1

Photosynthetic responses of aspen clones to simultaneous exposures of ozone and C02

O. Kull, A. Sober, M.D. Coleman, R.E. Dickson, J.G. Isebrands, Z. Gagnon, and D.F. Karnosky

Abstract: Current projections indicate steady increases in both trophospheric ozone and carbon dioxide well into the next century with concurrent increases in plant stress. Because information about effects of these interacting stresses on forest trees is limited, we have conducted ozone and carbon dioxide experiments using ozone-tolerant and ozone-sensitive trembling aspen (Populus tremuloides Michx.) clones (clones 216 and 259, respectively). Aspen plants were grown either in pots (square-wave study) or in the ground (episodic study) in open-top chambers. Plants in the square-wave study were exposed for a single growing season to charcoal-filtered air (CF) or to CF plus elevated carbon dioxide (C02 ), ozone (0 3 ), or 0 3 plus C0 2 (0 3 + CO,). Plants in the episodic study were exposed for three growing seasons to CF, twice simulated ambient (2x) 0 3 (2x 0 3 ), or 2x 0 3 plus CO, (2x 0 3 + CO,). Photosynthetic measurements were made either in the open-top chambers at treatment CO, concentrations or in controlled-environment cuvettes with various CO, concentrations, producing assimilation versus intercellular CO, concentration (A/C) curves. Ozone decreased photosynthetic rate and stomatal conductance and accelerated leaf senescence. Elevated CO, increased photosynthetic rate and decreased stomatal conductance when measured at treatment CO, concentrations, and exacerbated the negative effect of 03 on photosynthesis. For example, for clone 259, photosynthesis decreased 9% for the 0 3 treatment compared with the CF treatment, but decreased 24% for the 0 3 + C0 2 treatment compared with the CO, treatment. Similar decreases for clone 216 of 2% and 6% for 0 3 and 0 3 + CO,, respectively, were not significant. A/C { curves showed that 0 3 decreased carboxylation efficiency and maximum photosynthetic rate and that photosynthetic inhibition in response to 03 was greater with elevated C0 2 . The simultaneous declines in all factors of photosynthetic gas exchange measurements suggest that the equilibrium between stomatal conductance, carboxylation, and light harvesting systems was not disrupted by 03 and 0 3 X CO, interactions. Carbon dioxide did not ameliorate the detrimental effects of 0 3 on the leaf photosynthetic apparatus. In fact, the 0 3 -tolerant clone appeared more sensitive to 0 3 with elevated CO,. Rsum : Les projections actuelles prvoient une augmentation constante de l'ozone troposphrique et du CO, pour une bonne partie du sicle prochain avec une augmentation parallle des stress pour les plantes. tant donn que l'information sur les effets de ces stress et de leurs interactions sur les arbres forestiers est limite, nous avons ralis des expriences avec 0 3 et CO, en utilisant un clone tolrant (216) et un clone rsistant (259) de peuplier faux-tremble {Populus tremuloides Michx.). Les plants de peuplier ont t cultivs soit dans des pots (tude continue), soit dans des chambres ciel ouvert (tude pisodique). Les plants dans l'tude continue ont t exposs pendant une seule saison de croissance de l'air filtr au charbon de bois (CF), de l'air filtr au charbon de bois enrichi en dioxide de carbone (CO,), en 0 3 (0 3 ) et en 0 3 et CO, (0 3 + CO,). Les plants dans l'tude pisodique ont t exposs pendant trois saisons de croissance CF, 2 fois la concentration ambiante (2x) de 0 3 (2x 0 3 ) ou 2x 0 3 additionn de CO, (2x 0 3 + CO,). Les mesures de photosynthse ont t prises soit dans les chambres ciel ouvert aux concentrations du traitement CO,, soit dans des cuvettes environnement contrl avec diffrentes concentrations de CO, (courbes A/C,). L'ozone a diminu le taux de photosynthse et la conductance stomatale et acclr la snescence foliaire. Un enrichissement en CO, a augment le taux de photosynthse et diminu la conductance stomatale lorsque les mesures taient prises aux concentrations du traitement CO, et accentu l'effet ngatif de 0 3 sur la photosynthse. Chez le clone 259 par exemple, la photosynthse a diminu de 9% dans le traitement 0 3 comparativement au traitement CF mais a diminu de 24% dans le traitement 0 3 + CO, comparativement au traitement C0 2 . Des diminutions semblables de 2 et 6% respectivement pour les traitements 0 3 + CO, chez le clone 216 n'taient pas significatives. Les courbes AIC { ont montr que 0 3 diminuait l'efficacit de la carboxylation et le taux maximum de photosynthse et que l'inhibition de la photosynthse en raction 03 tait plus forte avec un enrichissement en C0 2 . La diminution simultane de tous les facteurs des mesures de l'change gazeux li la photosynthse suggre que l'quilibre entre la conductance stomatale, la carboxylation et les systmes de capture de la lumire n'tait pas affect O. Kull and A. Sober. Institute of Ecology, Estonian Academy of Sciences, Lai 40, 2400 Tartu, Estonia. M.D. Coleman,1 R.E. Dickson, and J.G. Isebrands. USDA Forest Service, North Central Forest Experiment Station, 5985 Highway K, Rhinelander, WI 54501, U.S.A. Z. Gagnon and D.F. Karnosky. Michigan Technological University, School of Forestry and Wood Products, 1400 Townsend Drive, Houghton, MI 49931, U.S.A. 1 Author to whom all correspondence should be addressed. Can. J. For. Res. 26: 639-648 (1996). Printed in Canada / Imprim au Canada par 0 3 et les interactions entre 0 3 et CO,. Le C0 2 n'a pas contrecarr les effets nocifs de 0 3 sur l'appareil photosynthtique des feuilles. En fait, le clone tolrant 0 3 a sembl plus sensible 0 3 avec un enrichissement en C0 2 . [Traduit par la Rdaction]

Can. J. For. Res. Vol. 26, 1996

Introduction
Human activities have varied the environment and affected plant growth for decades. Two such anthropogenic factors that have great influence are ozone (0 3 ) and carbon dioxide (CO,). Ozone concentrations in the plant environment may increase or decrease rapidly given its variable distribution in space and time. Carbon dioxide concentration in the environment increases slowly but continuously, with similar CO, concentrations throughout the globe. Both 0 3 and CO, concentrations may increase well into the future (Bowes 1993; Chameides et al. 1994). Thus, to predict the response of plants in the future, simultaneous 0 3 and CO, studies are needed. A few studies have been conducted with simultaneous 0 3 and CO, treatments with agricultural plants (Heck and Dunning 1967; Barnes and Pfirrmann 1992; Heagle et al. 1993), but relatively little is known about forest tree response (Kickert and Krupa 1991; Polle et al. 1993). Exposure to elevated C0 2 typically results in decreased sto-matal conductance and dark respiration, as well as increased photosynthetic rates, dry weight accumulation, leaf area, and leaf weight per unit area (Bowes 1993; Ceulemans and Mousseau 1994; Ceulemans et al. 1995). Because of these various responses to C0 2 , it is hypothesized that elevated CO, will ameliorate the detrimental effects of increased 0 3 (Allen 1990). Unfortunately, this hypothesis has not been verified. Plant exposure to 0 3 typically increases leaf senescence and decreases photosynthesis, stomatal conductance, and plant growth (Reich 1983; Wang et al. 1986; Pye 1988; Retzlaff et al. 1992; Sanders et al. 1992; Landry and Pell 1993; Coleman et al. 1995a). Additionally, 0 3 exposure may change the relative concentrations of leaf biochemical components such as starch and soluble sugars (Bucket" and Ballach 1992; Friend et al. 1992; Coleman et al. 1995>). Decreasing photosynthetic rates with 0 3 exposure may be caused by many factors, e.g., stomatal closure (Reich 1987), oxidant damage to the biochemical processes of light harvesting and carbon fixation (Floyd et al. 1989; Sasek and Richardson 1989; Sanders et al. 1992; Pell et al. 1994), or damage to membranes and other cellular components (Heath 1987; Matyssek et al. 1991). If 0 3 decreases photosynthetic rates, stomatal conductance may also decrease to maintain metabolic equilibrium and a constant intercellular CO, concentration to atmospheric CO, concentration (C/CJ ratio (Wong et al. 1979; Ball and Berry 1982). However, several experiments show that 0 3 affects stomata independently of mesophyll physiological processes (Moldau et al. 1991). Stomata may also close in response to increased external CO, concentration, thus keeping the C/C a ratio constant (Mott 1990); however, this effect may not persist in long-term treatments (Eamus and Jarvis 1989; Wullschleger et al. 1992). Estimates of stomatal and nonstomatal biochemical responses to environmental stress may be obtained from assimilation versus intercellular CO, concentration plots (A/C i curves) (Stitt 1991). Although these AIC X curves can provide information on major factors controlling photosynthesis, they will not completely define the biochemical effects because many biochemical reactions are involved in photosynthesis control. Measurement of several metabolites and enzyme systems would be necessary to more completely define response controls (Stitt 1991). Ozone exposure decreases carboxylation efficiency (measured as initial slope of the AIC X curve) and maximum photosynthetic rates (measured as plateau of the

A/C i curve) (Sasek and Richardson 1989; Moldau et al. 1991; Sasek et al. 1991; Matyssek et al. 1993). There is evidence that similar changes in AIC X curves may occur during plant acclimation to elevated CO,, but response depends on the species studied and environmental factors (Sage et al. 1989; Lawlor and Mitchell 1991; Stitt 1991). Ceulemans et al. (1995) found increased photosynthetic rates associated with increased CO, concentrations, and these increased rates were related to increases in both photochemical and biochemical processes of carbon fixation. Because plant reactions to changes in environmental factors are species specific, 0 3 and CO, response may differ among tree species (Pye 1988). Moreover, there are genotypes within species that differ in 0 3 sensitivity (Karnosky 1976; Berrang et al. 1989; Karnosky et al. 1992; Retzlaff et al. 1992) and in elevated CO, response (Woodward et al. 1991; Ceulemans et al. 1995"). To examine the effects of simultaneous increases of 0 3 and CO,, we conducted an open-top chamber study with two aspen (Populus tremuloides Michx.) clones that differ in sensitivity to 0 3 . These clones were exposed to both increased 0 3 and CO, to compare interactions among the treatment factors on different parameters of photosynthesis. Ozone exposure decreased photosynthetic activity and accelerated senescence of leaves, and added CO, did not ameliorate the effects of 0 3 on photosynthesis.

Materials and methods

Plant material Two aspen clones varying in sensitivity to 0 3 (216, 0 3 tolerant; 259, 0 3 sensitive) were selected for study based on foliar injury and decreases in dry weight after 0 3 exposure (Karnosky et al. 1996). Experimental plants were propagated vegetatively as softwood cuttings from vigorously growing stock plants. Rooted cuttings were transplanted into 6-L pots containing perlite -peat moss organic topsoil (1:1:1 by vol.) and 8 g slow-release fertilizer (17:6:12 N-P,0 5 -K,0, plus minor nutrients, 3- to 4-month release, Sierra Chemical Corp., Milpitas, Calif. U.S.A. 1 ). For the square-wave treatment, cuttings were transplanted into 6-L pots, grown about 6 weeks in a greenhouse (21C, 17 h day length) until they had 25-30 leaves, then transferred to field conditions under 50% shade. After 1 week, in June 1992, plants were moved from shaded field conditions to open-top chambers. Trees were watered daily. For the episodic treatments, cuttings were transplanted into 6-L pots under shaded field conditions and grown for 3 months. Twelve plants were then planted in the soil of each open-top chamber in September 1991. Trees were watered by precipitation and by hand as needed. They were in their second and third growing season, respectively, in 1992 and 1993 when measurements for this study took place. Six trees (three per clone) were harvested from each chamber (three chambers per treatment) in late August 1993 to measure leaf area, dry weight, and nitrogen content of leaves. Ozone and C02 exposures The square-wave open-top chamber treatments included (1) charcoal-filtered air (CF) or CF plus (2) 100 ppb O, for 6 h/day (08:00 to 14:00), 5 days per week (0 3 ), (3) 150 "(junol-mol - 1 CO, (C0 2 ), or (4) 0 3 plus elevated CO, (0 3 + CO,). There was one chamber per treatment. Ozone was generated (Griffin GTC-1A) from pure oxygen, dispensed

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Kull et al.

through manually adjusted needle valves and monitored using a time-shared Scanivalve system and a TECO model 49 ozone analyzer. The seasonal 0 3 exposure (24-h basis) for this experiment was about 55 ppm-h. Carbon dioxide was dispensed 24 h/day through manually adjusted valves and monitored using a time-shared Scanivalve system and a Valtronics model 2008SDL monitor. Episodic 0 3 treatments were derived from hourly ambient profiles constructed from 0 3 data collected in Michigan's Lower Peninsula (Karnosky et al. 1996). A sigmoidal weighing of peak levels in the ambient profile (Hogsett et al. 1988) was used to produce the twice ambient (2x) 0 3 treatment (2x 0 3 ). The twice ambient 0 3 plus CO, (2 0 3 + CO,) was produced by adding 150 fjLmolmol - 1 CO, in combination with the 2x 0 3 treatment. The cost of CO, application did not allow inclusion of the CO, only treatment. These treatments were added to charcoalfiltered air and were compared with a charcoal-filtered control (CF). Ozone was dispensed to match programmed profiles through computer-controlled mass-flow controllers. Ozone concentrations were monitored with a time-shared Scanivalve system using a TECO model 49 analyzer as described by Karnosky et al. (1996). Carbon dioxide was dispensed 24 h/day through manually adjusted valves and monitored using a time-shared Scanivalve system and a Valtronics model 2008SDL monitor. The seasonal 0 3 exposures (24-h basis) averaged about 83 ppm-h for the 2x 0 3 treatment and 87 ppm-h for the 2x 0 3 + CO, treatment for both the 1992 and 1993 seasons. There were three chambers per treatment.
JC

03 " 0 3 + CO, CF CO,

10.8+0 .3Z7 2398a 14.40.4a 1836> Ozone-sensitive clone 259 10.80.2Z> 16.90.7a 9.80.2 12.8+0 .7Z7 2426a 201 7Z7C 2084 ZP 1826c

0.670.02a 0.69+0.0 \a 0.670.0la 0.700.02a 0.690.01a 0.730.02a

o3"
0 3 + CO,

N o t e : First-year potted plants were grown in the square-wave

experiment and exposed to four different atmospheres: CF, CO,, 0 3 , and O, + CO,. For each clone, values in columns followed by the same letter are not significantly different ( a = 0.05) according to Tukey's honestly significant difference test. *Gas exchange was measured in August 1992 on recently mature leaves (-LPI 10). Values represent the mean standard error of four different trees in each exposure treatment.

Gas-exchange measurements Light-saturated photosynthetic CO, exchange rates were measured in the open-top chambers of both the squarewave and episodic studies using a LI-6200 portable photosynthesis system (LI-COR, Lincoln, Nebr., U.S.A.). Leaf temperatures ranged from 25 to 32C. In the episodic treatment chambers, measurements were taken after rain to ensure minimal leaf water deficits. AIC ) curves from plants within chambers from both studies were developed as CO, concentrations were drawn down through photosynthetic uptake in the LI-COR system (McDermitt et al. 1989). Leaf plastochron index (LPI) was used to describe the photosynthetic response to leaf maturity. With the LPI system, leaves are numbered from the top of the plant, with the youngest leaves having lowest LPI (Larson and Isebrands 1971; Ceulemans et al. 1988). Table 1. Gas-exchange parameters of two aspen clones differing in sensitivity to ozone.

In late August 1993, photosynthetic CO, response curves (A/C t curves) for trees in the episodic study were developed with a steady-state MPH-1000 gas-exchange system (Campbell Scientific, Logan, Utah, U.S.A.). Upper lateral branches were sampled from three different trees per clone for each treatment. Branches were cut from the trees and then recut and maintained in water during measurement. Cut branches maintain the same photosynthetic characteristics as those remaining on the tree (Koike and Sakagami 1984). Nitrogen and chlorophyll content were measured in leaves from the episodic plants. Leaf samples were taken in August at the same time and from similar branch positions as chosen for AIC { curves. Samples for nine trees per clone were analyzed from each treatment.

Results

Elevated CO, increased photosynthesis of recently mature leaves from plants in the square-wave experiment: 40% for the Oj-tolerant clone (216) and 55% for the 0 3 sensitive clone (259) compared with leaves from the CF treatment (Table 1). Ozone tended to decrease photosynthesis of recently mature leaves more so in the sensitive clone (259), and this response was most pronounced with elevated CO,; i.e., photosynthesis decreased 9% in the 0 3 treatment compared with the CF treatment, but decreased 24% in the 0 3 + CO, treatment compared with the CO, treatment. Similar decreases for clone 216 of 2% and 6% for 0 3 and 0 3 + CO,, respectively, were not significant. Stomatal conductance decreased in Stomatal Photosynthesis response to both elevated CO, and 0 3 (Table 1). The major conductance Treatment (umol-m~ 2 -s - 1 )* (umol-m^ 2 response was associated with C0 2 exposure. Stomatal conductance decreased 26% in clone 216 and 17% in s_l) C-JC a clone 259 with elevated CO,, but only 7% (216) and 14% Ozone-tolerant clone 216 (259) with the 0 3 treatment". The combination of 0 3 + CO, CF 11.00.2b 2574a 0.690.01a had little additional effect over CO, 15.40.9a 1895 ZP 0.720.02a Fig. 1. Carboxylation efficiencies for two aspen clones determined from photosynthetic carbon dioxide response curves. Ozone-tolerant (clone 216) and ozone-sensitive (clone 259) aspen plants were exposed to ozone (03 ), elevated carbon dioxide (CO,), or a combined treatment (0 3 + CO,) and compared with a charcoal-filtered control (CF). Carboxylation efficiencies were determined with plants from (A) square-wave treatments measured in August 1992 on recently mature leaves (LPI 6 to 8), (B) episodic treatments measured in July 1993 on recently mature leaves (LPI 6 to 8), and (C) episodic treatments measured in August 1993 on mature leaves (LPI 8 to 15). Values represent the mean and SE of three replicate trees measured per treatment. Bars with the same letter above are not significantly different (a = 0.05) according to Tukey's honestly significant difference test.
216 259

Clone
CO, alone in clone 216, but decreased conductance an additional 9% in clone 259. Because of similar decreases in photosynthetic rate and stomatal conductance, the C/C,

ratio remained relatively constant in these recently mature leaves (Table 1). Photosynthetic CO, response curves (A/C { curves) were used to determine whether gas-exchange responses to treatment could be attributed to nonstomatal effects. Carboxylation efficiency (CE), the initial slope of the A/C l curve, was lower for the 0 3 -tolerant clone (216) than

Can. J. For. Res. Vol. 26, 1996

for the 0 3 -sensitive clone (259) in both the CF and CO, treatments of the square-wave experiment (Fig. 1A). Carboxylation efficiency decreased with 0 3 exposure in the 0 3 -sensitive clone 259 much more than in the 0 3 tolerant clone 216. In contrast, the simultaneous 0 3 + CO, exposure decreased CE more than 0 3 alone in clone 216 but not in clone 259. This differential clonal response to treatments is statistically significant as shown by the clone X treatment interaction term (P = 0.0002) in the ANOVA (Table 2). Carboxylation efficiencies were also measured in the episodic experiments in July and August 1993 (Figs. IB and 1C). Clonal responses were similar to that found with the square-wave plants (Fig. 1A); clone 259 was more sensitive to the 0 3 treatment than clone 216, but gave little additional responses to the 0 3 + C0 2 treatment. In contrast, CE of clone 216 decreased more with the 0 3 + CO, treatment than with the 0 3 treatment alone (Figs. IB and 1C). Comparison of CE in leaves that range in age from LPI 6 to 8 (Fig. IB) and leaves that range in age from LPI 8 to 15 (Fig. 1C) shows that the negative effects of 0 3 and 0 3 + CO, increased with leaf age. Although measurements in Figs. IB and 1C were taken on different dates, the CF values are similar; therefore, the relative response to treatment can be compared. Photosynthetic CO, response curves (A/C l curves)

show the different responses of the two clones to 0 3 and 0 3 + C0 2 (Fig. 2). Carboxylation efficiency and maximum photosynthesis (P m ax ) were depressed in both clones with 0 3 and with 0 3 + CO,. In clone 216, however, 0 3 + C0 2 depressed the A/C ; curve (both CE and P m ax ) more than 0 3 alone. Changes in carboxylation efficiencies and maximum photosynthetic rates in response to 0 3 and 0 3 + C0 2 were highly correlated (Fig. 3). Both CE and P m a x for both clones decreased with the 0 3 and 0 3 + CO^ treatments, and these decreases were tightly coupled (R 2 = 0.995), indicating that these photosynthetic parameters responded proportionally to treatment. Nitrogen and chlorophyll contents determined at the time of the August 1993 A/Cj curve development for episodic plants had characteristic responses that were helpful in explaining the effect of treatments on photosynthetic response. Nitrogen content decreased in leaves in the 0 3 + C0 2 treatment compared with other treatments (Tukey's honestly significant difference pairwise comparison, a < 0.05), as did photosynthesis, especially in clone 216 (Fig. 4). But changes in nitrogen content do not explain all of the variation in photosynthetic parameters among the treatments. There were no differences in nitrogen content between the CF and 0 3 treatments in either clone (Tukey's honestly significant difference, a > 0.5), yet the

Kull et al

Fig. 2. The effects of ozone and carbon dioxide on photosynthetic carbon dioxide response curves of two aspen clones. Ozone-tolerant (clone 216) and ozone-sensitive (clone 259) aspen plants were treated with charcoal-filtered air (CF, ). 2x ozone (2x 0 3 , ). or ozone and elevated carbon dioxide (2x 0 3 + C0 2 , A). Measurements were taken with the steadystate Campbell instrument from leaves of age-class LPI 8 to 15 in August 1993. Individual points for each of three replicate measurements are shown. Nonlinear regressions were fitted using the model of Hanson et al. (1987). Corrected R 2 was equal to or greater than 0.86 for all but clone 216 treatment 2x 0 3 (R 2 = 0.77) and clone 259 treatment 2x 0 3 + C0 2 (R 2 = 0.46).

/'* ,J>
I I

CF 2x03
I

ZxO^ + CO^

I 259

I Clone

_________*
CF
A A

af
I

A.

2x03 + C02 " * 400 600 BOO

0 1000

200

Maximum Photosynthetic
1 1
I I

Clone 21 6 30 20 10 0

Intercellular C02 (yumol mol )

Rate

(yUimol - m

-s

O E

=1

0 3 effect on photosynthesis is especially apparent in clone 259 (Table 1, Figs. 1 and 2). Specific leaf weight, often associated 20 with changes in nitrogen content and photosynthetic rate, was not affected by treatment (average was 84 g-m~"). Because C >N 10 specific leaf weight was not affected by treatment, both the CO response of nitrogen content to treatment and the relationship o between carboxylation efficiency were the same when +- nitrogen was expressed on a weight or area basis (Fig. 4). 0 >O sz Chlorophyll content of recently mature leaves (LPI 8) of both clones, and that of mature leaves (LPI 25) of clone 216, were unaffected by treatment (average was 597 i^g-cm - "). In contrast, chlorophyll content decreased more than 48% in mature leaves of
CO

clone 259 in the 0 3 and 0 3 + C0 2 treatments (average was 345 (xgcm - 2 ), compared with the CF (670 ( ju -cm - 2 ). Photosynthetic g inhibition of clone 259 in 0 3 and 0 3 + C0 2 treatments reflects these lower chlorophyll concentrations (Fig. 2). Photosynthetic rates measured at various leaf ages demonstrated both clonal and treatment differences, but were very consistent among different branches and seasons (Fig. 5). Lightsaturated photosynthesis increased during

Leaf N Content (mg-cm

leaf development up to LPI 6 to 10, then remained almost constant in fully developed leaves to LPI 25 in plants from the CF treatment in both clones (Fig. 5). This pattern was

Fig. 5. Relationships between light-saturated photosynthesis and leaf plastochron index for ozone-tolerant (clone 216) and ozone-sensitive (clone 259) aspen clones. Treatments are as in Fig. 2. Relationships for both terminal and upper lateral shoots measured in August 1992 and August 1993 are displayed. Clone 21 6 20 in

Clone 259

15 10 5 0 20 15 10

E
_L
O

in in
CD

5 0

XZ
-t'

20 15

> ^
CO

10

Kull et al

o
-M

o
xz
Q_

5 0

Leaf Plastochron Index

unaffected by 0 3 treatment in clone 216, but photosynthesis of clone 259 steadily decreased from LPI 6-10 to LPI 25 in the 0 3 treatment. In the 2x 0 3 + C0 2 treatment, photosynthesis declined steadily in clone 216 as well as in clone 259 from LPI 6-10 to LPI 25. Terminal branches and upper lateral branches measured in August of both 1992 and 1993 had similar responses. A changing C/C, ratio can be used to determine whether the treatments affect stomata independent of nonstomatal mechanisms of photosynthesis. The C/C a ratio was somewhat higher for young developing leaves (LPI 3 to 5) than for mature leaves, perhaps indicating that the nonstomatal mechanisms of C0 2 fixation lag stomatal development (Fig. 6). The C/C a ratios for fully expanded leaves (LPI 6-10 and greater) ranged from 0.5 to 0.9, but were relatively constant for both clones and all treatments. These relatively constant ratios indicate that stomatal mechanisms and nonstomatal mechanisms of carbon fixation changed by about the same degree in response to the 0 3 and CO, treatments. because photosynthetic measurements were collected at treatment CO, concentrations and the increased CO, concentrations in the enriched treatment were higher, it was difficult to directly evaluate the interacting effect of elevated CO, and 0 3 on photosynthetic rate. Stomatal conductance typically decreases with both 0 3 and CO, exposure (Pye 1988; Lawlor and Mitchell 1991). These expected results were found in our study (Table 1), but the decrease in stomatal conductance with elevated CO, did not appear to provide protection from the negative effects of 0 3 on photosynthesis (cf. Allen 1990; Heagle et al. 1993)." To critically examine the interacting effect of elevated C0 2 and 0 3 on photosynthesis, we had to evaluate various components of carbon assimilation. The CJC a ratio reflects the interdependence among photosynthesis, stomatal conductance, and C { (Farquhar and Sharkey 1982; Mott 1990; Aphalo and Jarvis 1993). In our experiment, the C/C, ratios were in the range reported for other C 3 species, including trees (Wong et al. 1979; Matyssek et al. 1992). Stability of this ratio shows that decreases in the biochemical processes of photosynthesis with treatment are accompanied by similar decreases in stomatal conductance. The photosynthetic response to CO,, or the AIC t curve, is useful for characterizing some nonstomatal responses. The slope of the A/C^ curve reflects Rubisco carboxyla-tion efficiency (CE), and the plateau (P m ax ) reflects light harvesting reactions and the regeneration of ribulose-1,5-bisphosphate (Farquhar and Sharkey 1982; Stitt 1991; Bowes 1993). Elevated CO, alone had no effect on CE of plants from the square-wave treatments (Fig. 1A), but 0 3 decreased CE, particularly in the 0 3 -sensitive clone (259).

Discussion
Elevated CO, usually increases photosynthetic rate; however, this increase may not be proportional to the increase in CO, concentration (Eamus and Jarvis 1989; Mott 1990). In our experiment, however, we found that the increase was roughly proportional to the 40% increase in CO, concentration applied (Table 1). In comparison to the CO, effect, 0 3 decreased photosynthesis, especially in the 0 3 -sensitive clone. When the effect of 0 3 either with or without elevated CO, was examined, a greater 0 3 effect was observed with elevated CO,. However,

0.6 0.3

Kuli et al

0 0.9

0.3 0.0

Fig. 6. Relationships between C/C a ratio and leaf plastochron index for ozone-tolerant (clone 216) and ozone-sensitive (clone 259) aspen clones. Treatments are as in Fig. 2. Relationships for both terminal and upper lateral shoots measured in August 1992 and August 1993 are displayed. Clone 21 6 a i
i

Clone 259
i i i

CF 2x0 3
Lateral '92 ^ O Lateral '93 * '93 A 1 1 I I I Terminal '92 Terminal
1

0.0
i
1
1

i
1

i
1
1

2x0 3 +C0 2 ~ 10 15 20 25 0

20 5 10 15

25

Leaf Plastochron Index

In addition, there was no amelioration of the 0 3 effects on CE by elevated C0 2 in clone 259. In the 0 3 -tolerant clone (216), CE significantly decreased with 0 3 only with elevated C0 2 , suggesting that C0 2 was exacerbating the effects of 0 3 . The effect of 0 3 and elevated C0 2 on CE as measured with A/C t curves may be attributed to changes in total content or activity of the carboxylating enzyme Rubisco. Decreased Rubisco concentration has been measured in 0 3 -treated agronomic and woody plant species (Pell and Pearson 1983; Dann and Pell 1989; Landry and Pell 1993). Ozone sensitivity also has been correlated with increases in Rubisco degradation following 0 3 treatment that would lead to decreased concentrations (Enyedi et al. 1992). Elevated C0 2 may also decrease both Rubisco concentration and specific activity (Bowes 1993). Although we did not see decreased CE in these open-top chamber experiments, these clones had lower CE in growth chamber experiments when plants in elevated C0 2 were compared with those grown at ambient C0 2 concentrations (unpublished data). Therefore, it is possible that the negative effect of simultaneous 0 3 + C0 2 treatment on leaf photo-synthetic apparatus, which occurs in contrast with hypothesized expectations (cf. Allen 1990; Heagle et al. 1993), may be due to simultaneous negative effects on Rubisco by both 0 3 and elevated C0 2 . Also, as shown by the relationship between CE and P m a x (Fig. 3), the integrated photo-synthetic system (Evans and Farquhar 1991), carboxylation, light harvesting, and ribulose-l,5bisphosphate regeneration seem to be affected similarly by 0 3 and 0 3 + C0 2 . The content of nitrogen and chlorophyll helped explain some of the responses to treatment. Leaf nitrogen content usually decreases with elevated C0 2 (Norby et al. 1986; Ceulemans and Mousseau 1994). We found that elevated C0 2 decreased leaf nitrogen content, which may explain why carboxylation efficiencies and P m a x were lower for the 0 3 + C0 2 treatment than for the 0 3 treatment (Figs. 3 and 4). Photosynthetic rates are correlated with nitrogen concentration in the leaves at ambient CO, levels (Evans 1989), and this may be a factor in the 0 3 + C0 2 response. Ozone has been shown to severely decrease chlorophyll content in aspen leaves (Gagnon et al. 1992). Chlorophyll content was lower for both the 0 3 treatment and the 0 3 + CO, treatment in the sensitive clone (259). This may be a factor in the greater sensitivity of this clone to 0 3 compared with clone 216. The chlorophyll content of clone 216 shows little response to 0 3 even in the 0 3 + C0 2 treatment, but nitrogen concentration decreased. These results suggest that the exacerbation of the 0 3 effect on photosynthesis in clone 216 in the 0 3 + C0 2 treatment may reflect nitrogen nutrition, whereas the sensitivity of clone 259 may reflect decreased chlorophyll content. Response to 0 3 exposure was strongly related to leaf developmental stage (Fig. 5). Leaf photosynthetic rates of CF plants increased rapidly up to LPI 6-8 and then remained stable throughout the season. This stable photosynthetic response following total leaf expansion has been found in some field-grown hybrid poplars (Nelson and Ehlers 1984), but is in contrast with the more normal pattern of steady decline with increasing leaf age usually found for other poplars (Reich 1983; Nelson 1985; Michael et al. 1990; Coleman et al. 1995a). Although there were no apparent differences in the photosynthesis-LPI relationships between the two clones with the CF treatment, the two clones differed considerably with 0 3 treatment (Fig. 5). The relationship between photosynthesis and LPI remained unchanged compared with CF plants in the 0 3 tolerant clone. In contrast, photosynthesis of mature leaves decreased with increasing LPI in the 0 3 -sensitive clone. With 0 3 + C0 2 treatments, photosynthesis decreased with LPI in both clones. Again, the response of the 0 3 -tolerant clone was similar to that of the 0 3 -sensitive clone when 0 3 and CO-, were applied in combination. Unfortunately, we did not have an elevated CO, treatment alone in the episodic fumigation experiment. Thus, it is possible that the depression of photosynthesis of older leaves in the 0 3 -tolerant clone was caused by elevated CO, rather than a combined 0 3 and CO, response. Rapid senescence and shorter leaf retention time have been found with CO, enrichment in agricultural plants (Bhattacharaya et al. 1990) and trees (Ceulemans and Mousseau 1994), but leaf retention time increased in white oak (Quercus alba L., Norby et al. 1986). Differences between square-wave and episodic 0 3 treatments may influence plant response to 0 3 . Our experiments show that plant response was greater in the square-wave treatment than in the episodic treatment (Karnosky et al. 1996), but the mechanisms of response are similar (Coleman et al. 1995a). Both square-wave and episodic experiments show that CO, does not counteract the negative effect of 0 3 on carboxylation efficiency (Fig. 1). Because carboxy-lation efficiency response patterns are similar between square-wave and episodic treatments, comparisons between the two regimes are more plausible. Additionally, CO, is applied the

Can. J. For. Res. Vol. 26, 1996

same way in both square-wave and episodic experiments; therefore, it is reasonable to assume that the response to elevated CO, will also be the same, and extending mechanistic conclusions about elevated CO, effects from the square-wave experiments to the episodic experiment is quite reasonable. In addition, the cost of three extra CO, chambers in the episodic series was prohibitive. Three conclusions can be derived from our data. First, 0 3 impact on trees in these experiments can be explained by depression of photosynthetic activity and the accelerated senescence of leaves. Accelerated decline in photosynthesis and accelerated senescence of leaves under 0 3 treatment are well known (Reich 1983; Wang et al. 1986; Pell and Dann 1991; Retzlaff et al. 1992; Sanders et al. 1992; Landry and Pell 1993; Coleman et al. 1995a). Second, stomatal conductance, CE, light harvesting, and regeneration of ribulose-l,5-bisphosphate are affected similarly by the 0 3 and 0 3 + CO, treatments. Thus, aspen seems to maintain equilibrium among the various components of the photosynthetic apparatus with these stresses. Third, results of these experiments with aspen do not support the hypothesis that higher concentrations of CO, will ameliorate the detrimental effects of increased 0 3 on the photosynthetic apparatus of leaves. Moreover, a genotype considered tolerant with normal CO, levels appears to have decreased 0 3 tolerance with elevated CO,.

Acknowledgments
We appreciate the technical support of Mr. Priit Pechter on this study. Funding was provided by the USDA Forest Service's Northern Global Change Program and the National Council of the Paper Industry for Air and Stream Improvement, Inc.

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