . . . . 41

8 FINAL 4Ca CONTENT IN RESISTANT AND SUSCEPTIBLE
OAT ROOTS TREATED WITH VICTORIN. . . . . . 42

9 RETENTION OF 45Ca IN RESISTANT AND SUSCEPTIBLE
OAT ROOTS AFTER DESORPTION IN SOLUTIONS CONTAINING
VICTORIN, CaCl2, AND VICTORIN PLUS CaC12.. . . 44

10 FINAL 133Ba CONTENT IN RESISTANT AND SUSCEPTIBLE
OAT ROOTS TREATED WITH VICTORIA. . . . ... .. 46

11 INFLUENCE OF DURATION OF VICTORIN PRETREATMENT ON
FINAL 86Rb CONTENT IN SUSCEPTIBLE OAT LEAVES . . 47

12 FINAL 4Ca CONTENT IN RESISTANT AND SUSCEPTIBLE
OAT LEAVES TREATED WITH VICTORIN . . . . . 48







Table


Page


13 ROOT GROWTH OF RESISTANT AND SUSCEPTIBLE CULTIVARS
IN CALCIUM DEFICIENT SOLUTIONS. . . . . . 50

14 ROOT GROWTH INHIBITION BY EXTRACTS FROM VICTORIN-
TREATED RESISTANT AND SUSCEPTIBLE OAT ROOTS . . 52














LIST OF FIGURES


Figure Page

1 The retention of 86Rb in untreated oat root
tissue . . . . . . . . . . 30

2 Untreated susceptible oat root tissue (x27,500). 54

3 Higher magnification (x45,000) of untreated
susceptible oat root tissue. . . . . . 55

4 Susceptible oat root tissue treated for one
hour with one unit/ml victorin (x45,000) . 56

5 Higher magnification (x77,500) of susceptible
oat root tissue treated for one hour with one
unit/ml victorin . . .. . ... . . . 57

6 Susceptible oat root tissue grown in a calcium-
deficient solution (x18,750) showing areas of
plasma membrane invagination (indicated by
arrows). . . . . . . . .. . 58

7 Higher magnification (x37,500) of susceptible
root tissue grown in a calcium-deficient
solution showing unit structure in areas of the
plasma membrane. ... . . . . . . 59

8 Untreated resistant oat root tissue (x23,500). 61

9 Higher magnification (x45,000) of untreated
resistant oat root tissue showing regions of
unit structure in the plasma membrane. .. . 62

10 Resistant root tissue treated for one hour with
one unit/ml victorin (x24,500) . . . ... 63

11 Resistant oat root tissue treated for one hour
with one unit/ml victorin showing dense staining
areas in the cell walls (x18,750). . . . 64

12 Resistant root tissue grown in a calcium-deficient
solution showing areas of unit structure in the
plasma membrane and tonoplast (x52,500). . . 66


vii












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


PHYSIOLOGICAL AND ULTRASTRUCTURAL STUDIES OF
OAT MEMBRANES TREATED WITH
HELMINTHOSPORIUM VICTORIA TOXIN

By

Vernon Edward Gracen, Jr.

March, 1970



Chairman: Dr. S. H. West
Major Department: Agronomy

Helminthosporium victoria M.& M.produces a toxin,

victorin, which disrupts the permeability of susceptible

but not of resistant oat (Avena byzantina C. Koch) cultivars.

The mechanism of action of victorin is not known, but an

interaction between victorin and calcium has been suggested.

Calcium is believed to be essential for the maintenance of

membrane semipermeability. Physiological and ultrastructural

studies of resistant and susceptible oat root and leaf

tissues were made to determine whether differences in calcium

metabolism are related to the determination of resistance

or susceptibility to victorin.

Studies of 86Rb absorption and retention appeared to

provide a more sensitive method of measuring permeability

changes induced by victorin than previously used techniques

viii







Calcium was shown to interact with victorin in some way

to suppress its activity. The suppression appeared to

involve the binding of victorin to sites that normally

bind calcium in susceptible membranes. The effects of
45
victorin treatments on 4Ca absorption and retention

indicated that differences in the calcium metabolism of

resistant and susceptible tissues existed. Similar

differences were not revealed with another divalent cation,

133Ba, which did not suppress victorin's activity. EDTA

appeared to remove calcium more easily from susceptible

tissues than from resistant. The susceptible cultivar

was more sensitive to calcium deficiency than either

the resistant cultivar or two resistant mutants of the

susceptible cultivar. Ultrastructural studies revealed

that both victorin treatment and calcium deficiency induced

membrane changes in the susceptible but not in the resistant

root tissues.

The physiological and ultrastructural studies suggest

that victorin disrupts the permeability of susceptible

membranes by altering some essential calcium binding sites.

Such sites in the resistant membranes are either not altered

because of some functional and/or structural differences or

the sites are altered but are repaired very rapidly in the

resistant tissue. Although the possibility of rapid self-

repair of resistant tissue was not ruled out, the results of

this study favored the interpretation that resistant membranes

were not affected by victorin.












INTRODUCTION


The fungus, Helminthosporium victoria M. and M., is

the causal agent of the disease, Victoria blight of oats.

It produces a toxin, victorin, which induces all of the

known physical and biochemical.symptoms of the disease in

susceptible oat plants.

The earliest detectable symptom induced by victorin

is an abrupt loss of semipermeability in treated susceptible

but not in resistant tissues. This loss of permeability

can be suppressed by the addition of calcium to the victoria

solutions. Several lines of evidence indicate that an

interaction between victorin and calcium may occur. Such

an interaction between victorin and calcium that would

disrupt calcium binding sites in susceptible membranes

could cause the permeability changes observed.

The very rapid effect of victorin on the permeability

of susceptible but not of resistant tissues suggests that

differences in membrane structure and/or function may exist

before victorin treatments. No reports on the characteristics

of resistant and susceptible oat cell membranes before

victorin treatment have been made. Studies of resistant

and susceptible oat cell membranes, before and after

victorin treatments, were undertaken to determine whether
1





2

basic physiological and ultrastructural differences in

membranes of resistant and susceptible cells exist. A

comparison of the results of ultrastructural and physio-

logical investigations of oat tissues might yield additional

information on the possible mechanism of resistance, or

susceptibility, to victorin.













LITERATURE REVIEW


The disease, Victoria blight of oats, was first

identified in the United States by Meehan and Murphy

(25, 26). It has been demonstrated that the causal

organism, Helminthosporium victoria, produces a toxin

which is capable of inducing all the visible and known

biochemical symptoms of Victoria blight in susceptible

oat cultivars (16, 17, 18, 19, 26, 36, 49). Typical

visible symptoms are not induced in resistant oat cultivars

and all other non-host plants. Some physiological symptoms

have been induced in resistant and non-host plants by high

(100-200 units/ml) victorin concentrations (54,55). The

toxin produced by H. victoria was one of the first toxins

produced by any phytopathogen that was shown conclusively

to be a disease inducing agent.


Toxin Isolation and Chemical Characterization


The toxic principle has been isolated from culture

filtrates and named victorin by Wheeler and Luke (47). They

reported that culture filtrates also contained a secondary

toxin of low activity (19). They (19) also defined a unit

for the measurement of relative toxicity of victorin solutions.

Pringle and Braun further purified victorin (29). Chromato-

graphy of concentrated culture filtrates on acid alumina and







starch columns resulted in the recovery of fractions that

were active at 0.01 pg/ml. When these fractions were dried

and precipitated with ethanol and acetone, a toxic material

active at 0.0002 pg/ml was obtained. The toxin is relatively

stable in culture filtrates held at pH below 4.0 but is

inactivated at higher pH (7.0 14.0) values (18). The

purified toxin was reported to be highly unstable and

attempts to purify the toxin have.resulted in loss of

activity (29).

When purified victorin was treated with a saturated

sodium bicarbonate solution for 24 hours at room temperature,

two breakdown products were isolated (30). The first has

been characterized as a tricyclic, secondary amine which

was given the common name, victoxinine. The second product

was a polypeptide containing aspartic acid, glutamic acid,

glycine, valine, and one of the leucines (30). A quanti-

tative analysis of the peptide has not been reported.

Freshly purified victorin failed to give a positive

ninhydrin reaction, but the two breakdown products were

ninhydrin positive (29, 30). This has been interpreted as

an indication that the linkage between victoxinine and the

peptide involves the amino groups of both moieties (31, 32).

The exact nature of the bond between the base and the peptide

is not known.

The molecular weight of victorin, calculated from the

sum of the molecular weights of the constituents was

suggested to be 800 (30). These calculations were based on

the assumption that the victorin molecule contains equimolar







portions of victoxinine and a pentapeptide of the amino

acids detected (30). Since a quantitative analysis of

the peptide has not been reported, this must be taken as

a minimum estimate. Molecular sieving experiments indicated

that the toxin has a molecular weight of 2000 (32).

The tricyclic secondary amine, victoxinine, is a

general toxin to both resistant and susceptible tissues at

a concentration of 2.5 X 10-4M. Victorin is about 7500

times more toxic than victoxinine in susceptible tissue

(39). The empirical formula of victoxinine has been reported

to be C17H29NO (30). It has been isolated from cultures of

H. victoria which produced little or no victorin as well

as from sodium bicarbonate treated victorin solutions (30,

31). Victoxinine showed absorption in the ultra-violet

region with a maximum at 185 nm which is characteristic of

an isolated double bond (30). The compound was reported to

be colorless and to contain a single double bond. Its single

oxygen atom seems to be in an ether linkage because OH or

C=0 absorption was not detected in the infrared region (32).

The nitrogen atom appears to be a secondary amine since

greater than NH absorption was observed in the infrared

region and the compound gave a positive dithiocarbamate test

(32). The empirical formula and the presence of a single

double bond indicate that victoxinine is.a tricyclic compound.


Nature of Resistance or Susceptibility


Although knowledge of the exact structure of the victorin

molecule is desirable, difficulties in purification and







isolation of the compound have thus far prevented a

complete chemical characterization. Even though the

exact structure of the victorin molecule is unknown,

studies of the differential responses of oat tissues to

victorin have yielded a tremendous amount of information

on the nature of resistance, or susceptibility, to

victorin.

Several hypotheses to explain the nature of resistance

to victorin have been proposed. One hypothesis maintained

that victorin entered the cells of susceptible tissues but

was excluded from the cells of resistant tissues. This

hypothesis was based on the premise that victoxinine was

the toxic portion of the victorin molecule and that the

peptide determined specificity for susceptible tissue (3).

Evidence that victoxinine was not responsible for the

toxicity of victorin (39) and that victorin induced

physiological changes in resistant tissues at high concent-

rations (54) suggested that victorin enters resistant

tissues. One would expect that a molecule with the chemical

properties of victorin would be soluble in membranes of

both resistant and susceptible cultivars.

An alternative mechanism of resistance that has been

proposed was that victorin might be deactivated by resistant

but not by susceptible tissue (36). The original hypothesis

was based on data which indicated that victorin could be

recovered from susceptible but not from resistant tissue

(17, 36). Failure of other investigators to recover victorin

from either resistant or susceptible tissue seemed to







contradict the inactivation hypothesis (41). Experiments

that measure the recovery of victorin do not really test

the inactivation hypothesis. Failure to recover victorin

can not be taken as evidence that the molecule is inactivated.

A recent report in which measured quantities of victorin were

sealed in hollow coleoptile segments indicated that victorin

was either inactivated or firmly bound by both resistant and

susceptible tissue (46). This binding or apparent inactivation

proceeded at the same rate in both resistant and susceptible

tissues until susceptible cells were severely damaged. The

binding or inactivation then ceased in susceptible but not

in resistant tissues (46). The site of binding was not known

but the data indicated that resistant and susceptible tissues

both have sites that bind victorin.

The ultimate goal of studies of the response of oat

tissues to victorin is the determination of the mode of

action of victorin. Studies designed to resolve the mechanism

of action of victorin can be grouped into two general cate-

gories. The first category includes studies which attempted

to determine the nature of the physiological changes induced

by victorin treatment. The second category includes studies

designed to identify various substances that influence

victorin's activity. Identification of substances that are

either competitive or synergistic to victorin may indirectly

yield information on the mechanism of action of victorin.


Physiological Symptoms


Studies of victorin-induced physiological changes began

with investigations of the effect of victorin on respiration







of treated tissues. Victorin induced a marked increase in

the rate of respiration of treated susceptible tissue (11,

16, 35, 40, 49, 51). The respiration rate reached a max-

imum in four to ten hours after treatment and declined to

30 percent of the control rate by 24 hours after treatment

(16). During this period of increased respiration, toxin-

treated tissue failed to respond to dinitrophenol (DNP) at

concentrations that stimulated the respiration of controls

(11, 16). It was hypothesized that a direct primary effect

of victorin involved the uncoupling of oxidative phosphoryl-

ation. Subsequent experiments investigating the effect of

victorin on various Krebs cycle, glycolysis, and oxidative

enzymes failed to support this hypothesis (20,21,52).

Ascorbic acid oxidase was the only enzyme for which increased

activity was found. Its activity increased two to four

times over controls after victorin treatment (11, 16). This

increase in activity occurred after the maximum respiration

rate had been reached. No increase in the activity of

cytochrome oxidase, polyphenol oxidase, or catalase occurred

as a response to victorin treatment (16, 21).

Another physiological change induced by victorin treat-

ments has been reported. Victorin has been shown to disrupt

the semipermeability of treated susceptible tissues to

inorganic ions (48, 50). Susceptible cells that were treated

with victorin lost more electrolytes when shaken in distilled

water than treated resistant cells and controls (50). The

loss of electrolytes was detected by measuring increases in

the conductivity of the external solution in which the treated





9

tissue was shaken (50). Wheeler and Black (50) reported that

the magnitude of electrolyte loss varied directly with the

toxin concentration. The rate of electrolyte loss had a

low temperature coefficient typical of a physical process.

Permeability changes were induced by much lower con-

centrations of victorin than required to induce increased

respiration. Changes in permeability were detected in five

minutes compared to 30 minutes required to detect changes

in respiration (50). Therefore, victorin-induced perme-

ability changes seem to be induced before changes in

respiration. In studies designed to determine the mechanism

of action of victorin, it is desirable to identify the

earliest detectable physiological symptoms induced in

treated plants. The earliest detectable symptoms are

probably related to the primary changes induced by victorin

and not to secondary changes which may result when normal

metabolism is disrupted by any of a number of different

mechanisms.

Amador and Wheeler (1) reported that when victorin-

treated susceptible tissue was leached in distilled H20

after treatment, the duration of the elevated respiration

was reduced to about eight hours. Respiration of controls

was unaffected. Results with DNP indicated that respiratory

control was reestablished during leaching. Wheeler and Black

(48) suggested that changes in permeability, by affecting

the salt balance of cells, played a role in respiration

increases induced by victorin. This further indicated





10

that respiration increases were secondary effects of victorin

treatments and that studies of permeability changes would be

more suitable for attempts to determine the mechanism of

action of victorin.


Ultrastructural Studies


Luke et al. (22) examined the ultrastructural effects

of victorin treatment. They reported that victorin treat-

ment resulted in the appearance of a dark staining material

between the cell wall and plasma membrane. This was followed

by a partial separation of the membrane from the cell wall.

A general disruption of the internal membrane system was

finally seen. The endoplasmic reticulum, nuclear membranes

and chloroplast membranes were more severely damaged than

mitochondrial membranes. Membrane systems of resistant

varieties were not affected by toxin treatment.

Hanchey et al. (13) reported from further ultrastruc-

tural studies that changes induced in internal root cortex

cells of susceptible varieties after victorin treatment

closely resembled changes observed in untreated epidermal

root cap cells. They suggested that structural changes

seen in both untreated epidermal root cap cells and victorin-

treated internal cells were characteristic of cells destined

to undergo disintegration. The authors concluded from the

developmental sequence of the ultrastructural changes observed

that the initial effect of victorin was either on the inner

surface of the cell wall or the outer surface of the plasma

membrane (22).







Wheeler and Hanchey (53) have pointed out that the

plasmolysis observed by Singh et al. (42) and Luke et al.

(22) after victorin treatment was interesting since it

occurred in hypotonic solutions. This type of plasmolysis

was referred to as "false" or pseudoplasmolysis and this

effect has been recently examined and found to indicate

membrane damage (14).


Site of Action

Samaddar and Scheffer (38) reported that root hair

cells lost the ability to plasmolyze in hypertonic solutions

after 20 minutes exposure to victorin while resistant cells

retained plasmolytic activity even after three hours of

treatment with victorin. This suggested membrane damage

since cells with damaged membranes will not plasmolyze when

placed in hypertonic solutions. The apparent free space of

susceptible tissue increased after toxin treatment while

no increase was found in treated resistant tissue. Proto-

plasmic streaming stopped and plasma membranes of susceptible

protoplasts burst within one hour after cell-wall-free

protoplasts were exposed to victorin. Resistant protoplasts

were not affected significantly. Victorin decreased the

uptake of exogeneous amino acids and inorganic phosphate in

susceptible but not in resistant tissue. The data above

were taken to indicate that a primary effect of victorin was

on the membranes of susceptible cells. Cell walls did not

seem to be necessary for reaction to the toxin (38).





12

Luke and colleagues (23) suggested that the plasmalemma

was the site of action of victorin. This was suggested

because victorin treatment caused a leakage of labeled

phosphorylated hexoses from susceptible tissue. The rate

of loss of phosphorylated hexoses was directly proportional

to the concentration of toxin applied. Phosphorylated

hexoses will not pass through normally functioning membranes

(15).

Studies of physiological symptoms induced by victorin

have indicated that the toxin very rapidly affects the plasma

membrane,resulting in a loss of semipermeability. However,

these studies have not revealed how victorin disrupts semi-

permeability. In other words, the mechanism of action of

victorin has not been determined from these studies. A

second approach that has been used to try to determine the

mechanism of action of victorin has yielded additional

data. The second approach involved studies of various

substances known to influence victorin's toxicity.


Suppression of Toxicity


Relatively few substances have been found to influence

victorin's effect on susceptible tissue. Bisulfite was

reported to suppress toxic activity when partially purified

victorin was diluted with freshly prepared sulfite solutions

rather than with water (41). This does not appear to be a

pH effect because solutions of sodium sulfite and sodium

bisulfite both reduced toxicity. No decrease in toxicity







was seen when victorin was diluted with sodium barbital

solutions which were more alkaline than the sulfites. Also

when the sulfite and toxin solution was buffered at pH 7,

the suppression of toxicity was still apparent. If sulfite

was added to a concentrated victorin solution and left

overnight before being diluted with water, no suppression

of toxicity was observed. Pretreatment of seedlings with

sulfite did not affect the activity of victorin in suscept-

ible tissue. The relationship between sulfite suppression

and the mechanism of action of victorin is not clear,

although possible relationships have been discussed (28,

32, 53). The relationship between another substance that

is known to suppress victorin's toxicity and the mechanism

by which victorin disrupts permeability may be more apparent.

Doupnik (4) reported that under certain conditions,

calcium at a 0.1 M concentration suppressed typical victorin-

induced symptoms including leaf discoloration, wilting,

and permeability changes. Strontium was partially effective

in suppressing victorin-induced symptoms but manganese,

magnesium, sodium, potassium, and barium demonstrated no

protective effects. Calcium nutrition tests suggested that

the susceptible variety, Victorgrain, was more sensitive to

calcium deficiency than was the resistant variety, C. I.

7418 (4). Hanchey et el. (13) pointed out that ultrastruc-

tural changes induced by victorin in root cells of susceptible

oat tissue were similar to submicroscopic changes reported by

Marinos in calcium-deficient shoot apex cells of barley.







Therefore, not only did calcium suppress the toxicity of

victorin but a difference in calcium metabolism of resistant

and susceptible varieties was indicated.


Mechanism of Action


The suppressive effect of calcium on victorin-induced

permeability changes and the possible differences in calcium

metabolism of resistant and susceptible tissues are especially

interesting in view of Epstein's data showing the essentiality

of calcium for the maintenance of membrane semipermeability

(7). Epstein and co-workers have developed experimental

techniques using radioactive ions to study ion uptake and

retention in plant tissues (5, 6, 7, 8, 9). They have used

these techniques to study the mechanism of ion absorption

in plant root tissues (5, 7, 8, 33, 34).

Since the uptake and retention of ions depends on intact

membranes, the experimental techniques developed to study

ion retention can be used to measure membrane damage induced

by victorin and to study the possibility that an interaction

between victorin and calcium may be involved in victorin's

mechanism of action.

Epstein et el. (9) have developed a short-term ion

absorption technique for measuring the uptake of labeled ions

within cells that, with some modifications, would be perfect

for studying the effects of victorin on semipermeability.

Cations are known to occur in three different fractions in

plant tissue. One fraction of cations taken up by root tissue

occupies the apparent free spaces of the tissue. Another





15

fraction is adsorbed to negative point charges in cell wall

or the outside surface of the plasma membrane. Both of these

fractions are in equilibrium with the external solution. The

third fraction of ions taken up by root tissue is located

within cells of the tissue. Ions in this fraction are not

freely exchangeable with ions in the external solution. This

technique employed a "desorption" period designed to remove

essentially all of the labeled ions from the first two

fractions, thus allowing the measurement of labeled ions

actually taken up within the cells.

Since an interaction between victorin and calcium has

been indicated, studies of a third substance that can suppress

the toxicity of victorin are of interest. Uranyl ions which

are bound 100 times more firmly than calcium to the outer

surface of cells suppressed victorin-induced symptoms in

susceptible tissue when victorin was mixed with uranium

solutions (12, 37). Pretreatments with uranyl acetate or

uranyl nitrate solutions were also effective in suppressing

victorin's activity (12). Pretreatments with calcium were

not. Therefore, two positive cations were able to suppress

the activity of victorin. The cation which was bound more

firmly to cell surfaces (membranes and walls) showed the

stronger suppression of victorin's activity. This could be an

indication that the removal of cations from cell surfaces

may be involved in victorin's mechanism of action. The removal

of calcium from cell membranes is known to result in permea-

bility changes, a primary effect of victorin's treatments. The

difference between resistant and susceptible tissues, therefore,





16

may be related to differences in the calcium binding ability

of the tissues. The possibility that basic differences in

calcium retention may be related to the determination of

resistance or susceptibility to victorin has not been

investigated previously. A series of experiments designed

to study physiological and ultrastructural differences in

the different oat varieties before and after victorin treat-

ment has been undertaken and the results are reported in

this dissertation.














MATERIALS AND METHODS


1. Toxin purification

Crude culture filtrates of the fungus, Helminthosporium

victoria, were used as a source of the toxin, victorin. In

this study, a technique was designed for the preparation of

relatively large quantities of partially purified victorin

for use in physiological studies. The technique consists

of three steps: lyophilization, extraction with methanol,

and electrophoresis.

(a) Lyophilization and methanol extraction: Fifty ml

portions of crude culture filtrates were rapidly frozen and

dried under vacuum lyophilizedd) at -500 C. The dried

residue was extracted overnight at 40 C with dry methanol.

The methanol extracts were centrifuged at 15,000 rpm for ten

minutes to remove undissolved particles. The supernatant

was then evaporated to dryness, in vacuo, at room temperature

(270 C) on a rotary evaporator. The residue after methanol

evaporation was restored to the original volume by the

addition of 50 ml distilled, deionized water.

(b) Electrophoresis: The aqueous victorin solution was

further purified by electrophoresis at room temperature on a

Beckman Model CP continuous flow paper electrophoresis system

(200-400 volts). A 0.01 M citric acid solution (pH = 2.9)

17







was used an an electrolyte. The sample was continuously

applied to the center tab at the top of the filter paper

curtain. The bottom edge of the curtain was divided into

tabs which allowed for the collection of 32 different

fractions. The current was applied across the curtain so

that components in the sample were separated. The fractions

were assayed for toxicity by a root growth bioassay described

by Wallace et al. (45). The most active fractions were

refrigerated until used. Fractions were used within two

weeks after electrophoresis.

2. Growth of oat seedlings

Seeds of resistant and susceptible cultivars of Avena

byzantina C. Koch were used.in this study. The susceptible

cultivar was 'Victorgrain' and the resistant cultivar was

'Fulgrain', strain 2, C. I. 3423. Resistant mutants which

had been previously induced from the susceptible cultivar

with ionizing radiations (45) were also used in this study.

Seeds were soaked for two hours in distilled water and

germinated between moist blotter pads for 48 hours. After

germination, seedlings were placed on a layer of cheesecloth

stretched over a two liter plastic container. The seedlings

were placed so that their roots were submerged in a 10-4 M

CaSO4 solution. The CaSO4 solution was constantly aerated.

Seedlings were grown for seven to ten days in the laboratory

in moderate light.

3. Ion absorption and retention technique

(a) Preparation of roots: Roots were prepared for

radioactive uptake and retention studies by a modification of







the "teabag" technique described by Epstein et al. (9).

The roots were excised just below the cheesecloth,

blotted on cheesecloth, weighed into 200 mg samples on a

torsion balance. The samples were transferred to a single-

layer square of cheesecloth. The edges of the cheesecloth

were gathered together and tied with a length of cotton

string making a "teabag". The teabags of roots could be

easily transferred through the series of solutions described

below.

(b) Labeled cation uptake procedure: The teabags were

first placed in a 0.5 mM CaC12 "holding" solution for 30

minutes. The roots that received a victorin pretreatment

were treated during this "holding" period.

The samples were washed twice in 0.5 mM CaCl2 and

transferred to an "absorption" solution that contained

various labeled cations. The "absorption" period was 30

minutes for 86Rb uptake studies and one hour for 45Ca and

133Ba uptake studies.

After the absorption period, the samples were rinsed

twice in 0.5 mM CaCl2 and transferred to a "desorption"

solution for 30 minutes. During the "desorption" period,

the readily exchangeable fraction of labeled ions was

desorbed by exchange with unlabeled ions. Tissues that

received a victorin post-treatment were treated during the

desorption period.

All solutions were constantly aerated during the

experiments. Temperature was maintained at 30 + 0.20 C

by a Blue M constant temperature water bath. The absorption








solutions were contained in 500 ml wide mouth flasks. One

resistant cultivar was compared to the susceptible cultivar

in each experiment. Experiments which involved both victorin

pre- and post-treatments required 18 flasks. This allowed for

three replicates each of susceptible and resistant control,

pretreated and post-treated tissues. Some experiments

involved either pre- or post-treatments but not both. These

experiments required only 12 absorption flasks. Results

for each experiment were expressed as percent controls for

that experiment.

(c) Radioactive assay: After desorption, the tissue

was rinsed in deionized, distilled water and the teabags were

placed in metal planchets. Samples, ashed at 4800 C, were

spread in the planchets and counted in a Nuclear Chicago

thin-window Geiger counter with automatic sample changer

and scaler.

(d) Preparation of solutions: The holding solutions

contained 0.5 mM CaCl2 in all cases except for experiments

designed to determine the effects of higher calcium concen-

trationson victorin's activity. In victorin pretreatment

experiments, holding solutions which usually contained 1.0

unit of victorin/ml were used for treated tissues. In certain

designated experiments, the holding solution contained 0.01

to 0.1 mM EDTA.

The holding solutions were prepared in 1,000 ml volumes

in beakers. All of the glassware used in this study was

acid washed. The three replicates for each treatment were

placed in the same holding solution.





21

All absorption solutions contained 0.5 mM CaCl2 as has

been recommended by Epstein (6). In rubidium absorption

experiments, the absorption solutions contained 0.1 mM

RbCl labeled with approximately 5 X 10-4 to 5 X 10-3 pC

86Rb/ml. In calcium absorption experiments, the absorption

solutions contained 1.0 mM CaC12 labeled with approximately

2 X 10-4 to 2 X 10-3 C45Ca/ml instead of 0.5 mM CaCl2.

In the barium absorption experiments, absorption solutions

contained 1.0 mM BaC12 labeled with approximately 2 to 8

X 10-4 PC 133Ba/ml. The exact concentration of label

added to the absorption solutions was not determined.

Since all results were based on the relative differences in

label retained in treated versus control tissues, the exact

amount of label retained was not determined. The amount of

labeled cation added to each absorption solution was

negligible; therefore, the total cation concentration was

essentially unchanged.

Desorption solutions contained 0.5 mM CaC12 unless

otherwise stated. The desorption solutions were prepared in

3,000 ml volumes in beakers. The three replicates of each

treatment were desorbed in one beaker.

The 45 Ca desorption solutions contained 5.0 mM CaC12

instead of 0.5 mM CaCl2. The 86Rb desorption solutions

contained 5.0 mM KC1 in addition to 0.5 mM CaC12. In the

victorin post-treatment experiments, the desorption solutions

in which treated tissues were desorbed contained 1.0 unit

of victorin/ml.





22

In one series of rubidium absorption experiments, the

calcium concentration was varied and the effect of each

calcium concentration on victorin's activity was determined.

Calcium was applied at 0.5, 1.0, 10.0, 50.0 or 100.0 mM in

either holding or desorption solutions.

The solutions described above were not buffered; however,

the pH did not change appreciably during the experiments.

The initial pH values ranged from 5.5 to 5.8.

(e) Preparation of leaves: Leaves of seedlings grown

as described above were clipped at the base and weighed into

500 mg samples. The leaves were then cut transversely into

Imm strips and tied into teabags as described above for roots.

The leaf samples were then transferred to holding,

absorption, and desorption solutions as described for root

samples. The length of victorin treatment and absorption

times were increased. The leaf samples were ashed and

counted as described for roots.

4. 45Ca growth procedure

Seeds were germinated and placed on cheesecloth as

described above. The solution in which the roots were grown,

however, contained two liters of 10-4M CaS04 to which 40pC
45
45Ca was added. The seedlings were grown with constant

aeration for 7-10 days. The roots were harvested, weighed

into 100 mg samples and tied in cheesecloth teabags. The

roots were then desorbed for 30 minutes in one of the following

solutions: (1) 5.0 mM CaC12; (2) 0.5 units/ml victorin;

(3) 5.0 mM CaC12 plus 0.5 units/ml victorin; or (4) deionized

distilled water. The roots were then dried, ashed, and







counted as described above.

5. Calcium nutrition procedure

Seeds were soaked two hours in deionized, distilled

water and germinated between moist blotter pads for 48 hours.

The seedlings were then placed on fiberglass screen pads

over 2 liter plastic containers. The seedlings were placed

with their roots submerged in the growth solutions. The

growth solutions were 10-4M CaSO4 for normal and deionized,

distilled water for low calcium solutions. All solutions

were aerated continuously. The seedlings were grown for

3 weeks in the laboratory (25 270C).

After three weeks, root length of the seedlings was

measured as an indication of the differences in growth

rates in the two solutions. The root tips were then fixed

and embedded for electron microscopy. Some of the control

roots were victorin treated. Seedlings that were victorin

treated were transferred to a 1 unit/ml victorin solution

for 1 hour before fixation for electron microscopy.

6. Victorin reisolation procedure

Roots were grown and harvested as described in section

2 above. One gram samples of roots were tied into cheesecloth

bags and transferred to a victorin solution for one hour.

The victorin solution contained 1 unit of victorin/ml in

phosphate citrate buffer. Treated samples were rinsed twice

with the buffer and then transferred to a solution of the

buffer for various lengths of time. The samples were allowed

to dry and then were macerated with a mortar and pestle in

8 ml of deionized, distilled water. The root extracts were







centrifuged at 15,000 rpm for ten minutes (40 C). The

supernatant was assayed by a modification of the root growth

inhibition test (45). The extract was added to a divided

plastic petri dish. Roots of a susceptible cultivar were

assayed on one side of the dish and roots of a resistant

cultivar on the other.

7. Electron microscopic techniques

Roots, excised two to three mm back of the root tips

under a glutaraldehyde solution, were fixed for four hours

at 40 C in a 2.5 percent glutaraldehyde solution buffered at

pH 7.4 with sodium cacodylate. Roots were then rinsed twice

with buffer and washed overnight in buffer solution at 40 C.

Fixation and buffer solutions also contained 2.5 percent

sucrose.

Roots were post-fixed for 90 minutes in a 1 percent osmium

tetroxide solution buffered with the sodium cacodylate

buffer, rinsed twice in the buffer, and then dehydrated

and embedded in an Epon-Araldite mixture (27) according to

the following schedule:

25% ETOH 15 minutes
50% ETOH 15 minutes
75% ETOH & 2% Uranyl Overnight
Acetate
75% ETOH (2 washes) 20 minutes
95% ETOH 15 minutes
100% ETOH 15 minutes
100% ETOH 15 minutes
100% Acetone 15 minutes
100% Acetone 30 minutes
30% plastic-70% acetone 1 hour
70% plastic-30% acetone 1 hour
100% plastic (in capsule) 1 hour

After the tissue had been in the 100 percent plastic for

one hour, it was placed in a vacuum oven at 600C and the acetone

was boiled off. The tissue was transferred to a 600 C oven





25

for 24 hours and then to an 800 C oven for 24 hours.

The plastic embedded tissue was mounted on fiberglass

rods (7/8" diameter X 1/2" long) and faced for sectioning.

Sections that gave silver-gray interference colors under a

fluorescent light were picked up on 200 mesh copper grids

for examination. They were post-stained for 12 minutes in

0.5 percent uranyl acetate followed by five minutes in lead

citrate. The sections were examined on a Hitachi HS-8

electron microscope.














RESULTS


1. Toxin purification

Lyophilization of crude filtrates produced a sticky,

light brown residue. Methanol redissolved some of this

residue but some remained after methanol extraction. This

residue was removed from solution by the centrifugation

procedure. After methanol evaporation, a very thin, yellow-

orange layer of residue was left. This residue completely

redissolved in the deionized, distilled water.

The relative toxicity of crude culture filtrate before

and after lyophilization and methanol extraction was compared

(Table 1). The data presented are from an experiment in

which 50 ml of culture filtrate was lyophilized and extracted

with methanol. The methanol was then evaporated and the

residue redissolved in 50 ml of deionized, distilled water.

The lyophilization and methanol extraction did not decrease

the toxicity of the culture filtrate. In fact, the relative

toxicity increased.

The lyophilization and methanol extraction removed a

large portion of the electrolytes from the culture filtrates

as demonstrated by a decrease in conductivity. The conductivity

of the culture filtrate before lyophilization and methanol

extraction was 1200 micromhos when measured with a probe

26









Table 1


COMPARISON OF THE RELATIVE
TOXICITY OF
CULTURE FILTRATE AND PARTIALLY PURIFIEDa CULTURE FILTRATE

Victorin Bioassay


Victorin Culture Partially purified
dilutions filtrate culture filtrate


(MM)b (MM)b


10-1 4.0 + 2.6 ---

10-2 5.6 + 2.0 6.5 + 1.7

10-3 15.9 + 5.7 9.3 + 4.9

10-4 101.0 + 9.1 9.0 + 6.7

10-5 106.8 + 11.7 12.8 + 4.9

10-6 103.5 + 16.7 20.3 + 0.5

10-7 94.8 + 10.7 32.3 + 17.2

Buffer 112.0 + 14.9 88.0 + 19.3


a The culture filtrate was partially
and methanol extraction.


purified by lyophilization


b Values presented in each column represent the average length
of the primary root of 25 susceptible seedlings.








attached to an Industrial Instruments, Inc.,conductivity

bridge (model RC-16B2). The conductivity measured 450

micromhos after the partial purification.

Electrophoresis of the methanol extracts which contained

victorin gave additional purification of the toxin without

loss of activity. When seedlings were treated with the

methanol extracted victorin solutions diluted 1:10 with a

phosphate-citrate buffer (pH 5.8), the growth of resistant

and susceptible roots was inhibited. After electrophoresis

at 7.5 watts in a citric acid electrolyte solution,. fractions

were collected which maintained the same degree of inhibition

of susceptible root growth but which did not inhibit the

growth of resistant roots. Victoxinine and other antimetabo-

lites which have been reported to inhibit the growth of

resistant roots at high concentrations of culture filtrate

(39) appeared to be removed by electrophoresis.

The victorin purification technique used in this study

resulted in the partial purification of victorin solutions.

A complete purification was not attempted because victorin

was reported to be unstable when purified (29). This

technique resulted in the removal of a large portion of the

electrolytes from culture filtrates. This was desirable for

victorin solutions used in ion absorption experiments because

electrolytes in the solutions would interfere with the

absorption of labeled cations in victorin treated tissues.

The purification procedure also seemed to remove victoxinine

and other toxic antimetabolites present in culture filtrates.





29

This partial purification was accomplished without a signifi-

cant loss of toxicity.

The charge carried by the victorin molecule was readily

determined by electrophoresis. The victorin molecule was

positively charged at pH 2.9 (7.5 watts). When victorin

was electrophoresed at higher pH values in a phosphate-

citrate electrolyte solution, the molecule maintained a

positive charge at least up to pH 7.8. Electrophoresis at

higher pH values in the same electrolyte was not attempted.

No attempt to compare the strength of the charge at different

pH values was made.

2. Ion absorption and retention

(a) Linear uptake: Data for an experiment measuring

the total retention of 8Rb in oat root tissue allowed to

absorb label for various lengths of time are given in Figure 1.

The samples were desorbed in a non-labeled KC1 solution for

30 minutes after absorption of label. The linear uptake of

86Rb was similar to the uptake of 8Rb in barley root tissues

reported by Epstein et al. (9). The linear relationship

probably indicated that exchangeable fractions of label

had been removed and that label which was located in the

"inner" space fraction accounted for the total 86Rb retention.

A non-linear relationship between 86Rb retained and absorption

time was reported from non-desorbed tissues (9).

The values presented for final content of label in the

following sections always represent that inner space fraction

of label which was not removed by a 30 minute desorption

period. The difference in final content between treated and









600
C,


o 500
0


o
C.)

o 400


U
300


9)200
CO




100




o




10 20 30 40
Absorption Time, Minutes

Figure 1. The retention of 8Rb in untreated oat root tissue. Each
point represents average cpm for three replicates.








control tissues resulted from the ability of victorin to

decrease the amount of label absorbed and/or retained in the

inner space fraction.

(b) Rb absorption and retention in roots: Measure-

ments of the final content of 86Rb when root tissues were

treated with victorin before and after the absorption of

Rb were used to determine changes in semipermeability of

membranes (Table 2). In each experiment, three replicates

of untreated susceptible roots were compared with three

replicates of treated susceptible roots. In'addition, three

untreated and three treated replicates of roots of one of the

resistant cultivars were compared. Although the final content
86
of Rb varied from experiment to experiment, the percent

that remained in treated versus control tissue was relatively

constant. The amount of label remaining after victorin

treatments, in cpm per sample, was expressed as percent

control. Each sample was composed of 200 mg of root tissue.

A desorption period followed the absorption of 8Rb in every

case; thus the label retained was not readily exchangeable

with the external solution and was believed to be located

within cells.

The amount of 86Rb retained in susceptible roots treated

with victorin after the absorption of label ranged from 77

to 80 percent of that retained by untreated roots. When

susceptible roots were treated with victorin before the

absorption of label, the final content of 86Rb in the roots

ranged from 13 to 20 percent of the control value. The final






Table 2


FINAL 86Rb CONTENT OF RESISTANT
AND SUSCEPTIBLE
OAT ROOT TISSUE TREATED WITH VICTORIN

Pretreated with victorin Post-treated with victorin
Cultivar
treated Control Percent treated Control Percent
cpm cpm control cpm cpm control

Susceptible 62 + 8 495 + 22 12** 2514 + 50 3273 + 57 77**

219 + 15 1113 + 33 20** 4484 + 67 5714 + 75 78**

1328 + 36 6643 + 82 20** 9960 + 100 11958 + 109 81**

Resistant 2043 + 45 2072 + 46 99 3426 + 58 3023 + 55 113*

1020 + 32 931 + 30 110 5336 + 73 4975 + 70 113*

4401 + 66 4448 + 67 99 12150 + 110 10710 + 103 113*

Resistant 3360 + 58 3006 + 55 112 1262 + 36 1169 + 34 108
Mutant
2-5-1 3190 + 56 3155 + 56 101 3205 + 57 3111 + 56 103

Resistant 4142 + 64 3966 + 63 104 3557 + 60 3527 + 59 101
Mutant
500 ---- ---- --- 3127 + 56 2965 + 60 106

Resistant 4431 + 66 4127 + 64 107* 5531 + 74 5244 + 72 106
Mutant
78-1-1 4026 + 63 3819 + 62 105 -- --


86Rb absorption w
NJ


a Victorin treatments were applied either before or after
* Significant at 5% level
** Significant at 1% level







content of 86Rb was not reduced by victorin treatments

before or after the absorption of label in roots of the

resistant cultivar or any of the resistant mutants. Victorin

treatments before the absorption of 86Rb may have inhibited

the absorption of 86Rb into as well as induced its leakage

from roots after absorption. It was impossible to differ-

entiate between inhibition of uptake and induced leakage

by the technique employed here; therefore results of

victorin pretreatments were expressed as the amount of

label remaining within the tissues. In either case (reduced

absorption or induced leakage), this technique was useful

for measuring differences in membrane function because

resistant membranes were not affected by either treatment

while susceptible membranes were.

Victorin pretreatments very rapidly affected the final

content of 86Rb in susceptible roots (Table 3). The final

content of 86Rb in susceptible root tissue pretreated for

two minutes with victorin was 54 percent of the final content

of untreated tissue. When the pretreatment time was increased

to ten minutes, the final content of 86Rb in treated roots was

47 percent of control. The data presented in Table 3 are

from an experiment which included three replicate samples

for each treatment time.

Since calcium has been reported to suppress the activity

of victorin, the effect of various concentrations of calcium

in the victorin solutions (0.5 units/ml) on the activity of

victorin was measured (Table 4). The calcium concentrations









Table 3


INFLUENCE OF DURATION OF VICTORIN
PRETREATMENT ON FINAL 6Rb CONTENT IN
SUSCEPTIBLE ROOT TISSUEa

Pretreatment Final 86Rb content
retention Percent
time (min.) cpm control


0 3322 + 58 100

2 1790 + 42 54**

5 1647 + 41 50**

10 1550 + 39 47**



a Victorin treatments were applied before 86Rb absorption


** Significant at 1% level







Table 4


FINAL 8Rb CONTENT IN SUSCEPTIBLE OAT ROOTS
TREATED WITH VICTORIN SOLUTIONS
CONTAINING VARIOUS CALCIUM CONCENTRATIONSa
Victorin Victorin
pretreated post-treated
Calcium Control percent percent
concentration cpm cpm control cpm control


0.5 mM 3080 + 56 385 + 20 12** 2180 + 47 70**

567 + 24 126 + 11 22** 439 + 21 77*

10.0mM 2151 + 46 277 + 17 13** 1610 + 40 75**



50.0 mM 2180 + 47 621 + 25 28** 2640 + 51 121**

1940 + 44 540 + 24 28** 1841 + 43 95

100.0 mM 1250 + 35 600 + 30 48** -----

1460 + 38 710 + 27 49** ------


a Victorin treatments were applied either before or after 86Rb absorption

* Significant at 5% level

** Significant at 1% level





36

were varied in the victorin treatment solutions, i.e.,in the

holding solutions for victorin pretreatments and in the

desorption solutions for victorin post-treatments. Each

experiment included three replicates of untreated and

victorin-treated susceptible roots exposed to normal treat-

ment solutions (0.5 mM CaC12 with or without victorin) and

three replicates of untreated and treated susceptible roots

exposed to treatment solutions that contained a higher

calcium concentration.

An increase in the calcium concentration of the victorin

solutions gave a suppression of victorin activity. An

increase in calcium concentration from 0.5 mM to 50 mM gave

a definite suppression of victorin activity. There apparently

was complete suppression of the activity of victorin in

post-treatments because tissues post-treated with victorin

plus 50 mM CaC12 solutions retained as much label as controls.

However, victorin activity was only slightly suppressed by

increasing the calcium concentration to 50 mM in-pretreat-

ment solutions. An increase in calcium concentration to

100 mM further suppressed the activity of victorin in the

pretreatment solutions but still did not give full suppression.

It must be remembered that victorin pretreatments may have

inhibited 86Rb uptake as well as induced its leakage back

out of susceptible oat tissue. Part of the reduction in

final content of 86Rb in susceptible tissue pretreated with

victorin plus 100 mM CaC12 may have been due to inhibition of

86Rb uptake.





37

When roots of the susceptible cultivar were exposed to

high calcium concentrations and later treated with victorin

solutions containing 0.5 mM CaCI2, no suppression of victorin

activity was seen (Table 5). In these experiments, root

tissues were placed in the higher concentration calcium

solutions for 30 minutes during the holding period. The

roots were then transferred to normal absorption solutions

and finally were post-treated with victorin. Tissues

exposed to high calcium concentrations before victorin

treatments lost as much label after treatment as those

exposed to low (0.5 mM) calcium concentrations before

treatment (Table 5). Susceptible tissue that had been

previously exposed to 100.0 mM CaC12 retained 67 percent
86
as much Rb as controls after a victorin post-treatment.

This amount was well within the range (67 to 76 percent of

controls) of 86Rb retained in victorin post-treated tissues

that were not exposed to high calcium concentrations before

victorin treatment. If victorin's activity were suppressed

by high calcium pretreatments, more label would have been

retained in the treated tissues exposed to high calcium

concentrations.

Ethylenediaminetetraacetic acid (EDTA) has been reported

to affect membrane permeability (44). The effect of EDTA

pretreatments on 86Rb retention in susceptible and resistant

roots was measured to determine whether permeability of

resistant and susceptible membranes was differentially

affected (Table 6). EDTA at a 105M concentration had no

effect on the final content of Rb in roots of the susceptible










Table 5



RETENTION OF 86Rb AFTER VICTORIN POST-TREATMENT
IN SUSCEPTIBLE OAT ROOTS PREVIOUSLY
EXPOSED TO VARIOUS CALCIUM CONCENTRATIONSa

Calcium Victorin Percent
concentration Control post-treated control
cpm cpm

0.5 mM 3722 + 61 2514 + 50 68**

5863 + 77 4485 + 67 76**

1909 + 44 1280 + 36 67**

1.0 mM 16049 + 127 10006 + 100 62**



10.0 mM 6200 + 79 4732 + 69 76**

5130 + 72 3230 + 57 63**

100.0 mM 946 + 31 634 + 25 67**





a Victorin treatments were applied after 86Rb absorption


** Significant at 1% level









Table 6


FINAL CONTENT OF 86Rb IN RESISTANT AND SUSCEPTIBLE
OAT ROOTS AFTER EDTA TREATMENTa b

Control 10-5M EDTA 10-4M EDTA
Cultivar pretreatment pretreatment
Percent Percent
cpm cpm control cpm control


Susceptible 2806 + 54 2659 + 52 95 1825 + 43 65**

Resistant 2704 + 52 2735 + 52 101 2945 + 54 109*



a EDTA treatments were applied before 86Rb absorption

b Values presented are average cpm for three replicates of
each treatment

* Significant at 5% level

** Significant at 1% level







cultivar, but EDTA at a 10-4M concentration did. When

susceptible roots were pretreated with 10-4M EDTA, their

final 86Rb content was 65 percent of the control. EDTA

at both concentrations failed to reduce the final content

of 86Rb in roots of the resistant cultivar. The EDTA was

added in the holding solutions; therefore some of the

reduction in final 86Rb content may have been due to

inhibition of absorption.

The effects of EDTA, victorin, and victorin plus EDTA

pretreatments on the resistant cultivar and two resistant

mutants were measured to determine whether EDTA and victorin

together affected resistant tissues. The results presented

in Table 7 are from experiments in which three replicate

samples of untreated roots and samples of susceptible

untreated and victorin-treated roots were included in each

experiment to indicate the relative toxicity of victorin.

Neither EDTA (10-4M) nor victorin (1.0 unit/ml) gave a

significant reduction in the final 8Rb content in roots of

the resistant cultivar. When resistant root tissue was

pretreated with EDTA plus victorin, the final content of
86Rb ranged from 65 to 69 percent of control. Neither

EDTA, victorin, nor EDTA plus victorin reduced the final

content of 8Rb in roots of the two resistant mutants.
45
(c) 4Ca absorption and retention in roots: The

effects of victorin treatments on final content of 45Ca

differed from its effects on the final content of 86b in

susceptible and resistant oat roots (Table 8). Victorin
pretreatment did not reduce the final content of 45Ca in
pretreatment did not reduce the final content of Ca in






Table 7


FINAL CONTENT OF 86Rb IN RESISTANT ROOTS
PRETREATED WITH EDTA, VICTORIN, AND
S.... : EDTA PLUS VICTORINa : :


Cultivar


Resistant




Resistant
Mutant 2-5-1




Resistant
Mutant 78-1-1


Control

cpm


4449

2856

5556

2370

2586

216

3627

3819

344


+ 67

+ 53

+ 74

+ 41

+ 51

+ 15

+ 60

+ 62

+ 18


EDTA
retreated


cpm


4846

2895

5379

3324

2939

322

3324

3494

316


+ 70

+ 54

+ 73

+ 58

+ 54

+ 18

+ 58

+ 59

+ 18


Percent
control


109*

101

97

140**

114**

150**

92*

92*

92


Victorin
pretreated


Percent
cpm control cpm


4401

2657

5218

3360

3530

319

4251

4127

438


+ 66

+ 52

+ 72

+ 58

+ 51

+ 18

+ 65

+ 64

+ 21


99

93

94*

142**

136**

148**

117**

108*

127*


3058

1868

3674

2533

2766

230

3546

3813

338


EDTA and
victorin
retreated


+ 55

+ 43

+ 61

+ 50

+ 53

+ 15

+ 60

+ 62

+ 18


Percent


Percent
control

69**

65**

66**

107

107

107

99

100

98


Susceptible
check


Percent
retained

37

41

45

46

49

44

35



41
41


a All treatments were applied before 86Rb absorption

b The % retention of 86Rb in victorin pretreated versus untreated susceptible roots is
presented to indicate the relative activity of the victorin used in each experiment

,* Significant at 5% level

** Significant at 1% level












Table 8


FINAL _Ca CONTENT IN RESISTANT AND
SUSCEPTIBLE OAT ROOTS TREATED WITH VICTORIA

Victorin Victorin
Control pretreated post-treated
Cultivar
Percent Percent
cpm cpm control cpm control

Resistant 350 + 19 198 + 19** 56 335 + 18 96

324 + 18 196 + 14** 60 314 + 18 97

2730 + 52 1390 + 37** 51

Susceptible 346 + 19 372 + 19 108 231 + 15 67**

360 + 11 372-'+ 19 101 196 + 14 55**

1373 + 37 1369 + 37 100 817 + 39 60**
Resistant
Mutant
2-5-1 220 + 15 229 + 15 164 149 + 17 68*

1713 + 41 1704 + 41 99 1293 + 36 75**



a yictorin treatments were applied either before or after
Ca absorption

Significant at the 5% level


** Significant at the 1% level







roots of the susceptible cultivar or the resistant mutant

of the susceptible cultivar. However, when roots of the

resistant cultivar were pretreated, the final content of

45Ca in the roots ranged from 51 to 60 percent of that in

untreated roots. Part of the reduction in final 45Ca

content in the resistant cultivar after victorin pretreat-

ment may have been due to inhibition of 4Ca uptake.
45
Post-treated resistant roots retained as much 4Ca as

untreated controls, but post-treated susceptible roots

retained less 4Ca than controls. Roots of the resistant
45
mutant retained from 68 to 75 percent as much 4Ca as

controls after victorin post-treatment.

(d) Ca growth experiments: When susceptible and

resistant oat roots grown in the presence of 4Ca were

desorbed in various solutions, differences in the amounts

of label removed were observed (Table 9). The susceptible

roots lost more label than the resistant in all of the

desorption solutions. Calcium, victorin, and calcium plus

victorin solutions removed about the same amount of label

from the resistant roots. Victorin desorption removed

about 15 percent more label than calcium desorption from

the susceptible roots. Calcium plus victorin, however, did

not remove more label than calcium desorption alone.

Desorption in water removed less 4Ca than desorption in

non-labeled calcium. This was consistent with the results

of Epstein et al. (8) for desorption of previously absorbed

labeled cations.











Table 9


45
RETENTION OF 4Ca IN RESISTANT AND SUSCEPTIBLE
OAT ROOTS AFTER DESORPTION IN SOLUTIONS CONTAINING
VICTORIN, CaCl2, AND VICTORIN PLUS CaC12a

Desorption Solution
Cultivar

NO CaC12 Victorin CaC12 plus
desorption (5.0 mM) (1 unit/ml) victorin H20


Susceptible 2700 7868 6523 8851 14320
cpmb
Percent label
removed 0 72 86** 68 52

Resistant 10725 6364 6182 6404 8093
cpmb

Percent label
removed 0 40 42 40 25



a Root tissues were grown in 4Ca solutions

b Values are average cpm of six replicates

** Value is significantly different from calcium desorption
value at 1% level






133
(e) Ba absorption and retention: The effects of

victorin treatments on the final 133Ba content of roots

were measured to determine whether the effects of victorin

on final 45Ca content were specific for calcium. Barium

was chosen because it is a divalent cation that did not

suppress victorin's activity (4). The effects of victorin

treatments on final content of 133Ba and 8Rb were very

similar. There were some slight differences which were

probably due to differences in monovalent and divalent

cation absorption and retention. There was a much greater

difference between the effects of victorin treatments on

the final content of 4Ca and 133Ba (Table 10). The final

content of 133Ba ranged from 82 to 87 percent of controls

in pretreated and from 73 to 80 percent of controls in

post-treated susceptible roots. No significant reduction

was seen in the final 1Ba content of resistant roots

with either pre- or post-treatments.

(f) 86Rb absorption and retention in leaves: The

effects of victorin treatments on susceptible oat leaves

(Table 11) were much less drastic than on roots. Victorin

pretreatments for 30 minutes had no effect on the final
86Rb content of leaves of the susceptible cultivar. When

susceptible leaves were pretreated for one and two hours,

the final content of 86Rb ranged from 74 to 84 percent of

controls and from 70 to 77 percent of controls, respectively.

(g) 45Ca absorption and retention in leaves: Victorin

treatments of susceptible leaves for two hours gave results

similar to those observed in susceptible roots (Table 12).











Table 10


FINAL 133Ba CONTENT IN RESISTANT AND
SUSCEPTIBLE OAT ROOTS TREATED WITH VICTORINa
Victorin Percent Victorin Percent
Cultivar Control pretreated control post-treated control
.cpm cpm cpm_

Susceptible 524 + 23 428 + 21 82* 417 + 20 80*

477 + 22 413 + 20 87 351 + 19 73*

648 + 25 564 + 24 87 485 + 22 75**

Resistant 334 + 18 33Q+ 18 99 303 + 17 91

481 + 22 524 + 23 109 450 + 21 94
Resistant
Mutant 233 + 15 235 + 15 101 ----
2-5-1



a yistorin treatments were applied either before or after
Ba absorption

Significant at the 5% level


** Significant at the 1% level













Table 11


INFLUENCE OF DURATION OF VICTORIN
PRETREATMENT ON FINAL
86Rb CONTENT IN SUSCEPTIBLE OAT LEAVESa

Pretreatment Victorin
Control pretreated
Percent
time cpm cpm control


1/2 hour 612 + 25 618 + 25 101

746 j27 748 + 27 100

1 hour 363 + 19 268 + 16 74*

443 + 21 364 + 19 82*

2 hours 143 + 12 111 + 10 77

429 + 21 302 + 17 70**



86
a Victorin treatments were applied before 8Rb absorption

Significant at the 5% level

** Significant at the 1% level







Table 12


FINAL 45Ca CONTENT IN RESISTANT AND SUSCEPTIBLE
OAT LEAVES TREATED WITH VICTORINa

Victorin Percent Victorin Percent
Cultivar Control pretreated control Control post-treated control
cpm cpm cpm cpm


Susceptible 4570 + 68 4820 + 69 106 914 + 30 183 + 14 20**

144 + 12 139 + 12 96

Resistant 1083 + 33 846 + 29 78** 2682 + 16 2033 + 45 77**



a Victoria treatments were applied either before or after 45Ca abortion
a Victorin treatments were applied either before or after Ca absorption


** Significant at the 1% level







Victorin post-treatments reduced the total retention of

45Ca to 20 percent of controls but victorin pretreatments

gave no reduction in the final 4Ca content. Both victorin

pretreated and post-treated resistant leaves retained from
45
77 to 78 percent as much Ca as controls.

3. Calcium nutrition experiments

The susceptible cultivar was reported to be more

susceptible to calcium deficiency than a resistant cultivar

(4). To determine whether differences reported were the

result of varietal differences or whether they were related

to the response to victorin, the susceptible and resistant

cultivars and two resistant mutants were grown in calcium-

deficient solutions. The average length of the susceptible

roots grown on solutions containing essentially no calcium

was decreased significantly compared to roots grown in a

10-4M CaSO4 solution (Table 13). The root length of the

resistant cultivar and the two resistant mutants was not

reduced in the calcium-deficient solution.

4. Victorin extraction from root tissue:

It is not known whether or not victorin enters the

protoplasts of resistant oat cultivars. Because of its proposed

chemical structure (polar and nonpolar components) and

properties (solubility in aqueous and polar solvents), one

would assume that victorin would be readily bound to plant

cell membranes and walls. Since electrophoresis experiments

demonstrated that victorin had a positive charge at physiolog-

ical pH, water would not be expected to remove all of it

from the free spaces of root tissues. The fraction bound to











Table 13


ROOT GROWTH OF RESISTANT AND SUSCEPTIBLE
CULTIVARS IN
CALCIUM DEFICIENT SOLUTIONS

Growth solution
Cultivar
deionized, distilled
10-4 CaSO4 H20
mma mma

Susceptible 148 + 19 35 + 9**

Resistant 162 + 20 234 + 14

Resistant
Mutant 2-5-1 138 20 161 + 6

Resistant
Mutant 78-1-1 132 + 19 139 + 17


a Values presented are
of 25 seedlings


average length of the primary root


** Significant at the 1% level







negative charges within the tissue would not be removed by

water; therefore attempts to reisolate victorin from resistant

and susceptible roots after a desorption period in an

electrolyte solution were made. A modification of the ion

absorption technique described above was employed. This

technique included a desorption period which was designed to

wash out victorin in the free spaces and bound to negative

charges in the root tissue before extraction. Extracts of

victorin-treated and untreated roots of resistant and

susceptible cultivars were assayed (Table 14). Root growth

inhibition bioassays using resistant and susceptible seeds

were used to detect victorin in the extracts.

When roots were treated with victorin for one hour

and then desorbed for 30 minutes, the extracts of both

resistant and susceptible roots contained victorin. After

desorption for one hour, the extracts contained less victorin.

If the desorption period was increased to five hours, no

victorin activity was detected in root extracts of either

cultivar. This indicated that the victorin recovered after

the shorter desorption periods was probably located in the

free spaces of the root tissues. Victorin was recovered from

resistant as well as susceptible roots. Extracts from roots

of both cultivars that were not treated with victorin did

not inhibit root growth; thus the toxic effects of the

extracts from victorin-treated roots were definitely due to

victorin. The inhibition was not due to some toxic component

of the root tissue.







Table 14


ROOT GROWTH INHIBITION BY EXTRACTS
FROM VICTORIN-TREATED RESISTANT AND SUSCEPTIBLE OAT ROOTS
Desorption Time
Source 1/2 Hour 1 Hour 5 Hours
of Suscep- Resist- Suscep- Resist- Suscep- Resist-
Extract tible ant tible ant tible ant
Bioassay % Bioassay % Bioassay % Bioassay % Bioassay % Bioassay %
mm inh. mm inh. mm inh mm inh. mm inh. mm inh.

Victorin-
treated
susceptible
roots 5.1 80.0 51.9 0.0 30.4 45.1 47.6 1.5 33.5 0.0 2.8 0.0

Victorin-
treated
resistant
roots 5.9 76.8 58.5 0.0 22.1 60.1 53.4 0.0 29.6 6.3 27.6 0.0

Non-
treated
susceptible
roots 28.3 0.0 58.9 0.0 51.1 7.6 50.1 0.0 29.8 6.3 18.8 0.0

Non-
treated
resistant
roots 40.5 0.0 51.1 7.1 49.9 9.8 44.5 7.9 31.0 0.0 28.8 0.0


Values represent average root length of the primary root of ten seedlings

Expresses the percent inhibition of root growth as compared to growth in phosphate
citrate buffer.







5. Ultrastructural studies

Victorin and low calcium treatments caused similar

ultrastructural changes in susceptible oat root tissues

as shown by a comparison of Figures 4 and 5 to Figures

6 and 7. Membrane invaginations, some of which resembled

loamosomes, were observed in victorin-treated and calcium-

deficient susceptible roots but not in untreated roots.

The plasma membranes continued to exhibit unit membrane

structure in some areas after both treatments. Plasmolysis

and cell wall changes induced by victorin were not observed

in calcium-deficient roots.

Electron micrographs of untreated susceptible root

tip cells taken at two different magnifications have been

included in this report to show the general ultrastructural

characteristics of oat root cortex cells (Figures 2 and 3).

The nuclei contained dense areas of chromatin and were

enclosed by nuclear envelopes (Figure 2). The plasma

membranes were closely appressed to the cell walls.

Mitochondria, plastids, and dictyosomes were present. Densely

stained areas believed to represent DNA were observed in the

mitochondria and plastids. The root cells contained

numerous ribosomes and structures similar to spherosomes as

defined by Frederick et al. (10).

Sections from root tips that had been treated for one

hour with one unit/ml victorin solutions (Figures 4 and 5)

showed some changes that were previously reported in

KMnO4-fixed tissues after victorin treatments (13, 14, 22).






















































Key to labels (Figures 2 through 12) CW=cell wall; D = dictyosome,
M = mitochondrion, N = nucleus, NE = nuclear envelope, P = plastid,
PM = plasma membrane, RER = rough endoplasmic reticulum,
S = spherosome, SG = starch granule, T = tonoplast, V = vacuole.

Figure 2. Untreated susceptible oat root tissue (x27,500). Note
the general appearance of cell walls and cytoplasmic components
and the lack of plasmolysis.























































Figure 3. Higher magnification (x45,000) of untreated susceptible
oat root tissue. Note the absence of plasmolysis.





































with one unit/m victoria (x45, ). Note the invaginations
slightly densely stained.






.o r



















Fg re4 .'t. a
slihtl deslsand
































































Figure 5. Higher magnification (x77,500) of susceptible oat
root tissue treated for one hour with one unit/ml victorin
showing plasmolysis and densely stained cell walls. Note the
areas of unit structure in plasma membranes.


5* ^
*^


*y.
^A


r
~j~srYi~i'


ri-


c
, ft -1
~3~-
1:









































j -' > .


," *' -"i .' ", --' u :-
^ ",'*, 'v ,,, ....: .&


Figure 6. Susceptible oat root tissue grown in a calcium-deficient
solution (x18,750) showing areas of plasma membrane invagination
(indicated by arrows). Note the lack of densely stained cell
walls and lack of plasmolysis.

























c w










4.N


















tk*










Figure 7. Higher magnification (x37,500) of susceptible root
tissue grown in a calcium-deficient solution showing unit
structure in areas of the plasma membrane. Note the membrane
invaginations (indicated by the arrows) .
X v -,v .4

E-IS
RirfA


~-x*7







Cell walls often stained more densely in victorin-treated

cells (Figure 5). Plasma membranes pulled away from the

cell walls indicating slight plasmolysis and membrane

invaginations, some of which resembled loamosomes (indicated

by arrows), developed (Figures 4 and 5). Changes in the

Golgi complexes (dictyosomes) were not as apparent in this

study as those reported in KMn04-fixed tissues (13) although

dictyosomes in treated cells appeared to be somewhat more

densely stained. There was no indication that spherosome-

like structures were induced by victorin treatment in this

study.

Susceptible roots grown in calcium-deficient solutions

did not show plasmolysis like that observed in victorin-

treated roots but several similar membrane changes were

observed (Figures 6 and 7). Various types of membrane

invaginations, including some that resembled loamosomes

(indicated by arrows), were induced by calcium deficiency

(Figures 6 and 7). Cell walls did not stain more densely

in calcium deficient tissues.

Untreated resistant root tip cells (Figures 8 and 9)

,looked similar to untreated susceptible root tip cells

(Figures 2 and 3). Nuclei, plastids, mitochondria, and

dictyosomes were all similar in appearance to those in

untreated susceptible cells. Ultrastructural changes like

those observed in victorin-treated and calcium-deficient

susceptible roots, however, were not induced in resistant

roots either by the victorin treatment (Figure 10 and 11)






















































Figure 8. Untreated resistant oat root tissue (x23,500). Note
the similarity to untreated susceptible root tissue (Figures 2
and 3).























































Figure 9. Higher magnification (x45,000) of untreated resistant
oat root tissue showing regions of unit structure in the plasma
membrane.












































Figure 10. Resistant root tissue treated for one hour with one
unit/ml victorin (x24,500). Note the lack of plasmolysis and
plasma membrane invaginations.


&JW~




64










-lip





Ict




























Figure 11. Resistant oat root tissue treated for one hour with
one unit/mi victorin showing dense staining areas in the cell
walls (x18,750). Note the extensive rough endoplasmic reticulum
and difference in the appearance of mitochondria (compare to
Figure 8).
1 3






















and difrnei h perne fmtcodi cmaet
Figure 8)





65

or by calcium deficiency (Figure 12). Neither plasmolysis

nor extensive membrane invaginations were observed in resistant

tissues. Changes in appearance of mitochondriaa and the

appearance of dark staining blotches in cell walls were

observed in some victorin-treated resistant cells (Figure 11).

No evidence that these changes were direct responses of

victorin treatment was found. Large amounts of rough

endoplasmic reticulum were apparent in some cells in victorin-

treated resistant roots (Figure 11).























































Figure 12. Resistant root tissue grown in a calcium-deficient
solution showing areas of unit structure in the plasma memIbrane
and tonoplast (x52,500). Note the lack of plasmolysis and
membrane invaginations.


~k)ras~
P' ~J~gaBc~c~l;-














DISCUSSION


The primary effect of victorin appears to be the

disruption of membrane permeability in treated susceptible

tissue. The exact mechanism of action of victorin is

unknown but a relationship between the toxin and membrane-

bound calcium has been indicated in this study. Experiments

designed to study the characteristics of resistant and

susceptible membranes before and after victorin treatments

were conducted. The results of the experiments were

consistent with the hypothesis that victorin altered the

binding of calcium in susceptible membranes more easily

than in resistant membranes. Alteration of the calcium

binding sites could have induced the permeability changes

observed in susceptible tissues.

Victorin was extracted from the intercellular spaces of

roots of both resistant and susceptible cultivars in this

study. Victorin was also shown to disrupt the semipermea-

bility of susceptible tissues, probably by affecting the

function of the plasma membrane as has been previously

reported (23). Since the permeability of resistant tissues

was not disrupted, resistant membranes were either not

affected by victorin (because of some functional or structural

differences) or they were affected but were also capable of







rapidly repairing the damage induced by victorin. Several

reports have indicated that resistant membranes were affected

by very high victorin concentrations and that a self-

repairing process was involved in resistance to victorin in

oats and other plants (54,55). Self-repair would have to

occur very rapidly in resistant cultivars. Victorin

pretreatment times as short as two minutes were shown to

give approximately a 50 percent reduction in final 86Rb

content in susceptible roots. A repair mechanism would

have to function rapidly to overcome such effects in

resistant roots. The results of this study did not rule

out the possibility of induced repair in resistant cultivars;

however they were more consistent with the hypothesis that

differences in resistant and susceptible membranes existed.

There was little doubt that membranes of resistant

and susceptible cultivars functioned differently after

victorin treatment. After victorin treatment, the susceptible

membranes lost the ability to perform their most important

function, ion uptake and retention, while resistant membranes

did not. Whether this difference in function was entirely

a result of victorin treatment has not been determined.

The membrane differences may have existed before victorin

treatment and, therefore, may be related to the mechanism

of resistance or susceptibility.

Results of experiments designed to determine whether

resistant and susceptible membranes differed before victorin

treatments have not been previously reported. The results of

experiments measuring the effect of victorin on the final








content of 86Rb, like the results of previous experiments

that measured victorin-induced electrolyte losses (2,48),

indicated only that semipermeability was affected by victorin

treatments. These measurements gave no information about

whether membranes of resistant and susceptible cultivars

were functionally different before the victorin treatments.

As a result, other types of experiments had to be designed

to study whether a basic difference in membranes existed.

Changes in permeability in leaf tissue induced by

victorin appeared to be similar to those induced in root

tissues except that longer treatment and absorption times

were required with leaf tissues. The longer times were

presumably due to a slower absorption rate in the relatively

large leaf slices used in this study. To obtain maximum

absorption, leaf slides should have been so narrow that

all of the cells were directly exposed to the absorption

solution (43). These results indicated that root tissue

was better suited for studies designed to measure basic

differences in membrane structure and/or function than

leaf tissue.

The fact that calcium suppressed the activity of victorin

suggested that an interaction between calcium and victorin

occurred. Calcium was reported to be required to maintain

the semipermeability of membranes (6). Since victorin was

capable of destroying the semipermeability of membranes,

it seemed logical to propose that victorin may have interacted

with calcium in some way,resulting in the removal of calcium







from some vital spots in the membranes. This would result

in the loss of semipermeability in the victorin-treated

susceptible membranes. Differences in the ability of

membranes to hold calcium may, therefore, be related to

the determination of resistance or susceptibility to

victorin.

The nature of the interaction between victorin and

calcium is unknown. This interaction may be related to

the fact that the victorin molecule was positively charged

at physiological pH. One would have expected victorin to

be negatively charged at physiological pH if the carboxyl

groups in the molecule were free to disassociate. Since

victorin was positively charged, it could bind to the

negatively charged sites in the membranes. The binding

of calcium to negative sites in membranes is thought to

be important for the maintenance of semipermeability.

Binding of victorin to negative sites that disrupted the

normal calcium binding at the sites could have resulted in

permeability changes.

Since victorin appeared to disrupt permeability by

altering calcium binding of membranes of oat root tissues,

experiments measuring the effect of another substance on

membrane permeability were conducted. Ethylenediamine-

tetraacetic acid (EDTA) has been shown to induce the loss

of semipermeability of beet root tissues (44). The effects

of EDTA on semipermeability were reversed by the addition of

calcium. Membrane leakiness occurred when 69 to 76 percent

of the total calcium present in the tissue was removed.







The remaining calcium (24 to 31 percent) was not removed

and was believed to be present in a more stable complex

than EDTA-Ca (44). Equal amounts of calcium were removed

from the protoplasmic and cell wall fractions. The changes

in permeability induced by EDTA may have been similar to

the changes in permeability induced in susceptible oat

membranes by victorin. Both appeared to involve inter-

actions with calcium. Victorin also appeared to have an

effect on cell walls which may indicate an interaction

of victorin with calcium in the cell walls.

When resistant and susceptible oat tissues were

pretreated for 30 minutes with 10-4M EDTA, the final content

of 86Rb was reduced by 35 percent in susceptible roots but

not in resistant. If the permeability changes induced by

EDTA were the result of the removal of calcium from membranes,

then calcium appeared to be removed from susceptible membranes

more easily than from resistant membranes. This indicated

that calcium in resistant membranes was present in a more

stable complex than in susceptible membranes. At least

the calcium binding sites involved in the maintenance of

semipermeability were either altered more easily in susceptible

membranes or were repaired more rapidly in resistant membranes.

The calcium binding sites in resistant membranes may occur

in some configuration so that they are hidden or inaccess-

ible to victorin. Binding will be frequently used to

describe the ability of membranes to maintain calcium whether

this is accomplished by holding it tighter or replacing it







more rapidly. It is not used necessarily to refer to

membrane affinity for calcium.

Studies of the effect of EDTA plus victorin on resistant

root tissues were made to further investigate the ability

of resistant membranes to hold calcium. Neither victorin

(0.5 units/ml) nor EDTA (10-4M) reduced the retention of
86Rb in resistant roots. When the victorin and EDTA

solutions were combined, they gave a reduction of 30 to

35 percent in the final 86Rb content. This may be an

indication that victorin could bind to the negative sites

in resistant membranes once the calcium had been removed

(by EDTA) but that it could not remove the calcium itself.

The most direct way to measure differences in the

ability of resistant and susceptible membranes to hold

calcium was to study the absorption and retention of 45Ca.

The results of such studies indicated that resistant and

susceptible roots differed in their calcium metabolism.

The difference appeared to be specific for calcium. A

similar difference was not seen in experiments with another

divalent cation, barium. The difference was first observed

when the final 45Ca content was not reduced by a victorin

pretreatment in susceptible roots but was reduced by a

victorin post-treatment. Victorin pretreatments had

resulted in a larger reduction of final 86Rb content than

post-treatments. Since both victorin pre- and post-treat-

ments disrupted the permeability of susceptible tissues,

the lack of a reduction in final content of 4Ca by victorin

pretreatment was significant. These results, however, were








consistent with the report that victorin treatments failed

to induce.a measurable loss of calcium in oat leaf tissues

(2). Since victorin treatments which clearly disrupted

the permeability of oat membranes failed to induce leakage

of calcium, it appeared that there might not be much free

calcium present in oat tissues to leak out. The results

obtained with 45Ca studies, therefore, did not seem to

measure permeability changes directly. Most of the labeled

calcium studied was probably completed in cell membranes,

walls, and organelles and not found in the free state in

the cytoplasm. Victorin post-treatments removed 45Ca from

susceptible tissues,indicating that sites responsible for

binding calcium were altered. Victorin pretreatments also

may have altered calcium binding sites in susceptible

membranes; however when 45Ca was added after victorin

treatments, the calcium was bound to the membrane sites

anyway. The data indicated that victorin may have been

bound to sites in membranes that normally bind calcium.

This could result in changes in membrane structure resulting

in a loss of semipermeability. Neither victorin nor calcium

appeared to be bound very tightly in susceptible membranes.

Victorin post-treatments removed 45Ca from susceptible

roots and 4Ca post-treatments removed previously absorbed

victorin.

Suppression of victorin activity by calcium may be

explained by the hypothesis that victorin and calcium both

bind to the same membrane sites. If both victorin and calcium

competed for some sites on membranes but neither was bound






very tightly in susceptible membranes, then increasing the

calcium concentration of a victorin solution would favor

the binding of calcium to membranes. If calcium were bound

to the membranes then the semipermeability would be main-

tained and the apparent activity of the victorin solution

would be decreased. On the other hand, the removal of

calcium from crude culture filtrates should give an apparent

increase in the activity of victorin. Victorin fractions

with highest activity were obtained by techniques which

would serve to remove most of the calcium and other elec-

trolytes, i.e., drying and extraction in ethanol, methanol,

etc. Of course, an interaction between victorin and calcium

in solution which changed the activity of the victorin

molecule cannot be ruled out.

Treating cells with high calcium concentrations before

victorin treatment did not reduce the activity of the

victorin solutions. The calcium had to be added directly

to the victorin solution to suppress its activity. One might

expect that cells "loaded up" with calcium before victorin

treatment would maintain high enough levels of calcium to

suppress victorin activity. This was not the case. Roots

pretreated with high (0.1 M) calcium solutions would contain

essentially 0.1 M calcium concentration in their intercellular

spaces. Most of this calcium in the intercellular spaces

would be readily exchangeable with the external solution;

therefore when the roots were immersed in a victorin solution

that was relatively low in calcium, most of the calcium in

the intercellular spaces would rapidly move out into the








solution. This could account for the inability of calcium

pretreatments to suppress victorin activity. There would

not be enough calcium present in the victorin solution to

favor the binding of calcium to membranes instead of victorin.

Calcium and/or victorin were apparently more tightly

bound to resistant membranes than to susceptible. Victorin

post-treatments of resistant membranes did not-reduce the

total retention of 45Ca; therefore victorin either did not

remove calcium from the resistant membranes or resistant

membranes replaced the calcium, i.e., repaired the damage

rapidly.

Victorin pretreatments reduced the final content of

45Ca in resistant root tissue by approximately 45 percent,

which possibly indicated that if victorin was previously

bound to membrane sites then they were not available to

bind calcium. The resistant roots would have to have

calcium bound to a portion of the essential membrane sites

to maintain semipermeability necessary for growth. Therefore,

the sites involved in the binding of the victorin in resistant

roots probably represented only a portion of the total

calcium binding sites. These sites could have been located

in the cell walls and organelles as well as in membranes.

EDTA studies indicated that 67 to 76 percent of the

total calcium in beet root cells had to be removed before

semipermeability was affected. Lack of calcium at some

calcium binding sites, therefore, did not affect membrane

semipermeability. The resistant oat roots grown on the

10-4M CaSO4 solutions presumably did not have all of their







potential calcium binding sites covered with calcium.

Enough calcium was present to maintain semipermeability

but still there were some free sites. When victorin was

bound to the free sites (by pretreatment), the sites were
45
unavailable for binding 4Ca later. Hence, victorin
45
pretreatments reduced the total retention of Ca in

resistant roots. Again I should point out that resistant

roots were able to maintain calcium better than susceptible

roots. Whether this was because of different abilities

of the tissues to hold calcium or because of a rapidly

induced replacement of calcium in resistant tissues was

not shown.
45
When roots that had been grown in Ca solutions

were desorbed in various solutions, victorin removed 15

percent more calcium from the susceptible cultivar than

either calcium or calcium plus victorin solutions. The

results are consistent with the hypothesis that victorin

could remove calcium from susceptible tissues that was

bound in such a way that it was not readily exchangeable

with the external solution. It could not remove calcium

that was not readily exchangeable with the external solution

in resistant tissues. The addition of calcium to the victorin

solution again resulted in a suppression of victoria

activity. The results indicated that a larger portion of
45 45
the total 4Ca incorporated into roots grown on 4Ca was

more easily removed from susceptible than from resistant

roots.





77

Doupnik (4) has reported that the susceptible cultivar

was more sensitive to calcium deficiency than a resistant

cultivar. When seedlings of the susceptible cultivar,

the resistant cultivar, and two resistant mutants were

grown on solutions that contained essentially no calcium,

the results indicated the susceptible cultivar was more

sensitive to calcium deficiency than any of the other

three. If roots of the susceptible cultivar have more

trouble binding calcium than resistant roots, then one

might expect that a calcium deficiency would have more

effect on the susceptible cultivar.

Ultrastructural changes induced by victorin in suscep-

tible oat roots have been compared to changes observed in

calcium-deficient barley tissues (13). An ultrastructural

comparison of calcium-deficient and victorin-treated

susceptible oat roots 'as not previously been reported.

Such a comparison was made in this study in an attempt to

correlate ultrastructural and physiological effects of

victorin.

Some of the membrane changes induced by victorin

treatment (invaginations which formed loamosome-like

structures) were seen in calcium-deficient susceptible root

tissue. Other changes (plasmolysis and cell wall changes)

reported to be induced by victorin treatments (13, 22) were

not seen in calcium-deficient tissues.

The fact that some similar changes were induced by

calcium deficiency and by victorin treatment is probably not

significant in itself. The changes may be typical in cells







destined to undergo disintegration as has been suggested

(13). The significance of the data is that both treatments

induced changes in susceptible but not in resistant tissues.

This further supports the hypothesis that differences in

calcium metabolism exist between the two cultivars. Also,

since victorin-induced physiological symptoms may be related

to an interaction between victorin and calcium, the differ-

ences in calcium metabolism of the two cultivars may be

related to the mechanism of resistance. It was interesting

that the most noticeable early changes induced by victoria

involved cell walls and membranes. Calcium is known to

play important roles in both walls and membranes.

Several reports of ultrastructural changes induced by

victorin treatments in susceptible tissues have been published

(13, 14, 22). Electron micrographs of victorin-treated

resistant tissues have not been shown in the previous

reports. Electron micrographs of victorin-treated resistant

tissues were included in this report. The fact that victorin

did not induce the same ultrastructural changes in resistant

tissues that it did in susceptible tissues was significant.

The possibility that some of the ultrastructural features

of victorin-treated resistant cells (especially large

amounts or rough endoplasmic reticulum) may be an indication

of the mechanism of resistance (induced self-repair) should be

investigated.

Previous reports (13, 14, 22) have indicated that

KMnO4 fixation gave the best results with oat root tissues

(13, 22). The present results indicated that an improved







glutaraldehyde-osmium fixation technique gave good ultra-

structural preservation of oat root tissues. This was

significant because glutaraldehyde-osmium fixation is

superior to the KMn04 fixation for preserving nucleic

acids and for use in many histochemical techniques.

Several ultrastructural changes previously reported

to be induced by victorin were observed in this study;

however others were not. Some of the previously reported

changes may have been artifacts. Some of the previously

reported changes that were observed in victorin-treated

tissues in this study included plasmolysis in hypotonic

solutions (pseudoplasmolysis), membrane invaginations,

and densely staining cell walls. Victorin-induced modifi-

cations of Golgi complexes were not as apparent in this

study as in those previously reported.

Unlike the previous reports (13), spherosome-like

structures did not seem to be induced by victorin treatment

in this study. Spherosome-like structures were observed

in both control and treated tissues fixed as described in

the Materials and Methods section. Preservation of the

spherosomes which are believed to contain lipids (10) was

enhanced by dehydration in cold (4C) alcohol solutions.

Concentric arrays of membranes previously reported (13)

to be induced by victorin treatments were seen very rarely

in both treated and control cells.

The resistant mutants differed from the resistant and

susceptible cultivars. The retention of 86Rb in the resistant

mutants was not affected by victorin treatments. This was







contrary to previous results (45) which indicated that

two of the resistant mutants gave intermediate reactions

to victorin when electrolyte loss and root growth inhibition

were measured. Resistant mutant 500 was reported to lose

22 percent as much electrolytes as the susceptible cultivar

when treated at 220 C. Resistant mutant 78-1-1 lost only

27 percent as much. Both mutants were more resistant

when measured at 300 C (24). The two mutants did not

give an intermediate reaction when 86Rb absorption and

retention was measured at 30 C in this study.

The resistant mutants were not as sensitive to calcium

deficiency as the susceptible cultivar. However, the

resistant mutants responded more like the susceptible

cultivar than the resistant cultivar when the effect of

victorin treatments on 45Ca retention was examined. While

the nature of resistance in the resistant mutants was

not fully understood, it did not appear to be the same as

the mechanism of resistance in the resistant cultivar.

This indicated that the resistant mutants probably represent

mutations at some genetic locus (loci) other than the Hv

locus, the main locus involved in controlling the reaction

to victorin in oats. The resistant mutants did not seem to

represent back mutations of the Hv locus that restored

function to the product of the locus.














SUMMARY


The purpose of this investigation was to determine

whether basic differences in membrane structure and/or

function might be related to the differential responses

of resistant and susceptible oat cultivars to victorin.

Victorin used in these investigations was partially

purified by lyophilization, methanol extraction, and

electrophoresis of culture filtrates of the fungus,

Helminthosporium victoria M. and M. Victorin was shown

to be positively charged at physiological pH.

Decreases were observed in the total amount of 86b

remaining in victorin-treated susceptible but not in
86
resistant tissues. Studies of Rb retention probably

provided a more sensitive measurement of victorin-induced

permeability changes than previously used techniques,

but such studies failed to determine whether basic differ-

ences in resistant and susceptible membranes existed before

victorin treatments.

Calcium suppressed victorin-induced permeability

changes when added directly to the victorin solutions

but no suppression was observed when oat root tissues

were pretreated with high calcium solutions.







Studies of 45Ca absorption and retention in root tissues

revealed apparent differences in the ability of resistant

and susceptible membranes to hold calcium. Victorin

treatments appeared to alter the calcium binding sites of

susceptible membranes. Sites in the resistant membranes

were either not altered or were repaired rapidly. These

differences appeared to be specific for calcium since

they were not observed with barium, another divalent cation.

EDTA, which was reported to induce permeability

changes by removing calcium from membranes, affected the

permeability of susceptible root tissues more than resistant.

EDTA plus victorin solutions were shown to reduce the final
86Rb content of resistant tissues.

When root tissues grown in a 4Ca solution were desorbed

in various solutions, desorption in a victorin solution

removed more 45Ca from susceptible tissues than desorption

in a non-labeled calcium solution. Victorin did not

remove more 45Ca than non-labeled calcium from resistant

roots.

Ultrastructural studies revealed some similar changes

in membrane structure in calcium-deficient and victorin-

treated susceptible oat roots. Such changes were not

observed in untreated susceptible roots or in victorin-

treated or calcium-deficient resistant roots.

The results of this study were consistent with the

hypothesis that victorin altered calcium binding sites in

susceptible membranes which resulted in a loss of semi-

permeability. Membranes of the resistant cultivar were





83

either not altered by victorin or were altered and were

rapidly repaired. The mechanism of resistance in the

resistant mutants of the susceptible cultivar seemed to

differ from that of the resistant cultivar.












BIBLIOGRAPHY


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86


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88

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BIOGRAPHICAL SKETCH


Vernon Edward Gracen, Jr. was born July 31, 1945 in

Savannah, Georgia. He was graduated from R. W. Groves

High School, Savannah, Georgia in June, 1962. In

September, 1962, he entered the Georgia Institute of

Technology. He transferred to Georgia Southern College

in January, 1964, and received the degree of Bachelor

of Science in Education with a major in biology in

June, 1966. In June, 1966, he began studies toward

the Ph.D. degree in the Department of Agronomy, University

of Florida, and was supported by an NDEA Fellowship

beginning in September, 1966. Requirements for the Ph.D.

degree were completed in March, 1970.

Vernon Edward Gracen, Jr. is married to the former

Sharon Marx and is the father of one son, Michael. He

is a member of Alpha Zeta and Gamma Sigma Delta.












This dissertation was prepared under the direction of

the chairman of the candidate's supervisory committee and

has been approved by all members of that committee. It

was submitted to the Dean of the College of Agriculture

and to the Graduate Council, and was approved as partial

fulfillment of the requirements for the degree of Doctor

of Philosophy.



March, 1970






Dean, College of Agric~ tu




Dean, Graduate School



Supervisory Committee:




Chairman




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AUTHOR: Gracen, Vernon
TITLE: Physiological and ultrastructural studies of oat membranes treated with
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