Ecological Research Series
III LIBRARY
EWIR6IW8STAL FMTfOTIOH ASINCT
OZONE AND VASCULAR TISSUE
DIFFERENTIATION IN PLANTS
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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EPA-600/3-76-068
May 1976
OZONE AND VASCULAR TISSUE
DIFFERENTIATION IN PLANTS
By
John P. Rier, Jr.
Department of Botany
Howard University
Washington, D. C. 20059
Grant No. R801209
Program Element 1A1006/1HA323
Project Officer
Willie Ashley, Jr.
Office of Monitoring and Technical Support
Technical Support Division
U. S. Environmental Protection Agency
Office of Research and Development
Washington, D. C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. . Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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DISCLAIMER
This report has been reviewed by the Office of Research and
Development, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
ii
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ABSTRACT
The purpose of this research is to ascertain the possible influence of ozone on the
process of vascular tissue differentiation in plants and the protein changes associated
with it. Test materials were wounded plant intemodes and callus tissues grown,
exposed, and studied under laboratory conditions. Ozone was more effective in re-
ducing xylem regeneration in those internodes grown in Indoleacetic acid than in
Dichlorophenoxyacetic acid. Preliminary findings of the protein and enzyme patterns
in callus tissues exposed to ozone suggest that it has an influence on them. It is con-
cluded that plant internodes and callus tissues can be used to study the effects of ozone
on certain processes related to plant growth and development.
This report submitted in fulfillment of Project Number AP 01654 01-02-03 by the
Environmental Protection Agency. Work was completed as of May, 1974.
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A CKNOWLEDGMENT
The technical assistance of Mrs. Helene H. Cann (Rutgers University) and Miss
Vivian A. Owens (Howard University) is acknowledged with thanks and appreciation.
IV
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CONTENTS
Abstract iii
Acknowledgments iv
Sections
I. Conclusions 1
II. Recommendations 2
HI. Introduction 3
IV. Materials and Methods 5
Assembly of Ozonation Apparatus 5
Wounding of Internodes 5
Ozonation and Staining 6
Culture and Ozonation of Callus Tissues 8
Extraction of Protein from Callus Tissue 8
Electrophoresis of Extracts 9
Staining for Proteins and Peroxidase 10
V. Discussion 11
Xylem Regeneration in Internodes 11
Ozone Enhancement of Membrane Permeability 17
Reduction of Cell Division by Ozone 18
Oxidation of Lignin Forming Enzymes by Ozone 19
Ozone Affect on the Incorporation of Sucrose into Cellulose 20
Ozone Inactivation of Indole-3-acetic acid 21
Reductions in Regeneration Caused by the Rate of Diffusion of 22
Indole-3-acetic acid or 2, 4-Dichlorophenoxyacetic acid
Protein Patterns in Ozonated Callus Tissues 23
VI. References 24
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SECTION I
CONCLUSIONS
1. Ozone reduces xylem regeneration in wounded internodes.
2. The reduction in the number of new xylem elements formed around the wounds
was greater following immediate rather than delayed exposure of the internodes
to ozone.
3. Ozone-induced xylem regeneration was reduced more in internodes cultured in
sucrose plus Indole-3-acetic acid (IAA) than in sucrose or 2, 4-Dichloro-
phenoxyacetic acid (2,4-D) plus sucrose.
4. Basal applications of equimolar concentrations of Indole-3-acetic acid (2 ppm)
and 2, 4-Dichlorophenoxyacetic acid (2.5 ppm) does not induce a significant
number of new xylem in wounded isolated internodes of Cole us.
5. Basal applications of either Indole-3-acetic acid or 2, 4-Dichlorophenoxyacetic
acid in the presence of sucrose induces significant numbers of new xylem
around the wounds of isolated Cole us internodes.
6. Regeneration of new xylem failed to occur in internodes cultured in glass
distilled water.
7. Xylem regeneration occurred in internodes cultured in liquid media providing
the proper constituents are added.
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SECTION II
BECOMMENDATIONS
Along with the research on the role of oxidants in plant cell growth and plant vitality,
it is recommended that more attention be given to cell and organ differentiation and
the subtle biochemical changes associated with these processes.
The following lines of research should be followed in order to understand further the
impact of ozone on vascular tissue differentiation in plants:
1. The effect of ozone on phloem differentiation
2. The effectiveness of growth regulators in the presence of ozone
3. The effect of ozone on cellulose and lignin production
4. The effect of ozone on plant membrane integrity
5. The effect of ozone on the quality and quantity of proteins and enzymes
in growing and differentiating plant tissues.
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SECTION HI
INTBODUCTION
The purpose of this research was to determine whether plant tissues upon exposure to
ozone will show alterations in the quality and quantity of vascular tissues produced in
them when grown on a variety of media. Xylem regeneration around wounds was
studied in ozonated and nonozonated internodes of Coleus blumei (Benth) grown on
media containing 4% sucrose in combination with different growth regulators. Studies
were also made to determine the effect of ozone on peroxidase and the total protein
content in plant callus tissues from stems of Parthenocissus tricuspidata (veitchi)
which were also grown on a variety of media. One basic assumption of this study was
that the role of IAA in the biochemistry of xylogenesis could possibly be influenced by
a strong oxidant such as ozone which is a common air pollutant,, Ordin (1962) found
that ozone did indeed inactivate IAA in cell elongation studies. Furthermore, studies
with isolated internodes have shown that IAA influences differentiation and regenera-
tion of xylem, Jacobs (1952, 1954) and of phloem, Lamotte and Jacobs (1963)0 Addi-
tionally, IAA in combination with sucrose enhances differentiation and regeneration in
callus, Wetmore and Rier (1963) and Eier and Beslow (1967)0 A combination of
sucrose and IAA stimulated more regeneration in isolated Coleus internodes than
separate additions of either components, Beslow and Eier (1969)0 A logical extension
of the assumption in the above process is that perhaps ozone could modify IAA-
influenced xylogenesis0 Furthermore, current information in regards to changes in
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protein constituency and enzymic variations in relation to plant growth and development
support an examination of them in these studies on ozone and xylogenesis.
This report will show that ozone does influence the degree of xylogenesis in internodes
grown in media containing IAA and sucrose and that the substitution of 2,4-D for IAA
has a mitigating effect on the action of ozone in this process.
Experiments were done to determine the protein pattern of ozonated and non-ozonated
callus from stems of Boston Ivy, Parthenocissus tri cuspidate, (var. veitchi) which was
grown on a maintenance medium containing 1. 5% sucrose and 0.1 ppm IAA, Wetmore
and Rier (1963). Variations in the medium were made by increasing the IAA concen-
tration to 2 ppm, or by substituting an equimolar concentration of 2, 4-D (2.4 ppm) for
IAA. Previous studies have shown that modifications of the medium influenced xylo-
genesis. Changes in sucrose concentration will also influence the quantities of xylem
in these tissues, Bier and Beslow (1967). Furthermore, it has been shown in the
present report that ozone does have an effect on xylogenesis. It was thought that the
protein patterns and perhaps some of the enzymic systems have been correlated with
morphogenesis in plants and plant parts, Frenkel and Hess (1974), Juo and Stotzky
(1970), and Leshemet_al (1970).
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SECTION IV
MATERIALS AND METHODS
ASSEMBLY OF OZONATION APPARATUS
The ozone generator (Welsbach Ozonator - Model T 408) used in these experiments
was appropriately connected to a cold water source for cooling, Aviator's Breathing-
oxygen, and an exposure chamber. The flow of oxygen from a cylinder was con-
trolled by a regulator on the tank and by a pressure valve on the generator. Oxygen
was allowed to flow into the ozonator until a steady rate could be maintained. Subse-
quently, the voltage on the ozonator was adjusted to give an ozone concentration of
50 pphm, as measured by a Welsbach Ozone Meter, Model H-100 LC, and recorded
on a Modified Strip Chart Recorder. The excess ozone was passed into a KI solution
where it was reconverted into oxygen for disposal.
WOUNDING OF INTERNODES
Cuttings were taken from Cole us and placed in perlite. Roots developed on them within
5 to 7 days and they were subsequently potted in soil. All plants were watered daily
for one month and fertilized once weekly. Lateral shoots were removed as soon as
they appeared. At the end of the growth period, the apical half of the plant was re-
moved, washed in 10% chlorox, rinsed in three changes of sterile distilled water, and
debladed. The second internode was removed and wounded by cutting a V-notch in
one corner in such a manner as to sever a major vascular bundle. The apical end of
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the segment was cut at an angle for identification.
After wounding, ten segments were placed in petri dishes containing various media
and held in an upright position with polyethylene sponge discs. The aqueous media
contained one of the following: (1) 2 ppm IAA, (2) 2.4 ppm 2,4-D, (3) 4% sucrose,
(4) 2 ppm IAA plus 4% sucrose, and (5) 2.4 ppm 2,4-D plus 4% sucrose. Internodes
were cultured in glass distilled water for comparison. All internodes were cultured
under room conditions. A total of 2160 internodes were grown and used in this study.
OZONATION AND STAINING
A schedule of the culture and ozonation of the internodes were made to reveal the
degree of xylogenesis under the following conditions :
A. Ozone exposures were made at the noon hour. (It has been
determined that perhaps the ozone level of the atmosphere
is highest during the noon hours, Heggestad and Middleton,
1959).
B. Immediately upon wounding, internodes were exposed to
ozone at 50 pphm for one hour daily for one week and killed.
B . Control internodes were grown in the absence of ozone for
one week and killed.
C. Internodes were cultured as in B for one week in the presence
of ozone at 50 pphm for one hour daily and for an additional
week in the absence of ozone prior to killing.
D. Internodes were grown for two weeks. During the first week
they received no ozone. During the second week they were
exposed to ozone at 50 pphm for one hour daily.
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E. Segments were ozonated for the entire two week culture period.
F. Conditions Cp D.., and E^ were controls in which the internodes were
grown in the absence of ozone for two weeks.
Cultures under the above conditions were grown in each of the media listed above.
Preparation of materials for histological examination followed the procedure estab-
lished by Fuchs (1963). The segments were killed and fixed in formalin acetyl
alcohol (FAA), hydrated, and stained in fuchsin stain at 60 C for 10 to 14 hours.
The staining solution contained 10 grams of sodium hydroxide in 100 ml of distilled
water and 1 gram of basic fuchsin. After staining the segments were placed in
several changes of tap water and taken through dehydration to xylene. Each segment
was cut longitudinally at the corner immediately behind the wounded vascular bundle
and placed on the slide, wounded side facing up. This was done so that the entire
wound area was visible for, microscopic examination.
With the aid of a microscope and a tally denominator, counts of newly regenerated
xylem vessels were made in the wound area between two major vascular bundles and
to a distance approximately 2.0 to 2. 5 mm above and below the wound. Computations
were made in regards to the number of vessels regenerated in relation to the experi-
mental conditions. Statistical means and standard deviations were obtained and t-test,
were run to determine the significance between the controls and experimental treat-
ments. Graphs were made of these calculations.
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CULTUEE AND OZONATION OF CALLUS TISSUES
Subcultures were made of stem callus tissue stocks of Parthenocissus tricuspidata
var. veitchi that were grown on a maintenance medium. The experimental media
consisted of Kaden's media plus 1.5% sucrose and 2 ppm IAA and 4% sucrose and
2 ppm IAA (Rier and Beslow). They were placed in 2 ounce square bottles with
screw caps. Cultures were maintained at 25 C with a photoperiod of 8 hours light
and 16 hours darkness.
Following an adjustment period of 24 hours in the incubator, tissues were selected
and exposed to ozone. Bottles containing tissues were surfaced sterilized with 70%
ethyl alcohol and placed in sterile chamber for ozonation. Their caps were carefully
removed and placed in the chamber where they remained during ozonation. The
cultures received 50 pphm ozone for three hours once each of four successive weeks
and returned to the incubator following each treatment. Control tissues remained in
the incubator throughout the experimental period of six weeks.
EXTRACTION OF PROTEIN FROM CALLUS TISSUE
At the end of the growth period the ozonated tissues and controls were removed from
the bottles and washed with distilled water. Approximately 10 grams of the tissue were
placed in the refrigerator and allowed to cool. Extraction of protein and disc electro-
phoresis followed the methods of Caponetti (personal communication) with modifications.
Tissue was ground in a prechilled mortar which was kept cold on a stirrer cooler
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(Model SK 12 manufactured by Thermoelectrics Unlimited, Inc.). Proteins were
extracted with 6 ml of 0.1M Hepes buffer at pH 7.4, 8 drops of Cleland's reagent or
dithiothreitol (Dtt), and 1 gram of polyvinylpyrrolidone (PVP-AT) powder per 10
grams of tissue at a temperature of minus 2°C. After extraction the tissue was
forced through 4 layers of cheesecloth, previously soaked in Hepes buffer. The
strainate was centrifuged twice at 4°C at 20, 000 x g for 30 minutes each. The super-
natant was eluted from a 15 x 1. 5 cm column packed with Sephadex G50.
The Sephadex beads were previously soaked in distilled water for 3 days. The column
was flushed with the eluant, 0.05M Tris-HCl buffer, pH 8. 0 to remove the water and
allowed to settle for 12 to 24 hours. Approximately 100 ml of the extract and eluant
was collected and dialyzed against several changes of 0. 05M Tris-Glycine buffer,
pH 8.3, for 12 hours. The dialyzate was concentrated by rolling in dry Aquacide n
powder. The final volume of 1 or 2 ml was placed in small vials in the refrigerator.
ELECTROPHORESIS OF EXTRACTS
The apparatus used for disc electrophoresis and the power supply were manufactured
by Buchler Instruments. A volume of 0.35 ml of the extract was applied to gels for
separation. The gels were made in tubes 13 cm x 1/2 cm. The separation gel was
8 cm long with a pH of 8.9 and a pore size of 13 percent. The stacking gel was 2 cm
long with a pH of 5.5 and a pore size of 4.5 percent. The extract was placed on the
stacking gel and carefully layered with resevoir buffer, 0.01M Tris-Glycine, pH 8.3.
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A tracking or indicator dye, bromophenol blue, was placed in one of the tubes prior
to adding buffer. Electrophoresis was done in the cold at about 4 C and run at 3 mA
per tube.
STAINING FOE PROTEINS AND PEEOXIDASE
Gels were removed from the tubes and stained either for total protein with 0.1%
Amido Schwartz overnite and destained with several changes of 7% acetic acid or were
stained for peroxidase. The peroxidase stain (Yoneda and Endo, 1969) contained
equal volumes of:
0. 5M Na-Acetate buffer pH 4. 0
0.1% Benzidine HCl
0.3% H202
The sodium acetate buffer was substituted for Tris-acetic acid buffer used in this
study.
All gels were compared for similarity or differences in the banding pattern for total
protein and peroxidase of callus tissue grown on the various media in the presence
or absence of ozone.
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SECTION V
DISCUSSION
XYLEM REGENERATION IN INTERNODES
Regeneration of xylem elements was negligible in segments cultured in aqueous
media containing IAA or 2,4-D. The additions of sucrose to each medium increased
the amount of regeneration. Medium containing sucrose and distilled water pro-
duced a minimal response (Fig. 1). Xylem regeneration was increased three-fold by
a combination of sucrose and IAA (Fig. 2) and about twelve-fold by a combination of
sucrose and 2,4-D (Fig. 3).
Ozone effectively reduced the amount of xylem regeneration in Coleus segments when
cultured in either of two media, sucrose or sucrose plus IAA (Fig. 4). Suppression of
the regenerative response was greatest when the segments were cultured in these two
media and exposed to ozone immediately after wounding. For instance, regeneration
in segments grown under conditions A of the culture schedule was reduced by 34% when
grown in sucrose and by 46% when they were grown in sucrose plus IAA. Under condi-
tion C, regeneration was reduced by 26% when the segments were grown in sucrose and
65% when they were grown in sucrose plus IAA. When segments were exposed to ozone
for two weeks, condition E, regeneration was reduced by 22% in internodes cultured in
sucrose and 56% in those cultured in sucrose plus IAA. If cultures of internodes were
initiated and grown in sucrose fpr 1 week without ozonation and exposed to ozone during
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SUCROSE
120+
80+
40+
<=>
IX
Fig. 1. The mean number of xylem elements
regenerating in internodes cultured in 4% sucrose in
response to conditions of exposure to ozone. Vertical
bars (inserts) represent standard error.
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SUCROSE + IAA
500--
300
ix 100-
-Oj
+0,
Fig. 2. The mean number of xylem elements
regenerating in internodes cultured in 4% sucrose plus
2 ppm IAA and the affect of varying conditions of ozone
exposure. Vertical bars (inserts) represent standard
error.
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SUCROSE + 2,4-D ~°3
CO
3 2000• •
1500-
1000-
ix 500 -
2 WKS
Fig. 3. The mean number of new xylem elements
regenerating in internodes cultured in 4% sucrose plus 2.5 ppm
2,4-D under varying conditions of exposure to ozone. Vertical
bars (inserts) represent standard error.
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60
Z
o
D
O
ui
at
30
-30
OZONE
EXPOSURES
0 1 WEEK
• TT WEEK
2"° WEEK
A 2 WEEKS
SUCROSE SUCROSE • IAA SUCROSE'2,4-D
Fig. 4. Percent of reduction in the regeneration of xylem
elements for each exposure period. Intemodes were cultured in
sucrose, sucrose plus IAA, or sucrose plus 2,4-D. Negative
values represent increases.
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the second week as in condition D, there was no reduction in regeneration. There was
actually a 26% increase in regeneration. Under these same conditions (D) if they were
grown in the sucrose plus IAA medium, regeneration was reduced by 30 percent.
Suppression of regeneration was not as significant in segments which were cultured in
the medium containing sucrose and 2, 4-D regardless of the timing of the ozone exposure
(Fig. 4). For example, in those segments treated under exposure condition B, regenera-
tion was reduced by 1.5 percent. There was a 7% increase in regeneration in segments
receiving treatment C and a 17% decrease in those receiving treatment D (exposure to
ozone was during the second week of growth). When the segments received treatment E
(exposure was for two weeks) regeneration was reduced by 21 percent. There was little
difference in the number of xylem elements regenerated in segments in culture for 1 or
2 weeks in the absence of ozone regardless of the composition of the media.
Growth and development in plants is dependent upon cell division, cell enlargement, and
cytodifferentiation. A delay or alteration in either of these processes will ultimately
change the structure and physiology of the plant. Studies have shown that ozone will
indeed influence cell enlargement, Ordin (1962). Information is scant in regards to its
effect in cytodifferentiation or tissue differentiation. Fully and properly differentiated
cells and tissues in plants maximize their effectiveness in competing with the environ-
ment and functioning efficiently.
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Specifically, the vascular tissues, xylem and phloem, are the paramount conductors
of nutrients and water in plants. Reduction in the amount of these tissues could re-
duce the capacity of the plant to produce fruit, wood and other products.
There are many reports of the killing effect of ozone on plant life, Heggestad and
Middleton (1959), Ledbetter, et al. (1960), Hill, et al. (1961) among others. The
issue in this research, however, raises a more insiduous question. What are the
effects of ozone on plant life when from all appearances it is surviving and healthy ?
The work in this report suggests that possibly some damage is done to the biochemical
systems for xylem differentiation. The ultimate test for this will have to come in the
whole intact plant.
The effect of ozone on the processes of xylem regeneration and the suppression of this
affect by 2, 4-D is possibly related to chemical and physical changes in the plant. These
changes involve membrane permeability changes caused by ozone and IAA, the transport
of auxins and sucrose, cell division, processes of differentiation, and the affect of the
polarity of the auxins involved.
OZONE ENHANCEMENT OF MEMBRANE PERMEABILITY
Membrane permeability changes caused by ozone can affect the rate of regeneration.
Evans and Ting (1973) observed increases in the permeability of water and solutes in
leaf tissues exposed to ozone. Such enhanced permeability could account for the uptake
of ozone which would increase the peroxidation of lipids in membranes and decrease
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fatty acid synthesis, Tomlinson and Rich (1969, 1971). This decrease in lipid bio-
synthesis resulted from the relatively easy oxidation of sulfhydryl groups by ozone,
Tomlinson and Rich (1967, 1968, 1969, I970a, 1970b, 1971) and Treashow et al.
(1969). The extent to which ozone caused such changes in the membrane of parenchy-
ma cells of Coleus was probably minimal unless the gas was absorbed along with com-
ponents of the media. Whatever changes in the membranes did occur probably affected
the rate of diffusion of auxin as well as sucrose resulting in reduced regeneration. The
diffusion of 2,4-D and sucrose was apparently not affected by changes in permeability
since regeneration was only slightly reduced in cultures containing these substances.
REDUCTION OF CELL DIVISION BY OZONE
Another manner in which ozone influences regeneration is by interrupting the processes
of cell division which leads to a reduction in the number of cells ordinarily undergoing
division. The manner in which ozone could have interrupted these processes is by
oxidizing nucleic acids and proteins. Davis (1959) demonstrated the sensitivity of the
bases of nucleic acids to ozone. He found that thymidine was more sensitive to ozone
than cytosine or uracil. This shows that ozone also penetrates nuclear membranes.
Oxidations of nucleic acids would limit the synthesis of proteins but the protein mole-
cules can also be oxidized. In fact, increases in free pool amino acids of leaves ex-
posed to ozone suggested that such oxidations readily occur, Tomlinson and Rich
(I967a), Lee (1968), Ting and Mukerji (1971), and Craker and Starbuck (1972).
Additionally, Mudd et al. (1969) demonstrated the sensitivity of amino acids to ozone,
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cysteine being more sensitive than others. This amino acid accumulates in excess in
the walls of parenchyma cells undergoing meristematic activity and in the walls of new
xylem elements, Rier and Beslow (1967). Its oxidation by ozone could greatly limit
differentiation and reduce the regeneration of new xylem. Since cell division is a
necessary prerequisite of differentiation, Fosket (1968), reduction in the amount of
xylem in Cole us internodes may not necessarily have been caused by extensive oxida-
tion of cysteine but such oxidations could greatly limit the number of parenchyma cells
which differentiate directly into new wound vessel members. The lack of pronounced
reduction in segments cultured in 2,4-D plus sucrose is probably due to the fact that
the initiation of the regenerative process by 2,4-D was too rapid and had proceeded to
a level not readily affected by ozone by the time of exposure. Sucrose and 2,4-D had
previously negated the effect of smog, which contained ozone and hexene, in reducing
growth of coleoptile segments. This shows that at least the processes of cell division
and elongation was not affected by ozone, Koritz and Went (1953).
OXIDATION OF LIGNIN FORMING ENZYMES BY OZONE
Ozone can interfere with the processes of lignin formation, a process which is depen-
dent upon auxins in the differentiation of new xylem. For instance, the auxin activated
peroxidase enzyme, which stimulates lignin formation has been shown to be inactivated
by ozone. It has been successfully inactivated in vitro, Todd (1958) but not in vivo,
Dass (1972). In Dass's study, electrophoretic peroxidase actually increased, by one
band, in bean leaves exposed to ozone. Another enzyme, one involved in cell wall
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synthesis, phosphoglucomutase, is also inactivated in vitro but not in vivo, Ordin
(1965), by ozone. These unsuccessful in vivo oxidations show that for ozone to be
greatly effective in reducing lignin formation, it would have to penetrate the cells.
These authors did not observe such penetration. Extensive reduction in the number
of lignified elements was not observed in Cole us nor in maple seedlings treated with
2 to 3 ppb ozone when tested for changes in lignin content following exposure, Hibben
(1969)0
OZONE AFFECT ON THE INCOBPOEATION OF SUCROSE INTO CELLULOSE
Ordin and Skoe (1964) showed that ozone caused increased uptake of labelled glucose
by isolated coleoptile segments but incorporation of the label into cell wall components
was considerably reduced. They attributed this reduction to the sensitivity of glucose
pathways leading to cell wall formation to ozone. Glucose pathways leading to cellulose
formation were also found to be sensitive to ozone. This was indicated by pronounced
inhibition of cellulose synthesis. In this manner, ozone reduced these normally rapidly
elongating segments. Ozone could have similarly inhibited the synthesis of cellulose
in cells elongating in the wound area of Coleus intemodes. Such inhibition could prevent
differentiation in a number of cells resulting in reduced regeneration. Reduction in the
number of elements in segments grown in sucrose show that ozone could have affected
the utilization of sucrose in a manner similar to the change in the utilization of the
labelled glucose. In addition to reductions caused by sucrose alone, reduction in cellu-
lose and other processes of xylem regeneration could also result from changes in the
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utilization of IAA.
OZONE INACTIVATION OF IAA
The most probable factor responsible for reduction of xylem regeneration in Coleus
segments was inactivation of the IAA moleculeo Ordin and Propst (1962) observed
complete inactivation of IAA within one hour of ozone treatment and found that the
inactivation was caused by breaks in the indole ring part of the molecule, Inactivation
of IAA by ozone is more responsible than permeability changes in reducing regenera-
tion because many of the processes involved in regeneration are initiated by auxin.
Reduction in the amount of IAA, due to inactivation by ozone would subsequently reduce
the activity of these processes, cell division, lignin and cellulose formation,, Since an
hour was required for complete inactivation of the molecule, many of the remaining
active molecules probably acted with sucrose to stimulate regeneration regardless of
ozone treatmento If this were not so, the amount of regeneration in internodes grown
in the presence of sucrose, IAA, and ozone would be comparable to that in sucrose
alone Such similarity was never observed. Reduction in regeneration was more
pronounced in segments grown in sucrose and IAA than in any other media because
ozone could have affected the utilization of both materials. Minimal reductions in
regeneration in segments grown in media containing 2, 4-D and sucrose indicate that
2,4-D was not inactivated .by the ozone.
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REDUCTIONS IN REGENERATION CAUSED BY THE RATE OF DIFFUSION OF IAA
OR 2,4-D
The protective effects of 2,4-D and sucrose together against reductions in xylem
regeneration can also be due to the increased rate of absorption and translocation of
2,4-D over IAA. Hill (1965) showed how the difference between rates of translocation
of the two auxins affected the production of callus on isolated potato stems. Low con-
centrations of 2,4-D, Oo 5 ppm or less, produced callus only at the top of the stem
away from the medium,, Higher concentrations induced callus over the entire stem,,
On the other hand, IAA, regardless of concentration, produced callus only on the end
of the stem closest to the medium whether the stem was in the normal upright or in-
verted positiono This shows that IAA moved only in its established acropetal manner,
a very slow movement, and that 2,4-D moved in a very rapid acropetal fashion. This
type of polarity was expressed in Cole us internodes. That 2, 4-D was translocated
more extensively into the apical region than IAA was noted by the appearance of newly
differentiated xylem close to the apex of the stem. New xylem did not regenerate to
such a height in internodes cultured in IAA plus sucrose. Rapid translocation of the
auxin provided a means of establishing the processes of xylem regeneration prior to
exposure to ozone. Slow diffusion of IAA allows more of it to become inactivated by
ozone which decreases the amount of xylem regeneration,,
There is an apparent interaction between ozone, IAA, sucrose, and 2,4-D. These
interactions, though not fully understood, can modify, quantitatively, the amount of
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xylem regeneration around wounded Cole us internodes. The rates of translocation of
auxins and sucrose may be significant factors influencing the amount of xylem regen-
eration in wounded internodes and the inactivation if IAA may have been the major
factor reducing xylem regeneration in this study.
PROTEIN PATTERNS IN OZONATED CALLUS TISSUES
Preliminary findings suggests that bands for proteins and peroxidase from callus tissue
can be visualized in acrylamide gel electrophoresis. This is the first report of its kind
on electrophoresis of ozonated callus tissue. The present assessment of the banding
pattern and number of bands suggests that variation exists as a function of media com-
position and ozone exposure. A tendency for variation exists in the pattern and number
of bands in cultures containing IAA rather than in those containing 2,4-D. Given the
findings of several workers on studies made to correlate protein composition and
enzymic patterns with growth and development, research would be fruitful in regards
to ozone and its effects on growth and development. A callus tissue system is perhaps
a good one for studying the effects of ozone on differentiation.
23
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SECTION VI
REFERENCES
Beslow, D. T, and J, P, Rier. Sucrose Concentration and Xylem Regeneration in
Coleus Internodes in vitro. Plant and Cell Physiol. l£:69-77, 1969.
Caponetti, James D., Personal Communication. Department of Botany, University
of Tennessee, Knoxville, Tennessee, 1973.
Craker, L, E. and T. S. Starbuck. Metabolic Changes Associated with Ozone Injury
to Bean Leaves. Can. J. Plant Sci. 52^:589-597, 1972.
Dass, H. C. Enzymatic Changes in Intact Leaves of Phaseolus vulgaris Following
Ozone Fumigation. Atmos. Environ, j5:759-763, 1972.
Davis, I. The Survival and Mutability of Escherichia coli in Aqueous Solutions of
Ozone. Ph.D. Thesis, University of Pennsylvania Medical School,
Philadelphia. 1959.
Evans, L. S. and I. P. Ting. Ozone Induced Membrane Permeability Changes. Amer,
J. Bot. 60:155-162, 1973.
Fosket, D. E. Cell Division and the Differentiation of Wound Vessel Members in
Cultured Stem Segments of Coleus. Proceedings of the National Academy of
Sciences, £9:1081-1096, 1968.
Frenkel, C. and C.E. Hess. Isozymic Changes in Relation to Root Initiation in Mung
Bean. Can. J. Bot. 52_:295-297, 1974.
Fuchs, C. Fuchsin Staining with NaOH Clearing for Lignified Elements of Whole
Plants or Plant Organs. Stain Technol. 38:141-144, 1963.
Heggestad, H. E. and J. T, Middleton. Ozone in High Concentration as a Cause of
Tobacco Leaf Injury. Sci. IS2.:208-210, 1959.
Hibben, G.R. Sugar Maple - Ozone Toxicity. Phytopath. £9:1423-1427, 1969.
Hill, R.A. Polarity Studies in Roots of Ipomea batatas in vitro. Master's Thesis,
Howard University, Washington, D. C. 1965.
24
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Jacobs, W. P. The Role of Auxin in the Differentiation of Xylem Around a Wound.
Amer. J. Bot. 39:301-309, 1952.
Jacobs, W. P. Acropetal Auxin Transport and Xylem Regeneration - A Quantiative
Study. Amer. Nat. 88^327-337, 1954.
Juo, P. and G. Stotzky. Changes in Protein Spectra of Bean Seed During Germination.
Can. J. Bot. 48:1347-1350, 1970.
Koritz, H.G. and F. W. Went, The Physiological Action of Smog on Plants. I.
Initial Growth and Transpiration Studies. Plant Physiol. 2_8:50-62, 1953.
Lamotte, C. and W. P. Jacobs. A Role of Auxin in Phloem Regeneration in Cole us
Internodes. Develop. Biol. _8:80-98, 1963.
Lee, T. T. Effect of Ozone on Swelling of Tobacco Mitochondria. Plant Physiol.
43:133-139, 1968.
Leshem, Y. , A.W. Galston, R. Kaur-Sawhney, and L. M. Shih. Auxin Macromole-
cular Repressers and the Development of Isoperoxidases in Cultured Tobacco
Pith. In: Plant Growth Substances 1970, Carr, D.J. (ed.). New York,
Springer-Verlag Publishers, 1972. pp. 228-233.
Mudd, J. B., R. Leavitt, A. Ongun, and T.T. McManus. Reactions of Ozone with
Amino Acids and Proteins. Atmos. Environ. J.-699-682, 1969.
Ordin, L. and A. Altman. Inhibition of Phosphoglucomutase Activity in Oat Coleop-
tiles by Air Pollutants. Physiol. Planatarum. l_8:790-797, 1965.
Ordin, L. and B. Propst. Effect of Airborne Oxidants on Biological Activity of
Indoleacetic Acid. Bot. Gaz. 123:170-175, 1962.
Ordin, L. and B. P. Skoe. Ozone Effects on Cell Wall Metabolism of Avena Coleoptile
Sections. Plant Physiol. 39:751-753,1964.
35
Rier, J. P. and D. T. Beslow. The Incorporation of Cysteine S in Callus Tissue of
Parthenocissus tricuspidata. Plant and Cell Physiol. 8:799-781,1967.
Rier, J. P. and D. T. Beslow. Sucrose Concentration and the Differentiation of Xylem
in Callus. Bot. Gaz. 128:73-77, 1967.
25
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Ting, I. P. and S. K. Mukerji. Leaf Ontogeny as a Factor in Susceptibility to Ozone:
Amino Acid and Carbohydrate Changes During Expansion. Amer. J. Bot.
5_8:497-504, 1971.
Todd, G. W. Effects of Low Concentrations of Ozone on the Enzymes Catalase,
Peroxidase, Papain, and Urease. Physiol. Planturum. L1.-457-463, 1958.
Tomlinson, H. and S. Rich. A Comparison of the Effects of Ozone and Sulfhydryl
Reagents on Plants. Phytopathol. 57:834. (Abstr.) 1967.
Tomlinson, H. and S. Rich. The Ozone Resistance of Leaves as Related to their
Sulfhydryl and Adenosine Triphosphate Content. Phytopathol. 5_8:808-810,
1968.
Tomlinson, H. and S. Rich. Relating Lipid Content and Fatty Acid Synthesis to Ozone
Injury of Tobacco Leaves. Phytopathol. J59 .-1284-1287, 1969.
Tomlinson, H. and S. Rich. Bisulfides in Bean Leaves Exposed to Ozone. Phytopathol.
Notes. 60:1842-1843, 1970.
Tomlinson, H. and S. Rich. Lipid Peroxidation, a Result of Injury in Bean Leaves
Exposed to Ozone. Phytopathol. 6£:1531-l532, 1970.
Tomlinson, H. and S. Rich. Effects of Ozone on Sterols and Sterol Derivatives in
Bean Leaves. Phytopathol. 61.:1404-1405, 1971.
Wetmore, R.H. and J. P. Rier. Experimental Induction of Vascular Tissues in Callus
of Angiosperms. Amer. J. Bot. £0:418-430, 1963.
Yoneda, Y. and T. Endo. Effects of Low Concentration of Hydrogen Peroxide on
Indoleacetic Oxidase Zymogram in Pharbitis nil. Plant and Cell Physiol. 10;
235-237, 1969.
26
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-068
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Ozone and Vascular Tissue Differentiation
in Plants
5. REPORT DATE
May 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
John P. Rier, Jr.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Botany
Howard University
Washington, D.C. 20059
10. PROGRAM ELEMENT NO.
1A1006/1HA323
11. CONTRACT/GRANT NO.
R-801209
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
U. S. Environmental Protection Agency
Office of Research and Development
Office of Monitoring and Technical Support
Washington. D.C. 20460
in/77 - S/74
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This study is concerned with the influence of ozone on the process of vascular
tissue differentiation in plants and the concomitant changes in plant proteins.
The test materials consisted of wounded plant intermodes and callus tissues
grown, exposed, and studied under controlled laboratory conditions. Ozone was
more effective in reducing xylem regeneration in those internodes grown with
indole-3-acetic acid than with 2, 4-dichlorophenoxyacetic acid. From the results,
it was concluded that plant internodes and callus tissues can be used to study the
effects of ozoen on certain processes related to plant growth and development.
This report submitted in fulfillment of Grant Number R801209
by the Environmental Protection Agency. Work was completed as of May 1974.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
Plant Anatomy
Plant Chemistry
Ozone
Plant Physiology
Acetic Acid
Regeneration (Physiology
Proteins
Enzymes
Indole-3-acetic acid
2, 4-Dichlorophenoxyaceti
Plant Morphology
06F
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