Plant Physiol. (1988) 87, 638-646
0032-0889/88/87/0638/09/$Ol .00/0
Effects of Ozone and Peroxyacetyl Nitrate on Polar Lipids and
Fatty Acids in Leaves of Morning Glory and Kidney Bean
Received for publication May 15. 1987 and in revised form January 19, 1988
ISAMU NoUCHI* AND SUSUMU TOYAMA
Division of Agrometeorology, National Institute of Agro-Environmental Sciences, P. 0. Box 2,
Kannondai, Tsukuba-shi, Ibaraki 305, Japan (I.N); and Department of Biology, Faculty of Science,
Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112, Japan (S.T.)
ABSTRACT
To compare the effects of ozone and peroxyacetyl nitrate (PAN) on leaf
lipids, fatty acids and malondialdehyde (MDA), morning glory (Pharbitis
nil Choisy cv Scarlet O'Hara) and kidney bean (Phaseolus vulgaris L. cv
Gintebo) plants were exposed to either ozone (0.15 microliter per liter for
8 hours) or PAN (0.10 microliter per liter for up to 8 hours). Ozone
increased phospholipids in morning glory and decreased in kidney bean
at the initial stage (2-4 hours) of exposure, while it scarcely changed
glycolipids, the unsaturated fatty acids, and MDA in both plants. A large
reduction of glycolipids occurred 1 day after ozone exposure in both plants.
PAN caused marked drops in phospholipids and glycolipids in kidney
bean at relatively late stage (6-8 hours) of exposure, while it increased
phosphatidic acid and decreased the unsaturated fatty acids, an increase
which was accompanied by a large increase in MDA. These results suggest
that ozone may not directly oxidize unsaturated fatty acids at the initial
stage of exposure, but may alter polar lipid metabolism, particularly
phospholipids. On the other hand, PAN may abruptly and considerably
degrade phospholipids and glycolipids by peroxidation or hydrolysis at
the late stage of exposure. The present study shows that ozone and PAN
affect polar lipids in different manners.
Ozone and PAN' are the main phytotoxic components of photochemical air pollutants. In Japan, atmospheric oxidant (mostly
ozone) concentrations have often been recorded above 0.12 ,ul/
L, the standard value for issue of photochemical oxidant warning,
in urban areas (16). Atmospheric PAN concentrations averaged
0.001 to 0.002 ,ul/L or less, with occasional peaks of between
0.01 and 0.03 ,ul/L (17). Ozone and PAN cause visible injuries
to many susceptible plant species in Japan. Ozone causes bleached
spots, pigmentation stippling, and necrosis on the upper surface
of mature leaves, while PAN causes damage such as silvering,
metallic glazing, and bronzing on the lower surface of young
expanding leaves. The development of visible injury symptoms
results from biochemical and physiological changes. The initial
effects of oxidants are to alter the membrane permeability, allowing leakage of the cell contents into the extracellular spaces
and leading to an osmotic or ionic imbalance within the cell (7).
It has been considered that membrane lipids may be one of
the primary sites of ozone injury (7), since ozone in vitro rapidly
1 Abbreviations: PAN, peroxyacetyl nitrate; MGDG, monogalactosyl
diglyceride; DGDG, digalactosyl diglyceride; SQDG, sulfoqinovosyl diglyceride; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine;
PG, phosphatidyl glycerol; PI, phosphatidyl inositol; PA, phosphatidic
acid; MDA, malondialdehyde.
reacts with double bonds leading to the production of oxidation
products (7). Indeed, Pauls and Thompson (19, 20) have reported
that chemical and physical changes in membrane lipids such as
the breakdown of phospholipids, the loss of sterols from the
membrane, and the formation of gel phase lipid membrane were
induced by treating the isolated microsomal membrane from
bean leaves with ozone in solution. However, the results obtained
from isolated systems have not been confirmed in plant tissues.
PAN reacts with the sulfhydryl group to form either disulfide
or S-acetyl group and also reacts with olefine to form epoxides
(14). Such reactions could affect the proteins and the lipids of
membranes. Ozone not only destroys the double bond in unsaturated fatty acids, but also affects lipid biosynthesis (12, 13).
Lipid synthesis from acetate and acetyl-CoA (12) and the incorporation of galactose from UDP-galactose into galactolipids
(13) were inhibited by bubbling ozone through a chloroplast
suspension. PAN in vitro also inhibited the synthesis of fatty
acids from acetate (11). Thus, exposure to ozone and PAN seems
to alter the membrane lipids in leaf tissues.
The present study was initiated to compare the effects of ozone
and PAN on the leaf lipids, fatty acids, and MDA of two different
plants. Morning glory and 6 to 7 d old kidney bean plants were
exposed to either ozone (0.15 ,ul/L for 8 h) or PAN (0.10 ,ul/L
for 8 h) and then examined for changes in their fatty acids, polar
lipids, and MDA that occurred immediately after or 24 h after
the exposure periods.
Morning glory and kidney bean are plants susceptible to ozone,
and morning glory in particular has been used as an indicator
plant of photochemical oxidants (ozone) in Japan (16). Kidney
bean is also susceptble to PAN; for example, it was injured when
exposed to 0.030 to 0.045 ,ul PAN/L for 4 h (18). Therefore,
these two plants were selected as the testing materials. There
are several reports of the effects of ozone on lipids and fatty
acids in leaves exposed to very high concentrations such as 0.30
to 0.50 ,ul ozone/L (6, 23, 26, 28), but no reports of exposure to
low ozone concentrations. Therefore, 0.15 ,ul ozone/L was chosen in this study. On the other hand, there are no reports of
efects of PAN on the leaf lipids and fatty acids in leaves. Concentration of 0.10 ,ul PAN/L was chosen because it produced
injury on kidney bean from the moderate to the severe within 8
h exposure.
MATERIAL AND METHODS
Plant Materials. Two seeds of morning glory, Pharbitis nil
Choisy cv Scarlet O'Hara, were planted in each plastic pot (soil
surface area: 200 cm2, pot height: 19 cm) and one seed of kidney
bean, Phaseolus vulgaris L. cv Gintebo, was planted per plastic
pot (soil surface area: 100 cm2, pot height: 6 cm) containing a
commercial culture soil (Kureha Chemical Co. Ltd). Morning
638
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639
FOLIAR LIPIDS WITH 03 AND PAN
glory and kidney bean plants were grown in a naturally lit, environment-controlled glass chamber (2 m [d] x 2 m [w] x 1.8
m [h]) equipped with activated charcoal filters. The conditions
in the chamber were maintained at 27/22°C and 70/80% day/
night for temperature and relative humidity, respectively. Morning glory plants with 12 to 14 leaves about 5 weeks after germination were used for ozone exposure, and kidney bean plants
with primary leaves expanded fully at 6 to 7 d after germination
were used for ozone and PAN exposure.
Ozone and PAN Exposure Procedures. Ozone was generated
by passing air through a high voltage discharging tube and was
introduced into the naturally lit, environment-controlled glass
chamber in which the plants were placed. Environmental conditions during ozone exposure were the same as those for the
growing periods. The concentration of ozone in the glass chamber
was maintained at 0.15 + 0.01 AI/L and monitored with a UV
absorption ozone analyzer (Dashibi, model 1003AH) which was
dynamically calibrated with a 1 % neutral buffered KI method.
Synthesis of PAN and determination of PAN concentration
were by the methods described previously (18). PAN exposure
was made in an exposure chamber (0.6 m [d] x 1.0 m [w] x
1.1 m [h]) made of acryl resin. The chamber was installed in a
larger environment-controlled chamber (1.3 m [d] x 1.0 [w] x
1.1 m [h]) equipped with 9 metal halide lamps (Yoko Lamp 400
W: Toshiba). The light intensity was about 400 ,uE m-3 s-1 at
the center of the exposure chamber. Temperature in the exposure
chamber was kept at 27°C, but relative humidity varied from 60
to 80% by transpiration from leaves. The volume of incoming
air which contained PAN was approximately 15 L min- 1 and
the air velocity in the chamber was controlled at 0.5 ms-I by
two propellers. PAN concentration in the exposure chamber was
monitored by using a gas chromatograph with an electron capture
detector (Yanagimoto Co. Ltd, PAN monitor, model GPH-1OA).
A teflon tube, 450 mm long and 3 mm in diameter, and packed
with 5% polyethylene glycol on Chromosorb W (AW DMCS,
60-80 mesh) was used for the GC column.
Lipid Analysis. Twenty leaf discs (10 mm in diameter), cut
from interveinal areas of leaves, were excised either immediately
after or 24 h after the end of a given exposure. These leaf discs
were immersed 3 min in boiling water to inactivate lipases. Lipids
were extracted according to the method of Bligh and Dyer (3),
dissolved in 8 ml of chloroform containing 0.05% (w/v) atocopherol. and stored under N2 at -20°C. The crude lipid solution concentrated under N2 was spotted on a silica gel plate
(Merck; 5717 Silica Gel 60F254). The plate was developed in the
first direction with chloroform-methanol-water (65:25:4, v/v) and
in the second direction with chloroform-methanol-isopropylamine-ammonium hydroxide (65:35:0.5:5, v/v) (1). The chromatogram was viewed under UV after spraying with 0.01% (w/
v) primuline in 80% acetone. Individual polar lipids were identified by co-chromatography with authentic standards and by
their reaction with specific spray reagents of anthrone sulfuric
acid, Dittmer, Dragendorff, ninhydrin, and iron (III) chloride
specific to glycolipid, phospholipid, the choline group, the amino
group, and sterol, respectively. Each fluorescent lipid area was
scraped from the plate and subjected to methanolysis with 10%
(w/v) sulfuric acid in methanol at 40°C overnight with a given
amount of heptadecanoic acid (Sigma) as an internal standard.
A small amount of the lipid extract before TLC separation was
also subjected to methanolysis to determine the total fatty acid
content.
The fatty acid methyl esters formed were extracted with nhexane and chromatographed on a GLC (Shimadzu GC-9A)
equipped with a hydrogen flame ionization detector and an integrator (Shimadzu, Chromatopac C-RIB). Separations were
carried out at 200°C using a glass column (3 mm x 2 m) packed
with 5% Thermon-1000 (Wako Pure Chem.) on Chromosorb W
(AW DMCS, 80- 100 mesh). The fatty acids were identified by
their retention time relative to that of standard methyl ester
mixtures (Applied Science Laboratories). Quantitative analyses
of the fatty acids and their parent lipid were made according to
Allen and Good (1).
MDA Content. MDA content in leaves was assayed according
to the method of Heath and Packer (8). The discs excised from
leaves were homogenized in distilled water. Three ml of leaf
homogenate in distilled water were mixed with S ml of 0.5%
thiobarbituric acid in 20% trichloroacetic acid. The mixture was
incubated in a boiling water bath for 30 min. After centrifugation,
the MDA content in the supernatants was determined by using
the difference absorbance coefficient of 155 mM cm at 532
and 600 nm (8).
RESULTS
Effects of Ozone on Fatty Acid Content and Composition in
Morning Glory Leaves. Morning glory plants were exposed to
0.15 ,ul ozone/L for 0, 2, 4, 6, and 8 h under natural light. In
morning glory plants exposed to 0.15 Al ozone/L, water-soaked
spots appeared on the upper surface of leaves at 6 h after the
beginning of exposure and then spread over the interveinal area.
At 1 d after exposure, numerous small bleached spots had appeared on leaves exposed to ozone for 4, 6, and 8 h, and the
injured area was 40 to 60% of the leaf area. For the lipid assay,
the most sensitive leaves that were third or fourth from the
bottom were used.
Figure 1 shows the changes in the total fatty acid content and
composition in morning glory leaves immediately after and 24 h
after the end of 0.15 ,ul ozone/L exposure. The total fatty acid
content and composition in control leaves stayed almost constant
during the course of the experiment. The total fatty acid content
and composition in morning glory leaves exposed to ozone were
almost the same when compared to those of control until 6 h of
exposure, and then significantly decreased to 86% of the control
level at 8 h. At 8 h the decreased fatty acids, linoleic acid (C18:2)
and linolenic acid (C18:3), reached 82 and 72% of the control
level, respectively. Twenty-four h after exposure, the contents
of total fatty acids and C18:3 were similar to those immediately
after the end of exposure. But a slight increase in content of
palmitoleic acid (C16:1) in 2 to 8-h-exposed leaves and a reversion to control level of C18:2 in 8-h-exposed leaves were observed.
Effects of Ozone on Fatty Acid Content and Composition in
Kidney Bean Leaves. Ozone exposure and assays of fatty acids
and polar lipids of kidney bean leaves were carried out in the
same way as with morning glory. Obvious water-soaked spots
did not appear on kidney bean leaves during exposure. However,
when plants were left 1 d after the end of each period of exposure,
injury occurred for every period of exposure. Numerous bleached
spots appeared at 2, 4, and 6 h, and the percentage of foliar
injury was 20 to 30% for each. The percentage of foliar injury
at 8 h was about 50%, and injury symptoms were mixed with
bleached spots and reddish brown stipples on the upper surface
of the leaves.
The diurnal changes in the total fatty acid content and composition in control leaves of 6 to 7-d-old kidney bean were compared to the results obtained immediately after and 24 h after
the end of exposure (Fig. 2). The total fatty acid content remained approximately unchanged during the daytime under natural light, but its components varied; C18:2 began to increase
accompanied by a decrease of C18:3 from the morning to the
evening. During exposure to ozone, total fatty acid content and
composition were nearly equal when compared to those of control. Twenty-four h after the end of exposure, the total fatty acid
content also remained approximately unchanged, but a slight increase in content of C16:0 was observed in 6 to 8-h-exposed leaves.
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640640
Plant
NOUCHI AND TOYAMA
NOUCHI
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FIG. 1. Changes in total fatty acid content and composition of morning glory leaves immediately after and 24 h after the end of 0.15 A1d ozone/
L. C, 0 h and 24 h represent control, immediately following exposure, and 24 h after exposure, respectively. Each bar indicates total fatty acid
content and is the mean of three samples -+- SD. Numerals in each bar indicate percent molar concentrations of fatty acids. Differences in the
content of fatty acids between ozone treatment and control are significantly different at the 5% level (*) and 1%/c level (**) as determined by t test.
Effects of PAN on Fatty Acid Content and Composition in
Primary Leaves of Kidney Bean. Kidney bean plants were exposed to 0.10 ,ul PAN/L for 0, 1, 2, 4, 6, and 8 h under artificial
light. Kidney bean leaves showed water-soaked lesions on the
upper surface at 6 h. When the plants were left 1 d after the
exposure, foliar injury occurred even in those given 1 h exposure.
Leaves exposed to 0.10 ,ul PAN/L for 1 h showed bronzing over
about 70% of their lower surface area, those exposed for 2 h
showed bronzing over 100% of their lower surface area and about
20% bifacial bleached necrosis, and those exposed for 4 to 8 h
showed about 90 to 95% bifacial bleached necrosis, remaining
green at the large veins.
The total fatty acid content and composition in the primary
leaves of kidney bean plants during exposure to PAN and 1 d
after exposure for either 2 h (bronzing symptoms) or 8 h (bifacial
bleached necrosis) are shown in Figure 3. The fatty acid content
and composition in control leaves stayed almost constant during
the course of experiment. The fatty acid content during 8 h of
exposure to PAN showed little or no change till 4 h and then
began to decrease at 6 h (appearance of water-soaked lesions)
and reached 75% at 8 h. The fatty acid composition during PAN
exposure also showed little or no change compared with control
till 4 h. After this, both C18:2 and C18:3 began to decrease at
6 h and reached 77 and 66% of the control level at 8 h, respectively. Twenty-four h after the end of exposure, the content of
total fatty acids, C18:2 and C18:3, decreased more than the levels
during the exposure. C16:0 and C16:1 showed little or no change
after exposure to PAN.
Effects of Ozone and PAN on MDA Content in Leaves of Morning Glory and Kidney Bean. Changes in the MDA content in
leaves of morning glory and kidney bean plants during ozone or
PAN exposure are shown in Figure 4. During ozone exposure,
MDA content in morning glory remained unchanged till 6 h, and
then slightly increased to 134% of the initial level at 8 h, while
little or no change was observed in kidney bean plants. The MDA
content in kidney bean leaves during PAN exposure began to
increase to 240 and 296% of the initial level at 6 and 8 h, respectively. These increases of MDA content occurred only after
water-soaked symptoms had appeared.
Effects of Ozone on Polar Lipids in Morning Glory Leaves.
Changes in the glycolipid and phospholipid content during ozone
exposure are shown in Figure 5, A and B, respectively. There
were significant changes in the phospholipid content during exposure. PC, PG, PE, and PI rapidly increased at an initial exposure (2-4 h) and decreased at a late stage of exposure (6-8
h). The maximum PC, PG, and PE content reached about 175,
180, and 200% of the control levels, respectively, at 4 h. In
contrast, the glycolipid content remained unchanged or slightly
decreased during ozone exposure.
Changes in the glycolipid and phospholipid content 24 h after
the end of the exposure are shown in Figure 6, A and B, respectively. When plants were left 1 d to permit symptoms to
develop, the contents of MGDG, DGDG, and SQDG showed
a large drop. The phospholipid contents at 24 h after the exposure
were almost the same (PE, PI, and PA) or decreased (PC and
PG) when compared to the content immediately after exposure,
but all the levels were higher than those of the control.
Effects of Ozone on Polar Lipids in Primary Leaves of Kidney
Bean. Changes in the glycolipid and phospholipid content during
exposure are shown in Figure 7, A and B, respectively. The
change in patterns of MGDG, DGDG, and SQDG were similar
to those in morning glory. That is, MGDG and SQDG decreased
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Copyright © 1988 American Society of Plant Biologists. All rights reserved.
641
FOLIAR LIPIDS WITH 0, AND PAN
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FIG. 2. Changes in total fatty acid content and composition of kidney bean leaves immediately after and 24 h after the end of 0.15 ,ul ozone/L.
Conditions as described in Figure 1.
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Copyright © 1988 American Society of Plant Biologists. All rights reserved.
of
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642
NOUCHI AND TOYAMA
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8
FIG. 4. Changes in MDA content in leaves of morning glory and
kidney bean with exposure of 0.15 Al ozone/L or 0.10 ,ul PAN/L. (0),
Kidney bean for ozone; (0), kidney bean for PAN; (A), morning glory
for ozone.
a little, while DGDG remained unchanged. On the other hand,
changes in the phospholipid content differed remarkably from
those of morning glory. PC, PG, PE, and PI decreased after an
initial 2 h exposure, but PG, PE, and PI returned to control
levels at 4 to 6 h. At 8 h exposure, PE and PI had remarkably
increased and reached about 150% of the control levels, while
PC and PG were the same as control levels.
One day after exposure, MGDG rapidly decreased with the
increase of exposure duration, while DGDG and SQDG remained almost unchanged compared with control level (Fig. 8A).
In phospholipids, PG decreased at 6 to 8 h, but PC, PE, PI, and
PA increased at 4 to 8 h (Fig. 8B). The increase of PE and PI
content was particularly remarkable.
Effects of PAN on Polar Lipids of Primary Leaves of Kidney
Bean Leaves. Changes in the glycolipid and phospholipid content
during PAN exposure are shown in Figure 9, A and B, respectively. All polar lipids showed little change until 4 h (at which
time there was no occurrence of water-soaking symptoms), while
there was a marked drop in MGDG, DGDG, PC, PE, and PI
at 6 h. PA began to increase rapidly at 6 h and reached about 6
times the level of control levels at 8 h. Figure 10, A and B, show
the content of polar lipids in both mildly injured leaves (1 h
exposure) and severely injured leaves (8 h exposure) in plants
which were left 1 d after exposure to PAN. Glycolipid, MGDG
and DGDG, levels decreased in leaves showing bronzing symptoms, while the phospholipid levels remained stable. As for the
polar lipid content in bifacial bleached necrosis leaves, glycolipids
Plant
Physiol. Vol. 87,
1988
almost disappeared and phospholipids were reduced remarkably.
But PA increased even in necrotic leaves.
DISCUSSION
The fatty acid content and composition in kidney bean leaves
exposed to ozone showed little or no change during 8 h exposure
to a low concentration of ozone. This conforms to the results of
Tomlinson and Rich (28), Swanson et al. (26), and Fong and
Heath (6). In addition, MDA, which is formed both from ozonolysis and lipid peroxidation of unsaturated fatty acids (14), also
remained unchanged during ozone exposure. On the other hand,
fatty acid content in morning glory leaves significantly decreased
only after an 8 h exposure to ozone and was accompanied by a
concomitant slight accumulation of MDA (Fig. 4). Fatty acids
whose levels decreased were linoleic and linolenic acd (Fig. 1).
These results suggest that ozone or oxy-radical cause oxidation
of the unsaturated fatty acids in morning glory at the late stage
of exposure. Oxy-radical, which is formed by ozone decomposition in aqueous solution, enzymically, or the oxygen reduction
in the chloroplasts (27), can cause lipid peroxidation and lipid
deesterification (9). Peters and Mudd (21) pointed out that oxidation of unsaturated fatty acids did not occur, presumably because double bonds of the fatty acid are embedded in the bilayer
and protected by reduced glutathione or by proteins more susceptible to oxidation.
Although only the C18:2 and C18:3 showed the significant
difference among fatty acids upon the exposure of morning glory
to ozone for 8 h, a slight increase in the content of C16:1 in
morning glory and C16:0 in kidney bean were observed 24 h
after the end of exposure. These results may be explained by
alterations in lipid metabolism by the secondary damage.
The phospholipid and glycolipid content altered markedly with
ozone exposure in the leaves of morning glory and kidney bean.
Phospholipid content such as PC, PE, PG, and PI increased
remarkably in morning glory and decreased in kidney bean at a
2- to 4-h-period after the start of exposure (Figs. SB and 7B),
while unsaturated fatty acids in morning glory decreased only at
8 h (Fig. 1). A slight decrease also occurred in MGDG in both
the leaves of morning glory and kidney bean during ozone exposure (Figs. 5A and 7A). These results suggested that 0.15 ,ul/
L ozone may not directly oxidize unsaturated fatty acids of polar
lipids by ozonolysis or lipid peroxidation at the initial stage, but
it may affect enzymes relating to the lipid metabolism in leaves.
In kidney bean plants exposed to ozone, the decreases in phospholipid and MGDG content may be due to either the inhibition
of lipid synthesis, the increase of hydrolysis, or a combination
of these. It is well known that ozone inhibits lipid biosynthesis
in spinach chloroplast suspensions or mitochondrial or microsomal fractions from rat lung by way of oxidation of sulfhydryl
enzymes (12, 13, 21). Moreover, Mudd et al. (13) reported that
glycolipid biosynthesis was inhibited in chloroplasts treated with
ozone and the formation of DGDG was more strongly inhibited
than the formation of MGDG. However, the present study showed
that MGDG was more sensitive to ozone than was DGDG. It
is possible that ozone may enhance polar lipid hydrolysis. The
predominant loss of MGDG compared with DGDG in both leaves
of morning glory and kidney bean might be caused by ozoneinduced activation of galactolipase, which is known to be more
hydrolytic with MGDG than DGDG (2).
In morning glory exposed to ozone, the contents of the phospholipid components increased at 2 h, reached their maximum
levels at 4 h, and then decreased gradually until 8 h (Fig. 5A).
The large increase in the contents of phospholipid components
would suggest the synthesis of membranes and thereafter the
degradation of membranes. Thus, increases of phospholipid content at the initial stage as seen in morning glory exposed to ozone
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80
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643
FOLIAR LIPIDS WITH O AND PAN
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FIG. 5. Changes in polar lipid content of morning glory leaves during exposure to 0.15 ,ul ozone/L. A, Glycolipids and sulpholipids; B.
phospholipids. Straight lines: ozone treatment; dashed lines: control (air treatment). Each symbol is the mean of three samples, and the vertical
bar represents a standard deviation.
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FIG. 6. Changes in polar lipid content of morning glory leaves 24 h after the end of exposure t 0.15 ,ul ozone/L. A, Glycolipids and sulpholipids;
B, phospholipids. Legend as described in Figure 5.
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Copyright © 1988 American Society of Plant Biologists. All rights reserved.
NOUCHI AND TOYAMA
644
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1
PA
-
41
PA
0
I
0
I
I
Oc_
II
2
4
6
Exposure duration, hr
0
8
_I
0
O- , ..
2
4
6
Exposure duration, hr
8
FIG. 7. Changes in polar lipid content of kidney bean leaves during exposure to 0.15 ,ul ozone/L. A, Glycolipids and sulpholipids; B, phospholipids.
Legend as described in Figure 5.
have not been reported in other plant species previously (6, 23,
26), but increases in the phospholipid content have been observed as an adaptation to water stress in wheat and barley (4),
to salt stress in cotton (29), and to rust infection (10). In the
present study, it is impossible to explain the mechanism by which
the contents of individual phospholipid component increased uniformly during ozone exposure; such changes may be a speciesspecific injury response to ozone of morning glory alone.
MGDG, DGDG, and SQDG in morning glory and MGDG
in kidney bean leaves decreased markedly 1 d after exposure to
ozone. MGDG and DGDG are located in both the envelope
membrane and thylakoid membranes of chloroplasts and they
comprise about 80% of nonpigment lipids in the chloroplast of
higher plants (30). Therefore, a large reduction of these glycolipids in ozone-exposed leaves (1 d after exposure) suggested
that chloroplast membranes had collapsed. In fact, using morning
glory leaves 1 d after exposure to 0.15 p,l ozone/L, Nouchi et al.
(15) observed by electron microscopy that with an increasing
duration of ozone exposure the thylakoid membranes of chloroplasts collapsed, and the thylakoid membranes in leaves exposed for 8 h almost disappeared. Large reductions of glycolipids
occurred after ceasing ozone exposure, suggesting that the large
destruction of thylakoid membranes was not directly caused by
low concentrations of ozone, but that their destruction may result
from secondary damage of membrane permeability.
Compositional changes in phospholipids in leaves of morning
glory and kidney bean, especially the considerable increase of
PE and PI, were observed 24 h after the end of ozone exposure
(Figs. 6B and 8B). These changes of PE and PI and the preferential loss of MGDG rather than DGDG resemble the pattern
of changes in senescing leaves (5). Therefore, such lipid changes
be a generalized feature of cell deterioration.
Swanson et al. (26) showed by TLC that the amounts of the
glycolipids and phospholipids of tobacco leaf did not change
significantly immediately after 2 h exposure to 0.30 ,u1 ozone/L.
Similarly, Fong and Heath (6) reported that exposure of 15-dold bean leaves to 0.30 or 0.50 ,u1 ozone/L for 1 h caused small
alterations in PE, PG, and MGDG/DGDG molar ratio only in
severely injured leaves (0.50 ,ul ozone/L) several hours or 24 h
after exposure. On the other hand, Sakaki et al. (22, 23) reported
that the exposure of spinach to 0.50 ,ul ozone/L for 8 h produced
a marked decrease in MGDG, DGDG, and PC within 4 to 6 h.
The present results at low concentrations of ozone (0.15 ,ul/L)
were different from the findings of Swanson et al. (26), Fong
and Heath (6), and Sakaki et al. (23). This could be due either
to the different plant species, ages, or ozone dose or to a combination of these factors.
PAN can oxidize NADPH and prevent the incorporation of
acetate into long chain fatty acids (11), but little is known about
the effect of PAN on lipids or fatty acids. The present study
indicated that the exposure of kidney bean leaves to 0.10 ,ul
PAN/L caused a marked drop in phospholipids, glycolipids, and
total fatty acids. Little or no change occurred in phospholipids
and glycolipids till 4 h from the beginning of PAN exposure.
Thereafter, the content of PC, PG, PE, PI, MGDG, DGDG,
and unsaturated fatty acids decreased remarkably, while the content of PA and MDA showed a sharp increase, as water-soaking
and wiltings began to be observed 6 h after exposure (Fig. 9, A
and B). Since PA is a common breakdown product of phospholipids by phospholipase D, the large increase of PA suggested
may
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Copyright © 1988 American Society of Plant Biologists. All rights reserved.
645
FOLIAR LIPIDS WITH 03 AND PAN
gos
I
I
I
36
I
I
0
80
-
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E 50 F
._Q
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0
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E
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a-
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0
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*.
T
E
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32
1\
70 cm
I
I
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TMGDG
30 F
PE
?-12
~
-
-
1
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20 F
_
SQDG
lop ---0
*__
^I
Li ,
I
I
I
P4
--
-
4
,
PA
-.
-
I
8
4
0
6
4
6
8
2
2
Exposure duration, hr
Exposure duration, hr
FIG. 8. Changes in polar lipid content of kidney bean leaves 24 h after the end of exposure to 0.15 ,lI ozone/L. A, Glycolipids and sulpholipids;
B, phospholipids. Legend as described in Figure 5.
0
40
100
F-7
IB
T
361
TI
-
32
28 _-
E
o 60
50
a,% 40
75
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E
E
.
Eu
C
20 k
a
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T
U-
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-
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--
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*i-*.A.
.
I
I
I
- - V-o
-.
6
2
4
8
2
4
6
0
Exposure duration, hr
Exposure duration, hr
FIG. 9. Changes in polar lipid content of kidney bean leaves during exposure to 0.10 ,l PAN/L. A, Glycolipids and sulpholipids; B, phospholipids.
Straight lines: PAN treatment; dashed lines: control (air treatment). Each symbol is the mean of three samples, and the vertical bar represents a
standard deviation.
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Copyright © 1988 American Society of Plant Biologists. All rights reserved.
NOUCHI AND TOYAMA
646
36r
LITERATURE CITED
I
I--
B
32j
281E
u 24
E
E0
E
C
C
-
20 I-
-6
0.
m
06
0.
0
0.
16
PE
T
§-
12H
8
4
0
I
8
hr
Exposure duration,
1
0'
,*71,
* PA
I
0
Plant Physiol. Vol. 87, 1988
1
Exposure
'r
x
8
duration, hr
Changes in polar lipid content of kidney bean leaves 24 h
after the end of exposure to 0.10 ,ul PAN/L. A, Glycolipids and sulpholipids; B, phospholipids. Each symbol is the mean of three samples, and
the vertical bar represents a standard deviation.
FIG. 10.
that PAN may enhance the hydrolytic activity of phospholipase
D. In addition, a large reduction of unsaturated fatty acids such
as C18:2 and C18:3 occurred at 8 h during exposure to PAN
(Fig. 3), which was accompanied by a concomitant large accumulation of MDA (Fig. 4). These results suggested that PAN
may cause lipid peroxidation of unsaturated fatty acids at the
late stage of PAN exposure.
PAN produced singlet oxygen when decomposed with alkalisolution (25). Shimazaki et al. (24) have reported that singlet
oxygen plays a dominant role in the lipid peroxidation of SO,exposed leaves. Therefore, PAN might break down polar lipids
and fatty acids through its oxidizing property as a peroxide or
the toxicity of active oxygen. The massive decomposition of lipid
components, particularly of glycolipids such as MGDG and DGDG
by PAN, proceeded faster than that by ozone. It was assumed
that the PAN molecule itself or its breakdown product may directly and strongly attack the thylakoid membranes of chloroplasts and induce an abrupt collapse of the membrane structure.
Although the reason the effects of ozone and PAN on polar
lipids and fatty acids were very different is not known, these
results suggested that the attack of ozone and PAN on polar
lipids differed from each other.
The present study revealed that both ozone and PAN altered
the content and composition of the polar lipids of membranes.
In addition, there is a distinct difference in the mode of action
of the effect on lipids between ozone and PAN. The content and
composition of phospholipids was altered rapidly by ozone prior
to any visible injury appearing, while that of phospholipids and
glycolipids decreased markedly by PAN, which was concomitant
with the appearance of symptoms of water-soaking. Even if such
polar lipid changes are not the cause of initial damage, alterations
in the content and components of polar lipids by ozone and PAN
could affect the functions associated with membranes such as the
control of permeability, fluidity, and membrane-bound enzyme
activities.
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