PHYSIOLOGICAL AND ULTRASTRUCTURAL STUDIES
OF OAT MEMBRANES TREATED WITH
Helminthosporium victoria TOXIN
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
Dedicated to my
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
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
TABLE OF CONTENTS
ACKNOWLEDGMENTS . .
LIST OF TABLES . .
LIST OF FIGURES . .
ABSTRACT . . . .
INTRODUCTION .. .
LITERATURE REVIEW. .
MATERIALS AND METHODS.
RESULTS. . . . .
DISCUSSION . . .
SUMMARY . . . .
BIBLIOGRAPHY . . .
BIOGRAPHICAL SKETCH. .
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
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
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
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
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
Vernon Edward Gracen, Jr.
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
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
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
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.
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
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.
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
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
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.
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
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
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
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.
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
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).
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
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
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,
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
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,
(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)
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
(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
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
(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
(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.
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
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
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
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
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
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
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
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
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
COMPARISON OF THE RELATIVE
CULTURE FILTRATE AND PARTIALLY PURIFIEDa CULTURE FILTRATE
Victorin Culture Partially purified
dilutions filtrate culture filtrate
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.
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
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
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
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
FINAL 86Rb CONTENT OF RESISTANT
OAT ROOT TISSUE TREATED WITH VICTORIN
Pretreated with victorin Post-treated with victorin
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
2-5-1 3190 + 56 3155 + 56 101 3205 + 57 3111 + 56 103
Resistant 4142 + 64 3966 + 63 104 3557 + 60 3527 + 59 101
500 ---- ---- --- 3127 + 56 2965 + 60 106
Resistant 4431 + 66 4127 + 64 107* 5531 + 74 5244 + 72 106
78-1-1 4026 + 63 3819 + 62 105 -- --
86Rb absorption w
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
INFLUENCE OF DURATION OF VICTORIN
PRETREATMENT ON FINAL 6Rb CONTENT IN
SUSCEPTIBLE ROOT TISSUEa
Pretreatment Final 86Rb content
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
FINAL 8Rb CONTENT IN SUSCEPTIBLE OAT ROOTS
TREATED WITH VICTORIN SOLUTIONS
CONTAINING VARIOUS CALCIUM CONCENTRATIONSa
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
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
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
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
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
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
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
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
FINAL CONTENT OF 86Rb IN RESISTANT AND SUSCEPTIBLE
OAT ROOTS AFTER EDTA TREATMENTa b
Control 10-5M EDTA 10-4M EDTA
Cultivar pretreatment pretreatment
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
* 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.
(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
FINAL CONTENT OF 86Rb IN RESISTANT ROOTS
PRETREATED WITH EDTA, VICTORIN, AND
S.... : EDTA PLUS VICTORINa : :
cpm control cpm
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
FINAL _Ca CONTENT IN RESISTANT AND
SUSCEPTIBLE OAT ROOTS TREATED WITH VICTORIA
Control pretreated post-treated
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**
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
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.
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
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
RETENTION OF 4Ca IN RESISTANT AND SUSCEPTIBLE
OAT ROOTS AFTER DESORPTION IN SOLUTIONS CONTAINING
VICTORIN, CaCl2, AND VICTORIN PLUS CaC12a
NO CaC12 Victorin CaC12 plus
desorption (5.0 mM) (1 unit/ml) victorin H20
Susceptible 2700 7868 6523 8851 14320
removed 0 72 86** 68 52
Resistant 10725 6364 6182 6404 8093
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
(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).
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
Mutant 233 + 15 235 + 15 101 ----
a yistorin treatments were applied either before or after
Significant at the 5% level
** Significant at the 1% level
INFLUENCE OF DURATION OF VICTORIN
PRETREATMENT ON FINAL
86Rb CONTENT IN SUSCEPTIBLE OAT LEAVESa
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**
a Victorin treatments were applied before 8Rb absorption
Significant at the 5% level
** Significant at the 1% level
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
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
ROOT GROWTH OF RESISTANT AND SUSCEPTIBLE
CALCIUM DEFICIENT SOLUTIONS
10-4 CaSO4 H20
Susceptible 148 + 19 35 + 9**
Resistant 162 + 20 234 + 14
Mutant 2-5-1 138 20 161 + 6
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.
ROOT GROWTH INHIBITION BY EXTRACTS
FROM VICTORIN-TREATED RESISTANT AND SUSCEPTIBLE OAT ROOTS
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.
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
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
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
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
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.
Fg re4 .'t. a
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.
, ft -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.
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
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
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
Figure 9. Higher magnification (x45,000) of untreated resistant
oat root tissue showing regions of unit structure in the plasma
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.
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
and difrnei h perne fmtcodi cmaet
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
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
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
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
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
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
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
unavailable for binding 4Ca later. Hence, victorin
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
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
the total 4Ca incorporated into roots grown on 4Ca was
more easily removed from susceptible than from resistant
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
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
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.
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
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
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
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
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.
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on victorin-induced respiration of oat tissue.
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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
Dean, College of Agric~ tu
Dean, Graduate School
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TITLE: Physiological and ultrastructural studies of oat membranes treated with
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