Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol15.1995:
Tree Physiology 15, 799--805
© 1995 Heron Publishing----Victoria, Canada
Osmotic adjustment induced by elevated ozone: interactive effects of
acid rain and ozone on water relations of field-grown seedlings and
mature trees of Pinus ponderosa
B. MOMEN1,2 and J. A. HELMS1
1
Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA 94720, USA
2
Present address: MRC, Biology Department, Rensselaer Polytechnic Institute, Troy, New York 12180, USA
Received January 3, 1995
Summary We investigated the effects of simulated acid rain
and elevated ozone on tissue water relations of mature clones
of a fast-growing genotype of ponderosa pine (Pinus ponderosa
Dougl. ex Laws.) and their half-sib seedlings. Whole seedlings
and branches of mature trees were exposed to acid rain (pH 5.1
and 3.0) and ozone (ambient and twice-ambient) treatments in
open-bottomed chambers. The acid rain treatment was applied
to foliage weekly from January to April 1992. The ozone
treatment was applied daily from September 1991 to November
1992. The treatments had little effect on the water relations of
branches of mature trees. In contrast, water relations of seedlings were affected by the treatments, particularly by the twiceambient ozone treatment, and in many instances, there was a
significant acid rain × ozone interaction. A combination of
twice-ambient ozone and pH 3.0 rain lowered seedling solute
potential, turgor loss point and cell-wall elasticity but increased
pressure potential and symplastic water content, whereas total
water content was unchanged. We conclude that twice-ambient
ozone caused osmotic adjustment in seedlings, and the response was magnified by pH 3.0 rain. The long-term consequences of this response are unclear because although osmotic
adjustment and increased pressure potential might be advantageous to seedlings in resisting drought and maintaining growth,
the concurrent decrease in cell-wall elasticity might induce
adverse effects. We note that the effects of elevated ozone on
water relations of ponderosa pine seedlings were similar to the
effects of drought on water relations of many species.
Keywords: branches, cell wall elasticity, drought, pH, ponderosa pine, pressure potential, solute potential, symplastic water
content, turgor loss point.
Introduction
Elevated tropospheric ozone and acid rain are among the air
pollutants that can cause severe damage to terrestrial and
aquatic ecosystems (Miller et al. 1977, Hileman 1984, Chappelka et al. 1985, Reich et al. 1986, Pye et al. 1988, USNAPAP
1991). It has been hypothesized that acid rain enters plant cells
through the cuticle of the leaf surface, alters the permeability
of cell membranes, and produces osmotically active anions
leading to decreased cell solute potential (Heath 1980). Ozone
has been reported to cause a decrease in relative water content
(Evans and Ting 1974), no significant changes in total water
potential, solute potential and pressure potential of beech seedlings (Taylor and Dobson 1989), a temporary increase in cell
solute potential of water-stressed Norway spruce (Picea abies
(L.) Karst.) seedlings (Dobson et al. 1990), an increase in total
water potential of cotton (Temple 1990), and a decrease in total
water potential of orange trees (Olszyk et al. 1991). The conflicting results obtained from studies of the effects of ozone on
plant water relations may be related to the limited number of
water relations components examined or to the use of shortterm exposure to ozone under laboratory conditions, or both.
We investigated the long-term interactive effects of simulated acid rain and twice-ambient ozone on total water potential (Ψw), solute potential (Ψs), pressure potential (Ψp), turgor
loss point (TLP), bulk modulus of elasticity (ε), and total water
content (Vt ) and its osmotically active portion, symplastic
water content (Vs), of foliage of mature clones of ponderosa
pine (Pinus ponderosa Dougl. ex Laws.) and their half-sib
seedlings growing under field conditions.
Material and methods
Site
Field studies were conducted at the USDA Forest Service
Chico Tree Improvement Center (CTIC) in Chico, CA. The site
is located at the lower elevation of the natural range of ponderosa pine. The climate is Mediterranean with hot dry summers
and mild wet winters.
Plant material
Plants consisted of field-grown, grafted mature trees and corresponding half-sib seedlings of ponderosa pine, Clone 3087.
This clone was selected by the USDA Forest Service in the
mid-1970s to produce seed for future plantations in California.
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MOMEN AND HELMS
In the mid-1970s, scions were collected from a 70-year-old
tree growing at about 1200 m elevation on the El Dorado
National Forest in the central Sierra Nevada (USDA-FS Seed
Zone 526). Scions were grafted onto 3-year-old root stocks of
unknown genetic origin and transplanted to a seed orchard in
1980. In 1990, platforms were constructed around the grafted
trees (6- to 7-m tall) to facilitate access. In 1992, at the time of
experimental measurements, the grafted trees possessed morphological characteristics of 80-year-old mature trees as indicated by branch diameter, branch angle, needle length, bark
color and cone production.
Seed was collected at the same time as scions from the same
parent tree, stored until April 1989, and then grown in containers in a greenhouse. In February 1990, seedlings were transplanted to a site adjacent to the grafted mature trees.
After transplanting, both mature trees and seedlings were
watered every other week from May to October of both 1991
and 1992, and competing herbaceous species were removed
regularly.
Acid rain and ozone treatments
Air pollution treatments were applied in branch exposure
chambers (Houpis et al. 1991), which were installed in September 1991. A single chamber covered 3 years of annual
shoot growth of an individual branch of a mature tree, whereas
each seedling was entirely covered by a single chamber. Chambers were open-bottomed, 1.5 m long × 0.7 m diameter, and
were made of 5-mm Teflon sheeting supported by an aluminum frame covered by nonreactive Teflon tape. Teflon was
used because it is transparent to longwave heat radiation, does
not alter light quality, and is nonreactive to ozone. The chamber included a mixing region at the top, a main exposure region
in the middle, and an exhaust frustum at the bottom. A fan
connected to the mixing region at the top of the chamber
circulated ambient or ozonated air downward and prevented
variations in chamber temperature.
Treatments consisted of four combinations of two ozone
(ambient and twice-ambient) and two rain (pH 5.1 and 3.0)
regimes: (1) pH 5.1 rain and ambient ozone (pH5.1 + Oamb ), (2)
pH 5.1 rain and twice-ambient ozone (pH5.1 + Otwice), (3) pH
3.0 rain and ambient ozone (pH3.0 + Oamb ), and (4) pH 3.0 rain
and twice-ambient ozone (pH3.0 + Otwice). Because the pH of
natural rain in Sierra Nevada is 5.1 to 5.6, the pH5.1 + Oamb
treatment was considered the control chamber treatment.
Simulated rain regimes were applied weekly during the
normal winter precipitation period from January 17 until April
30, 1992, based on records of Leonard et al. (1981) and on
25-year precipitation records from the University of California
Blodgett Forest Research Station near Georgetown, CA. A
total of 17 rain events, with a deposition of 5 cm per event,
resulted in a total deposition of 85 cm. The rain solutions were
based on a 2/3 (equivalent basis) mix of H2SO4 and HNO3 plus
the kinds and amounts of ions needed to simulate the chemical
composition of normal and acidic rains in northern California
(McColl and Johnson 1983). Rain droplets with diameters of
0.1 to 1.0 mm were produced with a calibrated nozzle mounted
at the top of each branch exposure chamber. The solution
throughfall was collected in containers at the base of each
chamber.
Twice-ambient ozone was applied daily (0600 to 2000 h in
spring and summer, 0800 to 1700 h in fall and winter) from
September 1991 to November 1992. Ozone fumigation was
interrupted for 3 weeks in mid-December 1991 for system
maintenance and calibration, and again for 3 weeks in late June
1992 because of the break-down of the ozone generator. Twiceambient ozone was generated from compressed ambient air by
electrical discharge with an ozone generator (PCI Ozone and
Control Systems Inc., West Caldwell, NJ).
Ambient ozone concentration was monitored every 15 min
and logged by a data acquisition system that calculated the
twice-ambient value and transmitted signals to the ozone generator to produce twice-ambient ozone concentration within
the designated chambers. All fumigation and sample gases
were passed through Teflon tubing. All sampling instruments
were calibrated and serviced according to standard operating
procedures (Houpis et al. 1988). Ozone analyzers (Model
1003, Dasibi Environmental Corp., Glendale, CA) were calibrated against a California Air Resources Board certified transfer standard. Midday ambient ozone concentration increased
from 0.038 ppm in February to 0.058 ppm in June and August,
and then decreased to 0.020 ppm in November 1992. Midday
elevated ozone concentration inside the chambers increased
from 0.075 ppm in February to 0.115 ppm in June and August,
and then decreased to 0.044 ppm in November 1992.
Sampling and measurement techniques
In April, July and August 1992, fully expanded fascicles were
harvested 2 and 3 days after scheduled irrigation for seedlings
and mature trees, respectively. In April and July, 1-year-old
fascicles, and in November, current-year fascicles were examined. Fascicles were collected from the upper part of the
growing segments and were pulled so that a short strand
remained extended from the base. When pressurizing fascicles,
the extended strand prevented resin from obstructing the flow
of sap from the xylem. Samples were harvested at predawn to
prevent confounding effects of diurnal transpiration and kept
on ice in the dark until water relations measurements were
made.
Individual fascicles, each with three needles, were placed
inside two serially connected pressure chambers (Soil Moisture Equipment Corp., Santa Barbara, CA), and pressurized
with nitrogen gas. Bench-drying pressure-volume (P-V) techniques were used to construct P-V curves (Tyree and Hammel
1972, Cheung et al. 1976, Koide et al. 1989). The P-V measurements of all treatment combinations for seedlings or mature
trees were made within 8 h.
Data analysis
For seedlings, a 22 factorial experiment within a completely
randomized design was used with three replicates per treatment combination. To test for chamber effects, three replicates
of nonchambered nontreated seedlings were compared with
seedlings in the control (pH5.1 + Oamb) treatment. For mature
trees, the design slightly differed from that of seedlings and
OSMOTIC ADJUSTMENT IN RESPONSE TO OZONE
801
was a 22 factorial experiment within a split-plot, randomized
complete block setting. Four mature trees were assigned randomly to either of the two rain regimes. On each tree, three
similar branches were selected; two branches were assigned
randomly to either of the two ozone regimes, and the third
branch was used as a nonchambered notreated replicate (total
of four) to test for chamber effects.
The GLM (General Linear Model) procedure with SS III
option of the SAS System (SAS/STAT, SAS Institute Inc.,
Cary, NC) was used to analyze the data. When rain × ozone
interaction was not significant, the main-effect means were
compared. When rain × ozone interaction was significant,
ANOVA was performed on four simple-effect means, and
Student-Newman-Keuls (SNK) test was used for mutiple mean
comparison (Steel and Torrie 1980). Main- or simple-effect
means were declared significant at P < 0.05.
Results
There were no significant differences between the control
(pH5.1 + Oamb ) and nonchambered nontreated treatments for
any water relations components of either seedlings or mature
branches at any measurement date, indicating that the results
were not confounded by chamber effects.
Seedlings
Exposure of seedlings to acid rain and twice-ambient ozone
alone or in combination had no significant effect on predawn
Ψw in April, July or November 1992. Water potential values of
seedlings in all treatments were near maximum at about −0.5
MPa on all measurement dates (Figure 1).
Interactions between rain and ozone effects on Ψs of seedlings were significant in April (P = 0.02) and July (P = 0.006)
(Figure 1). In April, the simple-effect means of Ψs in the pH5.1
+ Oamb and pH5.1 + Otwice treatments were not significantly
different but they both differed (P < 0.05) from the simple-effect means of Ψs in the pH3.0 + Oamb and pH3.0 + Otwice treatments (Figure 1). Seedlings in the pH3.0 + Otwice treatment had
the lowest Ψs, whereas seedlings in the pH3.0 + Oamb treatment
had the highest Ψs. In July, simple-effect means of Ψs in the
pH5.1 + Oamb and pH3.0 + Oamb treatments did not differ significantly but they both differed (P < 0.05) from the simple-effect
means of Ψs in the pH5.1 + Otwice and pH3.0 + Otwice treatments.
The simple-effect mean of Ψs in the pH5.1 + Otwice treatment
was also significantly (P < 0.05) lower than the simple-effect
mean of Ψs in the pH3.0 + Otwice treatment. Because the acid
rain by ozone interaction was not significant in November, we
compared main-effect means for this date. The comparison
showed that Ψs was significantly (P = 0.014) decreased in
seedlings in the twice-ambient ozone treatment.
Acid rain and ozone effects on Ψp of seedlings were of
similar magnitude to those on Ψs but the direction of the
changes was reversed (Figure 1).
Interactions between rain and ozone effects on TLP of seedlings were significant in April (P = 0.026) and July (P = 0.009).
In April, the only significant difference was a lower TLP in
seedlings in the pH3.0 + Otwice treatment compared with seed-
Figure 1. Effects of acid rain and O3 on total water potential (Ψw),
solute potential (Ψs), pressure potential (Ψp), turgor loss point (TLP)
and bulk modulus of elasticity (ε) of ponderosa pine seedlings. Maineffect means are shown when rain × O3 interaction was not significant.
Simple-effect means are shown when rain × O3 interaction was significant. Different letters over bars indicate significant treatment effects
(ns = nonsignificant effects); bars = 1 SE.
lings in the pH3.0 + Oamb treatment (Figure 1). In July, acid rain
had no significant effect on TLP in seedlings exposed to
ambient ozone, whereas the twice-ambient ozone treatment
decreased (P < 0.05) TLP, and the decrease was greater (P <
0.05) in the presence of pH 5.1 rain than in the presence of pH
3.0 rain. In November, we compared main-effect means, because the acid rain × ozone interaction was not significant, and
found that the twice-ambient ozone treatment caused a significant decrease (P = 0.009) in TLP.
In April and November, signifiacnt interactions between rain
and ozone effects were detected on ε (P = 0.001 and 0.007,
respectively). The twice-ambient ozone treatment caused ε to
increase, particularly in seedlings exposed to pH 3.0 rain
(Figure 1). In July, ε was not significantly affected by acid rain
or twice-ambient ozone either alone or in combination.
Acid rain, ozone and a combination of the two treatments
had no significant effects on Vt in April, July or November
(Figure 2). Because there were no significant acid rain × ozone
interactions on Vs, we compared the main-effect means (Figure 2). There was no significant effect of acid rain or twice-ambient ozone on Vs in April. In July, Vs increased (P = 0.004) in
seedlings exposed to twice-ambient ozone, whereas in November, Vs increased in seedlings in both the pH 3.0 rain (P =
0.047) and twice-ambient ozone (P = 0.002) treatments.
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MOMEN AND HELMS
Figure 2. Effects of acid rain and O3 on total water content (Vt ) and
symplastic water (Vs) of ponderosa pine seedlings. Main-effect means
are shown because rain × O3 interaction was not significant. Different
letters over bars show significant treatment effects (ns = nonsignificant
effects); bars = 1 SE.
Mature trees
The only significant effects of acid rain and twice-ambient
ozone on tissue water relations of mature trees were (1) an
increase (P = 0.028) in ε in April caused by the main-effect of
pH 3.0 rain, and (2) rain × ozone interaction (P = 0.038) on ε
in November. There were no significant simple-effect mean
differences in November (Figures 3 and 4).
Discussion
Total water potentials of seedlings were near maximum (−0.5
MPa) throughout the study and, contrary to reports by Taylor
and Dobson (1989), they were not affected by the pollutant
treatments. Similar Ψw values indicate that the potential effects
of water deficit and transpiration were minimized as a result of
regular irrigation and the use of predawn measurements, respectively, and that confounding effects of differences in Ψw
on other water relations components were prevented.
Solute potential of seedlings was decreased by twice-ambient ozone, particularly in the presence of pH 3.0 rain. This
finding is contrary to previous reports (Taylor and Dobson
1989, Dobson et al. 1990) and differs from the expectation that
elevated ozone increases osmotic potential as a result of a
reduction in photosynthesis and, hence, cellular concentration.
Figure 3. Effects of acid rain and O3 on total water potential (Ψw),
solute potential (Ψs), pressure potential (Ψp), turgor loss point (TLP),
and bulk modulus of elasticity (ε) of mature ponderosa pine trees.
Main-effect means are shown when rain × O3 interaction was not
significant. Simple-effect means are shown when rain × O3 interaction
was significant. Different letters over bars show significant treatment
effects (ns = nonsignificant effects); bars = 1 SE.
It may be argued that Ψs decreased as a result of the movement
of N ions from the acid rain to leaf cells through the cuticle, or
that Ψs changed in response to the formation of N2O5 and
HNO3 during generation of ozone from ambient air via electric
discharge (Brown and Roberts 1988). However, we have previously found that foliar N concentration of seedlings and
mature trees either decreased or did not change in response to
elevated ozone or acid rain, respectively (Momen and Helms
1995).
Decreased Ψs might be caused by an increased concentration of osmotically active metabolites in response to an ozoneinduced increase in allocation of photosynthate to foliage at
the expense of decreased root growth. Alteration of carbon
partitioning by elevated ozone has been attributed to disturbed
phloem loading or increased accumulation of starch and sugar
in foliage (Hanson and Stewart 1970, McLaughlin et al. 1982,
Okano et al. 1984, Cooley and Manning 1987, Küppers and
Klumpp 1988, Gorissen and Van Veen 1988, Edwards 1991,
Gorissen et al. 1991, Olszyk et al. 1991, Smeulders et al.
1995). However, in this study, the decreased Ψs is unlikely to
be the result of an ozone-induced increase in the allocation of
photosynthate to foliage because only very small changes in
foliar C concentration occurred in response to elevated ozone
(Momen and Helms 1995). We hypothesize that Ψs was de-
OSMOTIC ADJUSTMENT IN RESPONSE TO OZONE
Figure 4. Effects of acid rain and O3 on total water content (Vt ) and
symplastic water (Vs) of mature ponderosa pine trees. Main-effect
means are shown because rain × O3 interaction was not significant.
Different letters over the bars show significant treatment effects (ns =
nonsignificant effects); bars = 1 SE.
creased by elevated ozone as a result of osmotic adjustment
(Morgan 1984, Salisbury and Ross 1985). Concurrent increases in sugar concentrations and decreases in starch concentrations in response to elevated ozone have been observed in
many studies (Barnes 1972, Koziol 1984, Ito et al. 1985,
Cooley and Manning 1987, Friend and Tomlinson 1992, Kelly
et al. 1993, Smeulders et al. 1995). In our study, osmotic
adjustment in response to elevated ozone is indicated by decreases in Ψs and TLP, and concurrent increases in Ψp and Vs
but unchanged Vt. Because osmotic adjustment facilitates
water extraction and turgor maintenance during drought
(Hsiao et al. 1976, Tyree et al. 1978, Turner and Jones 1980,
Tyree and Jarvis 1982, Morgan 1984, Turner 1986, Momen et
al. 1992, Momen et al. 1994), our findings support the hypothesis that elevated ozone enhances drought resistance of
plants (Temple 1990).
Ozone-induced osmotic adjustment and increases in Ψp
should enhance plant growth; however, many reports suggest
that plant growth is inhibited by elevated ozone. Growth or
plastic (irreversible) cell expansion depends partly on the rate
at which Ψp exceeds a threshold value that controls cell-wall
extensibility (Salisbury and Ross 1985). Our finding that elevated ozone caused an increase in ε may indicate an increase
in the threshold pressure required to permit cell expansion and
hence a decrease in growth rate. Decreased elasticity of guard
803
cell walls may also affect plant growth through changes in
stomatal conductance (Runeckles and Rosen 1977) and water
use efficiency. Therefore, the positive effects of osmotic adjustment and increased Ψp on plant growth might be offset by
the negative effect of decreased cell wall elasticity.
The interactive effects of acid rain and elevated ozone on ε
and Ψs could support the hypothesis (Heath 1980) that elevated
ozone reacts with the plasmalemma and increases the permeability of membranes thereby enhancing the movement of S
and N ions of acid rain into leaf cells leading to decreased Ψs.
However, analysis of foliar chemistry (Momen and Helms
1995) indicated no increase in foliar N concentration of ponderosa pine seedlings or mature trees in response to increased
rain acidity.
The effects of elevated ozone on water relations of ponderosa pine seedlings were similar to the effects of drought in
many species (cf. Chapin 1991). It seems unlikely that elevated
ozone induced water stress in seedlings as a result of decreased
root growth and water uptake, because Ψw of seedlings were
not affected by twice-ambient ozone and were near maximum.
Interactive effects of acid rain and elevated ozone indicate that
the effects of one pollutant depended on the level of the other,
and thus no accurate conclusion can be drawn from single
pollutant studies in regions exposed to multiple pollutants. The
large responses of seedling water relations to pollutants contrasted with those of mature trees and may indicate that the
parent genotype had adapted to environmental stresses. It is
possible that seedlings and mature trees differed in their responses to the treatments because seedlings were entirely
exposed to pollutants whereas mature trees were not, or because the branches of mature trees were not entirely autonomous with regard to plant water relations. However, cellular
damage by elevated ozone is local, and branches of mature
trees have been found autonomous with respect to carbon
transport (Cregg 1990, Mattson et al. 1990, Sprugel et al. 1991)
and water relations (Zimmermann 1983, Tyree 1988).
Acknowledgments
We thank P.D. Anderson and J.L.J. Houpis for their help throughout
the study. We also thank F.S. Chapin and G. Biging for comments on
an earlier version of this paper. This study was supported by the
California Air Resources Board through Contract A132-101 and by the
McIntire-Stennis project 4635-MS.
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© 1995 Heron Publishing----Victoria, Canada
Osmotic adjustment induced by elevated ozone: interactive effects of
acid rain and ozone on water relations of field-grown seedlings and
mature trees of Pinus ponderosa
B. MOMEN1,2 and J. A. HELMS1
1
Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA 94720, USA
2
Present address: MRC, Biology Department, Rensselaer Polytechnic Institute, Troy, New York 12180, USA
Received January 3, 1995
Summary We investigated the effects of simulated acid rain
and elevated ozone on tissue water relations of mature clones
of a fast-growing genotype of ponderosa pine (Pinus ponderosa
Dougl. ex Laws.) and their half-sib seedlings. Whole seedlings
and branches of mature trees were exposed to acid rain (pH 5.1
and 3.0) and ozone (ambient and twice-ambient) treatments in
open-bottomed chambers. The acid rain treatment was applied
to foliage weekly from January to April 1992. The ozone
treatment was applied daily from September 1991 to November
1992. The treatments had little effect on the water relations of
branches of mature trees. In contrast, water relations of seedlings were affected by the treatments, particularly by the twiceambient ozone treatment, and in many instances, there was a
significant acid rain × ozone interaction. A combination of
twice-ambient ozone and pH 3.0 rain lowered seedling solute
potential, turgor loss point and cell-wall elasticity but increased
pressure potential and symplastic water content, whereas total
water content was unchanged. We conclude that twice-ambient
ozone caused osmotic adjustment in seedlings, and the response was magnified by pH 3.0 rain. The long-term consequences of this response are unclear because although osmotic
adjustment and increased pressure potential might be advantageous to seedlings in resisting drought and maintaining growth,
the concurrent decrease in cell-wall elasticity might induce
adverse effects. We note that the effects of elevated ozone on
water relations of ponderosa pine seedlings were similar to the
effects of drought on water relations of many species.
Keywords: branches, cell wall elasticity, drought, pH, ponderosa pine, pressure potential, solute potential, symplastic water
content, turgor loss point.
Introduction
Elevated tropospheric ozone and acid rain are among the air
pollutants that can cause severe damage to terrestrial and
aquatic ecosystems (Miller et al. 1977, Hileman 1984, Chappelka et al. 1985, Reich et al. 1986, Pye et al. 1988, USNAPAP
1991). It has been hypothesized that acid rain enters plant cells
through the cuticle of the leaf surface, alters the permeability
of cell membranes, and produces osmotically active anions
leading to decreased cell solute potential (Heath 1980). Ozone
has been reported to cause a decrease in relative water content
(Evans and Ting 1974), no significant changes in total water
potential, solute potential and pressure potential of beech seedlings (Taylor and Dobson 1989), a temporary increase in cell
solute potential of water-stressed Norway spruce (Picea abies
(L.) Karst.) seedlings (Dobson et al. 1990), an increase in total
water potential of cotton (Temple 1990), and a decrease in total
water potential of orange trees (Olszyk et al. 1991). The conflicting results obtained from studies of the effects of ozone on
plant water relations may be related to the limited number of
water relations components examined or to the use of shortterm exposure to ozone under laboratory conditions, or both.
We investigated the long-term interactive effects of simulated acid rain and twice-ambient ozone on total water potential (Ψw), solute potential (Ψs), pressure potential (Ψp), turgor
loss point (TLP), bulk modulus of elasticity (ε), and total water
content (Vt ) and its osmotically active portion, symplastic
water content (Vs), of foliage of mature clones of ponderosa
pine (Pinus ponderosa Dougl. ex Laws.) and their half-sib
seedlings growing under field conditions.
Material and methods
Site
Field studies were conducted at the USDA Forest Service
Chico Tree Improvement Center (CTIC) in Chico, CA. The site
is located at the lower elevation of the natural range of ponderosa pine. The climate is Mediterranean with hot dry summers
and mild wet winters.
Plant material
Plants consisted of field-grown, grafted mature trees and corresponding half-sib seedlings of ponderosa pine, Clone 3087.
This clone was selected by the USDA Forest Service in the
mid-1970s to produce seed for future plantations in California.
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MOMEN AND HELMS
In the mid-1970s, scions were collected from a 70-year-old
tree growing at about 1200 m elevation on the El Dorado
National Forest in the central Sierra Nevada (USDA-FS Seed
Zone 526). Scions were grafted onto 3-year-old root stocks of
unknown genetic origin and transplanted to a seed orchard in
1980. In 1990, platforms were constructed around the grafted
trees (6- to 7-m tall) to facilitate access. In 1992, at the time of
experimental measurements, the grafted trees possessed morphological characteristics of 80-year-old mature trees as indicated by branch diameter, branch angle, needle length, bark
color and cone production.
Seed was collected at the same time as scions from the same
parent tree, stored until April 1989, and then grown in containers in a greenhouse. In February 1990, seedlings were transplanted to a site adjacent to the grafted mature trees.
After transplanting, both mature trees and seedlings were
watered every other week from May to October of both 1991
and 1992, and competing herbaceous species were removed
regularly.
Acid rain and ozone treatments
Air pollution treatments were applied in branch exposure
chambers (Houpis et al. 1991), which were installed in September 1991. A single chamber covered 3 years of annual
shoot growth of an individual branch of a mature tree, whereas
each seedling was entirely covered by a single chamber. Chambers were open-bottomed, 1.5 m long × 0.7 m diameter, and
were made of 5-mm Teflon sheeting supported by an aluminum frame covered by nonreactive Teflon tape. Teflon was
used because it is transparent to longwave heat radiation, does
not alter light quality, and is nonreactive to ozone. The chamber included a mixing region at the top, a main exposure region
in the middle, and an exhaust frustum at the bottom. A fan
connected to the mixing region at the top of the chamber
circulated ambient or ozonated air downward and prevented
variations in chamber temperature.
Treatments consisted of four combinations of two ozone
(ambient and twice-ambient) and two rain (pH 5.1 and 3.0)
regimes: (1) pH 5.1 rain and ambient ozone (pH5.1 + Oamb ), (2)
pH 5.1 rain and twice-ambient ozone (pH5.1 + Otwice), (3) pH
3.0 rain and ambient ozone (pH3.0 + Oamb ), and (4) pH 3.0 rain
and twice-ambient ozone (pH3.0 + Otwice). Because the pH of
natural rain in Sierra Nevada is 5.1 to 5.6, the pH5.1 + Oamb
treatment was considered the control chamber treatment.
Simulated rain regimes were applied weekly during the
normal winter precipitation period from January 17 until April
30, 1992, based on records of Leonard et al. (1981) and on
25-year precipitation records from the University of California
Blodgett Forest Research Station near Georgetown, CA. A
total of 17 rain events, with a deposition of 5 cm per event,
resulted in a total deposition of 85 cm. The rain solutions were
based on a 2/3 (equivalent basis) mix of H2SO4 and HNO3 plus
the kinds and amounts of ions needed to simulate the chemical
composition of normal and acidic rains in northern California
(McColl and Johnson 1983). Rain droplets with diameters of
0.1 to 1.0 mm were produced with a calibrated nozzle mounted
at the top of each branch exposure chamber. The solution
throughfall was collected in containers at the base of each
chamber.
Twice-ambient ozone was applied daily (0600 to 2000 h in
spring and summer, 0800 to 1700 h in fall and winter) from
September 1991 to November 1992. Ozone fumigation was
interrupted for 3 weeks in mid-December 1991 for system
maintenance and calibration, and again for 3 weeks in late June
1992 because of the break-down of the ozone generator. Twiceambient ozone was generated from compressed ambient air by
electrical discharge with an ozone generator (PCI Ozone and
Control Systems Inc., West Caldwell, NJ).
Ambient ozone concentration was monitored every 15 min
and logged by a data acquisition system that calculated the
twice-ambient value and transmitted signals to the ozone generator to produce twice-ambient ozone concentration within
the designated chambers. All fumigation and sample gases
were passed through Teflon tubing. All sampling instruments
were calibrated and serviced according to standard operating
procedures (Houpis et al. 1988). Ozone analyzers (Model
1003, Dasibi Environmental Corp., Glendale, CA) were calibrated against a California Air Resources Board certified transfer standard. Midday ambient ozone concentration increased
from 0.038 ppm in February to 0.058 ppm in June and August,
and then decreased to 0.020 ppm in November 1992. Midday
elevated ozone concentration inside the chambers increased
from 0.075 ppm in February to 0.115 ppm in June and August,
and then decreased to 0.044 ppm in November 1992.
Sampling and measurement techniques
In April, July and August 1992, fully expanded fascicles were
harvested 2 and 3 days after scheduled irrigation for seedlings
and mature trees, respectively. In April and July, 1-year-old
fascicles, and in November, current-year fascicles were examined. Fascicles were collected from the upper part of the
growing segments and were pulled so that a short strand
remained extended from the base. When pressurizing fascicles,
the extended strand prevented resin from obstructing the flow
of sap from the xylem. Samples were harvested at predawn to
prevent confounding effects of diurnal transpiration and kept
on ice in the dark until water relations measurements were
made.
Individual fascicles, each with three needles, were placed
inside two serially connected pressure chambers (Soil Moisture Equipment Corp., Santa Barbara, CA), and pressurized
with nitrogen gas. Bench-drying pressure-volume (P-V) techniques were used to construct P-V curves (Tyree and Hammel
1972, Cheung et al. 1976, Koide et al. 1989). The P-V measurements of all treatment combinations for seedlings or mature
trees were made within 8 h.
Data analysis
For seedlings, a 22 factorial experiment within a completely
randomized design was used with three replicates per treatment combination. To test for chamber effects, three replicates
of nonchambered nontreated seedlings were compared with
seedlings in the control (pH5.1 + Oamb) treatment. For mature
trees, the design slightly differed from that of seedlings and
OSMOTIC ADJUSTMENT IN RESPONSE TO OZONE
801
was a 22 factorial experiment within a split-plot, randomized
complete block setting. Four mature trees were assigned randomly to either of the two rain regimes. On each tree, three
similar branches were selected; two branches were assigned
randomly to either of the two ozone regimes, and the third
branch was used as a nonchambered notreated replicate (total
of four) to test for chamber effects.
The GLM (General Linear Model) procedure with SS III
option of the SAS System (SAS/STAT, SAS Institute Inc.,
Cary, NC) was used to analyze the data. When rain × ozone
interaction was not significant, the main-effect means were
compared. When rain × ozone interaction was significant,
ANOVA was performed on four simple-effect means, and
Student-Newman-Keuls (SNK) test was used for mutiple mean
comparison (Steel and Torrie 1980). Main- or simple-effect
means were declared significant at P < 0.05.
Results
There were no significant differences between the control
(pH5.1 + Oamb ) and nonchambered nontreated treatments for
any water relations components of either seedlings or mature
branches at any measurement date, indicating that the results
were not confounded by chamber effects.
Seedlings
Exposure of seedlings to acid rain and twice-ambient ozone
alone or in combination had no significant effect on predawn
Ψw in April, July or November 1992. Water potential values of
seedlings in all treatments were near maximum at about −0.5
MPa on all measurement dates (Figure 1).
Interactions between rain and ozone effects on Ψs of seedlings were significant in April (P = 0.02) and July (P = 0.006)
(Figure 1). In April, the simple-effect means of Ψs in the pH5.1
+ Oamb and pH5.1 + Otwice treatments were not significantly
different but they both differed (P < 0.05) from the simple-effect means of Ψs in the pH3.0 + Oamb and pH3.0 + Otwice treatments (Figure 1). Seedlings in the pH3.0 + Otwice treatment had
the lowest Ψs, whereas seedlings in the pH3.0 + Oamb treatment
had the highest Ψs. In July, simple-effect means of Ψs in the
pH5.1 + Oamb and pH3.0 + Oamb treatments did not differ significantly but they both differed (P < 0.05) from the simple-effect
means of Ψs in the pH5.1 + Otwice and pH3.0 + Otwice treatments.
The simple-effect mean of Ψs in the pH5.1 + Otwice treatment
was also significantly (P < 0.05) lower than the simple-effect
mean of Ψs in the pH3.0 + Otwice treatment. Because the acid
rain by ozone interaction was not significant in November, we
compared main-effect means for this date. The comparison
showed that Ψs was significantly (P = 0.014) decreased in
seedlings in the twice-ambient ozone treatment.
Acid rain and ozone effects on Ψp of seedlings were of
similar magnitude to those on Ψs but the direction of the
changes was reversed (Figure 1).
Interactions between rain and ozone effects on TLP of seedlings were significant in April (P = 0.026) and July (P = 0.009).
In April, the only significant difference was a lower TLP in
seedlings in the pH3.0 + Otwice treatment compared with seed-
Figure 1. Effects of acid rain and O3 on total water potential (Ψw),
solute potential (Ψs), pressure potential (Ψp), turgor loss point (TLP)
and bulk modulus of elasticity (ε) of ponderosa pine seedlings. Maineffect means are shown when rain × O3 interaction was not significant.
Simple-effect means are shown when rain × O3 interaction was significant. Different letters over bars indicate significant treatment effects
(ns = nonsignificant effects); bars = 1 SE.
lings in the pH3.0 + Oamb treatment (Figure 1). In July, acid rain
had no significant effect on TLP in seedlings exposed to
ambient ozone, whereas the twice-ambient ozone treatment
decreased (P < 0.05) TLP, and the decrease was greater (P <
0.05) in the presence of pH 5.1 rain than in the presence of pH
3.0 rain. In November, we compared main-effect means, because the acid rain × ozone interaction was not significant, and
found that the twice-ambient ozone treatment caused a significant decrease (P = 0.009) in TLP.
In April and November, signifiacnt interactions between rain
and ozone effects were detected on ε (P = 0.001 and 0.007,
respectively). The twice-ambient ozone treatment caused ε to
increase, particularly in seedlings exposed to pH 3.0 rain
(Figure 1). In July, ε was not significantly affected by acid rain
or twice-ambient ozone either alone or in combination.
Acid rain, ozone and a combination of the two treatments
had no significant effects on Vt in April, July or November
(Figure 2). Because there were no significant acid rain × ozone
interactions on Vs, we compared the main-effect means (Figure 2). There was no significant effect of acid rain or twice-ambient ozone on Vs in April. In July, Vs increased (P = 0.004) in
seedlings exposed to twice-ambient ozone, whereas in November, Vs increased in seedlings in both the pH 3.0 rain (P =
0.047) and twice-ambient ozone (P = 0.002) treatments.
802
MOMEN AND HELMS
Figure 2. Effects of acid rain and O3 on total water content (Vt ) and
symplastic water (Vs) of ponderosa pine seedlings. Main-effect means
are shown because rain × O3 interaction was not significant. Different
letters over bars show significant treatment effects (ns = nonsignificant
effects); bars = 1 SE.
Mature trees
The only significant effects of acid rain and twice-ambient
ozone on tissue water relations of mature trees were (1) an
increase (P = 0.028) in ε in April caused by the main-effect of
pH 3.0 rain, and (2) rain × ozone interaction (P = 0.038) on ε
in November. There were no significant simple-effect mean
differences in November (Figures 3 and 4).
Discussion
Total water potentials of seedlings were near maximum (−0.5
MPa) throughout the study and, contrary to reports by Taylor
and Dobson (1989), they were not affected by the pollutant
treatments. Similar Ψw values indicate that the potential effects
of water deficit and transpiration were minimized as a result of
regular irrigation and the use of predawn measurements, respectively, and that confounding effects of differences in Ψw
on other water relations components were prevented.
Solute potential of seedlings was decreased by twice-ambient ozone, particularly in the presence of pH 3.0 rain. This
finding is contrary to previous reports (Taylor and Dobson
1989, Dobson et al. 1990) and differs from the expectation that
elevated ozone increases osmotic potential as a result of a
reduction in photosynthesis and, hence, cellular concentration.
Figure 3. Effects of acid rain and O3 on total water potential (Ψw),
solute potential (Ψs), pressure potential (Ψp), turgor loss point (TLP),
and bulk modulus of elasticity (ε) of mature ponderosa pine trees.
Main-effect means are shown when rain × O3 interaction was not
significant. Simple-effect means are shown when rain × O3 interaction
was significant. Different letters over bars show significant treatment
effects (ns = nonsignificant effects); bars = 1 SE.
It may be argued that Ψs decreased as a result of the movement
of N ions from the acid rain to leaf cells through the cuticle, or
that Ψs changed in response to the formation of N2O5 and
HNO3 during generation of ozone from ambient air via electric
discharge (Brown and Roberts 1988). However, we have previously found that foliar N concentration of seedlings and
mature trees either decreased or did not change in response to
elevated ozone or acid rain, respectively (Momen and Helms
1995).
Decreased Ψs might be caused by an increased concentration of osmotically active metabolites in response to an ozoneinduced increase in allocation of photosynthate to foliage at
the expense of decreased root growth. Alteration of carbon
partitioning by elevated ozone has been attributed to disturbed
phloem loading or increased accumulation of starch and sugar
in foliage (Hanson and Stewart 1970, McLaughlin et al. 1982,
Okano et al. 1984, Cooley and Manning 1987, Küppers and
Klumpp 1988, Gorissen and Van Veen 1988, Edwards 1991,
Gorissen et al. 1991, Olszyk et al. 1991, Smeulders et al.
1995). However, in this study, the decreased Ψs is unlikely to
be the result of an ozone-induced increase in the allocation of
photosynthate to foliage because only very small changes in
foliar C concentration occurred in response to elevated ozone
(Momen and Helms 1995). We hypothesize that Ψs was de-
OSMOTIC ADJUSTMENT IN RESPONSE TO OZONE
Figure 4. Effects of acid rain and O3 on total water content (Vt ) and
symplastic water (Vs) of mature ponderosa pine trees. Main-effect
means are shown because rain × O3 interaction was not significant.
Different letters over the bars show significant treatment effects (ns =
nonsignificant effects); bars = 1 SE.
creased by elevated ozone as a result of osmotic adjustment
(Morgan 1984, Salisbury and Ross 1985). Concurrent increases in sugar concentrations and decreases in starch concentrations in response to elevated ozone have been observed in
many studies (Barnes 1972, Koziol 1984, Ito et al. 1985,
Cooley and Manning 1987, Friend and Tomlinson 1992, Kelly
et al. 1993, Smeulders et al. 1995). In our study, osmotic
adjustment in response to elevated ozone is indicated by decreases in Ψs and TLP, and concurrent increases in Ψp and Vs
but unchanged Vt. Because osmotic adjustment facilitates
water extraction and turgor maintenance during drought
(Hsiao et al. 1976, Tyree et al. 1978, Turner and Jones 1980,
Tyree and Jarvis 1982, Morgan 1984, Turner 1986, Momen et
al. 1992, Momen et al. 1994), our findings support the hypothesis that elevated ozone enhances drought resistance of
plants (Temple 1990).
Ozone-induced osmotic adjustment and increases in Ψp
should enhance plant growth; however, many reports suggest
that plant growth is inhibited by elevated ozone. Growth or
plastic (irreversible) cell expansion depends partly on the rate
at which Ψp exceeds a threshold value that controls cell-wall
extensibility (Salisbury and Ross 1985). Our finding that elevated ozone caused an increase in ε may indicate an increase
in the threshold pressure required to permit cell expansion and
hence a decrease in growth rate. Decreased elasticity of guard
803
cell walls may also affect plant growth through changes in
stomatal conductance (Runeckles and Rosen 1977) and water
use efficiency. Therefore, the positive effects of osmotic adjustment and increased Ψp on plant growth might be offset by
the negative effect of decreased cell wall elasticity.
The interactive effects of acid rain and elevated ozone on ε
and Ψs could support the hypothesis (Heath 1980) that elevated
ozone reacts with the plasmalemma and increases the permeability of membranes thereby enhancing the movement of S
and N ions of acid rain into leaf cells leading to decreased Ψs.
However, analysis of foliar chemistry (Momen and Helms
1995) indicated no increase in foliar N concentration of ponderosa pine seedlings or mature trees in response to increased
rain acidity.
The effects of elevated ozone on water relations of ponderosa pine seedlings were similar to the effects of drought in
many species (cf. Chapin 1991). It seems unlikely that elevated
ozone induced water stress in seedlings as a result of decreased
root growth and water uptake, because Ψw of seedlings were
not affected by twice-ambient ozone and were near maximum.
Interactive effects of acid rain and elevated ozone indicate that
the effects of one pollutant depended on the level of the other,
and thus no accurate conclusion can be drawn from single
pollutant studies in regions exposed to multiple pollutants. The
large responses of seedling water relations to pollutants contrasted with those of mature trees and may indicate that the
parent genotype had adapted to environmental stresses. It is
possible that seedlings and mature trees differed in their responses to the treatments because seedlings were entirely
exposed to pollutants whereas mature trees were not, or because the branches of mature trees were not entirely autonomous with regard to plant water relations. However, cellular
damage by elevated ozone is local, and branches of mature
trees have been found autonomous with respect to carbon
transport (Cregg 1990, Mattson et al. 1990, Sprugel et al. 1991)
and water relations (Zimmermann 1983, Tyree 1988).
Acknowledgments
We thank P.D. Anderson and J.L.J. Houpis for their help throughout
the study. We also thank F.S. Chapin and G. Biging for comments on
an earlier version of this paper. This study was supported by the
California Air Resources Board through Contract A132-101 and by the
McIntire-Stennis project 4635-MS.
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