Association of Microbodies with Induced
Resistance to Tobacco Mosaic Virus in
Hypersensitive Tobacco

By

MARVIN EDWARD MILLER

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

1971

AC KN OWLEDGMENTS

I wish to thank Dr. D. A. Roberts, Chairman of the
Supervisory Committee, for his guidance and advice through-
out this study; and for his critical assistance in the
pieparatlon of 'chis manuscript. I also wish to thank the
other members of my Committee, Drs. J. W. Kimbrough,

R. C. Smith, and F. W. Zettlor for their critical reviovr
of this manuscript.

Acknowledgments are also extended to Miss Betty
Cordon for her technical assistance and to Mr. Richard
Christie for his generous advice on staining techniques.
Thanks are also due Dr. J. R. Edwardson for the use of
his equipment.

ii

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS. ................. ii

LIST OF FIGURES. ................. iv

ABS TRACT .... ................. v

INTRODUCTION .................... 1

METHODS OF INOCULATION .............. 3

-IDENTITY OF MICROBODIES IN HEALTHY AND VIRUS-IN-
FECTED TOBACCO. ................ 5

Staining and Counting Microbodies ...... 5

Catalase Activity in Microbodies of Epider-
mal Cells ® e . . e . . e . e . e . e . * c 9

THE INCREASE IN NUMBERS OF MICROBODIES IN RELA-
TION TO SYSTEMIC INDUCED RESISTANCE OF TOBACCO

TO TMV. .................... 13

DISCUSSION .................... 22

SUMMARY. ..................... 26

LITERATURE CITED ................. 28

BIOGRAPHICAL SKETCH. ... ...... 30

iii

LIST OF FIGURES

Figure Page

1. Crystalloid inclusions of microbodies (M) in
epidermal cells that had not been treated

with Triton X-100 to remove chloroplasts. . . . 6

2. Crystalloid inclusions of microbodies (M) in

epidermal cells treated with Triton X-100 to
remove chloroplasts 8

3. Crystalloid inclusions of microbodies (M }

ti'eated with diaminobenzidine and OsOjj. for
Localization of catalase activity ....... 11

h. Average number of microbodies in 40 cells
from two epidermal strips of leaves of
control plants which were not inoculated. ... 14

5. Average number of microbodies in 6 0 cells
from three epidermal strips of leaves of
control plants which were only inoculated

on the three lower leaves with TMV (10"') ... 15

6. Average number of microbodies in 140 cells
from seven epidermal strips of leaves of
plants which were inoculated on the three
lower leaves with TMV (10“ ■) and seven days
later inoculated on the two upper leaves with

TMV (10“5). .................. 17

7. Average number of microbodies in 80 cells
from four epidermal sti'ips from leaves of
plants which were only inoculated on the

upper two leaves with TMV (10~-5), ....... 18

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

ASSOCIATION OF MICROBODIES WITH INDUCED
RESISTANCE TO TOBACCO MOSAIC VIRUS IN
HYPERSENSITIVE TOBACCO

By

Marvin Edward Miller
June, 1971

Chairman: Dr. D. A. Roberts

Major Department: Plant Pathology

Numbers of microbodies in lower epidermal cells of
upper leaves of hypersensitive tobacco increased just
before reduction in the rate of enlargement of lesions
induced by tobacco mosaic virus. In control plants,
inoculated only on two upper leaves of decapitated plants,
there was a 1.6-fold increase in the number of microbodies
four days following inoculations. In challenge plants,
inoculated first on three lower leaves, and seven days
later inoculated on the two upper loaves, there was a
1.5-fold increase in microbody-count in upper leaves only
one day following the second inoculation; numbers of
microbodies almost doubled within U-8 hours of the second
inoculation. In control leaves, the diameters of the lesions
increased more than 0.2 mm per day for four days following
the appearance of the lesions; but lesions in challenged
leaves increased less than 0.2 mm per day following the

v

day the lesions appeared. In both control and challenge
leaves, the number of rnicrobodies increased prior to the
reduction in lesion growth, but both the increase of
microbodies and the reduction of lesion growth occurred
three days earlier in challenge leaves than in control
leaves. For this reason it is thought that rnicrobodies
may be associated with the development of induced resistance
in challenge leaves, and that induced resistance is but an
acceleration of the normal plant processes that restrict
lesion size in a hypersensitive host.

vi

INTRODUCTION

Infection in one part of a hypersensitive host plant
induces localized and systemic resistance in noninoculat ed
parts of that plant. This resistance is characterized by
the failure of localized lesions induced by subsequent
viral inoculations to develop to normal size (1,13,14,15)"

This failure is thought to result from enhancement of the
plant-defense reactions that normally restrict lesion size
in a hypersensitive host ( 1 5 )•

Numerous attempts have been made to determine differences
between plants that exhibit induced resistance and those
that do not. Simons and Ross (17, 18) reported increased
peroxidase and catalase activities prior to lesion forma-
tion in the systemically resistant upper leaves of plants
whose lower leaves had been previously inoculated with a
local-lesion strain of a virus. They suggested that the
increased peroxidase activity might play a role in the de-
velopment of systemic acquired resistance (1?).

Israel and Ross (8) reported finding single membrane-
bounded organelles, which contained crystalloid inclusions,
in a zone of cells of the palisade parenchyma surrounding
the lesions in leaves exhibiting systemic acquired resistance.
These organelles were not found in parenchymatous cells

1

2

surrounding lesions in leaves that did not exhibit systemic
acquired resistance. They suggested the possibility that
these organelles might play some role in the mechanism of
resistance. Zimmer (21) also found similar organelles,
which he referred to as crystal-containing microbodies, in
cells in the region where exclusionary-seedling-rust
resistance was operative in safflower. He suggested that
the crystal-containing microbodies might play an important
role in the rust-resistance mechanism in safflower.

The organelles referred to by Israel and Ross (8) and
by Zimmer (21) are apparently the same as those reported
by Frederick et^ al . , who first referred to them as cyto-
somes (6), and later as microbodies (5,7). Mollenhauer
et al . (11) reported that cytosomes, which fit the descrip-
tion of the microbodies clescribed by Frederick and Newcomb
(5>7)> are widespread throughout the plant kingdom and are
located in many different organs of healthy plants.

Because of the reports that cellular organelles, which
resemble the microbodies described by Frederick £t al . (5)
and Frecerick and Newcomb (7) might be involved in plant
defense reactions, a study was undertaken to determine if
numbers of microbodies were affected by the acquisition of
systemic resistance to tobacco mosaic virus (TMV) in hy-
persensitive tobacco.

METHODS OF INOCULATION

The rest plant used in all experiments was Nicotiana
L* ' Samsun NN* , which is hypersensitive to infection
by TMV . Plants were grown in composted soil in four-inch
pots kept in a greenhouse until ready for inoculation.

Vigorously growing plants with five or six well expanded
leaves were decapitated and trimmed to five adjacent leaves
as described by Ross (14). The lower three leaves were
dusted with 600~mesh Carborundum and inoculated by the
gauze-pad method with TMV in the juice of systemically in-
fected Turkish tobacco (N. tabacum): crude juice was di-
luted to 10-4 in o.l M neutral potassium phosphate buffer.
Seven days later, the two upper leaves were similarly
inoculated with TMV in juice diluted to 10“5 in the same
buffer. This inoculum caused approximately 100-150 discrete
lesions to form on each of the upper leaves.

Similar control plants were decapitated and trimmed as
the treated plants, and received the following treatments*

1) the three lower leaves were rubbed with 0.1 M potassium
phosphate buffer, and seven days later, the two upper leaves
were challenge-inoculated with TMV ( 1 0 “5 ) ; 2) the lowor

three leaves were neither inoculated nor rubbed with buffer,
but the upper two leaves were inoculated with TMV seven days
afuer decapitation; 3) the lower three leaves were inoculated

3

4

with TMV (10“4) } but the upper two leaves were neither inocu-
lated nor rubbed with phosphate buffer; and 4) leaves were
neither inoculated nor rubbed with phosphate buffer after
decapitation .

All experimental plants were kept in an air-conditioned
greenhouse at approximately 230c.

IDENTITY OF MICROBODIES IN HEALTHY AND VIRUS-INFECTED TOBACCO

Before investigating the possible correlation betv/een
cytoplasmic microbodies and the induction of acquired systemic
resistance, it was necessary to have reliable methods of
detecting and counting these organelles. Lower epidermal
cells were selected because the microbodies in these cells
could be viewed with the light microscope and the day-to-
day variations in numbers of microbodies could be readily
determined .

Staining and C ounting Microbodies

Crystal-containing microbodies were detected in the
cytoplasm of epidermal cells prepared by the method of
Christie (2). Strips of tissue removed manually from the
lower epidermis of tobacco were first submerged for 10-15
minutes in a 5% aqueous solution of octyphenoxypolyethoxyetha-
nol (Triton X-100) to disrupt the chloroplast membranes so
the chloroplasts would not interfere with viewing of the
microbodios (Figure 1). After treatment with Triton X-100,
the epidermal strips were stained 10-15 minutes in a dye
solution containing bromphenol blue in 2 % gluteraldehyde
and 0.2 M citrate buffer at pH 3*5*

Epidermal strips taken from upper leaves of decapitated
tobacco plants were examined daily for 14 days following the

5

Figure lc Crystalloid inclusions of microbodies
( M) in epidermal cells that had not been treated
with Triton X-100 to remove chloroplasts.

/

7

initial treatments. The numbers of mierobodies in each of
20 cells per epidermal strip were counted.

It was evident from preliminary observations that the
staining technique desciibed above made possib3.e the ready
detection of microbodies in cells from healthy and TMV-
infected plants (Figure 2). Reliability of the technique,
however, depended upon whether numbers of microbodies per
cell varied widely within a single leaf or within a single
treatment. Results of preliminary experiments, in which
mierobodies were counted in cells of different epidermal
strips from the same leaf, showed that only insignificant
variations occurred within a leaf. For example, the
average numbers of mierobodies per cell in each of four
strips from one healthy leaf were 19, 18, 18, and 17.

Data from experiments described later in this dissertation,
moreover, show that no significant variations in numbers of
mierobodies occurred within a single treatment.

Numbers of mierobodies per cell did vary within a
single epidermal strip, but this variation was positively
correlated with cell size. Consequently, mierobodies were
counted only in cells of similar size.

Reliability of the previously described staining tech-
nique for quantitative measurements remained in doubt until
it could be ascertained whether the early treatment of epi-
dermal cells with Triton X-100 had an effect on the numbers
of detectable mierobodies. It was possible that these cells

8

Figure 2c Crystalloid inclusions of microbodies
(M) in epidermal cells treated with Triton X-100
to remove chloroplasts*

9

might contain microbodies which did not contain crystalloid
inclusions and the chemical might destroy these microbodies
or interfere with their staining as a consequence of its
disruptive effect upon their membranes* Consequently,
epidermal strips were removed from the same healthy tobacco
leaf, and half were treated with Triton X-100. Treated and
nontreat ed scrips of cells were stained and later examined
for microbodies. That Triton X-100 did not interfere with
detection of microbodies was indicated by the fact that
about the same number of microbodies por cell wore observed
in treated and nontreated ones. In four epidermal strips
from the same leaf, counts in treated cells were 19 and 20;
those in nontreated cells were 1 6 and 19.

Reliability of staining and counting techniques having
been established it remained to test the observed inicrobodies
for enzymatic activity in order to identify them with pre-
viously described organelles (?).

Catalaso Activity in Microbodies of Epidermal C ells

The crystal-containing organelles found in epidermal
cells of ‘Samsun NN* tobacco were 0. 5-2.0 u in diameter and
were identical in appearance to the microbodies described
by Fredrick and Newcomb (5,?). Since most or all catalase
activity of the cell resides in microbodies (3), the crystal-
containing bodies in tobacco epidermal cells should be rich
in catalase if they are indeed identical to microbodies.

10

In two experiments, a total of six epidermal strips
from tobacco were treated so as to detect the site of
catalase activity in the individual cells.

Lower epidermal strips were fixed in Karnovslcy’s
solution (9), pH 7.2, for 1-1. 5 hours. After fixation,
the strips were rinsed for 15-20 minutes in three changes
of 0.01 M neutral potassium phosphate buffer. The strips
were then placed in a solution containing 10 mg of 3,3'-
diaminobenzidine (DAB), 5 ml of 0.05 M propanediol buffer
(2-amino-2-methyl-l, 3-propanediol) at pH 10.0, and 0.1 ml
of 3^ HgOg • The pH of the solution was adjusted to 9*0
prior to the addition of the epidermal strips. The strips
were incubated in covered petri dishes for 30 minutes at
37°C.

The epidermal strips were postfixed for 1 hour in Vjo
OSO4 in 0.01 M neutral potassium phosphate buffer. They
were then dehydrated in alcohol and mounted in Euparal for
examination under the light microscope.

Crystal-containing bodies in epidermal cells of ’Samsun
NN* tobacco always stained dark reddish-brown in DAB and
0sCi| (Figure 3)» indicating strong catalase activity (7).

The crystal-containing bodies did not stain in DAB and OsO^,
provided epidermal strips had been pre-treated with 3-a-inino-l,
2,4-triazole, a known inhibitor of catalase activity (10).
These results provide further evidence that the crystal-
containing bodies in epidermal cells of tobacco are the

Figure 3« Crystalloid inclusions of microbodies
(M) treated with diaminobenzidlne and OsO; for
localization of catalase activity. 4

12

same as the catalase-rich microbodies characterized by
Frederick and Newcomb (7).

THE INCREASE IN NUMBERS OF MICROBODIES IN RELATION

TO SYSTEMIC INDUCED RESISTANCE OF TOBACCO TO TMV

Now that the presence of microbodies in healthy plants
has been demonstrated (5?6j 7,11), and since staining tech-
niques described earlier make possible quantitative deter-
minations of microbodies through light microscopy, it becomes
possible to test the hypothesis of Israel and Ross (8) that
microbodies may be correlated with the development of systemic
induced resistance in hypersensitive hosts.

Microbodies were stained and counted as described
earlier, and were countod daily for 14 days after the first
inoculation or treatment in 20 cells per epidermal strip
from the two upper leaves. The experiment was performed
four times over a period of six months. All experimental
plants were inoculated or inoculations were simulated as
described in the section on methods of inoculation.

The numbers of microbodies in noninoculat ed control
plants did not vary significantly over a 14-day period
after decapitation of the plants. An average of 14 to 19
microbodies per cell occurred in lower epidermal strips of
these plants (Figure 4). Over the same l4-day period,
moreover, only 14-18 microbodies were observed in resistant
upper leaves that were not challenge-inoculated (Figure 5)*

In contrast, numbers of microbodies increased dramatically

13

14

DAYS AFTER TREATMENT

Figure 4. Average number of rnicrobodies in 40 cells
frotn two epidermal strips of leaves of control plants
which were not inoculated.

35

30

25

20

15

10

5

15

DAYS AFTER TREATMENT

Figure 5* Average number of microbodies in 6 0 cells
from three epidermal strips of leaves of control plants
which wero only inoculated on the three lower leaves
with TMV (10~^). Arrow indicates day of inoculation.

16

in challenge-inoculated upper leaves in which resistance
had been induced as a result of earlier inoculations of
lower leaves. For example, 2 6 microbodies per cell were
detected only one day after challenge-inoculation and 3®
per cell were counted on the second day after the challenge
inoculation (Figure 6). The numbers of microbodies per
cell thus almost doubled in resistant leaves within 1-2
days of the second, or challenge, inoculation.

Numbers of microbodies also increased in epidermal
cells of TMV-inoculated nonresistant leaves, but this
increase did not occur until at least four days after
inoculation of these leaves. Each cell contained 1.4-19
microbodies until the fourth day after inoculation, when
the number increased about 1.6-fold to 29 (Figure 7).

The same general trend was also found in plants where the
lower three leaves were rubbed with 0.1 M potassium phos-
phate buffer and the upper two leaves inoculated with
TMV . Rubbing the lower leaves with buffer thus does not
affect the number of microbodies in the upper leaves.

Results of these experiments suggest that microbodies
increase in cells of hypersensitive tobacco prior to the
reduction of lesion enlargement in both resistant and non-
resistant leaves.

In resistant leaves the lesions increased very little
in size, less than 0.2 mm per day, after the second day
following the challenge-inoculation (Table 1). This is

17

DAYS AFTER TREATMENT

Figure 6. Average number of mierobodies in 140 cells
from seven epidermal strips of leaves of plants which
were inoculated on the three lower leaves with TMV
(10-4) and seven days later inoculated on the two
upper leaves with TMV (10“5). Arrows indicate days
of inoculation.

18

t

i i i \ i i i i 1 1 1 t ( p

2 4 6 8 10 12 14

DAYS AFTER TREATMENT

Figure 7. Average number of microbodies in 80 cells
from four epidermal strips from leaves of plants which
were only inoculated on the upper two leaves with
TMV (10“5), Arrow indicates day of inoculation.

I

19

Table 1. Daily increase in lesion size in upper leaves
following inoculation with TMV (10“5).a

Day

1

2

3

k

5

6

7

Resistant
Leaves'3

0.7°

0.1

0.2

0.1

0.1

0.0

Nonresistant
Leaves^ __

0.9

9

o

0.5

0.3

0.2

0.1

a Increase is expressed in mm/day.

b Lower leaves were inoculated with TMV (10"^) and
upper leaves inoculated with TMV (10~5) seven days
later.

c Average of at least $0 lesions.

^ Only upper leaves were inoculated with TMV (10**5).

20

in contrast to the nonresistant leaves where the lesions
continued to increase more than 0.2 mm per day in size
for five days following inoculation.

If there is any association between the increase of
the microbodies and the mechanism that limits lesion size,
then the microbodies should increase in number prior to
the reduction in lesion growth. As was pointed out earlier,
tho numbers of microbodies in the resistant leaves began
to increase one day following the challenge-inoculation,
and there was a substantial reduction in lesion growth on
the third day. In nonresistant ieaves, the microbodies
did not begin to increase in number until four days following
the inoculation, and there was not a substantial reduction

in lesion growth until the sixth day. In both resistant

•- %

and nonresistant leaves, numbers of microbodies began to
increase two days prior to any substantial reduction in
lesion growth. In resistant leaves, however, the increase
in numbers of microbodies and the reduction in lesion
growth occurred three days earlier than in nonresistant
leaves .

This correlation of numbers of microbodies with growth
of lesions suggests that microbodies are somehow involved
in or stimulated by the normal mechanisms that limit lesion
development in hypersensitive hosts. The fact that numbers
of microbodies increase almost immediately after challenge-
inoculation of resistant leaves is consistent with Ross’s

21

hypothesis that induced resistance results from enhancement
of the normal resistant mechanisms of hypersensitive hosts

(15)-

DISCUSSION

Pathogens that cause a localized necrosis in plants
induce a systemic resistance to subsequent infection by
viruses that cause local lesions (1,13,14,15). It has
been suggested that the nature of the induced resistance
is the result of the enhancement of the plant defense
reactions which normally limit lesion size (15).

Israel and Ross (8) suggested that crystal-containing
bodies in a zone of palisade parenchyma cells surrounding
TMV-lesions in challenge-inoculated leaves ma3r be involved
in defense reactions. Simons and Ross (18) later demon-
strated a greater and earlier increase in peroxidase ac-
tivity in upper leaves of challenge plants than of control
plants. They suggested that the early increase of peroxi-
dase activity in challenge leaves may play some role in
the enhancement of the normal plant defense reactions.

Except for these reports, no other substantial suggestions
have been made that could account for the differences in
lesion size in challenge and control leaves.

Simons and Ross (18) also reported that catalase
activity increased prior to lesion formation in the challenge
leaves. Since most or all of the catalase activity is lo-
calized in the microbodies (3), the increase in catalase
activity is most logically explained by the increase in

I

22

23

the number of microbodies prior to lesion formation in the
- challenge leaves.

The increase in numbers of microbodies in the challenged
leaves begins before lesion formation and reaches a peak
about the time of lesion formation. In control plants, the
microbodies do not significantly increase in numbers in the
upper leaves until the fourth day after inoculation.

Lesions in control leaves continue to increase in size for
five days after their appearance; but in challenge leaves
there is no substantial increase in lesion size following
the day they appear. The numbers of microbodies increased
three days earlier in challenge leaves than in control
leaves. In both instances, however, the microbodies in-
crease before any substantial decrease in the rate of
lesion growth. The fact that lesions increase in size
very little following the increase in numbers of microbodies
suggests that the build-up of the microbodies may be asso-
ciated with the enhancement of the normal defense reactions.

At this point it is not possible to determine a specific
function of the microbodies in systemic acquired resistance.
It has been shown that peroxisomes (microbodies) contain, in
addition to catalase, the enzyme systems involved with gly-
colate metabolism (12), which is a byproduct of photosynthe-
sis. It has also been pointed out by de Duve (4) that the
respiratory system of peroxisomes (microbodies) is not
coupled to any mechanism for the conservation of energy,

2h

and it catalyses an essentially wasteful form of respira-
tion. This may be significant if there is in fact a rapid
increase of microbodies in virus-infected cells immediately
pi'ior to lesion formation in cells surrounding a lesion.

Ross and Israel (1 6) have suggested that cells surround-
ing a lesion are sensitive to collapse and the increased
sensitivity of these cells to collapse may be instrumental
in viral localization in plants. It is not known if the
increase of microbodies plays a role in making a cell more
sensitive to collapse following viral replication in that
cell} but it is interesting that the respiratory system of
microbodies is not linked to the energy-conserving steps
found in some metabolic pathways. Israel and Ross (8) also
pointed out that cells in a zone surrounding a lesion re-
semble cells that are in an active metabolic state s which
is energy-requiring. If there is an increase of microbodies ,
whose enzyme systems are breaking down products without the
usual energy return, then cells surrounding the lesion may
reach a state that would make them more sensitive to collapse.

Although Simons and Ross (18) reported an increase in
catalase activity in challenge leaves, they did not find a
corresponding increase in glycolic acid oxidase. This result
is somewhat puzzling since glycolic acid oxidase is also
associated with microbodies (12,19,20). Tolbert et al^ (19)
reported that glycolate oxidase activity was increased by
30-40';o when the peroxisomal (microbody) fraction was treatod

25

with Triton X-100. It may be that increases in glycolate
oxidase activity cannot be accurately determined unless
the microbody membranes are solubilized.

The role of microbodies in systemic induced resistance
is certainly not yet understood. But results reported
herein clearly associate microbodies in whatever defensive
mechanism hypersensitive tobacco has to limit the enlarge-
ment of lesions caused by TMV .

SUMMARY

Crystal-containing bodies in lower epidermal cells
of * Samsun NN* tobacco are identical to ,,microbodies,,
("peroxisomes, cytosomes, crystal-containing bodies”)*
Identity was based on the positive staining reaction for
catalase activity with diaminobenzidine and the similari-
ties in the size of the crystal-containing bodies and mi-
crobodies*

Treatment of epidermal cells with Triton X-100 to
disrupt the chloroplasts did not affect the number of
microbodies per cell.

There was a three-day lag between the time microbodies
began to increase in challenge plants and control plants.

In challenge plants, inoculated with tobacco mosaic virus
(TMV) first on the lower three leaves and seven days later
inoculated on the upper two leaves, there was a 1.5-f°ld
increase in the microbodies only one day following challenge-
inoculation and a 1.9-fold increase on the second day. In
control plants, inoculated with TMV only on the upper two
leaves, the microbodies did not begin to increase until
four days after inoculation.

There was a • substantial decrease in the rate of lesion
growth in challenge leaves two days after the second, or

26

I

2?

challenge, inoculation. In control leaves, there was no
substantial decrease in rate of lesion growth until the
sixth day after inoculation. In both challenge and control
leaves, there was an increase in the number of microbodies
prior to the time of a substantial decrease in lesion growth.

It is thought that: 1) the increase of microbodies

may play a role in the restrictions of lesion growth, in
challenge and control leaves; and 2) that the earlier
increase of microbodies in challenge leaves may have some
role in the earlier reduction of rate of lesion enlargement
in these leaves than in control leaves.

LITERATURE CITED

1. Bozarth, R. F., and A. F. Ross. 1964. Systemic

resistance by localized virus infections: Ex-
tent of changes in uninfected plant parts.

Virology 24: 446-455*

2. Christie, R. G. 1971* (Personal Communication).

3« do Duvo, C., and P. Baudhuin. 1966. Peroxisomes
(microbodies and related particles). Physiolo-
gical Reviews 46: 323-357*

I

4. de Duve, C. 1969* The peroxisome: a new cytoplas-
mic organelle. Proc. Roy. Soc. (bond.) 173s
71-83.

5* Frederick, S. E., E. H. Newcomb, E. L. Vigil, and

V/. P. Wergin. 1968. Fine structural characteri-
zations of plant microbodies. Planta 81: 229-252.

6. Frederick, S. E«, and E. H. Newcomb. 1968. Micro-
body-like organelles in leaf cells. Science 163:
1353-1355.

7* Frederick, S. E. , and E. II. Newcomb. 1969* Cytochemi-
cal localization of catalase in leaf microbodies
(peroxisomes). J. Cell Biol. 43: 343-353*

8. Israel, II. V.’., and A. F. Ross. 1967* The fine struc-

ture of local lesions induced by tobacco mosaic
virus in tobacco. Virology 33: 272-286.

9. Karnovslcy, M. J. 1965* A formaldehyde-glutaraldehyde

fixative of high osmolality for use in electron
microscopy. J. Cell Biol. 27s 137A-138A.

10. Hargoliash, E., and A. Novogrodsky. 1958. A study

of the inhibition of catalase by 3~a.mino~l : 2 : 4-
triazole. Biochem. J. 68: 468-475*

11. Mollenhauer, II . H., D. J. Moore, and A. G. Kelley.

1966. The widespread occurrence of plant cytosomes
resembling animal microbodies. Protoplasma 62:
44-52.

12. Kisaki, T., and N. E. Tolbert. 1969* Glycolate and

glyoxylate metabolism by isolated peroxisomes or
chi oroplasts . Plant Physiol. 44: 242-250.

28

29

13-

14.

15-

16.

1?.

18.

19.

20.

21 .

Rosss A. F« , and R. F. Bozartb. i960. Resistance
induced in one plant part as a result of virus
infection in another part. Phytopathology 50t
652. (Abstract).

Ross, A. F. 1961. Systemic acquired resistance
induced by localized virus infections in plants.
Virology 14: 3^0-358.

Ross, A. F. 1966. Systemic effects of local lesion
formation, p. 127-150. In A. B. R. Beemster and
Jeanne Dijkstra ( ed . ) . Viruses of Plants. North-
Holland Publ , Co., Amsterdam.

Ross, A. F., and H. V. Israel. 1970. Use of heat
treatments in the study of acquired resistance to
tobacco mosaic virus in hypersensitive tobacco.
Phytopathology 60; 755“??° *

Simons, T. J., and A. F. Ross. 1970* Enhanced
peroxidase activity associated with induction
of resistance to tobacco mosaic virus in hy-
persensitive tobacco. Phytopathology 60: 383**

384.

Simons, T. J., and A. F. Ross. 1971. Metabolic
changes associated with systemic induced re-
sistance to tobacco mosaic virus in Samsun NN
tobacco. Phytopathology 6 1 : 293-300.

Tolbert, N. E. , A. Oeser, R. K. Yamazaki, R. H. Ilageman,
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Phytopathology 60: 1157-1163.

BIOGRAPHICAL SKETCH

Marvin Edward Miller was born February 27, 1941 > at
Dawson, Georgia. He attended grammar school at Dawson
and graduated in 1959 from North Marion High School at
Reddick, Florida. Ho attended Abraham Baldwin Junior
College at Tifton, Georgia for one year. From i960 un-
til 1964 he served as an electronics technician in the
United States Navy and was stationed at Pensacola, Florida.
Following his release from the Navy he entered the Uni-
versity of Florida, where he received the dogree of Bachelor
of Science in Agriculture in August, 1967 and the degree
of Master of Science in March, 1969* In April, 1969, he
began studies toward the degree of Doctor of Philosophy.

He is a member of the American Phytopathological Society,
Alpha Zeta, and Gamma Sigma Delta.

Marvin Edward Miller is married to the former Maxine
Childress of Fairhope, Alabama.

30

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.

Daniel A. Roberts,
Chairman

Profes’sor of Plant
Pathology

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope /Snd quality,
as a dissertation for the degree of Doctor o/f jPhiJJ^©f>Oi$y .

Francis W. Zettler
Assistant Professor of
Plant Pathology

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.

O'/

Japes dVj Kimbrough
Assistant Professor of
Botany

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,

This dissertation was submitted to the Dean of the College
of Agriculture and to the (graduate Council, and was accepted
as partial fulfillment of the requirements for the degree
of Doctor of Philosophy.

June, 1971

Agriculturecy

Dean, Graduate School