Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol16.1996:
Tree Physiology 16, 597--605
© 1996 Heron Publishing----Victoria, Canada
Growth responses and related biochemical and ultrastructural changes
of the photosynthetic apparatus in birch ( Betula pendula) saplings
exposed to low concentrations of ozone
E. PÄÄKKÖNEN,1 J. VAHALA,2 T. HOLOPAINEN,1 R. KARJALAINEN2 and
L. KÄRENLAMPI1
1
2
Department of Ecology and Environmental Science, University of Kuopio, P.O. Box 1627, 70211 Kuopio,
Finland
Department of Plant Biology, Plant and Forest Pathology Section, University of Helsinki, 00014 Hels
inki, Finland
Received July 25, 1995
Summary Saplings of ozone-sensitive and ozone-tolerant
birch (Betula pendula Roth.), clones B and C, respectively,
were exposed to ozone concentrations that were 1.7-fold higher
than ambient for one growing season under open-field conditions. Ambient air was used as the control treatment. In the
ozone-sensitive clone B, there was an initial stimulation of leaf
area growth in response to the ozone treatment, but further
ozone exposure caused reductions in leaf and stem biomass
growth, Rubisco and chlorophyll a contents, net photosynthesis, water use efficiency and chloroplast size. It also caused an
alteration in chloroplast shape and injury to thylakoid membranes. In the ozone-tolerant clone C, ozone fumigation did not
affect growth rate, and there were no consistent changes in
chlorophyll content, photosynthesis or water use efficiency.
There were also fewer ultrastructural abnormalities in the
chloroplasts of clone C than of clone B. Based on the observed
biochemical, physiological and structural changes in chloroplasts of clone B in response to low concentrations of ozone,
we conclude that the increasing concentration of tropospheric
ozone represents a risk to natural birch populations.
Keywords: Betula pendula, growth ultrastructure, ozone, photosynthetic machinery.
Introduction
Negative effects of ozone on growth have been reported in
several tree species (e.g., Darrall 1989, Chappelka and
Chevone 1992). However, the mechanisms responsible for
these growth reductions are not completely understood. Impaired photosynthesis, decreased activity and quantity of Rubisco, decreased chlorophyll content and decreased stomatal
conductance are often reported in ozone-stressed plants (e.g.,
Reich 1983, Darrall 1989, Matyssek et al. 1991, Pell et al.
1992). In addition, many symptoms have been observed at the
ultrastructural level, primarily in chloroplasts, in the early
phases of ozone injury (Holopainen et al. 1992, Pääkkönen et
al. 1995).
During leaf aging and senescense, ozone has been reported
to accelerate the normal decline in chlorophyll content and
photosynthesis (e.g., Thomas and Stoddardt 1980, Reich 1983)
and in the activity and quantity of Rubisco (Pell et al. 1992,
Landry and Pell 1993). Decreased content of Rubisco protein
may be caused by inhibition of protein synthesis or enhanced
proteolysis, or both (Landry and Pell 1993, Eckardt and Pell
1994). Structural modification of the Rubisco molecule has
been proposed as an explanation for the decreased activity of
Rubisco preceding proteolytic degradation in ozone-stressed
cuttings of hybrid poplar (Landry and Pell 1993). It has been
suggested that, during ozone stress, free radicals are generated
that result in increased oxidation of biological macromolecules leading to structural modifications of Rubisco and chlorophyll (Pell and Dann 1991, Landry and Pell 1993).
The susceptibility of plants to ozone attack seems to depend
on the developmental stage of the leaves (Pell et al. 1992,
Eckardt and Pell 1994). In hybrid poplar, for example, the
deleterious effects of ozone on Rubisco were most apparent at
full leaf expansion, whereas during the early developmental
stages, the leaf was capable of synthesizing new enzyme and
therefore partially able to replace the ozone-damaged Rubisco
(Pell et al. 1992). Similarly, in potato, exposure to ozone
resulted in a significant decline in Rubisco of mature leaves,
whereas immature leaves were unaffected (Eckardt and Pell
1994). Chronic ozone exposure throughout the life span of the
leaves was necessary for continuous decline in Rubisco in
hybrid poplar and radish (Pell et al. 1992) and potato (Eckardt
and Pell 1994).
Physiological, anatomical and ultrastructural studies of five
birch clones have revealed that clones differ in their sensitivity
to ozone (Pääkkönen et al. 1993, 1995). In the most ozone-sensitive clone B, exposure to ozone resulted in decreased height
growth and leaf biomass, leaf chlorosis and vein yellowing and
increased numbers of necrotic flecks on the leaves. In the most
598
PÄÄKKÖNEN ET AL.
ozone-tolerant clone C, exposure to ozone had no effect on
height growth or the amount of visible foliar injury (Pääkkönen et al. 1993). However, ozone induced ultrastructural
injuries in leaf mesophyll cells of both clones, including
abnormal chloroplast morphology, dense stroma, curling and
swelling of thylakoids, increased disintegration of the mitochondrial matrix, a reduction of cristae, decreased amounts of
tannin and increased amounts of cytoplasmic lipids (Pääkkönen et al. 1995). These changes were regarded as signs of
accelerated senescence, because similar changes occurred
more slowly during normal leaf aging.
This study was undertaken to determine whether ozone-induced responses in stem and leaf growth and ultrastructural
changes in chloroplasts are related to the functioning of the
photosynthetic apparatus in two birch clones (B and C) showing different susceptibilities to ozone. The experiment was
carried out in an open-air fumigation system in an attempt to
provide realistic information about the extent of ozone effects
under field conditions.
Materials and methods
Plant material
Two-year-old saplings of clones KL-5-M (B) and KL-2-M (C)
of European white birch (Betula pendula Roth.) were planted
individually in 7.5-liter pots filled with fertilized sphagnum
peat and garden soil (10/1, v/v). The saplings were watered as
needed and fertilized weekly with 0.2% (19,5,20 N,K,P) nutrient solution.
Ozone fumigation
The saplings were transferred to the open-air exposure field at
Kuopio Botanical Garden (62°13′ N, 27°13′ E) in central
Finland on May 12, 1993. Ten saplings per clone per treatment
were arranged in two blocks per treatment. The control saplings were grown in ambient air, whereas the ozone-treated
saplings were fumigated continuously with ozone concentrations that were 1.7-fold higher than ambient air. The seedlings
were exposed to ozone in the natural microclimate. The computer-controlled release of ozone, which was generated from
pure oxygen, was from perforated tubes in up-wind positions.
The ozone concentration was maintained continuously as a
constant multiple of the ambient concentration, to mimic the
natural hourly and seasonal variation. The fumigation system
has been described in detail by Wulff et al. (1992) and Pääkkönen et al. (1993). The cumulative ozone exposures (ppm-h)
for the periods between the sampling dates for Rubisco analysis are given in Table 1. The starting date for the biochemical
and ultrastructural studies was June 28, when the leaves under
investigation emerged.
Growth measurements
Immediately after planting, the height of each sapling was
determined. All the seedlings were measured for height, number of leaves, individual leaf size (mean) and foliage area (total
leaf area) on July 12, July 27, August 9 and August 23. Foliage
area was estimated by multiplying the number of leaves by the
mean outline area of the individual leaves, measured from 10
average-sized leaves per sapling. On September 13, dry
weights for stem and roots were determined.
Electron microscopy studies
For the ultrastructural studies, leaf samples were collected on
July 12, August 2, August 16 and August 30 from each sapling
(10 saplings per clone per treatment). The samples were taken
from among the leaves that emerged in the last week of June
by cutting 5-mm strips each 1--2 cm in length from the leaf
apex. These leaf strips were immediately placed in a fixative
solution of 2.5% (v/v) glutaraldehyde in phosphate buffer
(0.1 M, pH 7.0). In the laboratory, 1.5 mm2 square pieces were
cut with a razor blade from the strips maintained under a drop
of 2.5% glutaraldehyde fixative. Leaf pieces were postfixed in
1% buffered OsO4 solution, dehydrated in an ethanol series
and embedded in LX 112 epon. Thin sections were stained
with uranyl acetate and lead citrate and were examined by
means of a JEOL 1200 EX electron microscope operating at
80 kV.
Twenty randomly selected cross sections of chloroplast palisade and spongy mesophyll cells per clone and treatment were
photographed. The length and width of chloroplasts, the number of small (< 10 thylakoids) and large (> 10 thylakoids)
grana, and the number and size of plastoglobuli were measured
from the photographs. The proportion of the chloroplast area
occupied by starch grains was calculated by a point analysis
method, where a cross-hatched grid with random points was
positioned over the photograph and the points within starch
grains were counted to determine the relative area of starch in
relation to chloroplast area. The changes in chloroplast shape,
density of stroma and appearance of thylakoids and mitochondrial matrix were determined by classifying the twenty photographed chloroplasts and cells per tissue per sample into injury
classes 0--3 (0 = not injured, 1 = slightly injured, 2 = clearly
injured, 3 = severely injured). The amounts of tannin, cytoplasmic lipids and microbodies were classified by visual observation, and the electron density of plastoglobuli was determined.
Biochemical analyses
Leaves selected for the determination of Rubisco protein and
chlorophyll were taken from the same position on the sapling
as leaves used for the electron microscopy assays. Leaf samples were collected on July 12, July 28, August 9 and August 25 from five saplings per clone per treatment. After
excision, the samples were weighed, and the outlines of the
leaves drawn on plastic film in the field. The final leaf areas
were determined from the plastic films with an LI-3000-A
portable area meter (Li-Cor, Inc., Lincoln, NE).
A crude leaf extract was prepared by grinding 0.1 g of frozen
leaf tissue in liquid nitrogen. Cold extraction buffer was added
to the fine leaf powder according to Gezelius and Hallen
(1980), except that Tween 80 was replaced with Tween 20.
Rubisco concentrations in the leaf were determined by PAGE
according to Rintamäki et al. (1988). Each gel contained a
purified spinach Rubisco standard. The total Rubisco content
RESPONSES OF BIRCH TO OZONE
599
Table 1. Cumulative ozone exposures (ppm-h) of the studied leaves. SUM00 accumulates all 1-h average concentrations and SUM40 accumulates
those above the 40 ppb base-line.
Period
Ambient ozone
Elevated ozone
Nighttime
Daytime
Night + Day
Nighttime
Daytime
Night + Day
SUM00
June 28--July 12
July 13--July 28
July 29--August 9
August 10--August 18
August 19--August 25
Total SUM00
1.12
1.28
1.16
1.52
0.46
4.30
4.23
3.50
1.94
1.16
5.43
5.51
4.66
3.46
1.62
20.68
1.89
1.93
1.81
2.51
0.71
7.32
6.91
6.36
3.45
2.15
9.21
8.85
8.17
5.96
2.86
35.05
SUM40
June28--July 12
July 13--July 28
July 29--August 9
August 10--August 18
August 19--August 25
Total SUM40
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.07
0.28
0.00
0.44
0.20
0.61
0.12
0.00
0.44
0.20
0.68
0.40
0.00
1.72
was determined by scanning the gels and measuring the areas
and intensities with the Pharmacia ImageMaster program (Version 1.0). For analysis of Rubisco small and large subunits,
SDS-PAGE analyses (Laemmli 1970) were performed on each
sample and the concentrations of large and small subunits were
determined as for the PAGE gels. The concentrations of chlorophyll a and b were determined by Arnon’s (1949) method.
Rubisco and the chlorophyll concentrations were calculated on
a fresh weight basis.
Gas exchange measurements
Net photosynthesis and transpiration measurements were carried out with a closed-loop Li-Cor LI-6200 portable photosynthesis system. The fifth emergent leaf was selected for the
measurements of gas exchange from 4--6 saplings per clone
per treatment. Field measurements were made in saturating
sunlight, over 900 µmol m −2 s −1 (PFD). Supplementary halogen light (Sylvania professional, FTY/50W/8o) was used
whenever natural irradiance was below 900 µmol m −2 s −1.
Measurements were taken on the same leaves on six dates
between August 3 and September 15.
Statistical methods
No significant block effects within the ozone and control
treatments were revealed by ANOVA, and therefore data for
these two blocks were pooled for further analysis. The Student’s t-test was used to test the differences in growth, parametric ultrastructural characteristics, Rubisco and chlorophyll
contents, photosynthesis, transpiration and WUE between
ozone-treated and control saplings. Differences in the relative
amounts of Rubisco subunits between treatments were tested
by the nonparametric Mann-Whitney U-test. Differences were
considered significant at P < 0.05.
Results
Growth responses
Ozone fumigation had no significant effect on any of the
growth parameters measured in saplings of clone C (Table 2).
For saplings of clone B, mean leaf size and foliage area were
significantly larger in saplings exposed to ozone compared
with control saplings on July 1, but on July 27 and August 9,
mean leaf size and foliage area were significantly smaller in
ozone-fumigated saplings than in control saplings (Table 2).
Based on data pooled for all dates, the ozone-induced decrease
in leaf area production was almost 10% greater in clone B than
in clone C (Table 2). Ozone-fumigated saplings of clone B had
significantly less (22.7%) stem dry weight than control saplings, whereas root dry weight was unaffected by the ozone
treatment (Table 2).
Ultrastructural changes
The responses of palisade and spongy mesophyll cells to ozone
were qualitatively similar, although some ozone-induced
changes were more pronounced in spongy mesophyll cells.
Ozone-induced ultrastructural changes were most evident in
the ozone-sensitive clone B in late summer, but several ozoneinduced structural injuries were observed in the ozone-tolerant
clone C.
In clone B, the width of chloroplasts was significantly
greater in ozone-exposed saplings than in control saplings on
July 12, whereas the length of chloroplasts was significantly
less in ozone-exposed saplings than in control saplings on
August 2 (Table 3). Accompanying these ozone-induced
changes in chloroplast size, the relative amount of starch was
significantly increased. In contrast to clone B, ozone-fumigated saplings of clone C had significantly narrower chloroplasts and less starch than control saplings on August 2
(Table 3). The number of large grana in ozone-treated saplings
decreased significantly on July 12 and August 2 in clone B and
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PÄÄKKÖNEN ET AL.
Table 2. Effects of ozone on growth parameters of 2-year-old seedlings ofBetula pendula, clones B and C. Height growth indicates growth since date
of planting on May 10. Values are means ± SE. Data are pooled for all measurement dates, when final ozone effects as a percent increaseor decrease
compared to control saplings are presented. Student’s t-test: ** = significant at the 1% level, *** = significant at the 0.1% level, n = 10.
Date
Clone B
Clone C
Control
Ozone
Control
Ozone
Height growth, cm
July 12
July 27
August 9
August 23
Final effect
15.2 ± 2.0
27.6 ± 3.6
32.4 ± 4.1
32.9 ± 4.9
18.2 ± 1.5
29.1 ± 2.9
34.4 ± 3.4
36.9 ± 4.4
+10.0%
19.3 ± 2.9
36.3 ± 2.7
42.8 ± 3.0
43.8 ± 3.2
27.1 ± 5.5
41.3 ± 4.7
49.1 ± 4.6
53.4 ± 4.9
+19.9%
Number of leaves
July 12
July 27
August 9
August 23
Final effect
72 ± 3
101 ± 5
91 ± 5
75 ± 5
79 ± 5
112 ± 7
86 ± 5
84 ± 7
+6.3%
92 ± 7
128 ± 10
120 ± 8
116 ± 10
100 ± 7
129 ± 7
120 ± 7
115 ± 4
+1.7%
Individual leaf size, cm2
July 12
July 27
August 9
August 23
Final effect
15.3 ± 0.9
31.5 ± 1.6
37.3 ± 1.7
21.1 ± 0.8
20.5 ± 1.3**
20.3 ± 1.8***
29.4 ± 1.1**
20.9 ± 1.0
−13.3%
16.2 ± 0.8
21.0 ± 1.1
30.5 ± 1.7
20.9 ± 0.5
15.4 ± 1.7
20.5 ± 0.9
29.8 ± 1.6
19.7 ± 0.9
−3.6%
Foliage area, cm2
July 12
July 27
August 9
August 23
Final effect
1102 ± 126
3182 ± 236
3390 ± 201
1583 ± 114
1620 ± 95**
2274 ± 229**
2528 ± 202**
1756 ± 143
−11.7%
1490 ± 146
2688 ± 324
3660 ± 290
2424 ± 209
1540 ± 138
2645 ± 111
3576 ± 283
2266 ± 145
−2.3%
Stem dry weight, g
Sep 16
22.0 ± 4.2
17.0 ± 3.1*
18.2 ± 2.8
21.5 ± 4.3
Root dry weight, g
Sep 16
13.2 ± 2.5
12.2 ± 3.6
12.5 ± 4.0
14.1 ± 3.6
on August 16 in clone C, followed by a significant increase on
August 16 in clone B and on August 30 in clone C. Ozone
fumigation significantly increased the number of small grana
on August 2 in clone B and on August 16 in clone C, but later
the ozone treatment caused a decreased in the number of small
grana in both clones and the decrease was significant on
August 16 in clone B. On July 12, the ozone treatment significantly reduced the total number of grana and significantly
increased the size of the plastoglobuli of saplings of clone B.
Ozone-fumigated saplings had significantly more plastoglobuli than control saplings from August 16 onward in clone
B and on August 30 in clone C.
In general, abnormal and spherically shaped chloroplasts
were more common in ozone-exposed saplings than in controls
in both clones. Ozone treatment resulted in approximately
10% more injured chloroplasts in clone B saplings than in
clone C saplings during late summer (Table 4). An ozone-induced increase in the density of stroma was observed after
August 2 in clone B, whereas clone C showed slight increases
in stroma density at all sampling dates. Ozone fumigation of
saplings resulted in increased swelling and curling of thyla-
koids in clone B after August 2, but not until August 30 in clone
C. In both clones, disintegration of the mitochondrial matrix
and a reduction of cristae of ozone-fumigated saplings were
occasionally observed throughout the summer. In response to
ozone fumigation, cytoplasmic lipids increased in clone B,
whereas lipid was more abundant in early summer in clone C
(Table 4). Ozone had no effect on the number of microbodies,
the electron density of plastoglobuli or the amount of tannin in
either clone.
Rubisco
Rubisco formed 42--57% of the total soluble protein in birch
leaves and this proportion remained constant during the experiment. There were no statistically significant differences
between the treatments in total Rubisco content in either clone.
However, after July 12, the total amount of Rubisco in ozonetreated seedlings of clone B was consistently lower than in
control saplings (Figure 1a). Ozone exposure changed the
relative proportions of the small and large subunits of Rubisco
in both clones; however, few of the changes were statistically
significant (Table 5).
RESPONSES OF BIRCH TO OZONE
601
Table 3. Effects of ozone on the ultrastructure of spongy mesophyll cells of clones B and C ofBetula pendula. Values are means ± SE. Student’s
t-test: * = significant at the 5% level, ** = significant at the 1% level, *** = significant atthe 0.1% level, n = 20.
Date
Clone B
Control
Clone C
Ozone
Control
Ozone
Chloroplast length (mm)
July 12
August 2
August 16
August 30
4.7 ± 0.2
4.9 ± 0.1
4.7 ± 0.2
4.8 ± 0.3
4.9 ± 0.2
4.4 ± 0.2*
4.6 ± 0.2
4.5 ± 0.2
4.7 ± 0.2
5.1 ± 0.2
4.8 ± 0.2
5.2 ± 0.2
4.2 ± 0.2
4.9 ± 0.2
5.2 ± 0.1
5.3 ± 0.2
Chloroplast width (mm)
July 12
August 2
August 16
August 30
2.0 ± 0.1
2.6 ± 0.1
2.2 ± 0.1
3.1 ± 0.1
2.4 ± 0.1*
2.9 ± 0.2
4.8 ± 1.9
3.0 ± 0.1
3.8 ± 1.2
3.1 ± 0.1
3.1 ± 0.2
3.3 ± 0.2
2.3 ± 0.1
2.2 ± 0.1***
2.9 ± 0.2
3.1 ± 0.2
24.2 ± 2.2
18.2 ± 2.8
48.0 ± 3.1
68.9 ± 4.1
42.2 ± 4.3**
37.2 ± 3.4***
46.7 ± 4.1
60.2 ± 3.2
37.2 ± 4.7
56.7 ± 4.4
59.2 ± 5.7
72.1 ± 3.0
48.3 ± 4.0
28.9 ± 0.7***
51.6 ± 4.3
62.8 ± 3.4*
Number of small grana (< 10 thylakoids)/chloroplast cross section
July 12
8.9 ± 0.3
August 2
4.6 ± 1.4
August 16
7.5 ± 0.7
August 30
3.7 ± 0.4
8.5 ± 0.5
9.6 ± 0.9**
5.4 ± 0.4**
3.9 ± 0.4
10.1 ± 1.2
3.9 ± 0.4
4.2 ± 0.6
5.4 ± 0.5
10.1 ± 2.9
5.0 ± 0.4
6.6 ± 0.5**
4.2 ± 0.5
Nunber of large grana (>10 thylakoids)/chloroplast cross section
July 12
1.2 ± 0.2
August 2
4.1 ± 0.5
August 16
0.5 ± 0.2
August 30
0.2 ± 0.1
0.2 ± 0.1***
1.8 ± 0.5**
1.9 ± 0.4**
0.2 ± 0.1
1.8 ± 0.7
5.1 ± 0.7
4.3 ± 0.9
0.1 ± 0.1
1.0 ± 0.3
4.0 ± 0.6
1.0 ± 0.2**
1.9 ± 0.3**
Starch (% of chloroplast area)
July 12
August 2
August 16
August 30
Total number of grana
July 12
August 2
August 16
August 30
10.1 ± 0.4
8.7 ± 1.3
8.0 ± 0.7
3.9 ± 0.5
8.7 ± 0.5*
11.4 ± 0.9
7.2 ± 0.4
4.1 ± 0.4
11.9 ± 0.8
9.0 ± 0.7
8.4 ± 0.6
5.5 ± 0.5
11.0 ± 2.9
9.0 ± 0.5
7.6 ± 0.5
6.1 ± 0.3
Number of plasto-globuli/chloroplast cross section
July 12
4.3 ± 0.5
August 2
5.8 ± 0.5
August 16
2.4 ± 0.4
August 30
3.5 ± 0.8
4.3 ± 0.4
4.7 ± 0.4
4.5 ± 0.6**
6.6 ± 0.8**
5.0 ± 0.7
4.8 ± 0.8
5.5 ± 0.9
2.9 ± 0.5
3.7 ± 0.5
6.5 ± 0.6
4.0 ± 0.6
4.7 ± 0.6*
Size of plastoglobuli (nm)
July 12
August 2
August 16
August 30
143 ± 7***
284 ± 14
413 ± 36
564 ± 27
116 ± 16
230 ± 11
326 ± 21
286 ± 16
109 ± 6
366 ± 10
331 ± 17
380 ± 24
87 ± 8
592 ± 25
203 ± 11
366 ± 26
Chlorophyll content and a/b ratio
After July 12, the total quantities of chlorophyll a and b and
the chlorophyll a/b ratio were lower in ozone-treated saplings
than in control saplings of clone B on most sampling dates
(Figures 1b and 1c). In clone C, there were no consistent
differences in chlorophyll a and b contents or chlorophyll a/b
ratios between treatments (data not shown).
Net photosynthesis and transpiration
In clone B, net photosynthesis declined throughout most of the
experiment and at all times it was lower in ozone-fumigated
saplings than in control saplings (Figure 2a). The average
photosynthetic rate was 1.2 µmol CO2 m −2 s −1 lower in ozonetreated saplings than in control saplings throughout the experimental period. The ozone treatment had little effect on
transpiration rate of saplings of clone B except at the beginning
of the measuring period (August 10) when transpiration rate of
ozone-treated saplings was significantly greater than that of
control saplings (Figure 2b).
In clone C, ozone exposure did not significantly reduce
photosynthetic capacity and transpiration rate was also unaffected throughout the experiment (data not shown).
602
PÄÄKKÖNEN ET AL.
Table 4. Percentage increase or decrease (−) in ultrastructural symptoms in spongy mesophyll cells of clones B and C ofBetula pendula in response
to ozone treatment compared with control saplings (n = 20).
Symptoms
Clone B
Clone C
July 12
August 2
August 16
August 30
July 12
10.3
13.0
4.2
12.8
14.0
4.7
10.0
15.9
14.0
14.5
25.4
16.3
5.2
6.7
6.7
2.5
4.3
5.3
1.7
2.0
5.4
−0.6
4.6
6.6
1.1
1.7
4.6
16.7
17.5
20.0
25.1
25.9
1.0
1.5
1.9
2.5
2.1
3.3
15.0
13.6
Other organelles
Disintegration of
mitochondrial matrix
17.5
Amount of cytoplasmic lipids 3.3
42.6
39.0
18.6
25.0
4.9
25.7
1.2
9.2
19.8
6.5
24.7
3.8
−0.5
3.6
Chloroplast
Abnormal shape
Spherical shape
Stroma density
Thylakoid
Swelling
Curling
Table 5. Effects of ozone on relative amounts (%) of Rubisco small
subunit as a proportion of total Rubisco content in clones B and C of
Betula pendula. Values are means ± SE. Mann-Whitney’s U-test: * =
significant at the 5% level, n = 10.
Date
July 12
July 28
August 9
August 18
August 25
Clone B
Clone C
Control
Ozone
Control
Ozone
26.0 ± 3.2
25.8 ± 2.2
30.2 ± 1.1
27.9 ± 0.9
29.8 ± 2.9
33.8 ± 1.6
23.9 ± 2.2
26.8 ± 1.7
24.8 ± 0.4*
25.4 ± 1.8
26.6 ± 1.3
29.7 ± 2.2
31.0 ± 1.7
26.0 ± 3.2
32.3 ± 2.8
21.5 ± 1.2*
23.1 ± 1.4*
30.1 ± 2.1
30.2 ± 1.0
32.3 ± 2.4
Water use efficiency (WUE)
There were significant differences in water use efficiency
(WUE) between ozone-fumigated saplings and control saplings (Figure 2c). In clone B, ozone caused a reduction in WUE
throughout the experiment and the decreases were significant
on August 10 and September 15. In clone C, significantly
higher WUE was observed in ozone-treated saplings than in
control saplings on August 11, but on other dates WUE was
higher in control saplings than in ozone-treated saplings (data
not shown).
Discussion
Exposure to low ozone concentrations over one growing season resulted in many structural and physiological changes in
the ozone-sensitive clone B. The changes led to enhanced
senescence and reductions in leaf and stem growth that appeared to be related to biochemical and structural changes of
the cellular photosynthetic machinery.
The negative effects of ozone on final stem biomass production in clone B were in accordance with earlier results with this
clone (Pääkkönen et al. 1993), but the initial stimulation of leaf
growth by ozone was not observed in the previous study. The
size of the grana was influenced by fumigation in both clones.
August 2
August 16
August 30
Initially, ozone exposure increased the proportion of small
grana, whereas in the late summer a decrease was observed,
possibly indicating enhanced stacking of thylakoids in response to long-term ozone stress.
There were large differences in ozone-induced ultrastructural changes between the clones (Table 3), which supports
earlier findings (Swanson et al. 1973, Pääkkönen et al. 1993,
1995). Changes in amount of starch and structure of grana
occurred within two weeks of the start of ozone fumigation.
Enhanced stacking of grana under ozone stress occurred two
weeks earlier in clone B than in clone C, and may be associated
with more rapid leaf development and aging processes in clone
B compared with clone C (Pääkkönen et al. 1993, 1995).
Generally, the ozone-induced changes in chloroplast structure paralleled the ozone-induced changes in Rubisco and
chlorophyll contents and net photosynthesis. Concomitant
with the transient stimulation of growth in the ozone-treated
clone B, increases were observed in chloroplast diameter, and
in contents of starch, Rubisco and chlorophyll. Later, in conjunction with the reduced rate of shoot growth of clone B, we
observed chloroplasts that were smaller in diameter, and also
reductions in Rubisco and chlorophyll contents and net photosynthetic rates. However, in clone C, ozone fumigation had no
effect on biomass production, but resulted in smaller diameter
chloroplasts and reduced amounts of starch and the small
subunit of Rubisco. Ozone induced ultrastructural changes,
including abnormal and spherically shaped chloroplasts, swelling and curling of thylakoid membranes, and increased density of stroma were observed in both clones, although the
changes were more evident in the ozone-sensitive clone B than
in the ozone-tolerant clone C.
Lehnherr et al. (1987) and Pell et al. (1992) reported that the
decline in net photosynthesis in ozone-treated wheat and hybrid poplar was correlated with decreased activity and quantity
of Rubisco. Concomitant with lower photosynthesis, lower
Rubisco content and reduced WUE, decreases particularly in
chlorophyll a were observed in clone B. Previously, reduced
WUE in saplings exposed to 2--4-fold higher ozone concentra-
RESPONSES OF BIRCH TO OZONE
Figure 1. Time course of effects of elevated ozone concentrations on
Rubisco content (a); chlorophyll content (b); and chlorophyll a/b ratio
(c) of the ozone-sensitive clone B of Betula pendula (continuous line
= control plants; broken line = ozone-fumigated plants). Each point
represents the mean ± SE of the third leaf of the emerged shoot
collected from five plants per treatment. Student’s t-test, P < 0.05.
tions than in our experiment has been reported, e.g., in birch
by Matyssek et al. (1991) and in hybrid poplar by Reich and
Lassoie (1984). Reduced content of chlorophyll a has been
observed earlier, e.g., in hybrid poplar (Reich 1983), in red
spruce (Rebbeck et al. 1992) and in Norway spruce (Robinson
and Wellburn 1991) after exposure to 2--5 fold higher ozone
concentrations than in our experiment. This suggests that the
reduction in net photosynthesis of the ozone-sensitive clone of
birch could be explained by changes in stomatal action result-
603
Figure 2. Time course of effects of elevated ozone concentrations on
net photosynthesis (a), transpiration rate (b), and water use efficiency
(WUE) (c) of clone B of Betula pendula (in (a) and (b), continuous
line = control plants; broken line = ozone-fumigated plants). Each
point represents the mean value ± SD of the fifth leaf of the emerged
shoot measured on four to six independent plants per treatment. The
value of P according to the Student’s t-test is indicated as follows:
* ≤ 0.05, ∗∗ ≤ 0.01.
ing in reduced WUE, and Rubisco and chlorophyll a contents
of ozone-treated saplings.
The temporal pattern in the quantity of Rubisco protein was
in accordance with the temporal pattern of growth rate of
leaves in the ozone-sensitive clone B. Stimulation of leaf
growth at the beginning of the experiment was accompanied
by increased Rubisco, followed by reduced growth rate and
604
PÄÄKKÖNEN ET AL.
lower Rubisco content. The ozone-induced reduction of the
Rubisco small subunit is in agreement with the recent studies
on potato by Reddy et al. (1993) and by Eckardt and Pell
(1994), where ozone exposure reduced the amount of the
Rubisco small subunit more than that of the large subunit. Pell
et al. (1994) considered that reduced amounts of mRNA for the
small subunit of Rubisco reduced the potential for synthesis of
the Rubisco protein. In hybrid poplar, ozone was found to
increase the loss of large subunits and to result in accumulation
of Rubisco large subunit aggregates (Landry and Pell 1993).
The early decline in Rubisco mRNA immediately after ozone
exposure (Reddy et al. 1993) indicates that ozone may be
capable of directly affecting synthesis of Rubisco. In addition,
Mehta et al. (1992) showed that Rubisco protein is highly
sensitive to oxidative stress in vivo, which affects its translocation and degradation as well as cross-linking of the large
subunit.
We conclude that ozone impaired the photosynthetic capacity of the ozone-sensitive birch clone as a result of multiple
injuries. Chloroplast injuries and reductions in Rubisco content were the main cause of reduced rates of photosynthesis
and leaf and stem growth and accelerated rates of senescencerelated changes in the ozone-sensitive clone. The low ozone
stress treatment had little effect on the structure, physiology
and biochemistry of the chloroplast of the ozone-tolerant birch
clone, which exhibited normal growth and leaf senescence.
The cumulative ozone exposures during the periods between
observations were low (range of SUM40 was 0.00--0.68). The
UN-ECE workshop in Bern concluded that a SUM40 of 10
ppm-h was the critical ozone exposure for trees (Fuhrer and
Achermann, unpublished observations). We conclude that this
ozone exposure threshold is too high, because our evidence
suggests that ozone exposures between 0.0 and 0.68 ppm-h
have more harmful impacts on ozone-sensitive individual trees
and species in northern Europe than in more southerly latitudes
(Beck and Grennfelt 1994).
Acknowledgments
This study was supported by the Maj and Tor Nessling Foundation. We
thank Timo Oksanen for valuable assistance in ozone data collection.
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© 1996 Heron Publishing----Victoria, Canada
Growth responses and related biochemical and ultrastructural changes
of the photosynthetic apparatus in birch ( Betula pendula) saplings
exposed to low concentrations of ozone
E. PÄÄKKÖNEN,1 J. VAHALA,2 T. HOLOPAINEN,1 R. KARJALAINEN2 and
L. KÄRENLAMPI1
1
2
Department of Ecology and Environmental Science, University of Kuopio, P.O. Box 1627, 70211 Kuopio,
Finland
Department of Plant Biology, Plant and Forest Pathology Section, University of Helsinki, 00014 Hels
inki, Finland
Received July 25, 1995
Summary Saplings of ozone-sensitive and ozone-tolerant
birch (Betula pendula Roth.), clones B and C, respectively,
were exposed to ozone concentrations that were 1.7-fold higher
than ambient for one growing season under open-field conditions. Ambient air was used as the control treatment. In the
ozone-sensitive clone B, there was an initial stimulation of leaf
area growth in response to the ozone treatment, but further
ozone exposure caused reductions in leaf and stem biomass
growth, Rubisco and chlorophyll a contents, net photosynthesis, water use efficiency and chloroplast size. It also caused an
alteration in chloroplast shape and injury to thylakoid membranes. In the ozone-tolerant clone C, ozone fumigation did not
affect growth rate, and there were no consistent changes in
chlorophyll content, photosynthesis or water use efficiency.
There were also fewer ultrastructural abnormalities in the
chloroplasts of clone C than of clone B. Based on the observed
biochemical, physiological and structural changes in chloroplasts of clone B in response to low concentrations of ozone,
we conclude that the increasing concentration of tropospheric
ozone represents a risk to natural birch populations.
Keywords: Betula pendula, growth ultrastructure, ozone, photosynthetic machinery.
Introduction
Negative effects of ozone on growth have been reported in
several tree species (e.g., Darrall 1989, Chappelka and
Chevone 1992). However, the mechanisms responsible for
these growth reductions are not completely understood. Impaired photosynthesis, decreased activity and quantity of Rubisco, decreased chlorophyll content and decreased stomatal
conductance are often reported in ozone-stressed plants (e.g.,
Reich 1983, Darrall 1989, Matyssek et al. 1991, Pell et al.
1992). In addition, many symptoms have been observed at the
ultrastructural level, primarily in chloroplasts, in the early
phases of ozone injury (Holopainen et al. 1992, Pääkkönen et
al. 1995).
During leaf aging and senescense, ozone has been reported
to accelerate the normal decline in chlorophyll content and
photosynthesis (e.g., Thomas and Stoddardt 1980, Reich 1983)
and in the activity and quantity of Rubisco (Pell et al. 1992,
Landry and Pell 1993). Decreased content of Rubisco protein
may be caused by inhibition of protein synthesis or enhanced
proteolysis, or both (Landry and Pell 1993, Eckardt and Pell
1994). Structural modification of the Rubisco molecule has
been proposed as an explanation for the decreased activity of
Rubisco preceding proteolytic degradation in ozone-stressed
cuttings of hybrid poplar (Landry and Pell 1993). It has been
suggested that, during ozone stress, free radicals are generated
that result in increased oxidation of biological macromolecules leading to structural modifications of Rubisco and chlorophyll (Pell and Dann 1991, Landry and Pell 1993).
The susceptibility of plants to ozone attack seems to depend
on the developmental stage of the leaves (Pell et al. 1992,
Eckardt and Pell 1994). In hybrid poplar, for example, the
deleterious effects of ozone on Rubisco were most apparent at
full leaf expansion, whereas during the early developmental
stages, the leaf was capable of synthesizing new enzyme and
therefore partially able to replace the ozone-damaged Rubisco
(Pell et al. 1992). Similarly, in potato, exposure to ozone
resulted in a significant decline in Rubisco of mature leaves,
whereas immature leaves were unaffected (Eckardt and Pell
1994). Chronic ozone exposure throughout the life span of the
leaves was necessary for continuous decline in Rubisco in
hybrid poplar and radish (Pell et al. 1992) and potato (Eckardt
and Pell 1994).
Physiological, anatomical and ultrastructural studies of five
birch clones have revealed that clones differ in their sensitivity
to ozone (Pääkkönen et al. 1993, 1995). In the most ozone-sensitive clone B, exposure to ozone resulted in decreased height
growth and leaf biomass, leaf chlorosis and vein yellowing and
increased numbers of necrotic flecks on the leaves. In the most
598
PÄÄKKÖNEN ET AL.
ozone-tolerant clone C, exposure to ozone had no effect on
height growth or the amount of visible foliar injury (Pääkkönen et al. 1993). However, ozone induced ultrastructural
injuries in leaf mesophyll cells of both clones, including
abnormal chloroplast morphology, dense stroma, curling and
swelling of thylakoids, increased disintegration of the mitochondrial matrix, a reduction of cristae, decreased amounts of
tannin and increased amounts of cytoplasmic lipids (Pääkkönen et al. 1995). These changes were regarded as signs of
accelerated senescence, because similar changes occurred
more slowly during normal leaf aging.
This study was undertaken to determine whether ozone-induced responses in stem and leaf growth and ultrastructural
changes in chloroplasts are related to the functioning of the
photosynthetic apparatus in two birch clones (B and C) showing different susceptibilities to ozone. The experiment was
carried out in an open-air fumigation system in an attempt to
provide realistic information about the extent of ozone effects
under field conditions.
Materials and methods
Plant material
Two-year-old saplings of clones KL-5-M (B) and KL-2-M (C)
of European white birch (Betula pendula Roth.) were planted
individually in 7.5-liter pots filled with fertilized sphagnum
peat and garden soil (10/1, v/v). The saplings were watered as
needed and fertilized weekly with 0.2% (19,5,20 N,K,P) nutrient solution.
Ozone fumigation
The saplings were transferred to the open-air exposure field at
Kuopio Botanical Garden (62°13′ N, 27°13′ E) in central
Finland on May 12, 1993. Ten saplings per clone per treatment
were arranged in two blocks per treatment. The control saplings were grown in ambient air, whereas the ozone-treated
saplings were fumigated continuously with ozone concentrations that were 1.7-fold higher than ambient air. The seedlings
were exposed to ozone in the natural microclimate. The computer-controlled release of ozone, which was generated from
pure oxygen, was from perforated tubes in up-wind positions.
The ozone concentration was maintained continuously as a
constant multiple of the ambient concentration, to mimic the
natural hourly and seasonal variation. The fumigation system
has been described in detail by Wulff et al. (1992) and Pääkkönen et al. (1993). The cumulative ozone exposures (ppm-h)
for the periods between the sampling dates for Rubisco analysis are given in Table 1. The starting date for the biochemical
and ultrastructural studies was June 28, when the leaves under
investigation emerged.
Growth measurements
Immediately after planting, the height of each sapling was
determined. All the seedlings were measured for height, number of leaves, individual leaf size (mean) and foliage area (total
leaf area) on July 12, July 27, August 9 and August 23. Foliage
area was estimated by multiplying the number of leaves by the
mean outline area of the individual leaves, measured from 10
average-sized leaves per sapling. On September 13, dry
weights for stem and roots were determined.
Electron microscopy studies
For the ultrastructural studies, leaf samples were collected on
July 12, August 2, August 16 and August 30 from each sapling
(10 saplings per clone per treatment). The samples were taken
from among the leaves that emerged in the last week of June
by cutting 5-mm strips each 1--2 cm in length from the leaf
apex. These leaf strips were immediately placed in a fixative
solution of 2.5% (v/v) glutaraldehyde in phosphate buffer
(0.1 M, pH 7.0). In the laboratory, 1.5 mm2 square pieces were
cut with a razor blade from the strips maintained under a drop
of 2.5% glutaraldehyde fixative. Leaf pieces were postfixed in
1% buffered OsO4 solution, dehydrated in an ethanol series
and embedded in LX 112 epon. Thin sections were stained
with uranyl acetate and lead citrate and were examined by
means of a JEOL 1200 EX electron microscope operating at
80 kV.
Twenty randomly selected cross sections of chloroplast palisade and spongy mesophyll cells per clone and treatment were
photographed. The length and width of chloroplasts, the number of small (< 10 thylakoids) and large (> 10 thylakoids)
grana, and the number and size of plastoglobuli were measured
from the photographs. The proportion of the chloroplast area
occupied by starch grains was calculated by a point analysis
method, where a cross-hatched grid with random points was
positioned over the photograph and the points within starch
grains were counted to determine the relative area of starch in
relation to chloroplast area. The changes in chloroplast shape,
density of stroma and appearance of thylakoids and mitochondrial matrix were determined by classifying the twenty photographed chloroplasts and cells per tissue per sample into injury
classes 0--3 (0 = not injured, 1 = slightly injured, 2 = clearly
injured, 3 = severely injured). The amounts of tannin, cytoplasmic lipids and microbodies were classified by visual observation, and the electron density of plastoglobuli was determined.
Biochemical analyses
Leaves selected for the determination of Rubisco protein and
chlorophyll were taken from the same position on the sapling
as leaves used for the electron microscopy assays. Leaf samples were collected on July 12, July 28, August 9 and August 25 from five saplings per clone per treatment. After
excision, the samples were weighed, and the outlines of the
leaves drawn on plastic film in the field. The final leaf areas
were determined from the plastic films with an LI-3000-A
portable area meter (Li-Cor, Inc., Lincoln, NE).
A crude leaf extract was prepared by grinding 0.1 g of frozen
leaf tissue in liquid nitrogen. Cold extraction buffer was added
to the fine leaf powder according to Gezelius and Hallen
(1980), except that Tween 80 was replaced with Tween 20.
Rubisco concentrations in the leaf were determined by PAGE
according to Rintamäki et al. (1988). Each gel contained a
purified spinach Rubisco standard. The total Rubisco content
RESPONSES OF BIRCH TO OZONE
599
Table 1. Cumulative ozone exposures (ppm-h) of the studied leaves. SUM00 accumulates all 1-h average concentrations and SUM40 accumulates
those above the 40 ppb base-line.
Period
Ambient ozone
Elevated ozone
Nighttime
Daytime
Night + Day
Nighttime
Daytime
Night + Day
SUM00
June 28--July 12
July 13--July 28
July 29--August 9
August 10--August 18
August 19--August 25
Total SUM00
1.12
1.28
1.16
1.52
0.46
4.30
4.23
3.50
1.94
1.16
5.43
5.51
4.66
3.46
1.62
20.68
1.89
1.93
1.81
2.51
0.71
7.32
6.91
6.36
3.45
2.15
9.21
8.85
8.17
5.96
2.86
35.05
SUM40
June28--July 12
July 13--July 28
July 29--August 9
August 10--August 18
August 19--August 25
Total SUM40
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.07
0.28
0.00
0.44
0.20
0.61
0.12
0.00
0.44
0.20
0.68
0.40
0.00
1.72
was determined by scanning the gels and measuring the areas
and intensities with the Pharmacia ImageMaster program (Version 1.0). For analysis of Rubisco small and large subunits,
SDS-PAGE analyses (Laemmli 1970) were performed on each
sample and the concentrations of large and small subunits were
determined as for the PAGE gels. The concentrations of chlorophyll a and b were determined by Arnon’s (1949) method.
Rubisco and the chlorophyll concentrations were calculated on
a fresh weight basis.
Gas exchange measurements
Net photosynthesis and transpiration measurements were carried out with a closed-loop Li-Cor LI-6200 portable photosynthesis system. The fifth emergent leaf was selected for the
measurements of gas exchange from 4--6 saplings per clone
per treatment. Field measurements were made in saturating
sunlight, over 900 µmol m −2 s −1 (PFD). Supplementary halogen light (Sylvania professional, FTY/50W/8o) was used
whenever natural irradiance was below 900 µmol m −2 s −1.
Measurements were taken on the same leaves on six dates
between August 3 and September 15.
Statistical methods
No significant block effects within the ozone and control
treatments were revealed by ANOVA, and therefore data for
these two blocks were pooled for further analysis. The Student’s t-test was used to test the differences in growth, parametric ultrastructural characteristics, Rubisco and chlorophyll
contents, photosynthesis, transpiration and WUE between
ozone-treated and control saplings. Differences in the relative
amounts of Rubisco subunits between treatments were tested
by the nonparametric Mann-Whitney U-test. Differences were
considered significant at P < 0.05.
Results
Growth responses
Ozone fumigation had no significant effect on any of the
growth parameters measured in saplings of clone C (Table 2).
For saplings of clone B, mean leaf size and foliage area were
significantly larger in saplings exposed to ozone compared
with control saplings on July 1, but on July 27 and August 9,
mean leaf size and foliage area were significantly smaller in
ozone-fumigated saplings than in control saplings (Table 2).
Based on data pooled for all dates, the ozone-induced decrease
in leaf area production was almost 10% greater in clone B than
in clone C (Table 2). Ozone-fumigated saplings of clone B had
significantly less (22.7%) stem dry weight than control saplings, whereas root dry weight was unaffected by the ozone
treatment (Table 2).
Ultrastructural changes
The responses of palisade and spongy mesophyll cells to ozone
were qualitatively similar, although some ozone-induced
changes were more pronounced in spongy mesophyll cells.
Ozone-induced ultrastructural changes were most evident in
the ozone-sensitive clone B in late summer, but several ozoneinduced structural injuries were observed in the ozone-tolerant
clone C.
In clone B, the width of chloroplasts was significantly
greater in ozone-exposed saplings than in control saplings on
July 12, whereas the length of chloroplasts was significantly
less in ozone-exposed saplings than in control saplings on
August 2 (Table 3). Accompanying these ozone-induced
changes in chloroplast size, the relative amount of starch was
significantly increased. In contrast to clone B, ozone-fumigated saplings of clone C had significantly narrower chloroplasts and less starch than control saplings on August 2
(Table 3). The number of large grana in ozone-treated saplings
decreased significantly on July 12 and August 2 in clone B and
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PÄÄKKÖNEN ET AL.
Table 2. Effects of ozone on growth parameters of 2-year-old seedlings ofBetula pendula, clones B and C. Height growth indicates growth since date
of planting on May 10. Values are means ± SE. Data are pooled for all measurement dates, when final ozone effects as a percent increaseor decrease
compared to control saplings are presented. Student’s t-test: ** = significant at the 1% level, *** = significant at the 0.1% level, n = 10.
Date
Clone B
Clone C
Control
Ozone
Control
Ozone
Height growth, cm
July 12
July 27
August 9
August 23
Final effect
15.2 ± 2.0
27.6 ± 3.6
32.4 ± 4.1
32.9 ± 4.9
18.2 ± 1.5
29.1 ± 2.9
34.4 ± 3.4
36.9 ± 4.4
+10.0%
19.3 ± 2.9
36.3 ± 2.7
42.8 ± 3.0
43.8 ± 3.2
27.1 ± 5.5
41.3 ± 4.7
49.1 ± 4.6
53.4 ± 4.9
+19.9%
Number of leaves
July 12
July 27
August 9
August 23
Final effect
72 ± 3
101 ± 5
91 ± 5
75 ± 5
79 ± 5
112 ± 7
86 ± 5
84 ± 7
+6.3%
92 ± 7
128 ± 10
120 ± 8
116 ± 10
100 ± 7
129 ± 7
120 ± 7
115 ± 4
+1.7%
Individual leaf size, cm2
July 12
July 27
August 9
August 23
Final effect
15.3 ± 0.9
31.5 ± 1.6
37.3 ± 1.7
21.1 ± 0.8
20.5 ± 1.3**
20.3 ± 1.8***
29.4 ± 1.1**
20.9 ± 1.0
−13.3%
16.2 ± 0.8
21.0 ± 1.1
30.5 ± 1.7
20.9 ± 0.5
15.4 ± 1.7
20.5 ± 0.9
29.8 ± 1.6
19.7 ± 0.9
−3.6%
Foliage area, cm2
July 12
July 27
August 9
August 23
Final effect
1102 ± 126
3182 ± 236
3390 ± 201
1583 ± 114
1620 ± 95**
2274 ± 229**
2528 ± 202**
1756 ± 143
−11.7%
1490 ± 146
2688 ± 324
3660 ± 290
2424 ± 209
1540 ± 138
2645 ± 111
3576 ± 283
2266 ± 145
−2.3%
Stem dry weight, g
Sep 16
22.0 ± 4.2
17.0 ± 3.1*
18.2 ± 2.8
21.5 ± 4.3
Root dry weight, g
Sep 16
13.2 ± 2.5
12.2 ± 3.6
12.5 ± 4.0
14.1 ± 3.6
on August 16 in clone C, followed by a significant increase on
August 16 in clone B and on August 30 in clone C. Ozone
fumigation significantly increased the number of small grana
on August 2 in clone B and on August 16 in clone C, but later
the ozone treatment caused a decreased in the number of small
grana in both clones and the decrease was significant on
August 16 in clone B. On July 12, the ozone treatment significantly reduced the total number of grana and significantly
increased the size of the plastoglobuli of saplings of clone B.
Ozone-fumigated saplings had significantly more plastoglobuli than control saplings from August 16 onward in clone
B and on August 30 in clone C.
In general, abnormal and spherically shaped chloroplasts
were more common in ozone-exposed saplings than in controls
in both clones. Ozone treatment resulted in approximately
10% more injured chloroplasts in clone B saplings than in
clone C saplings during late summer (Table 4). An ozone-induced increase in the density of stroma was observed after
August 2 in clone B, whereas clone C showed slight increases
in stroma density at all sampling dates. Ozone fumigation of
saplings resulted in increased swelling and curling of thyla-
koids in clone B after August 2, but not until August 30 in clone
C. In both clones, disintegration of the mitochondrial matrix
and a reduction of cristae of ozone-fumigated saplings were
occasionally observed throughout the summer. In response to
ozone fumigation, cytoplasmic lipids increased in clone B,
whereas lipid was more abundant in early summer in clone C
(Table 4). Ozone had no effect on the number of microbodies,
the electron density of plastoglobuli or the amount of tannin in
either clone.
Rubisco
Rubisco formed 42--57% of the total soluble protein in birch
leaves and this proportion remained constant during the experiment. There were no statistically significant differences
between the treatments in total Rubisco content in either clone.
However, after July 12, the total amount of Rubisco in ozonetreated seedlings of clone B was consistently lower than in
control saplings (Figure 1a). Ozone exposure changed the
relative proportions of the small and large subunits of Rubisco
in both clones; however, few of the changes were statistically
significant (Table 5).
RESPONSES OF BIRCH TO OZONE
601
Table 3. Effects of ozone on the ultrastructure of spongy mesophyll cells of clones B and C ofBetula pendula. Values are means ± SE. Student’s
t-test: * = significant at the 5% level, ** = significant at the 1% level, *** = significant atthe 0.1% level, n = 20.
Date
Clone B
Control
Clone C
Ozone
Control
Ozone
Chloroplast length (mm)
July 12
August 2
August 16
August 30
4.7 ± 0.2
4.9 ± 0.1
4.7 ± 0.2
4.8 ± 0.3
4.9 ± 0.2
4.4 ± 0.2*
4.6 ± 0.2
4.5 ± 0.2
4.7 ± 0.2
5.1 ± 0.2
4.8 ± 0.2
5.2 ± 0.2
4.2 ± 0.2
4.9 ± 0.2
5.2 ± 0.1
5.3 ± 0.2
Chloroplast width (mm)
July 12
August 2
August 16
August 30
2.0 ± 0.1
2.6 ± 0.1
2.2 ± 0.1
3.1 ± 0.1
2.4 ± 0.1*
2.9 ± 0.2
4.8 ± 1.9
3.0 ± 0.1
3.8 ± 1.2
3.1 ± 0.1
3.1 ± 0.2
3.3 ± 0.2
2.3 ± 0.1
2.2 ± 0.1***
2.9 ± 0.2
3.1 ± 0.2
24.2 ± 2.2
18.2 ± 2.8
48.0 ± 3.1
68.9 ± 4.1
42.2 ± 4.3**
37.2 ± 3.4***
46.7 ± 4.1
60.2 ± 3.2
37.2 ± 4.7
56.7 ± 4.4
59.2 ± 5.7
72.1 ± 3.0
48.3 ± 4.0
28.9 ± 0.7***
51.6 ± 4.3
62.8 ± 3.4*
Number of small grana (< 10 thylakoids)/chloroplast cross section
July 12
8.9 ± 0.3
August 2
4.6 ± 1.4
August 16
7.5 ± 0.7
August 30
3.7 ± 0.4
8.5 ± 0.5
9.6 ± 0.9**
5.4 ± 0.4**
3.9 ± 0.4
10.1 ± 1.2
3.9 ± 0.4
4.2 ± 0.6
5.4 ± 0.5
10.1 ± 2.9
5.0 ± 0.4
6.6 ± 0.5**
4.2 ± 0.5
Nunber of large grana (>10 thylakoids)/chloroplast cross section
July 12
1.2 ± 0.2
August 2
4.1 ± 0.5
August 16
0.5 ± 0.2
August 30
0.2 ± 0.1
0.2 ± 0.1***
1.8 ± 0.5**
1.9 ± 0.4**
0.2 ± 0.1
1.8 ± 0.7
5.1 ± 0.7
4.3 ± 0.9
0.1 ± 0.1
1.0 ± 0.3
4.0 ± 0.6
1.0 ± 0.2**
1.9 ± 0.3**
Starch (% of chloroplast area)
July 12
August 2
August 16
August 30
Total number of grana
July 12
August 2
August 16
August 30
10.1 ± 0.4
8.7 ± 1.3
8.0 ± 0.7
3.9 ± 0.5
8.7 ± 0.5*
11.4 ± 0.9
7.2 ± 0.4
4.1 ± 0.4
11.9 ± 0.8
9.0 ± 0.7
8.4 ± 0.6
5.5 ± 0.5
11.0 ± 2.9
9.0 ± 0.5
7.6 ± 0.5
6.1 ± 0.3
Number of plasto-globuli/chloroplast cross section
July 12
4.3 ± 0.5
August 2
5.8 ± 0.5
August 16
2.4 ± 0.4
August 30
3.5 ± 0.8
4.3 ± 0.4
4.7 ± 0.4
4.5 ± 0.6**
6.6 ± 0.8**
5.0 ± 0.7
4.8 ± 0.8
5.5 ± 0.9
2.9 ± 0.5
3.7 ± 0.5
6.5 ± 0.6
4.0 ± 0.6
4.7 ± 0.6*
Size of plastoglobuli (nm)
July 12
August 2
August 16
August 30
143 ± 7***
284 ± 14
413 ± 36
564 ± 27
116 ± 16
230 ± 11
326 ± 21
286 ± 16
109 ± 6
366 ± 10
331 ± 17
380 ± 24
87 ± 8
592 ± 25
203 ± 11
366 ± 26
Chlorophyll content and a/b ratio
After July 12, the total quantities of chlorophyll a and b and
the chlorophyll a/b ratio were lower in ozone-treated saplings
than in control saplings of clone B on most sampling dates
(Figures 1b and 1c). In clone C, there were no consistent
differences in chlorophyll a and b contents or chlorophyll a/b
ratios between treatments (data not shown).
Net photosynthesis and transpiration
In clone B, net photosynthesis declined throughout most of the
experiment and at all times it was lower in ozone-fumigated
saplings than in control saplings (Figure 2a). The average
photosynthetic rate was 1.2 µmol CO2 m −2 s −1 lower in ozonetreated saplings than in control saplings throughout the experimental period. The ozone treatment had little effect on
transpiration rate of saplings of clone B except at the beginning
of the measuring period (August 10) when transpiration rate of
ozone-treated saplings was significantly greater than that of
control saplings (Figure 2b).
In clone C, ozone exposure did not significantly reduce
photosynthetic capacity and transpiration rate was also unaffected throughout the experiment (data not shown).
602
PÄÄKKÖNEN ET AL.
Table 4. Percentage increase or decrease (−) in ultrastructural symptoms in spongy mesophyll cells of clones B and C ofBetula pendula in response
to ozone treatment compared with control saplings (n = 20).
Symptoms
Clone B
Clone C
July 12
August 2
August 16
August 30
July 12
10.3
13.0
4.2
12.8
14.0
4.7
10.0
15.9
14.0
14.5
25.4
16.3
5.2
6.7
6.7
2.5
4.3
5.3
1.7
2.0
5.4
−0.6
4.6
6.6
1.1
1.7
4.6
16.7
17.5
20.0
25.1
25.9
1.0
1.5
1.9
2.5
2.1
3.3
15.0
13.6
Other organelles
Disintegration of
mitochondrial matrix
17.5
Amount of cytoplasmic lipids 3.3
42.6
39.0
18.6
25.0
4.9
25.7
1.2
9.2
19.8
6.5
24.7
3.8
−0.5
3.6
Chloroplast
Abnormal shape
Spherical shape
Stroma density
Thylakoid
Swelling
Curling
Table 5. Effects of ozone on relative amounts (%) of Rubisco small
subunit as a proportion of total Rubisco content in clones B and C of
Betula pendula. Values are means ± SE. Mann-Whitney’s U-test: * =
significant at the 5% level, n = 10.
Date
July 12
July 28
August 9
August 18
August 25
Clone B
Clone C
Control
Ozone
Control
Ozone
26.0 ± 3.2
25.8 ± 2.2
30.2 ± 1.1
27.9 ± 0.9
29.8 ± 2.9
33.8 ± 1.6
23.9 ± 2.2
26.8 ± 1.7
24.8 ± 0.4*
25.4 ± 1.8
26.6 ± 1.3
29.7 ± 2.2
31.0 ± 1.7
26.0 ± 3.2
32.3 ± 2.8
21.5 ± 1.2*
23.1 ± 1.4*
30.1 ± 2.1
30.2 ± 1.0
32.3 ± 2.4
Water use efficiency (WUE)
There were significant differences in water use efficiency
(WUE) between ozone-fumigated saplings and control saplings (Figure 2c). In clone B, ozone caused a reduction in WUE
throughout the experiment and the decreases were significant
on August 10 and September 15. In clone C, significantly
higher WUE was observed in ozone-treated saplings than in
control saplings on August 11, but on other dates WUE was
higher in control saplings than in ozone-treated saplings (data
not shown).
Discussion
Exposure to low ozone concentrations over one growing season resulted in many structural and physiological changes in
the ozone-sensitive clone B. The changes led to enhanced
senescence and reductions in leaf and stem growth that appeared to be related to biochemical and structural changes of
the cellular photosynthetic machinery.
The negative effects of ozone on final stem biomass production in clone B were in accordance with earlier results with this
clone (Pääkkönen et al. 1993), but the initial stimulation of leaf
growth by ozone was not observed in the previous study. The
size of the grana was influenced by fumigation in both clones.
August 2
August 16
August 30
Initially, ozone exposure increased the proportion of small
grana, whereas in the late summer a decrease was observed,
possibly indicating enhanced stacking of thylakoids in response to long-term ozone stress.
There were large differences in ozone-induced ultrastructural changes between the clones (Table 3), which supports
earlier findings (Swanson et al. 1973, Pääkkönen et al. 1993,
1995). Changes in amount of starch and structure of grana
occurred within two weeks of the start of ozone fumigation.
Enhanced stacking of grana under ozone stress occurred two
weeks earlier in clone B than in clone C, and may be associated
with more rapid leaf development and aging processes in clone
B compared with clone C (Pääkkönen et al. 1993, 1995).
Generally, the ozone-induced changes in chloroplast structure paralleled the ozone-induced changes in Rubisco and
chlorophyll contents and net photosynthesis. Concomitant
with the transient stimulation of growth in the ozone-treated
clone B, increases were observed in chloroplast diameter, and
in contents of starch, Rubisco and chlorophyll. Later, in conjunction with the reduced rate of shoot growth of clone B, we
observed chloroplasts that were smaller in diameter, and also
reductions in Rubisco and chlorophyll contents and net photosynthetic rates. However, in clone C, ozone fumigation had no
effect on biomass production, but resulted in smaller diameter
chloroplasts and reduced amounts of starch and the small
subunit of Rubisco. Ozone induced ultrastructural changes,
including abnormal and spherically shaped chloroplasts, swelling and curling of thylakoid membranes, and increased density of stroma were observed in both clones, although the
changes were more evident in the ozone-sensitive clone B than
in the ozone-tolerant clone C.
Lehnherr et al. (1987) and Pell et al. (1992) reported that the
decline in net photosynthesis in ozone-treated wheat and hybrid poplar was correlated with decreased activity and quantity
of Rubisco. Concomitant with lower photosynthesis, lower
Rubisco content and reduced WUE, decreases particularly in
chlorophyll a were observed in clone B. Previously, reduced
WUE in saplings exposed to 2--4-fold higher ozone concentra-
RESPONSES OF BIRCH TO OZONE
Figure 1. Time course of effects of elevated ozone concentrations on
Rubisco content (a); chlorophyll content (b); and chlorophyll a/b ratio
(c) of the ozone-sensitive clone B of Betula pendula (continuous line
= control plants; broken line = ozone-fumigated plants). Each point
represents the mean ± SE of the third leaf of the emerged shoot
collected from five plants per treatment. Student’s t-test, P < 0.05.
tions than in our experiment has been reported, e.g., in birch
by Matyssek et al. (1991) and in hybrid poplar by Reich and
Lassoie (1984). Reduced content of chlorophyll a has been
observed earlier, e.g., in hybrid poplar (Reich 1983), in red
spruce (Rebbeck et al. 1992) and in Norway spruce (Robinson
and Wellburn 1991) after exposure to 2--5 fold higher ozone
concentrations than in our experiment. This suggests that the
reduction in net photosynthesis of the ozone-sensitive clone of
birch could be explained by changes in stomatal action result-
603
Figure 2. Time course of effects of elevated ozone concentrations on
net photosynthesis (a), transpiration rate (b), and water use efficiency
(WUE) (c) of clone B of Betula pendula (in (a) and (b), continuous
line = control plants; broken line = ozone-fumigated plants). Each
point represents the mean value ± SD of the fifth leaf of the emerged
shoot measured on four to six independent plants per treatment. The
value of P according to the Student’s t-test is indicated as follows:
* ≤ 0.05, ∗∗ ≤ 0.01.
ing in reduced WUE, and Rubisco and chlorophyll a contents
of ozone-treated saplings.
The temporal pattern in the quantity of Rubisco protein was
in accordance with the temporal pattern of growth rate of
leaves in the ozone-sensitive clone B. Stimulation of leaf
growth at the beginning of the experiment was accompanied
by increased Rubisco, followed by reduced growth rate and
604
PÄÄKKÖNEN ET AL.
lower Rubisco content. The ozone-induced reduction of the
Rubisco small subunit is in agreement with the recent studies
on potato by Reddy et al. (1993) and by Eckardt and Pell
(1994), where ozone exposure reduced the amount of the
Rubisco small subunit more than that of the large subunit. Pell
et al. (1994) considered that reduced amounts of mRNA for the
small subunit of Rubisco reduced the potential for synthesis of
the Rubisco protein. In hybrid poplar, ozone was found to
increase the loss of large subunits and to result in accumulation
of Rubisco large subunit aggregates (Landry and Pell 1993).
The early decline in Rubisco mRNA immediately after ozone
exposure (Reddy et al. 1993) indicates that ozone may be
capable of directly affecting synthesis of Rubisco. In addition,
Mehta et al. (1992) showed that Rubisco protein is highly
sensitive to oxidative stress in vivo, which affects its translocation and degradation as well as cross-linking of the large
subunit.
We conclude that ozone impaired the photosynthetic capacity of the ozone-sensitive birch clone as a result of multiple
injuries. Chloroplast injuries and reductions in Rubisco content were the main cause of reduced rates of photosynthesis
and leaf and stem growth and accelerated rates of senescencerelated changes in the ozone-sensitive clone. The low ozone
stress treatment had little effect on the structure, physiology
and biochemistry of the chloroplast of the ozone-tolerant birch
clone, which exhibited normal growth and leaf senescence.
The cumulative ozone exposures during the periods between
observations were low (range of SUM40 was 0.00--0.68). The
UN-ECE workshop in Bern concluded that a SUM40 of 10
ppm-h was the critical ozone exposure for trees (Fuhrer and
Achermann, unpublished observations). We conclude that this
ozone exposure threshold is too high, because our evidence
suggests that ozone exposures between 0.0 and 0.68 ppm-h
have more harmful impacts on ozone-sensitive individual trees
and species in northern Europe than in more southerly latitudes
(Beck and Grennfelt 1994).
Acknowledgments
This study was supported by the Maj and Tor Nessling Foundation. We
thank Timo Oksanen for valuable assistance in ozone data collection.
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