Plant Physiol. (1978) 61, 80-84
Chlorophyll Fluorescence Assay for Ozone Injury in Intact
Plants'
Received for publication June 29, 1977 and in revised form September 27, 1977
ULRICH SCHREIBER
Department of Plant Biology, Carnegie Institution of Washington, Stanford, California 94305
WILLIAM VIDAVER
Department of Biological Sciences, Simon Fraser University, Burnaby, B. C., Canada VSA 1S6
VICTOR C. RUNECKLES AND PETER ROSEN
Department of Plant Science, University of British Columbia, Vancouver, B. C. Canada V6T I W5
MATERIALS AND METHODS
Bush bean (Phaseolus vulgaris L. cv. Pure Gold Wax) was
planted four seeds/pot, 2 cm deep, in soil. They were kept in
the greenhouse at 20/15 C, on an 11-hr photoperiod. Relative
humidity varied between 55 and 80%. Pots were watered to
capacity on alternate days. Ozone was applied either to the
whole plant in fumigation chambers or to portions of single
leaves using a gas exchanger attached to the fluorometer (18).
Ozone for the chambers was generated by passing a stream of
air through a corona discharge tube. For single leaf fumigation
a Triton Aquatics (model S-11) ozonizer was used. Ozone
concentration was monitored with a Mast (model 724) ozone
meter. No nitrogen dioxide was detected applying the method
of Saltzman (16).
Chl fluorescence was excited by a broad blue band isolated
from a movie projector lamp (Sylvania, type DL6) with a 10mm Corning 4-96 filter. Light intensity was 0.5 mw/cm2 sec if
not stated otherwise. Illumination and collection of fluorescence
from the leaf surface were achieved with bifurcated fiberoptics
previously described (19). Fluorescence at X > 660 nm was
detected by a photomultiplier (EMI 9658B) protected by a 3mm Corning 2-64 filter. The experiments of Figures 1 and 2
were carried out with a recently developed portable fluorometer
(18). Fast fluorescence transients were recorded on a storage
oscilloscope (Tektroniz 5103N), and slow transients on a strip
chart recorder (Metrohm, Hirisau, Switzerland). If not otherwise stated, samples were dark-adapted for 1 to 2 hr. All
experiments were carried out at room temperature (22-26 C).
Light intensity between 400 and 750 nm was 3.4 mw/cm2
during fumigation in the chambers.
Injury was assessed by visual rating of percentage leaf area
necrosis (% LAN) (15) and determination of leaf dry wt to
fresh wt ratio. Measurements, if not stated otherwise, were on
fully expanded primary leaves, between 17 and 20 days after
planting.
ABSTRACT
A chlorophyll fluorescence induction (Kautsky effect) asy predicted
ozone-induced injuy in bean leaves (Phaseolus vulgaris) at least 20
hous before any visible sign of leaf necrosis. The extent of injury,
which could be predicted during exposure to ozone, depended on
concentration, exposure time, and leaf development stage. Much more
injuy occurred In lght than in darkness and long exposures to lower
ozone concentrations were more injurious than brief exposres to
higher ones. The fist detectable effect was on the photosynthetic
water-splttng enzyme systems, folowed by inhibition of electron tansport between the photosystems. The fluorescence amy provides a
simple, rapid, nondestructive method for observing effects of ozone on
plants.
Ozone is a serious and widespread air pollutant, causing
among other physiological changes a repression of photosynthetic activity in plants (7, 10, 14). Ozone effects within the
photosynthetic apparatus are variously attributed to damage of
the CO2 fixation sites (22), damage of the Chl pigment system
(4), and, according to Chimiklis and Heath (3) in studies with
Chlorella, membrane damage causing leakage and subsequent
ionic imbalance.
Chl fluorescence is a sensitive indicator of photosynthetic
energy conversion (for a review see ref. 13). Partial reactions of
photosynthesis are reflected in parts of the complex fluorescence
induction curves displayed upon a dark-light transition. Changes
of the fluorescence induction pattern in an aqueous "model
system" with ozone treatment have been reported for Chlorella
(2).
The present report describes ozone effects on Chi fluorescence
induction in intact bean leaves and demonstrates the applicability of the fluorescence assay for ozone. This approach became
possible with the development of methods to measure fluorescence induction from the illuminated surface of dense leaf
material (18, 20). The results suggest that the photosynthetic
water-splitting enzyme system may be the primary site of ozone
damage. Marked dependence of injury on the light conditions
under which ozone is applied and on the developmental state of
the treated plants is shown.
I
Supported in part by National Research Council of Canada operating grants to W. V. and V. C. R.
RESULTS AND DISCUSSION
When a dark-adapted leaf is suddenly exposed to strong
continuous light the Chl fluorescence yield shows characteristic
changes in the time region from msec to min (8). For a healthy
plant under given conditions the fluorescence time course has a
characteristic pattern, which is altered by any change in the
photosynthetic apparatus. Figure 1 compares fast and slow
fluorescence transients in leaves of a control plant and of plants
20 hr after treatment for 6 hr with 0.3 or 0.5 ,ul/l ozone.
Although there was some plant to plant variability, the effect of
80
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Copyright © 1978 American Society of Plant Biologists. All rights reserved.
Plant
Physiol. Vol. 61,
1978
81
FLUORESCENCE ASSAY FOR OZONE INJURY
each treatment on at least 20 leaves was assayed. The curves higher doses of ozone, because with lower ozone concentrations
depicted represent typical responses. Fluorescence generally (e.g. 0.125 ul/l for 2 hr) we observed a remarkable reversal of
rises rapidly from an initial level (0) to an intermediate level the ozone effect within the first few hr after treatment, as
(I), then somewhat more slowly to a peak (P), from where it shown in Figure 3. While after 1-hr dark recovery the typical
decays to a relatively stationary yield (S). On a much slower suppression of P and a slow M-T decay indicate injury, after 4
time scale fluorescence rises again to a maximum (M) from hr the fluorescence characteristics appear close to normal. Thus,
where it decays to a terminal level (T). Ozone treatment the fluorescence assay reveals incipient ozone injury levels.
In Figure 4 data are presented which suggest that ozone
changes this pattern substantially. The part of the curve which
is most affected is the I-P-S transient, which in this experiment injury does not follow a simple dose response. Equal doses
were applied over varying times, with the greater injury indiwas virtually abolished by treatment with 0.3 ,ul/l ozone. The
rate of the M-T decay is also affected by ozone, particularly at cated at the lower concentrations applied over the longer
0.5 ,lI/l where the T level is substantially raised. These changes periods. This is in agreement with the observation of Heck et
can be generally interpreted as reflecting inhibition of partial al. (6) of an initial sigmoid phase in a plot of injury versus
photosynthetic reactions. Their significance is discussed below. exposure time. This behavior makes very short exposures to
In Table I the values of the fluorescence characteristics, P level
and M-T slope, are compared for each plant with injury assessed
b
a
visually (% LAN), and the dry wt to fresh wt ratio. There is
good agreement between damage estimated by the fluorescence
5assay and the visual assays. Not only can the extent of injury be
assayed from the fluorescence curves, but as discussed below,
the injury sites within the photosynthetic apparatus can be
I'
identified. In addition, use of the portable fluorometer allows
3immediate in situ assay of ozone injury. Important advantages
of the fluorescence method are thus its immediacy and nondestructiveness.
In the experiment of Figure 1 there was a 20-hr interval
between ozone treatment and the fluorescence assay to allow
direct comparison with visible injury. However, fluorescence
characteristics are significantly affected immediately on ozone
treatment, approximately 20 hr before any injury can be detected visually (Fig. 2). The changes observed in fluorescence
TIME
immediately after ozone treatment at 0.30 ul/l are essentially
FIG. 2. Ozone effect on fast (b) and slow (a) fluorescence transients
the same as those seen after 20 hr. This may be true only for assayed shortly after fumigation. Ozone concentration: 0.3 ,l/l applied
4
z
w
0
for 6 hr. Curves recorded after 30 min of darkness following fumigation
):
in the light. Portable fluorometer; conditions as in Figure 1. (
control; --- ): treated sample.
TIME min
2
2
0
P
4
control
M
0l3
05
Itl/l
pJ/
5-
~ ~~bb
3-
ui
0
.4-
Ccnu0w
rD
~~~~~~~~~~~~~~~~b
Li
2-
-i
aaa
JL
01
.
03
4
0
0
02
0
04
0
.
02
0.2
0.3
0.4
0
0.1
0.2
0.3
0.4
0
0.1
0.2
.0T
light on
0
0.1
0
0.4
0.3
z~~~
~
3i
TIME, sec
FIG. 1. Effect of ozone treatment on fast (b) curves and slow (a)
Chl fluorescence induction. At time zero continuous illumination
is started after 30 min of darkness. Notation for the characteristic
fluorescence levels: 0: initial fluorescence; I: intermediate level (not
visible on slow traces); P: peak; S: quasistationary level; M: second
maximum; T: terminal level. Curves recorded 20 hr after fumigation,
with the portable fluorometer (see ref. 18). Light intensity, 104 erg/
cm2 *sec. Ozone was applied for 6 hr.
o-1
curves
0
mi-
TIME
FIG. 3. Reversal of ozone effect on fluorescence induction after
treatment with subacute dose. a: control; b: plant exposed for 6 hr to
0.125 ,ul/l ozone, 1 hr dark recovery time; c: as (b) but 4 hr recovery
time.
Table I
Visible
ozone
injury compared to changes in chlorophyll fluorescence induction
Ozone Concentration
Assays for
injury a
1D
I-P, amplitudec
M-T, maxlmumC
slope
LANZ
DW/FW
x
aconditions
breplicate
101
as in
100
100
99
88
107
106
0
0
0
0
102
98
104
0.54l/1
0.33pl/l
Mean
4
98
97
103
Control
3
2
1
2
3
4
Mean
1
2
3
4
24
58
72
43
49
26
61
30
144
43
85
304
42
100
59
64
46
45
54
100
0
100
76
30
151
73
45
136
63
45
154
53
66
45
60
239
170
36
80 75
206 201
Fig. 1
plants
relative units, normalized at 100 for
mean
of control
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Copyright © 1978 American Society of Plant Biologists. All rights reserved.
Mean
67
214
82
Plant Physiol. Vol. 61, 1978
SCHREIBER ET AL.
high ozone concentrations less effective than the calculated
dose would suggest.
In the experiments reported above ozone was applied in the
light. It is known that ozone uptake is consistently higher in
bright light, when stomates are open (7). To achieve injury
comparable to that in Figures 1 to 4 by fumigation in the dark,
a)6
at least 10 times higher concentrations had to be applied. The
Li
dark sample showed only a small effect compared to the control
z 5while in the light sample there was almost a complete loss of
w
photosynthetic activity, in spite of the lack of visible injury
OD 4 M -T \ *
,
o
symptoms at the time at which the fluorescence measurements
0
160
3
,
0.
were taken (data not shown).
LL
In order to investigate the gradual change of the fluorescence
2induction change with increasing exposure to ozone, we devised
an experiment in which fluorescence of the same leaf section
1
was measured after different times of fumigation. A sample,
C
enclosed in a gas exchange adaptor (18), was first illuminated
10
3'0 45 6'0 75 90 105 120
in air with 1-min light/15-min dark cycles until fluorescence
TIME, min
induction was reproducible. Then ozone was added to the air
FIG. 6. Changes in amplitude of fluorescence transients during fustream and the change of fluorescence behavior recorded in
subsequent 1-min illumination periods every 15 min (Fig. 5). In migation. Amplitudes determined from curves in Figure 6.
Figure 6, the amplitudes of the major fluorescence characteristics (0, 0-I, I-P, P-S, M-T; see Fig. 1) are plotted versus time
of exposure to 2.5 ,ul/1 ozone.
Figure 7 shows a current scheme representing the primary
variable
photosynthetic reactions and the mechanisms of fluorescence
fluorescence
quenching. Our interpretation of the data of Figures 5 and 6 in
accordance with the scheme is as follows:
The 0 level is practically constant throughout the first 60 min
5-
(D
a
b
c
d
Q-quenching
ADP Pi
4-4
Calvin
cycle
Li
0
U,
X-1 min
TIME
FIG. 4. Exposure time-concentration relationship. Replicate plants
were exposed to the same dose of ozone applied as (b) 0.125 ,ul/l for 2
hr; (c) 0.25 1.l/l for 1 hr; (d) 0.5 ,ul/l for 30 min; (a): control. Recorded
1 hr after fumigation.
FIG. 7. Current scheme of primary photosynthetic reactions and
mechanisms of fluorescence quenching. Absorbed quanta are funneled
into reaction centers, where light energy is converted into chemical
energy. The probability of loss of energy as fluorescence increases when
reaction centers are blocked, primarily when the PSII primary electron
acceptor Q is in the reduced state. The reduction of Q depends both on
the efficiency of electron donation from H20 and the efficiency of
electron transport via PSI to NADP. Independently of the redox state
of Q, fluorescence is also quenched by the formation of the "high
energy state" during photophosphorylation.
and then declines slightly. This indicates that ozone does not
directly affect PSII reaction centers. The slight decline in 0
level at longer exposures may reflect Chl destruction.
The 0-I rise is only weakly affected. For exposures longer
0
than
60 min the rise rate is slowed (Fig. 5), but leads to a
5
15
Fluorescence recordedin1-min light/15-mindarkcycles.Afterr30 higher I level. This again argues for only a marginal effect of
ozone on the primary reaction of PSII. The slower rate at
longer exposures also suggests the injury expressed by the lower
60
curve (0 min ozone)wasreached.75 O level, indicating a decrease in absorbed light energy. The
cycles in air a constant control
LU
~~~~~~~~~~~~~~~~~~~~90
increase in I, in view of the fact that 0 and P are low at the
~~~~~~~~~~~~~~~~~~~~~105
0
D
min
same time, reflects
a partial loss of electron transport capacity
~~~~~~~~~~~~~~~~~~~~~~~12o
ozone
between Q2 and PSI.
-1secThe I-P rise is markedly suppressed by ozone treatment. As
0 15 30 45 60 75 90 105 120 min
the major decay in the P level occurs between 15 and 60 min,
~~~~~~~~~~ozone
ol
when 0-I is practically unaffected, decreased quantum absorption by PSII appears unlikely. This is at variance with a
TIME
FIG. 5. Change of fluorescence induction during ozone treatment. hypothesis advanced by Coulson and Heath (4) that the primary
Fluorescence recorded in 1-mmn light/15-mmn dark cycles. After four effect of ozone in the chloroplast is to disrupt the normal
cycles in air a constant control curve (0 min ozone) was reached. pathway of energy flow from excited Chl into the photochemical
Ozone, 2.5 pilll, was applied to an approximately 1-cm2 section of a
single primary leaf with use of a fluorometer gas exchange adaptor
2 Abbreviations:
(18). Plant age: 12 days after seeding. For clarity the right hand curves
Q: primary electron acceptor of photosystem II;
are displaced upward with approximately
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equalfrom
intervals
between
them.- Published
PQ: plastoquinone.
Copyright © 1978 American Society of Plant Biologists. All rights reserved.
Plant
Physiol. Vol. 61, 1978
83
FLUORESCENCE ASSAY FOR OZONE INJURY
events by a disruption of the components of the membrane.
While this kind of damage can be ruled out by our data as being
the primary effect of ozone (15- to 16-min exposure), it may
well be responsible for the effects of longer exposure. The
suppression of the I-P rise indicates the gradual loss of H20splitting activity. With decreasing rates of H20-splitting, electron pressure from the PSII donor side becomes insufficient to
reach a transient high reduction level of Q during the induction.
This is a general phenomenon observed with a number of other
treatments which block H20 -splitting, such as heat treatment
(17), prolonged anaerobiosis (20), tris treatment (1), and hydrostatic pressure (19).
The P-S decline is suppressed concomitantly with the I-P rise.
In separate experiments (not shown in the figures) we observed
that the half-decay time was slightly shortened by ozone treatment during the first 60 min. Thus, the reduction of the P-S
transient is not due to inhibition of the reaction which initiates
the decay but simply to the fact that it is preceded by a smaller
I-P rise. The P-S decline presumably reflects reoxidation of Q
with the Calvin cycle as the terminal electron acceptor. Accordingly, the sequence of electron transport from PSII through PSI
and NADP to CO2 appears unaffected by ozone at a time when
H20-splitting is already severely curtailed.
The S-M rise increases with ozone treatment. It is difficult to
decide from Figure 6 whether this is mainly caused by the
disappearance of the I-P-S transient or by a true stimulation of
S-M. Other data, not shown here, have convinced us that M is
indeed stimulated by ozone treatment. The significance of M is
not fully understood but it has been shown that uncoupling of
photophosphorylation leads to stimulation of M (11). The
stimulation of M by ozone may correspond to a dissipation of
the high energy state associated with the photophosphorylation
(9, 12, 23). This would occur parallel to the block of H20splitting. At present it is difficult to decide whether all of the
ozone effect is due to a block of H20-splitting and the concomitant electron transport to which photophosphorylation is coupled or whether there is also some uncoupling of photophosphorylation. Both inhibition of H20-splitting and uncoupling
could be caused by the same damage to the thylakoid mem-
In the course of this study we encountered occasional variabilozone susceptibility from leaf to leaf and day to day,
which resulted from differences in the developmental stages of
the leaves under investigation. Detailed investigation of this
parameter was beyond the scope of the present study, but our
observations led to the conclusion that Phaseolus vulgaris, at
the given growth conditions, displays a distinct peak in susceptibility to ozone 12 days after seeding. Figure 9 shows a plot of
the maximum suppression of the P level observed during identical 2-hr ozone treatments of leaves of different ages. The
treatment was as in the experiment of Figure 6. Because only
small leaf sections were involved in this series no correlation
between injury suggested by the fluorescence assay and visible
injury after 24 hr was attempted. We observed that after 24 hr
the fumigated section of the primary leaf of a 12-day-old plant
was severely necrotic, while no necrosis developed in leaves of
plants 11 days old or less. Between 50 and 100% of the treated
leaf area of 12-day-old and older plants became necrotic.
The observation that ozone injury depends on leaf age is not
new. Other investigators have reported that 8-day-old bean
leaves do not develop visible injury, while 14- to 15-day-old
leaves show maximum sensitivity (5, 21).
ity in
CONCLUSIONS
Effects of ozone on the leaves of whole plants can be
determined using Chl fluorescence induction as an assay. Such
determinations show that:
(a) Fluorescence induction undergoes characteristic modifications on ozone treatment. (b) The extent of the changes is
correlated with the injury indicated by visual assays. (c) The
fluorescence assay is capable of detecting injury during ozone
exposure, approximately 20 hr before visible assessment is
possible. (d) The way in which fluorescence induction is affected
I
I
uLi
z 5
C')
41
I
brane.
0
3
The M-T decay is slowed down, with the T level increasing in
-j
zn
LL
two waves. The first wave occurs in the time region where the
the
and
loss of I-P indicates gradual inhibition of H20-splitting,
z
stimulation of M suggests loss in photophosphorylation. In this
0
to
be
T
case the slower M-T decay and elevated level appear
--l
caused by a decrease in energy-dependent quenching. The
U) 01
0 20 40 60 80 100 120 140
additional
to
an
second wave, on the other hand, may be related
TIME, min
decline in electron transport rate between the two photosystems,
FIG. 8. Change of T level (stationary) fluorescence yield
expowhich leads to a general increase in the Q reduction level and sure to 2.5 1l/l ozone. T level recorded continuously
mw/cm2 *
hence in fluorescence yield.
blue excitation light; 14 days after sowing.
Although our analysis of the changes is to some extent
speculative, there is no doubt that the gradual changes in the
50
50~~ ~~
fluorescence parameters accompany progressive ozone injury in
the plant. For example in the experiment of Figures 5 and 6,
81 40
_
we observed 80% LAN after 24 hr within the leaf region
exposed to ozone for 120 min. The greatest amount of fluorescence information about the development of ozone injury can
be obtained with the light-dark cycle method described above.
A simpler way is by continuously recording the T level during
fumigation. In addition to being less time-consuming and requircL 100
ing less sophisticated equipment, this has the advantage of
monitoring the ozone response with the plant in the lightadapted state. Figure 8 shows a continuous T level recording
10 11
16
for a primary leaf from a 14-day-old plant. If our above analysis
AGE, days
is correct, the first increase in T accompanies a decrease in the
depression
FIG. 9. Effect of leaf age on ozone damage expressed
over-all photophosphorylation rate, parallel to a decrease in of P level during 2-hr ozone treatment.
1-min
Figure
H20-splitting activity, and the second increase in T reflects light/15-min dark sequence during fumigation.
PSI and PSII. The final 6. Maximum depression of P is plotted per
amplitude
inhibition of electron transport between
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of Chl.
fumigation.All rights reserved.
decrease goes along with destruction
Copyright
© 1978 American Society of before
Plant Biologists.
2
-.
with
in 0.5
sec
30-
j20-
w
12
13
14
15
as
P level was measured
Conditions as
as
cent
of
P
in a
in
84
SCHREIBER ET AL.
suggests sites of ozone damage within the photosynthetic apparatus. (e) The fact that the P level is substantially suppressed
before any effect on 0-I occurs argues for initial damage to the
PSII donor site (H20-splitting enzyme system) prior to any
decrease in energy transfer efficiency within the pigment system.
(f) With increasing exposure to ozone, the electron transport
from PSII to PSI also becomes inhibited, as indicated by an
increased I level. (g) Exposure of whole bean leaves to ozone
only marginally affects the pigment system and PSII reaction
centers, as can be concluded from the relative insensitivity of
the 0 level to ozone. (h) Important parameters determining
the degree of change of fluorescence characteristics with ozone
treatment are ozone concentration, exposure time, recovery
time, light conditions, and leaf age. These fluorescence results
are in agreement with conclusions drawn from extensive studies
using other assays.
The fluorescence assay clearly provides an easy and rapid
way of studying ozone effects in whole plants. It is conveniently
applied both in the laboratory and in field experiments. Its
nondestructive nature and the simplicity of the method, together
with the relatively low cost instrumentation involved are important advantages of the fluorescence assay. The technique should
also be of value in the study of other stress and pollution
factors.
LITERATURE CITED
1. ARENZ H 1970 Chlorophyllfluoreszenz und photochemische Primarprozesse in Chloroplastensuspensionen. Doctoral thesis. Rheinisch Westfalische Techniche Hochschule, Aachen
Germany
2. CHIMIKLIS PE, RL HEATH 1975 Fluorescence transients of 03 gassed Chlorella. Plant
Physiol 56: S-23
3. CHIMIKLIS PE, RL HEATH 1975 Ozone-induced loss of intracellular potassium ion from
Chlorella sorokiniana. Plant Physiol 56: 723-727
4. COULSON C, RL HEATH 1974 Inhibition of the photosynthetic capacity of isolated
chloroplasts by ozone. Plant Physiol 53: 32-38
Plant Physiol. Vol. 61, 1978
5. FONG F, RL HEATH 1975 Phospholipid metabolism in ozone-treated leaves. Plant Physiol
56: S-5
6. HECK WW, JA DUNNING, IJ HINDAWI 1966 Ozone: nonlinear relation of ozone and
injury in plants. Science 151: 577-578
7. HILL AC, N LrrrLEFIELD 1969 Ozone. Effect on apparent photosynthesis, rate of
transpiration, and stomatal closure in plants. Environ Sci Technol 3: 52-56
8. KAUTSKY H, U FRANCK 1943 Chlorophyllfluoreszenz und Kohlensaure assimilation.
Biochem Z 315: 139-232
9. KRAUSE GH 1973 The high-energy state of the thylakoid system as indicated by chlorophyll
fluorescence and chloroplast shrinkage. Biochim Biophys Acta 292: 715-728
10. MACDOWALL FDH 1965 Stages of ozone damage to respiration of tobacco leaves. Can J
Bot 43: 419-427
11. MELCAREK PK 1974 Microfluorimetric investigations of the chlorophyll fluorescence
induction phenomenon in leaves. PhD thesis. Yale University
12. MURATA N, K SUGAHARA 1969 Light-induced decrease of chlorophyll a fluorescence
related to photophosphorylation system in spinach chloroplasts. Biochim Biophys Acta
189: 182-192
13. PAPAGEORGIOU G 1975 Chlorophyll fluorescence: an intrinsic probe of photosynthesis. In
Govindjee, ed, Bioenergetics of Photosynthesis. Academic Press, New York, pp 319-371
14. PELL EJ, E BRENNAN 1973 Changes in respiration, photosynthesis, adenosine 5'-triphosphate and total adenylate content of ozonated pinto bean foliage as they relate to
symptom expression. Plant Physiol 51: 378-381
15. RUNECKLES VC, PM ROSEN 1974 Effects of pretreatment with low ozone concentrations
on ozone injury to bean and mint. Can J Bot 52: 2607-2610
16. SALTZMAN BE 1954 Colorimetric microdetermination of nitrogen dioxide in the atmosphere. Anal Chem 26: 1949-1955
17. SCHREIBER U 1971 Der Einfluss des Sauerstoffs auf die Chlorophyll-fluoreszenzanderungen
in der lebenden Pflanze. Doctoral thesis. Rheinisch Westfalische Technische Hochschule,
Aachen Germany.
18. SCHREIBER U, L GROBERMAN, W VIDAVER 1975 Portable, solid-state fluorometer for the
measurement of chlorophyll fluorescence induction in plants. Rev Sci Instrum 46: 538542
19. SCHREIBER U, W VIDAVER 1973 Hydrostatic pressure: a reversible inhibitor of primarn
photosynthetic processes. Z Naturforsch 28c: 704-709
20. SCHREIBER U, W VIDAVER 1974 Chlorophyll fluorescence induction in anaerobic Scenedesmus obliquus. Biochim Biophys Acta 368: 97-112
21. TING IP, R SUTrON 1975 Repair of ozone induced alterations in membrane permeability.
Plant Physiol 56: S-5
22. WILKINSON TG, RL BARNES 1973 Effects of ozone on "CO2 fixation patterns in pine. Can
J Bot 51: 1573-1578
23. WRAIGHT CA, AR CRoFrs 1970 Energy-dependent quenching of chlorophyll a fluorescence
in isolated chloroplasts. Eur J Biochem 17: 319-327
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Copyright © 1978 American Society of Plant Biologists. All rights reserved.