Localized ozone fumigation system for studying ozone effects on ...

Tree Physiology 25, 1523–1532 © 2005 Heron Publishing—Victoria, Canada

Localized ozone fumigation system for studying ozone effects on photosynthesis, respiration, electron transport rate and isoprene emission in field-grown Mediterranean oak species VIOLETA VELIKOVA,1 TSONKO TSONEV,1 PAOLA PINELLI,2 GIORGIO A. ALESSIO 2 and FRANCESCO LORETO 2,3 1

Bulgarian Academy of Sciences, Institute of Plant Physiology, Acad. G. Bonchev Str., Sofia, Bulgaria

2

CNR – Istituto di Biologia Agroambientale e Forestale, Via Salaria Km. 29, 300 00016, Monterotondo Scalo (Roma), Italy

3

Corresponding author ([email protected])

Received January 12, 2005; accepted May 3, 2005; published online September 1, 2005

Summary We used a localized ozone (O3) fumigation (LOF) system to study acute and short-term O3 effects on physiological leaf traits. The LOF system enabled investigation of primary and secondary metabolic responses of similarly and differently aged leaves on the same plant to three different O3 concentrations ([O3]), unconfounded by other influences on O3 sensitivity, such as genetic, meteorological and soil factors. To simulate the diurnal cycle of O3 formation, current-year and 1-year-old Quercus ilex (L.) and Quercus pubescens (L.) leaves were fumigated with O3 at different positions (and hence, different leaf ages) on the same branch over three consecutive days. The LOF system supplied a high [O3] (300 ± 50 ppb) on leaves appressed to the vents, and an intermediate, super-ambient [O3] (varying between 120 and 280 ppb) on leaves less than 30 cm from the vent. Leaves more than 60 cm from the O3 vent were exposed to an [O3] comparable with the ambient concentration, with a 100 ppb peak during the hottest hours of the day. Only leaves exposed to the high [O3] were affected by the 3-day treatment, confirming that Mediterranean oak are tolerant to ambient and super-ambient [O3], but may be damaged by acute exposure to high [O3]. Stomatal and mesophyll conductance and photosynthesis were all reduced immediately after fumigation with high [O3], but recovered to control values within 72 h. Both the intercellular and chloroplast CO2 concentrations ([CO2]) remained constant throughout the experiment. Thus, although treatment with a high [O3] may have induced stomatal closure and consequent down-regulation of photosynthesis, we found no evidence that photosynthesis was limited by low [CO2] at the site of fixation. One-year-old leaves of Q. ilex were much less sensitive to O3 than current-year leaves, suggesting that the low stomatal conductance observed in aging leaves limited O3 uptake. No similar effect of leaf age was found in Q. pubescens. Dark respiration decreased during the treatment period, but a similar decrease was observed in leaves exposed to low [O3], and therefore may not be an effect of O3 treatment. Light respiration, on the other hand, was mostly constant in ozone-treated leaves and increased only in leaves in which photosynthesis was temporarily inhibited by high [O3], pre-

venting them from acting as strong sinks that recycle respiratory CO2 in the leaves. There was no evidence of photochemical damage in Q. ilex leaves, whereas Q. pubescens leaves exposed to a high [O3] showed limited photochemical damage, but recovered rapidly. Biochemical markers were affected by the high [O3], indicating accumulation of reactive oxygen species (ROS) and increased denaturation of lipid membranes, followed by activation of isoprene biosynthesis in Q. pubescens leaves. We speculate that the high isoprene emissions helped quench ROS and normalize membrane stability in leaves recovering from O3 stress. Keywords: diffusive, isoprenoids, metabolic and photochemical limitations of photosynthesis, oxidative stress, stomatal conductance.

Introduction Ozone (O3) formation occurs predominantly in highly populated and industrialized areas of the world, but the discovery that O3 may also be formed by interactions between anthropogenic and biogenic precursors in part explains why O3 formation also occurs in some rural areas around the world (Trainer et al. 1987). Irrespective of the origin, O3 concentrations ([O3]) nowadays often rise above phytotoxic thresholds (Heck et al. 1986, Ormrod and Hale 1995, Fuhrer 2000), leading to visible foliar injuries and reduced plant productivity. At the biochemical level, O3 produces reactive oxygen species (ROS) (Guderian 1985, Emberson et al. 2000) that damage cellular membranes and impair the main metabolic functions (Pell et al. 1997). Stomatal closure, which reduces leaf O3 uptake, is the first line of defense against O3 (Mansfield and Freer-Smith 1984, Reiling and Davison 1995). It is unclear, however, whether stomatal closure is a direct response to O3, or an indirect effect of a primary stress response inducing inhibition of photosynthesis. Although past studies have tried to relate O3 damage to the external [O3] (Fuhrer et al. 1997), more recent studies have shown that leaf O3 uptake is a more suitable

1524

VELIKOVA, TSONEV, PINELLI, ALESSIO AND LORETO

index of damage (Pasqualini et al. 2002). The low stomatal conductance of Mediterranean plants may be associated with their characteristic O3 tolerance (Nali et al. 1998). Biochemical mechanisms are the second line of defense against O3. When O3 penetrates and forms ROS, antioxidants rapidly remove the resulting compounds (Musselman and Massman 1999). Some of these antioxidant mechanisms have been elucidated (Pell et al. 1997), whereas others, such as the antioxidant action of volatile isoprenoids (Loreto and Velikova 2001), are still being debated. Repair mechanisms are the final line of defence, reconstituting structures (e.g., protein assemblies within membranes) temporarily damaged by acute O3 exposure (Pell et al. 1997). Ozone effects on vegetation are generally assessed by fumigating parts of plants or entire plants with O3. The simplest method is to fumigate a small part of the plant (a leaf, or part of it), enclosed in a cuvette, where environmental parameters can be optimally controlled (Loreto and Velikova 2001). This method allows excellent mechanistic interpretations, but is not realistic in that the whole plant is not exposed to the stressor. At the other extreme, whole plants have been grown in large enclosures (growth chambers, greenhouses, open-top chambers) where they are exposed to O3 fumigation (Pleijel et al. 1991, Paakkonen et al. 1997). This method allows more realistic O3 exposure, but control of environmental parameters may not always be adequate for the study of metabolic processes, especially in plants exposed to acute O3 episodes. The enclosed system may significantly alter the environment compared with open air, and it is usually difficult to distinguish between effects attributed to acute O3 exposure and those associated with the control of growth conditions. To study the effects of acute and prolonged O3 exposure on some physiological traits of Mediterranean vegetation, we adopted an approach that allowed localized O3 fumigation (LOF) of single leaves in an oak canopy that was not maintained in an enclosed system. This approach is similar to the “zonal air pollution system” (ZAPS) (Runeckles et al. 1990), which comprises a series of pipes that continuously release O3 over a crop. Others have used similar systems (e.g., Tjoelker et al. 1994, Pepin and Körner 2002). Like those systems, LOF allows precise fumigation of target leaves of field-grown plants without interference from enclosure systems, allowing field studies of O3 exposure gradients in leaves of similar or different ontogeny within the same canopy, while maintaining other parts of the plant free from the pollutant. Our specific objectives were: (1) to determine whether primary (photosynthesis, respiration) and secondary (isoprenoid emission) metabolism of Mediterranean oak are sensitive to acute and prolonged (several days), but not chronic, super-ambient or high [O3]; and (2) if leaf ontogeny plays a role in establishing O3 sensitivity in the plant canopy. Materials and methods Plant materials and weather conditions We studied 5-year-old Quercus ilex (L.) and 3-year-old Quer-

cus pubescens (L.) seedlings planted in a sandy, alluvional soil at the CNR experimental station, Monterotondo Scalo, Rome. Plants were about 2 m tall and were pruned to three main branches. Experiments were run in the summer, under typically clear, dry and hot Mediterranean conditions. Plants were irrigated daily with tap water and fertilized weekly with half-strength Hoagland’s solution to avoid drought and nutritional stress during the experiments. Air temperature during the measurements was 30 – 34 °C, air humidity was around 40%, and solar irradiance at the canopy level was greater than 1000 µmol m – 2 s – 1. Experiments were performed in the absence of wind (< 0.5 m s – 1) to minimize O3 transport beyond the fumigation site. Wind was reduced by a natural windbreak formed by Eucalyptus plants surrounding the experimental site at an average distance of 200 m from the experimental plants. Climatic data were continuously monitored by a weather station installed 50 m away from the experimental site. The weather station included an anemometer and sensors for measuring air temperature, relative humidity, global radiation and photosynthetic photon flux (SIAP, Bologna, Italy). Localized ozone fumigation system Ozone was generated by pumping air with a diaphragm air pump through a winding quartz glass illuminated with a UV light source (Helios Italquartz, Milan, Italy). The amount of O3 generated was regulated by changing either the brightness of the UV light source or the rate of airflow. Ozonated air was supplied to the plants through Teflon tubes (0.5 cm internal diameter) (Figure 1), which were wrapped around the main tree branches and perforated with 0.2 mm holes adjacent to selected fully expanded leaves. Leaves were held at a distance of 0.5 cm from the holes through which O3 was released. No external mounting was needed to maintain this distance. The [O3] applied to different parts of the plant was continuously monitored with a photometric O3 analyzer (1008 Dasibi Environmental, Glendale, CA). Sampling lines were fixed at different distances from the O3 vents, that is, (1) directly fumigated leaves (0.5 cm from the O3 vent), (2) leaves in close proximity (< 30 cm to the O3 vent), and (3) leaves 60 cm from the O3 vent. Another sampling line was located on a non-fumigated tree (control). Each sampling line was connected to the O3 analyzer for 15 min before switching to another line by operating manual Teflon valves. Ozone fumigation was carried out for 9 h per day (0830 –1730 h) for three consecutive days (July 19 – 22, 2004). Each line was sampled nine times during each experiment day. Gas exchange measurements We measured the exchange of carbon dioxide (CO2) and water (H2O) between leaves and air with two LI-6400 portable gas exchange systems equipped with a red/blue light source (LiCor, Lincoln, NE). Photosynthesis, dark respiration (Rd), transpiration, stomatal conductance to CO2 and H2O, and intercellular CO2 concentration (Ci ) were calculated with the instrument software. The central part of a leaf was clamped in the LI-6400 gas exchange cuvette and exposed to a flux (0.5

TREE PHYSIOLOGY VOLUME 25, 2005

LOCALIZED OZONE FUMIGATION AND OZONE EFFECTS ON OAK PHYSIOLOGY

1525

Figure 1. Sketch of the localized ozone fumigation system (LOF) and ozone concentrations ([O3]) measured during a single day on leaves apressed to the O3 vents (A), at distances of < 30 cm from the O3 vents (B) and controls and leaves > 60 cm from the O3 vents (C). Values are means of [O3] ± SE (n = 6 measurements: two leaves on each of three plants for A and B, one leaf on three plants + one leaf on three control plants which were not subjected to LOF for C). Black bars represent [O3] during fumigation and white bars represent the background [O3] measured adjacent to control plants.

liters min – 1) of artificial air created by mixing O2, N2 and CO2 (20 or 2, 80 and 0.037%, respectively) from tanks of pure gases. Oxygen concentration was lowered to 2% when testing fluorescence and gas exchange of leaves under non-photorespiratory conditions. During all measurements, the cuvette was maintained at 30 °C and the leaf was illuminated at 1000 µmol m – 2 s – 1 (or darkened for at least 15 min for Rd measurements). Simultaneous with the CO2 and H2O gas exchange measurements, isoprene emission from Q. pubescens leaves was determined by connecting the outflow of the LI-6400 cuvettes to a portable gas chromatograph (Syntech GC 855 Syntech Spectras, Groningen, The Netherlands) as described by Loreto and Velikova (2001). Dark respiration was not measured in Q. pubescens. Light respiration (Rl), another component of the total CO2 exchange between leaf and air, was measured in Q. ilex leaves only by detecting 12CO2 release from leaves maintained in air with 13CO2, as described by Pinelli and Loreto (2003). Gas exchange was measured on intact leaves at the four above-mentioned distances from the O3 vent, before and immediately after O3 treatment (0 h), and during recovery (72, 168 and 288 h in Q. ilex and 72 and 288 h only in Q. pubescens). During the recovery period, plants were maintained at ambient [O3]. Measurements were made at the same time each day (1730–1830 h) to minimize physiological and environmental changes. Fluorescence measurements and chloroplastic CO2 concentration Chlorophyll fluorescence properties were measured in vivo simultaneously with the gas exchange measurements using the fluorometer incorporated in the head of the leaf chamber (LI-6400-40). The maximal quantum yield of photosystem II (PSII) was measured as the ratio of variable and maximal fluorescence in dark-adapted leaves (Fv /Fm), and the actual quan-

tum yield of PSII in illuminated leaves was measured by the fluorescence parameter ∆F / Fm′, the ratio between the increase over steady-state fluorescence following a saturating pulse of light (10,000 µmol m – 2 s – 1) and maximal fluorescence. The fluorescence traces were recorded as described by Van Kooten and Snel (1990). The electron transport rate (Jf) driving photosynthesis and photorespiration was estimated by multiplying ∆F/Fm′ by the absorbed irradiance (0.84 as independently measured on other leaves of the same plants; Fabrizio Pietrini, CNR-IBAF, Rome, Italy, personal communication) and by a correction factor, taking into account the percent (about 50%; see Laisk and Loreto 1996) of light impinging on PSII antennas. Internal conductance to CO2 diffusion, the inverse of the total resistance encountered by CO2 across the leaf mesophyll, was calculated by assuming that the electron transport rates calculated by gas exchange and fluorescence match in the absence of internal resistances (cf. Loreto et al. 1994). The actual [CO2] at the chloroplast site (Cc) was then calculated from the internal conductance (Harley et al. 1992). Biochemical assays Concentrations of hydrogen peroxide ([H2O2]) and the membrane peroxidation marker malonyldialdehyde ([MDA]) were determined in leaves that were cut and rapidly frozen in liquid nitrogen. The two compounds were measured only in leaves directly fumigated with O3 and in control leaves of plants unexposed to O3 fumigation. Leaves fumigated with O3 were sampled immediately and 288 h after the O3 fumigation in Q. ilex and an additional sampling 24 h after O3 fumigation was made in Q. pubescens. Hydrogen peroxide concentrations were determined spectrophotometrically (absorbance at 390 nm), as described by Velikova et al. (2000). We determined MDA as the end product of lipid peroxidation by the

TREE PHYSIOLOGY ONLINE at http://heronpublishing.com

1526


thiobarbituric acid (TBA) test (Heath and Parker 1968). Experimental design and statistics The experiment was carried out on three plants of each Quercus species. On each plant, only one branch was subjected to LOF and two leaves were directly exposed to O3 from the vents. In Q. ilex plants, current-year and 1-year-old leaves were selected. In Q. pubescens plants, the third and fifth leaves from the apical, fully expanded leaf were chosen. The data presented are means from at least three replications.

Results The LOF system allowed leaves from the same plant to be exposed to three O3 concentrations. A high [O3] was experienced by leaves apressed to the O3 vents. We were able to adjust the O3 formation supply so as to expose these leaves to 300 ± 50 ppb of O3, throughout the experiment (Figure 1). An intermediate, super-ambient [O3] that varied between 120 and 280 ppb (mean concentration over the whole treatment period = 188 ppb) was supplied to leaves located < 30 cm from the O3 vent. Leaves of branches 60 cm from the O3 vent were exposed to O3 concentrations ranging between 30 and 100 ppb, which were comparable to those observed around non-fumigated trees, reflecting the natural variation in ambient [O3] (Figure 1). Because the ambient O3 was photochemically generated, it likely increased as temperatures increased during the day and with the transport of photochemical smog from urban areas in Rome. Quercus ilex Measurements made immediately after fumigation revealed that photosynthesis decreased in Q. ilex leaves exposed to high

[O3] (Figure 2A), and inhibition of photosynthesis in current-year leaves was greater than in 1-year-old leaves. The inhibition was only transient, however, and leaf photosynthetic rates 72 h after fumigation had returned to control values. The intermediate [O3] had a small negative effect on photosynthesis immediately after the fumigation period (P ≤ 0.10). The low [O3] did not significantly affect leaf photosynthesis: these leaves had similar photosynthetic rates as leaves measured before the fumigation and leaves of untreated control plants measured during the 288-h experiment. Stomatal conductance was inhibited by the highest [O3], and, to a minor extent, by the intermediate [O3], and recovery from fumigation followed the same trend observed for photosynthesis (Figure 2B). Stomatal conductance was inherently lower in 1-year-old leaves than in current-year leaves, and the 1-year-old leaves showed a smaller reduction in stomatal conductance in response to O3 stress. The Ci was not significantly affected by the treatments (Figure 2C), although a trend of decreasing Ci with time was observed in all leaves, including leaves of control plants. Internal conductance to CO2 diffusion was unaffected by the O3 fumigation and, consequently, there were no significant treatment differences in Cc (Figure 2D). In contrast, Rd decreased significantly in all ozone-fumigated leaves, and the decrease occurred most rapidly in current-year leaves exposed to the high [O3] (Figure 3A). Respiration in light varied between 30 and 80% of Rd and was not affected significantly by low or intermediate [O3]. In leaves exposed to a high [O3], Rl increased significantly relative to control values, but the increase was observed immediately after treatment (Figure 3B). Fluorescence yield in dark-adapted leaves was unaffected by the O3 treatments (Figure 4A), but Jf showed a trend similar to that of photosynthesis and was significantly inhibited in leaves exposed to a high [O3] immediately after treatment. The

Figure 2. Photosynthesis (Pn ) (A), stomatal conductance (gs) (B), intercellular carbon dioxide concentration (Ci) (C) and chloroplast carbon dioxide concentration (Cc) (D) of Q. ilex leaves before the ozone (O3) treatment (control) and 0, 72, 168 and 288 h after the O3 treatment. Symbols: 䊊, 䊉 = current-year leaves; and 䉭, 䉱 = 1-year-old leaves. Open and closed symbols denote leaves exposed to an intermediate and a high [O3], respectively. Leaves exposed to a low [O3] served as controls and are not shown. Values are means ± SE (n = 4). Asterisks indicate significant differences of the means with respect to controls for P = 0.05 (**), or P = 0.10 (*), according to the Tukey’s test.



1527

greatest inhibition of Jf was observed in current-year leaves, but the inhibition was transient, with values recovering to control rates within 72 h (Figure 4B). The ratio between quantum yield of PSII, estimated by fluorescence, and quantum yield of CO2 fixation, calculated by gas exchange, was higher when measured under photorespiratory (21% O2) conditions than under non-photorespiratory (2% O2) conditions (Figure 5). However, in both cases, the response of leaves exposed to the O3 treatment fit the line for control leaves (r 2 = 0.78 under both photorespiratory and non-photorespiratory conditions with a confidence interval probability > 95%). The amount of H2O2 generated by leaves exposed to a high [O3] and assayed immediately after the treatment was significantly increased compared with control values, and a similar increased [H2O2] was also detected 288 h after the end of the treatment (Figure 6A). The [MDA] was slightly enhanced in leaves assayed immediately after fumigation with a high [O3], but 288 h after the treatment, the [MDA] was significantly lower than the control concentration (Figure 6B).

Figure 4. Ratio between variable and maximal fluorescence (Fv /Fm), indicating maximal photosystem II efficiency (A) and electron transport rate calculated from the fluorescence yield (Jf ) in illuminated (1000 µmol photons m – 2 s – 1 ) Q. ilex leaves (B). Symbols: 䊊, 䊉 = current-year leaves; and 䉭, 䉱 = 1-year-old leaves. Open and closed symbols denote leaves exposed to an intermediate or a high [O3], respectively. Leaves exposed to a low [O3] served as controls and are not shown. Values are means ± SE (n = 4). Asterisks indicate significant differences of the means relative to controls for P = 0.05 (**), or P = 0.10 (*), according to the Tukey’s test.

Quercus pubescens

Figure 3. Dark (A) and light respiration (B) in Q. ilex leaves before the ozone (O3) treatment (control) and 0, 72, 168 and 288 h after the O3 treatment. Symbols: 䊊, 䊉 = current-year leaves; and 䉭, 䉱 = 1-yearold leaves. Open and closed symbols denote leaves exposed to an intermediate and a high [O3], respectively. Leaves exposed to a low [O3] served as controls and are not shown. Values are means ± SE (n = 4). Asterisks indicate significant differences of the means relative to controls for P = 0.05 (**), or P = 0.10 (*), according to the Tukey’s test.

Ozone fumigation caused similar physiological impairments to leaves in Q. pubescens as in Q. ilex. Inhibition of photosynthesis, stomatal conductance and Jf were more evident and statistically significant in Q. pubescens leaves exposed to high [O3] than to intermediate [O3], but all parameters recovered to control values within 72 h of the treatment (Table 1), with the exception of photosynthesis. Photosynthetic rates of leaves exposed to a high [O3] were still significantly lower than in controls 72 h after treatment, although some recovery had occured. The maximal quantum yield of PSII in dark-adapted leaves was significantly affected by fumigation with a high [O3], but it also recovered to control values within 72 h of the treatment. Destructive leaf samplings indicated that exposure to a high [O3] stimulated the production of H2O2 and MDA in Q. pubescens leaves (Figures 6C and 6D). The production of both stress


1528


the treatment compared with control leaves, but a spike in isoprene emission was found at the last sampling (288 h after treatment) (Table 1). Exposure to an intermediate [O3] did not affect isoprene emission. Discussion Assessment of the LOF system

Figure 5. Relationship between the quantum yield of photosystem II (PSII), as measured by fluorescence in illuminated (1000 µmol photons m – 2 s – 1) leaves, and the quantum yield of CO2 fixation (φCO2), calculated by dividing the photosynthetic rate by the absorbed irradiance. Open symbols denote measurements made under non-photorespiratory conditions (O2 = 2%) and closed symbols denote measurements made under ambient conditions (O2 = 21%).

markers was sustained for 24 h following the O3 treatment, but concentrations were similar to those observed in control leaves 288 h after the treatment. Isoprene emission from Q. pubescens leaves was affected by O3 fumigation. Exposure to a high [O3] significantly reduced isoprene emission by leaves sampled immediately after

Ozone is a reactive gas, making the study of plant responses to elevated [O3] difficult. Free-air O3 enrichment can now be performed and is the most suitable technique for studying the effects of chronic exposure to O3 on vegetation (Karnosky et al. 2003). Whole plant (Pasqualini et al. 2001) and single leaf (Loreto et al. 2004) laboratory O3 fumigation experiments have, nevertheless, provided insight into the physiological and biochemical changes induced by acute and short-term exposure to elevated [O3]. Experimental fumigation systems used in the laboratory, however, are often not transferable to field conditions. The LOF system is a promising compromise that allows study of O3 effects on plant physiology under field conditions, by providing acute or short-term fumigation of individual leaves on field-grown plants with a minimal O3 source. We used the LOF system to simulate three O3 concentrations in three parts of the same tree. Under wind-free conditions, the system supplied O3 at three target values to leaves on the same tree over a 3-day experimental period (Figure 1). Thus, we demonstrated that the LOF system avoids the influence of genetic diversity on plant responses to O3 either by revealing the effect of different [O3] on leaves at the same ontogenetic stage

Figure 6. Biochemical stress markers in leaves of Q. ilex (A, B) and Q. pubescens (C, D) exposed to high ozone (O3) concentrations. Hydrogen peroxide (H 2O2) (A, C) and malonyldialdehyde (MDA) (B, D) were measured in control leaves and at 0 and 288 h (Q. ilex), or 0, 24 and 288 h (Q. pubescens) after recovery from the O3 treatment.



1529

Table 1. Photosynthesis (Pn ), stomatal conductance (gs), intercellular CO2 concentration (Ci ), chloroplast CO2 concentration (Cc), maximal photosystem II efficiency in dark-adapted leaves (Fv /Fm ), electron transport rate calculated from the fluorescence yield in illuminated leaves (Jf) and isoprene emission rate in Q. pubescens leaves at 0, 72 and 288 h after exposure to three ozone concentrations ([O3]) with the localized O3 fumigation (LOF) system. Parameters of control plant leaves exposed to ambient [O3] are shown. Values are means ± SE (n = 6 measurements: two leaves from each of three plants). Asterisks indicate significant differences between mean values for treated and control leaves: P = 0.05 (**), or P = 0.10 (*), according to the Tukey’s test. Parameter

Time (h)

High [O3]

Medium [O3]

Low [O3]/control

Control

Pn (µmol m – 2 s – 1)

0 72 288

6.1 ± 0.3 ** 8.5 ± 0.3 * 10.9 ± 0.5

7.8 ± 0.4 * 8.7 ± 0.3 10.5 ± 0.2

9.7 ± 0.4 10.3 ± 0.8 10.9 ± 0.8

9.9 ± 0.1 10.3 ± 0.5 10.4 ± 0.6

gs (mol m – 2 s – 1)

0 72 288

0.064 ± 0.002 ** 0.089 ± 0.001 * 0.116 ± 0.006

0.089 ± 0.003 * 0.086 ± 0.003 0.114 ± 0.003

0.108 ± 0.009 0.116 ± 0.011 0.142 ± 0.018

0.106 ± 0.006 0.113 ± 0.010 0.127 ± 0.009

Ci (ppm)

0 72 288

204 ± 11 208 ± 5 207 ± 3

219 ± 5 197 ± 4 211 ± 2

220 ± 7 213 ± 8 235 ± 7

222 ± 9 220 ± 3 230 ± 9

Cc (ppm)

0 72 288

213 ± 10 180 ± 6 178 ± 4

191 ± 9 173 ± 4 173 ± 1

190 ± 8 189 ± 10 197 ± 11

184 ± 7 186 ± 9 194 ± 5

Fv /Fm

0 72 288

0.66 ± 0.03 ** 0.79 ± 0.01 0.81 ± 0.04

0.77 ± 0.03 0.80 ± 0.01 0.81 ± 0.01

0.81 ± 0.07 0.82 ± 0.06 0.81 ± 0.05

0.80 ± 0.09 0.80 ± 0.07 0.81 ± 0.07

Jf (µmol m – 2 s – 1)

0 72 288

59 ± 1 ** 86 ± 3 105 ± 6

79 ± 5 91 ± 1 102 ± 2

93 ± 5 94 ± 8 93 ± 6

90 ± 4 93 ± 8 94 ± 8

Isoprene (nmol m – 2 s – 1)

0 72 288

18.9 ± 2.2 ** 25.6 ± 2.5 31.8 ± 3.0 *

25.5 ± 2.4 24.3 ± 2.9 25.8 ± 3.1

26.4 ± 2.3 25.9 ± 3.0 27.1 ± 2.4

26.9 ± 2.7 26.9 ± 3.2 26.5 ± 2.8

on the same tree, or by studying the effect of the same [O3] on leaves of different ages on the same tree. In both Quercus species, exposure to a high [O3] decreased photosynthesis and stomatal conductance. Exposure to an intermediate [O3] had a negligible effect on the measured parameters, confirming that Mediterranean oak, especially Q. ilex, are tolerant of ambient [O3] (Manes et al. 1998). We found an age-dependent sensitivity to O3 in Q. ilex leaves, with 1-yearold leaves being less sensitive to O3 than current-year leaves. This indicates that, although the youngest, most productive fraction of the canopy (based on photosynthetic rates) is negatively affected by high [O3] exposure, a large fraction of the leaves of this species may be tolerant to acute high [O3] exposure. Sensitivity to O3 does not reflect aging of current-year leaves, however, because in current-year leaves of Q. pubescens, the same age-dependent O3 effect was observed independent of leaf branch position. Decreases in photosynthesis and stomatal conductance of leaves exposed to high [O3] were transient, with full recovery observed in Q. ilex leaves 72 h after exposure and in Q. pubescens 288 h after exposure. This suggests that the decrease in photosynthesis during fumigation with a high [O3] in these oak species did not involve damage to biochemical processes, but was a result of alterations in processes, such as increasing re-

sistance to CO2 diffusion, caused by the transient decrease in stomatal conductance. However, calculated Ci did not change significantly in leaves exposed to high [O3], indicating that stomatal closure and inhibition of photosynthesis matched each other and that low Ci immediately after the O3 treatment did not reduce photosynthesis. Ozone may temporarily impair K+ channels involved in stomatal opening (Torsethaugen et al. 1999), resulting in down-regulation of photosynthesis rather than permanent inhibition. Because a reduction of the internal conductance to CO2 and, consequently, of the flux of CO2 to the chloroplasts, is often observed in aging (Loreto et al. 1994) or stressed leaves (Delfine et al. 1999), we determined whether a high [O3] decreases CO2 concentration at the chloroplast site. We found that Cc was unaffected by high [O3] and therefore, a decrease in Cc cannot account for the observed reduction in leaf photosynthesis in leaves fumigated with a high [O3]. Although diffusive resistances played no a role in limiting photosynthesis of leaves sensitive to O3, the same resistances may have restricted O3 uptake in 1-year-old Q. ilex leaves. These leaves showed inherently higher resistances (lower conductances) than current-year leaves, which might have considerably reduced leaf O3 uptake. The concept that O3 uptake (i.e., the [O3] inside leaves) is a more useful indicator of damage


1530


than external [O3] was first postulated by Reich (1986) and has recently received additional experimental support (Pasqualini et al. 2002, Matyssek et al. 2004). Respiration was extremely sensitive to O3; however, this parameter also decreased in control leaves and in leaves exposed to a low [O3] during the experiment. Thus, we were unable to demonstrate an effect of O3 on mitochondrial functions; however, previous studies indicate that mitochondrial respiration should be unaffected (Tjoelker et al. 1993, Wullschleger et al. 1996) or even stimulated (Tjoelker et al. 1993) by O3 exposure. Respiration in light increased immediately after treatment with a high [O3], both in current-year and 1-year-old Q. ilex leaves. Light respiration is the fraction of respiration that contributes to the total exchange of CO2 between illuminated leaves and atmosphere, together with photosynthesis and photorespiration. It is generally lower than Rd, being inhibited or recycled in illuminated leaves. The increase in Rl might have contributed to the observed decrease in photosynthesis in response to high [O3]. On the other hand, it has been shown that Rl is inversely related to photosynthesis, a fact that is explained on the basis that respiratory CO2 is not inhibited in light, but is recycled by photosynthesis (Pinelli and Loreto 2003). This could also explain why Rl increases significantly in parallel with the O3-driven reduction in photosynthesis. Photochemical reactions were temporarily damaged by exposure to high [O3] in Q. ilex because (1) the maximal efficiency of PSII was unaffected by the O3 treatment, as shown by the fluorescence yield in the dark; and (2) the fluorescence yield in illuminated leaves maintained a constant relationship with the CO2 fixation quantum yield in controls, treated and recovering leaves. This indicates that, irrespective of the O3 treatment, the same number of electrons were required to carry out photosynthesis under non-photorespiratory conditions (about five, which is close to the theoretical yield), and photosynthesis and photorespiration under ambient conditions (about 12). In Q. pubescens leaves, the photochemistry of photosynthesis was significantly inhibited by high [O3], as indicated by the significant drop in Fv / Fm in dark-adapted leaves and Jf in illuminated leaves (Table 1). This effect was observed only immediately after the treatment, however, indicating that fast repair of the photochemical machinery in this species, as previously found in O3-resistant clones of poplar (Nali et al. 1998). Quercus pubescens was more sensitive to O3 than Q. ilex, perhaps reflecting the slightly higher conductance to gases (including O3) of Q. pubescens, which is less sclerophyllous than Q. ilex. The complete recovery of photosynthetic properties after termination of O3 fumigation indicates that the biochemistry of photosynthesis in our Mediterranean oak was not permanently affected by even the highest [O3]. However, we observed significant variation in [H2O2] and [MDA], the biochemical markers, indicating a persistent accumulation of ROS after the high [O3] treatment. As predicted, the increase in ROS was associated with an increased denaturation of lipids in membranes (Loreto and Velikova 2001), but the lipid denaturation marker MDA returned to control values by 288 h after

treatment. These results indicate that, although some damage occurred to the biochemistry of oak leaves, it was not sufficient to reduce photosynthetic rates under ambient conditions, and that mechanisms protecting membranes from denaturation are activated to cope with the persistently high abundance of ROS caused by exposure to O3. The activation of these mechanisms may be triggered slowly during recovery, eventually reestablishing MDA concentrations similar to those of control leaves. We observed a decrease in H2O2 production 288 h after termination of high [O3] fumigation in Q. pubescens. Whether this was a consequence of the activation of defense mechanisms or the reason why lipid peroxidation reverted to control values remains to be determined. Isoprene, a volatile molecule emitted in significant quantities by many plant species, may have antioxidative properties, scavenging ROS or stabilizing membranes during stress (Loreto and Velikova 2001, Affek and Yakir 2002). We therefore measured isoprene emission rates in Q. pubescens, in which species isoprene emission rates are normally high. Isoprene emission rates were unaffected by fumigation with the low and the intermediate O3 concentrations, but emission rates were significantly depressed immediately after leaves were exposed to high [O3]. Because isoprene is predominantly derived from photosynthetic intermediates (Sharkey and Yeh 2001), we suggest that the temporary inhibition of photosynthesis accounts for the observed inhibition of isoprene emission and its subsequent fast recovery. However, isoprene emission rates increased significantly 288 h after termination of the high [O3] treatment and this increase was not associated with increasing photosynthetic rates. It has been previously observed that O3 (Loreto et al. 2004), as well as environmental stresses in general (Sharkey and Loreto 1993), can stimulate isoprenoid synthesis. Our results suggest that isoprene stimulation occurs only when the stress first affects photosynthesis and is delayed relative to the onset of O3 stress, probably reflecting the activation of a whole class of constitutive and induced genes (Sharkey et al. 2005). The stimulation of isoprene emission occurs when the [H2O2] and [MDA] start to be quenched, corroborating the idea that isoprene might be involved in ROS quenching and in the protection of membranes against oxidative stress. In conclusion, the LOF system facilitated the study of the effects of acute and short-term exposures to high and moderately high (super-ambient) [O3] on genetically similar and both ontogenetically similar and ontogenetically dissimilar leaves of plants grown under field conditions. We obtained evidence that Mediterranean oak are generally resistant to short-term high [O3] exposure, because no permanent damage was observed during the 3-day-long fumigation. Carbon assimilation of current-year leaves was temporarily affected by exposure to high [O3], but recovery was rapid and may have involved increasing resistance to CO2 diffusion (Q. ilex) and photochemical damage (Q. pubescens). Biochemical stress markers were affected only by high O3 concentrations, indicating that biochemical changes occurred in the absence of significant physiological changes, but may influence the development and performances of leaves exposed to O3 stress in the long term.


LOCALIZED OZONE FUMIGATION AND OZONE EFFECTS ON OAK PHYSIOLOGY Acknowledgments Giuseppe Santarelli provided weather data and Domenico Tricoli helped with LOF design and implementation. This study was supported by The European Commission (contract MC-RTN-CT-2003504720, “ISONET”), by the European Science Foundation scientific programme VOCBAS, by the Ministero dell’Ambiente Project “Effetti degli stress ambientali con particolare riguardo all’ozono troposferico sulla vegetazione naturale ed in area Mediterranea” (OZONIT 2) and by the Italian Ministry for Research project FIRBRBAU018FWP: “Attivita’ antiossidante degli isoprenoidi volatili e loro ruolo nella protezione delle piante dagli stress abiotici.” Part of the research was also supported by the bilateral project within the framework agreement between the Italian National Research Council and Bulgarian Academy of Sciences.

References Affek, H.P. and D. Yakir. 2002. Protection by isoprene against singlet oxygen in leaves. Plant Physiol. 129:269 – 277. Delfine, S., A. Alvino, M.C. Villani and F. Loreto. 1999. Restrictions to CO2 conductance and photosynthesis in spinach leaves recovering from salt stress. Plant Physiol. 119:1101–1106. Emberson, L.D., M.R. Ashmore, H.M. Cambridge, D. Simpson and J.P. Tuovinen. 2000. Modelling stomatal ozone flux across Europe. Environ. Pollut. 109:403–413. Fuhrer, J. 2000. Introduction to the special issue on ozone risk analysis for vegetation in Europe. Environ. Pollut. 109:359 – 360. Fuhrer, J., L. Skarby and M.R. Ashmore. 1997. Critical levels for ozone effects on vegetation in Europe. Environ. Pollution 97: 91 – 106. Guderian, R. 1985. Air pollution by photochemical oxidants. Formation, transport, control and effects on plants. Ecol. Stud. 52:346. Harley, P.C., F. Loreto, G. Di Marco and T.D. Sharkey. 1992. Theoretical considerations when estimating the mesophyll conductance to CO2 flux by analysis of the response of photosynthesis to CO2. Plant Physiol. 98:1429 – 1436. Heath, R.L. and L. Parker. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125:189–198. Heck, W.W., A.S. Heagles and D.S. Shriner. 1986. Effects on vegetation: native, crop, forests. In Air Pollution. 3rd Edn. Ed. A.C. Stern. Academic Press, New York, pp 248–333. Karnosky, D.F., D.R. Zak, K.S. Pregitzer et al. 2003. Tropospheric O3 moderates responses of temperate hardwood forests to elevated CO2: a synthesis of molecular to ecosystem results from the Aspen FACE project. Funct. Ecol. 17:289–304. Laisk, A. and F. Loreto. 1996. Determining photosynthetic parameters from leaf CO2 exchange and chlorophyll fluorescence: Rubisco specificity factor, dark respiration in the light, excitation distribution between photosystems, alternative electron transport rate and mesophyll diffusion resistance. Plant Physiol. 110: 903 – 912. Loreto, F. and V. Velikova. 2001. Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol. 127:1781–1787. Loreto, F., G. Di Marco, D. Tricoli and T.D. Sharkey. 1994. Measurements of mesophyll conductance, photosynthetic electron transport and alternative electron sinks of field grown wheat leaves. Photosynth. Res. 41:397 – 403.

1531

Loreto, F., P. Pinelli, F. Manes and H. Kollist. 2004. Impact of ozone on monoterpene emission and evidence for an isoprene-like antioxidant action of monoterpenes emitted by Quercus ilex leaves. Tree Physiol. 24:361–367. Manes, F., M. Vitale, E. Donato and E. Paoletti. 1998. O3 and O3+CO2 effects on a Mediterranean evergreen broadleaf tree, holm oak (Quercus ilex L.). Chemosphere 36:801 – 806. Mansfield, T.A. and P.H. Freer-Smith. 1984. The role of stomata in resistance mechanisms. In Gaseous Air Pollutants and Plant Metabolism. Eds. M.J. Koziol and F.R. Whatley. Butterworths, London, pp 131 – 145. Matyssek, R., G. Wieser, A.J. Nunn et al. 2004. Comparison between AOT40 and ozone uptake in forest trees of different species, age and site conditions. Atmos. Environ. 38:2271 – 2281. Musselman, R.C. and W.J. Massman. 1999. Ozone flux to vegetation and its relationship to plant response and ambient air quality standards. Atmos. Environ. 33:65 – 73. Nali, C., L. Guidi, F. Filippi, G.F. Soldatini and G. Lorenzini. 1998. Photosynthesis of two poplar clones contrasting in O3 sensitivity. Trees 12:196–200. Ormrod, D.P. and B.A. Hale. 1995. Physiological responses of plants and crops to ozone stress. In Handbook of Plant and Crop Physiology. Ed. M. Pessarakli. Marcel Dekker, New York, pp 735–760. Paakkonen, E., T. Holopainen and L. Karaelampi. 1997. Variation in ozone sensitivity among clones of Betula pendula and Betula pubescens. Environ. Pollut. 95:37 – 44. Pasqualini, S., P. Batini, L. Ederli, A. Porceddu, C. Piccioni, F. De Marchis and M. Antonielli. 2001. Effects of short-term ozone fumigation on tobacco plants: response of the scavenging system and expression of the glutathione reductase. Plant. Cell Environ. 24: 245 – 252. Pasqualini, S., M. Antonielli, L. Ederli, C. Piccioni and F. Loreto. 2002. Ozone uptake and its effect on photosynthetic parameters of two tobacco cultivars with contrasting ozone sensitivity. Plant Physiol. Biochem. 40:599 – 603. Pell, E.J., C.D. Schlagnhaufer and R.N. Arteca. 1997. Ozone induced oxidative stress: mechanisms of action and reaction. Physiol. Plant. 100:264–273. Pepin, S. and C. Körner. 2002. Web-FACE: a new canopy free-air CO2 enrichment system for tall trees in mature forests. Oecologia 113:1–9. Pinelli, P. and F. Loreto. 2003. 12CO2 emission from different metabolic pathways measured in illuminated and darkened C3 and C4 leaves at low, atmospheric, and elevated CO2 concentrations. J. Exp. Bot. 54:1761 – 1769. Pleijel, H., L. Skarby, G. Wallin and G. Selden. 1991. Yield and grain quality of spring wheat (Triticum aestivum L. cv. Drabant) exposed to different concentrations of ozone in open top chambers. Environ. Pollut. 69:151 – 168. Reich, P.B. 1986. Quantifying plant response to ozone: a unifying theory. Tree Physiol. 3:63 –91. Reiling, K. and A.W. Davison. 1995. Effects of ozone on stomatal conductance and photosynthesis in populations of Plantago major L. New Phytol. 12:587 – 594. Runeckles, V.C., E.F. Wright and D. White. 1990. A chamberless field exposure system for determining the effects of gaseous airpollutants on crop growth and yield. Environ. Pollut. 63:61–77. Sharkey, T.D. and F. Loreto. 1993. Water stress, temperature, and light effects on the capacity for isoprene emission and photosynthesis of kudzu leaves. Oecologia 95:328 – 333. Sharkey, T.D. and S. Yeh. 2001. Isoprene emission from plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52:407 – 436.


1532


Sharkey, T.D., S. Yeh, A.E. Wiberley, T.G. Falbel, D. Gong and D. Fernandez. 2005. Evolution of the isoprene biosynthetic pathway in kudzu. Plant Physiol. 137:700 – 712. Torsethaugen, G., E.J. Pell and S.M. Assmann. 1999. Ozone inhibits guard cell K+ channels implicated in stomatal opening. Proc. Natl. Acad. Sci. USA 96:13,577–13,582. Trainer, M., E.J. Williams, D.D. Parrish, M.P. Buhr, E.J. Allwine, H.H. Westberg, F.C. Fehsenfeld and S.C. Liu. 1987. Models and observations of the impact of natural hydrocarbons on rural ozone. Nature 329:705 – 707. Tjoelker, M.G., J.C. Volin, J. Oleksyn and P.B. Reich. 1993. Light environment alters response to ozone stress in seedlings of Acer saccharum Marsh and hybrid Populus L. 1. In situ net photosynthesis, dark respiration and growth. New Phytol. 124:627–636.

Tjoelker, M.G., J.C. Volin, J. Oleksyn and P.B. Reich. 1994. An open-air system for exposing forest canopy branches to ozone pollution. Plant Cell Environ. 17:211 – 218. Van Kooten, O. and J.F.H. Snel. 1990. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth. Res. 25:147–150. Velikova, V., I. Yordanov and A. Edreva. 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Protective role of exogenous polyamines. Plant Sci. 151:59 – 66. Wullschleger, S.D., P.J. Hanson and G.S. Edwards. 1996. Growth and maintenance respiration in leaves of northern red oak seedlings and mature trees after 3 years of ozone exposure. Plant Cell Environ. 19:577–584.
