Growth and visible injuries of four Centaurea jacea L. ecotypes exposed to elevated ozone and carbon dioxide

Environmental and Experimental Botany 58 (2006) 287–298

Growth and visible injuries of four Centaurea jacea L. ecotypes exposed to elevated ozone and carbon dioxide Kaisa R¨am¨o a,∗ , Hannele Slotte a , Teri Kanerva a , Katinka Ojanper¨a b , Sirkku Manninen a a

University of Helsinki, Department of Biological and Environmental Sciences, P.O. Box 65, FIN-00014 University of Helsinki, Finland b MTT, Agrifood Research Finland, Environmental Research, FIN-31600 Jokioinen, Finland Received 11 April 2005; received in revised form 23 June 2005; accepted 30 September 2005

Abstract We studied the variation in the responses of brown knapweed (Centaurea jacea) ecotypes from Finland and Switzerland to elevated O3 and CO2 and, the possible role of CO2 as a factor modifying O3 sensitivity. Individuals of four C. jacea ecotypes were exposed to elevated O3 (40 ppb) and CO2 (450 ppm) alone or in combination in open-top chambers (OTC) in the summer 2003. Open-field plots served as controls for the chamber effect. Ozone effects were mainly manifested as visible injuries: O3 -specific light brown flecks and non-specific purple pigmentation. The proportion of O3 -specific visible injuries was highest in the Swiss plants, where they correlated positively with early flowering, high dry matter production and high stem dry weight. CO2 ameliorated the severity of O3 -specific visible injuries only in the most O3 -sensitive ecotype, but diminished the differences between the different ecotypes. Ozone exposure also accelerated plant senescence, which was seen as enhanced development of purple pigmentation. The lack of growth responses may be explained by the low exposure concentrations. The intraspecific differences in O3 sensitivity may limit the use of C. jacea as a bioindicator for intact vegetation. © 2005 Elsevier B.V. All rights reserved. Keywords: Brown knapweed; Ozone; Carbon dioxide; Ecotype; Phenology; Visible injuries

1. Introduction There has been growing interest in the effects of ozone (O3 ) and carbon dioxide (CO2 ) on wild plants. This is an important area of research since the concentrations of tropospheric O3 and CO2 are rising due to human activities (IPCC, 2001; Vingarzan, 2004), and especially the effects of O3 on wild plants are relatively poorly known (Black et al., 2000; Fuhrer et al., 2003). Carbon dioxide has mainly beneficial effects on plant growth (Bazzaz, 1990; Jablonski et al., 2002), whereas O3 is a toxic compound known to cause visible injuries, accelerated senescence and severe reductions in photosynthesis, growth and seed production in a variety of wild species (Davison and Barnes, 1998; Black et al., 2000). Although the general trend suggests growth reductions, not all species respond similarly to O3 . In fact, previous studies have revealed considerable variation between and even ∗

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0098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2005.09.002

within wild species (e.g. Franzaring et al., 2000; Bassin et al., 2004). Furthermore, observations made in field conditions show that even individuals within the same population vary in their O3 sensitivity (Nebel and Fuhrer, 1994; Findley et al., 1997; Bungener et al., 2003; Davison et al., 2003). Scientists are currently trying to identify the most O3 sensitive taxa, in order to establish critical O3 levels for native vegetation and emission restrictions for O3 precursors, such as nitrogen oxides (NOx ). Assessing visible O3 injuries is an easy way to detect detrimental O3 concentrations, but finding a suitable O3 bioindicator for wild plants has been rather difficult (Bungener et al., 2003). One suggested species is brown knapweed (Centaurea jacea L.), which shows leaf injury under moderate O3 concentrations (Bungener et al., 1999a, 2003). The visible injuries in C. jacea are mainly manifested as O3 -specific light brown flecks, but enhanced non-specific purple pigmentation has also been proposed to be related to elevated O3 (Bungener et al., 1999a; Manning et al., 2002; Manning and Godzik, 2004). Although visible injuries are commonly used in mapping the occurrence

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of harmful O3 concentrations, they do not always correlate negatively with growth (Bergmann et al., 1995; Pleijel and Danielsson, 1997). Furthermore, some plants may show O3 induced growth reductions without exhibiting visible injuries (Reiling and Davison, 1992a; Mortensen and Nilsen, 1992). The characteristics that make a species or population sensitive to O3 are still under debate, and several hypotheses have been put forward. The first hypothesis assumes that taxa with a high relative growth rate are more sensitive to O3 than slowgrowing species (e.g. Bungener et al., 1999b; Franzaring et al., 2000). The high gas exchange rate of fast-growing individuals is thought to be accompanied by high O3 uptake (Becker et al., 1989; Nebel and Fuhrer, 1994; Bungener et al., 1999a). The differences in O3 uptake and, thus, sensitivity may also be due to differences in leaf morphology, such as stomatal density, proportion of mesophyll cells, leaf weight and leaf area (Franzaring et al., 2000; Ferdinand et al., 2000). The study of Bassin et al. (2004) on five populations of C. jacea from different European countries suggested that the extent of visible injuries was attributable to differences in plant development, and that individuals were most sensitive to O3 at the reproductive stage. One hypothesis suggests that populations of some species differ in O3 sensitivity in such a way that individuals originating from regions with relatively high O3 concentrations show greater resistance to O3 than populations from regions with low O3 concentrations (Pearson et al., 1996; Whitfield et al., 1997; Manninen et al., 2003). Prediction of the sensitivity of a species to O3 is further complicated by numerous abiotic and biotic factors. One of the major factors contributing to O3 uptake is relative air humidity (Benton et al., 2000). When air humidity is high, stomatal conductance increases and abundant O3 flux is allowed to enter the leaves, whereas when air humidity is low, conductance is reduced and higher O3 concentrations are required for O3 injury to appear (Benton et al., 2000). Other factors, such as temperature, soil water content, soil nutrient status and competition, are also known to alter plant responses (e.g. Davison and Barnes, 1998; Bungener et al., 1999a,b). Interactions with CO2 and other gases may also modify the species-specific responses to O3 . The responses of wild plants to a combination of elevated O3 and CO2 are largely unstudied and controversial, but because elevated CO2 generally increases C fixation, decreases stomatal conductance and enhances stress tolerance, elevated CO2 can be expected to protect plants from the negative effects of O3 (Allen, 1990). Thus far, empirical evidence has shown amelioration in some studies (Mulholland et al., 1997a,b; Volin et al., 1998) but not in others (Balaguer et al., 1995; Barnes et al., 1995). We wanted to study the O3 responses of two Finnish and two Swiss C. jacea ecotypes at northern latitudes (i.e. normal photoperiod, air humidity, etc.). Our specific aims were to study: (1) whether and how different C. jacea ecotypes vary in their response to elevated O3 in regard to visible injuries and growth. The Finnish ecotypes were selected to assess the

possibility of using local intact plants in biomonitoring, and the Swiss ecotypes were chosen as they have been proposed as biomonitors for O3 injury at a European scale. In addition, we wanted to learn (2) whether CO2 modifies the O3 responses of plants and (3) can some growth variables of C. jacea ecotypes be linked to the differences in sensitivity within the species.

2. Materials and methods 2.1. Plant material Four different ecotypes of brown knapweed (C. jacea L.) were used in this experiment. The two Southern Finnish populations originated from Korppoo (coastal, 61◦ 11 N, 21◦ 38 E) and S¨alink¨aa¨ (inland, 60◦ 43 N, 25◦ 14 E), and the two Swiss ones originated from Neuchˆatel (47◦ 00 N, 06◦ 58 E). The Swiss ecotypes have been used in the ICP vegetation biomonitoring studies and have previously been ranked as resistant (R) and sensitive (S). The Finnish seeds were obtained from the botanical garden of the University of Turku, and they had been collected from wild populations. The Swiss seeds were provided by the ICP coordination centre (UN-ECE, 2003). The two Finnish ecotypes were selected to originate from slightly different locations and environments, as there is a south–north gradient in O3 concentrations in Finland, and especially the southern coast receives occasional O3 pulses brought in by southern air masses from Central Europe (Laurila et al., 2004). Accumulated exposure over threshold concentration 40 ppb (AOT40) in April–September for coastal Southern Finland (Ut¨o; 59◦ 78 N, 21◦ 37 E) is approximately 7000 ppb h, for inland Southern Finland (Jokioinen; 60◦ 82 N, 23◦ 50 E) approximately 5500 ppb h (Finnish Meteorological Institute, 2005) and for Switzerland (Payerne; 46◦ 49 N, 06◦ 57 E) approximately 13,000 ppb h (Bassin, personal communication). The seeds of each ecotype were sterilized on 29 April 2003, pre-chilled and sown according to the standard experimental protocol outlined by ICP Vegetation (UN-ECE, 2003). Germination took place after approximately a week. On 19 May 2003, the seedlings were transplanted into small pots containing a mixture of peat and sand, and later, on 4 June, the seedlings were transferred to the experimental field and transplanted into 7.5 l pots (26 cm Ø) containing a mixture of peat:sand:local soil (in a ratio 1:1:1). Before the onset of the experiment 10 individuals per type were destructively harvested to obtain the mean initial above-ground biomass of the seedlings. All seedlings were watered with tap water when it was not raining. As most natural plants grow on soils that are nutrient-limited, no fertilizers were added. 2.2. Experimental design The open-top chamber (OTC) experiment was carried out in Jokioinen (60◦ 49 N, 23◦ 28 E), SW Finland, at 100 m above sea level. The experiment lasted from 4 June to 28

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August 2003. The plants were distributed into 12 open-top chambers (OTC) and 3 open-field plots (AA). One individual of each four ecotypes was placed in each OTC and AA plot. The OTC treatments were as follows (three replicates in each): non-filtered ambient air (NF), NF + O3 (1.5*ambient O3 ), NF + CO2 (1.3*ambient CO2 ) and NF + O3 + CO2 (1.5*ambient O3 and 1.3*ambient CO2 ). The three open-field plots (AA) served as controls for the chamber effect. The O3 and CO2 concentrations were chosen to simulate the predicted ambient concentrations in the year 2050, with a yearly increase of 0.5–2% in O3 (Vingarzan, 2004) and a moderate 0.5% increase in CO2 (IPCC, 2001). The plants were fumigated between 10 a.m. and 7 p.m. on 7 days a week except on days with heavy rain and ambient O3 concentrations below 20 ppb. The details of the experimental procedures and OTC design have been reported by Kanerva et al. (2005). Ozone was generated by electric discharge using pure oxygen with the Fischer 502 O3 generator, and the gaseous CO2 was distributed into the air circulation system of each CO2 chamber using Kyt¨ol¨a EK-8937 rotameters for CO2 . The gases (O3 , CO2 , NO2 and NO) were monitored at approximately 1 m above soil surface. Daily precipitation (mm), relative air humidity (%), temperature (◦ C) and global radiation (W m−2 ) were measured from two NF OTCs and one AA plot. 2.3. Visible injuries Visible O3 injury was recorded weekly from 12 June to 27 August. Ozone-related injuries were manifested on the leaves as either light brown flecks or purple pigmentation. These two injury types were recorded separately. Both the number of injured individuals per plant type in each treatment and the percentage of injured leaves in each individual were recorded. Visible injuries were scored using the following ICP injury scale (UN-ECE, 2003): (0) no injury, occurrence of the first symptoms; (I) very slight injury; (II) slight injury, 1–5% of the leaves with injury; (III) moderate injury, 5–25% of the leaves with injury; (IV) heavy injury, 25–50% of the leaves injured; (V) very heavy injury, 50–90% of the leaves injured; (VI) total injury, 90–100% of the leaves injured. For statistical analysis, a continuous variable for the class of visible injuries was formed from the categorical variable using the following median values for the classes: I, 0.5%; II, 2%; III, 15%; IV, 37.5%; V, 70%; VI, 95%. 2.4. Stomatal conductance and photosynthesis Stomatal conductance and photosynthesis were measured with a portable Infra Red Gas Analyser (LCA-3, ADC Ltd.). Gas exchange was measured on three occasions: 24 June, 18 July and 7 August 2003. Measurements were only conducted on bright days when radiation exceeded 800 ␮mol m−2 s−1 in order to measure maximum photosynthesis. The leaves chosen for measurement were to be larger than 6.1 cm−2 (area of the leaf cuvette). Photosynthesis and stomatal conductance

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were measured on 24 June from a random rosette leaf from each individual in the experiment (n = 3 for each type per treatment). When these parameters were measured again on 17 July, the sixth fully developed leaf counted from the tip was chosen, but due to the variable cloudiness we were able to measure only two-third of the plants (i.e. n = 2 for each type per treatment). On 7 August, photosynthesis and stomatal conductance were measured from the eighth fully developed leaf counted from the tip, and once again only two-third of the plants were measured (n = 2 for each type per treatment). 2.5. Growth and reproduction Reproductive development was monitored on 12 occasions between 21 July and 28 August. On each occasion, the numbers of fresh and wilted flowers per individual were counted. The experiment was terminated before the flowering of the Finnish types ended. After the experiment, the number and height of floral shoots and the number of leaves per individual were measured. The leaf area of the longest leaf per individual was also documented. The above-ground biomass of each individual was harvested and dried to constant weight at 60 ◦ C for at least 72 h. The dry weights of rosette leaves, floral shoots and reproductive organs were measured separately. Mean relative growth rate was calculated according to Hunt (1990). The average weight of individual rosette leaves was calculated from the rosette leaf dry weight and the number of rosette leaves. 2.6. SPAD Leaf senescence was recorded by a soil plant analysis development (SPAD) chlorophyll meter (Rexolin Tracer Chlorophyll meter). On the first occasion, two leaves per individual (random rosette leaf and the second youngest leaf) were tagged, and measurements were made repeatedly on the same leaves on four occasions: 30 July, 6 August, 15 August and 28 August 2003. 2.7. Statistical analyses Factor analysis of variance (ANOVA) was used to analyse the effects of treatment variables (O3 , CO2 and OTC), plant ecotype and their interaction with the response variables of C. jacea (SPSS-Windows programmes). Ambient air plots (AA) were tested against all OTC treatments. Repeated measures ANOVA was used to evaluate the differences between the ecotypes and treatments in the time-repeated measurements of flower production, SPAD values, gas exchange and visible injuries. Treatment effects within each type were analysed using ANOVA and LSD post hoc test. Visible injuries were analysed with non-parametric Kruskall–Wallis test. In the case of statistically significant differences, pairwise comparisons were conducted by Mann–Whitney U-test. Analyses between the different types were performed by one-way ANOVA and Tukey’s post hoc test. Logarithmic transfor-

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Table 1 Gas exposure and microclimate data for the different treatments in June–August 2003 Variable

NF

AA

NF + O3

NF + CO2

NF + O3 + CO2

O3 AOT 40 ppb h O3 9-h (10–19) mean (ppb) O3 1-h maximum (ppb)b CO2 9-h (10–19) mean (ppm) Mean temperaturec (◦ C) Relative humidityc (%) Global radiationc (W m−2 )

85 25 49 354 18.1 75.6 163

456 31 54 352 17.3 76.6 177

4782 40 76 357

93 24 49 455

5014 40 74a 449

a b c

On 5 June, a peak concentration of 114.0 ppb was measured, but it was attributable to the onset of the fumigations and thus not discussed. Measured during the fumigation hours (10.00–19.00). Measured from two NF OTCs and one AA plot.

mations (log 10) were used when necessary to normalize the data. In order to estimate the relevance of several plant growth variables for the extent of visible injuries at the end of the experiment, non-parametric Spearman’s correlations were performed between the visible injuries and the different plant characteristics. The results at P < 0.05 were considered significant.

3. Results

the statistically significant injuries in the Finnish ecotypes appeared later and were fewer in number than those in the Swiss ecotypes (Fig. 1A; Table 2). At the end of the experiment, the differences in the percentage of injured leaves per individual plant between the four ecotypes were marked (Fig. 1A; Table 3). The highest fraction of injured leaves was recorded in the Swiss plants, especially in ecotype S (86%). Ecotype S¨alink¨aa¨ exhibited the least visible injuries (2.5%). The 1-h daily maximum O3 concentrations did not play an important role in the development of visible injury (data not shown).

3.1. Climate and gas exposure Climate and gas exposure data are shown summarized in Table 1. The growing season 2003 was slightly warmer (1.7 ◦ C) and wetter (11.7 mm/month higher precipitation) than the long-term average (years 1971–2000) for the site (Finnish Meteorological Institute, 2002). Microclimate in the OTC’s did not differ markedly from that on the AA plots in terms of air temperature, precipitation or relative air humidity (for details, see Kanerva et al., 2005). Mean 9-h O3 concentrations were approximately 40 ppb in the treatments receiving supplemental O3 and 24–31 ppb in the other treatments. Cumulative AOT40 indexes were 4782 and 5014 ppb h in the NF + O3 and NF + O3 + CO2 treatments, respectively, which did not differ from each other statistically. Daily 1-h maximum O3 concentrations varied within 11–76 ppb in the treatments receiving supplemental O3 and within 11–54 ppb in the treatments with ambient O3 . The NF treatment had a slight, non-significant O3 filtering effect compared to the AA plots (Table 1). The 9-h mean CO2 concentrations were approximately 450 ppm in the treatments receiving supplemental CO2 and slightly over 350 ppm in the other treatments. 3.2. Visible leaf injuries All individuals of the four ecotypes of C. jacea developed light brown flecks on their leaves in the NF + O3 and NF + O3 + CO2 treatments. The first light brown flecks were recorded in the ecotypes R and S¨alink¨aa¨ on 4 July, a month from the onset of the experiment, at an AOT40 around 1500 ppb h (Fig. 1). In the NF + O3 treatment, the four ecotypes showed dissimilar development of light brown flecks:

Fig. 1. Development of O3 -specific injury (light brown flecks) per individual plant in the four ecotypes of C. jacea in (A) the NF + O3 and (B) NF + O3 + CO2 treatments. Letters indicate statistically significant differences (P < 0.05) between the different ecotypes in each treatment at the end of the experiment (August 2003). Symbols indicate the onset of flowering in each ecotype exposed to NF + O3 treatment.

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Table 2 Repeated measures ANOVA (P-values) for the ecotype and treatment (O3 , CO2 and open-top chamber) effects on light brown flecks, purple pigmentation, flowering, net photosynthesis and stomatal conductance for all data Source

Light brown flecks

Purple pigmentation

Flowering

Net photosynthesis

Stomatal conductance

Ecotype O3 CO2 OTC O3 *CO2 Ecotype*OTC Ecotype*O3 Ecotype*CO2 Ecotype*O3 *CO2 Time Time*O3 Time*CO2 Time*OTC Time*ecotype Time*O3 *ecotype Time*O3 *CO2 Time*CO2 *ecotype Time*O3 *CO2 *ecotype

0.004 <0.001 n.s. n.s. 0.024 n.s. <0.001 0.003 0.012 <0.001 <0.001 n.s. n.s. 0.002 <0.001 0.034 <0.001 0.021

<0.001 0.001 n.s. n.s. n.s. n.s. n.s. n.s. n.s. <0.001 <0.001 0.010 n.s. <0.001 n.s. n.s. n.s. n.s.

<0.001 n.s. n.s. n.s. n.s. n.s. 0.020 n.s. n.s. <0.001 n.s. n.s. n.s. <0.001 0.006 n.s. n.s. n.s.

n.s. n.s. 0.027 n.s. n.s. n.s. n.s. n.s. n.s. 0.015 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

0.049 0.038 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.041 0.009 n.s. n.s. n.s. 0.017 n.s. n.s.

The presence of CO2 altered the O3 response of the plants by diminishing the differences between the ecotypes (Fig. 1B; Table 3). At the end of the experiment, the Finnish ecotypes seemed to exhibit more light brown flecks in the NF + O3 + CO2 treatment than in the NF + O3 treatment, whereas the opposite was true of the Swiss ecotypes. The individuals of type S benefited from supplemental CO2 so markedly that there were no differences between the NF and NF + O3 + CO2 treatments (P > 0.05; data not shown). Ozone also seemed to enhance non-specific purple pigmentation (P < 0.001, factor ANOVA, Table 2), and all individuals of the four ecotypes of C. jacea developed purple pigmentation on their leaves in the NF + O3 and NF + O3 + CO2 treatments. The proportion of leaves with purple pigmentation was higher in the Swiss ecotypes than in the Finnish ecotypes (P = 0.001), but due to the high variation within treatments, there were no ecotype-specific differences in the response to O3 exposure (Tables 2 and 3). Elevated CO2 delayed the appearance (P = 0.010) and decreased the extent of purple pigmentation especially in the Finnish ecotypes (Fig. 2). However, there were no statistically significant differences between the treatments when the types were analysed separately.

3.3. Stomatal conductance and net photosynthesis Averaged over the whole data, net photosynthesis was mainly affected by CO2 (P = 0.027) and stomatal conduc-

Table 3 Simplified three-way ANOVA (P values) for the final assessment on the ecotype, O3 and CO2 effects on visible injuries (light brown flecks and purple pigmentation) for all data Source

Light brown flecks

Purple pigmentation

Ecotype O3 CO2 O3 *CO2 Ecotype*O3 Ecotype*CO2 Ecotype*O3 *CO2

0.006 <0.001 n.s. n.s. 0.001 0.006 n.s.

<0.001 <0.001 0.016 n.s. n.s. n.s. n.s.

Fig. 2. Development of purple pigmentation per individual plant in the four ecotypes of C. jacea in (A) the NF + O3 and (B) NF + O3 + CO2 treatments. Letters indicate statistically significant differences (P < 0.05) between the ecotypes in each treatment at the end of the experiment (August 2003).

55.59 (4.00) a 58.30 (2.50) a 38.64 (1.47) b 42.38 (5.85) b 50.31 (11.99) a 49.84 (21.05) a 29.48 (7.75) b 27.54 (12.13) b 4.67 (0.80) b 8.53 (0.90) a 4.87 (0.98) b 0.87 (0.41) c 1.73 (0.28) ab 2.73 (0.30) a 1.53 (0.27) ab 0.73 (0.21) b 2.08 (1.09) ab 3.47 (1.56) b 1.25 (0.55) a 0.86 (1.17) a

Flowers Floral shoots Stems Total

7.16 (0.31) ab 8.61 (0.59) a 5.92 (0.24) bc 5.32 (0.39) c S R Korppoo S¨alink¨aa¨

SPAD Height of the tallest floral shoot (cm) Leaf area of the longest leaf Number of Dry weight (g)

The ecotypes differed on most of the measured growth variables (Tables 4 and 5) and on an average the Swiss ecotypes resembled each other more than the Finnish ones, and vice versa. Averaged over all data, total dry weight was not affected by the elevation of either O3 or CO2 , but was higher in the OTCs (P = 0.013) compared to the AA plots. Ecotypes Korppoo and S¨alink¨aa¨ had higher total dry weights in the NF treatment than on the AA plots (28% and 77%, respectively; data not shown), although the difference in ecotype Korppoo was not significant (P = 0.066). The OTCs increased RGR (P = 0.011), and when the ecotypes were analysed separately, differences were found in ecotype S¨alink¨aa¨ (P = 0.037), which showed higher RGR in the NF treatment than in the AA (data not shown). The other ecotypes exhibited no change. Averaged over all data, dry weight of the leaves did not differ between the treatments or the ecotypes, but the four ecotypes differed in their O3 and CO2 responses (Table 5). For instance, the NF + O3 treatment reduced leaf dry weight in ecotype S¨alink¨aa¨ compared to the NF treatment, whereas individuals of ecotype R had the lowest leaf dry weight in the NF treatment. The NF + O3 + CO2 treatment reduced the leaf dry weight of the individuals of ecotype Korppoo but increased the leaf dry weight of the individuals of ecotype R. Leaf number was strongly enhanced by CO2 (P = 0.014) and reduced by O3 (P = 0.032). Nevertheless, when the ecotypes were analysed separately, no significant treatment effects were seen.

Ecotype

3.5. Growth

Table 4 Averages of several measured parameters (s.d.) at the end of the experiment (28 August 2003) in different ecotypes irrespective of treatment

Averaged over all data, neither elevated O3 nor CO2 had an effect on the flower development of C. jacea (Table 2). There were, however, significant differences between the ecotypes (P < 0.001) and their response to O3 . The Finnish ecotypes began flowering later than the Swiss ecotypes, and the experiment was terminated before their flowering ended. Elevated O3 seemed to delay the onset of flowering in ecotype R, while an opposite reaction was recorded for ecotype S. When the ecotypes were analysed separately, however, no differences between the different treatments were found (data not shown).

18.93 (5.50) b 19.55 (4.95) b 30.20 (6.17) a 29.04 (5.00) a

3.4. Flower development

Statistical differences (P < 0.05) between types are indicated by different letters. Values of stomatal conductance and net photosynthesis are means of all measurements (all times and all treatments).

0.065 (0.002) ab 0.067 (0.003) a 0.064 (0.002) b 0.061 (0.003) c

RGR (% day−1 )

tance by O3 (P = 0.038) (Table 2). Elevated CO2 increased net photosynthesis. Different ecotypes responded similarly to the gas exposures. The stomatal conductance of ecotype Korppoo (1.54 mol m−2 s−1 ) was significantly higher (P = 0.049) than that of ecotype S (0.93 mol m−2 s−1 ; Table 4), whereas the rates of net photosynthesis did not vary between the ecotypes (Table 2). On the whole, the levels of stomatal conductance remained stable throughout the three measurement occasions, while the rates of photosynthesis principally declined over time (P = 0.015). When the ecotypes were analysed separately, no differences between the different treatments were found (data not shown).

0.93 (0.41) a 1.11 (0.48) ab 1.54 (0.60) b 1.34 (0.67) ab

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Table 5 Four-factor ANOVA results (P values) of ecotype and treatment (O3 , CO2 and open-top chamber) effects on selected characteristics for all data Source

Dry weight (g)

RGR (% day−1 )

No. of leaves

Total

Leaves

Stems

Reproductive organs

Ecotype O3 CO2 OTC

<0.001 n.s. n.s. 0.013

n.s. n.s. n.s. n.s.

<0.001 n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. 0.011

0.003 0.032 0.014 n.s.

O3 *CO2 Ecotype*O3 Ecotype*CO2 Ecotype*OTC Ecotype*O3 *CO2

n.s. n.s. n.s. n.s. n.s.

n.s. 0.034 0.007 n.s. n.s.

n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s.

Treatments did not have an effect on stem dry weight, the weight of the reproductive organs (Table 5), the average area of individual leaves, the height and number of floral stems or the number of produced flowers (data not shown), either, when the whole data set was analysed, or when the types were analysed separately. 3.6. Relative chlorophyll content Neither elevated O3 nor elevated CO2 had effects on the SPAD values when whole data set was analysed. The Swiss ecotypes showed consistently lower SPAD values than the Finnish ones at each measurement time (P < 0.001; data not shown) and at the end of the experiment (Table 4). There were no treatment effects when tested irrespective of treatment.

3.7. Correlations Strong correlations were found between the extent of the two kinds of visible O3 injury (light brown flecks and nonspecific purple pigmentation) at the end of the experiment and several other plant characteristics (Table 6). The percentage of leaves exhibiting visible O3 injuries correlated most strongly with the appearance of the first flowers (see also Fig. 1), stem dry weight and the number of produced flowers. In addition, O3 injuries correlated positively with plant dry weight and, to some extent, with the relative growth rate, the leaf area of the longest leaf and the height of the longest floral shoot. The extent of purple pigmentation also strongly correlated with the relative chlorophyll concentration (SPAD). Many of the growth variables correlated also significantly with each other (data not shown). Stomatal con-

Table 6 Spearman’s correlation coefficients, with the proportion of leaves per individual showing O3 -specific visible injuries (light brown flecks) and non-specific purple pigmentation with other plant growth variables in the NF + O3 and NF + O3 + CO2 treatments measured at the end of the experiment (28 August) unless stated otherwise Light brown flecks

Light brown flecks Purple pigmentation Stomatal conductance (24 June) Net photosynthesis (24 June) RGR Total dry weight Leaf dry weight Stem dry weight Dry weight of reproductive organs Number of leaves Leaf area of an individual leaf Weight of an individual leaf Number of flowers Number of floral shoots Height of the longest floral shoot Date of appearance of first flowers SPAD value Asterisks denote two-tailed significances. (*) P < 0.1. * P < 0.05. ** P < 0.01. *** P < 0.001.

Purple pigmentation

N

r

r

24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24

– 0.611** −0.087 −0.154 0.392(*) 0.438* −0.022 0.552** −0.136 −0.073 0.403(*) 0.144 0.441* 0.397 0.496* −0.689*** −0.396(*)

0.611** – 0.094 −0.057 0.482* 0.579** 0.199 0.537** 0.226 −0.236 0.503* 0.347 0.567** 0.521** 0.399(*) −0.749*** −0.552**

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ductance, individual leaf weight, leaf dry weight or number of leaves did not correlate with specific visible O3 injuries (P > 0.05).

4. Discussion 4.1. Ozone effects The cumulative O3 exposure AOT40 values of 4700–5000 ppb h in the NF + O3 and NF + O3 + CO2 treatments exceeded the critical level (3000 ppb h over a 3-month period) for protecting semi-natural vegetation proposed by the UN-ECE convention (Karlsson et al., 2003). Significant O3 -specific visible injuries were manifested in all individuals exposed to elevated O3 alone and in combination with elevated CO2 . The development and the proportion of injured leaves varied significantly between the Swiss and Finnish types, depending on whether O3 was given alone or in combination with CO2 . The O3 -specific visible injuries were mainly light brown flecks, which have previously been identified by Bungener et al. (1999a) and Bassin et al. (2004). The first signs of visible injuries developed at AOT40 of 1540 ppb h, which is lower than reported previously for C. jacea (Bungener et al., 2003; Bassin et al., 2004) and several other wild species (Power and Ashmore, 2002). Bungener et al. (2003) and Bassin et al. (2004) who grew C. jacea types in Switzerland observed the first visible injuries at AOT40 of 2240 ppb h and 3350 ppb h, respectively. The study periods in the present study and the experiment A (21 May–20 August 2002) of Bassin et al. (2004) are highly comparable, with only a few weeks of difference, but the cumulative AOT 40 indexes over the experimental periods were markedly different, with 19,300 ppb h in Switzerland and 4730 ppb h in the present study. Although the O3 concentrations were measured from different heights (4.5 m in Switzerland versus 1 m in Finland), the difference is clear-cut. Therefore, it is interesting that the rate of injured plants was higher in the present study. One possible explanation for the responsiveness of the plants in the present study could be attributed to stomatal conductances, which were comparatively high in the present study (Bungener et al., 1999a). High stomatal conductances may result from the reasonably high relative air humidity (Krupa et al., 1995; Benton et al., 2000) and indeed, the highest O3 fluxes have been reported to occur in Northern Europe as opposed to the areas with the highest O3 concentrations (Pleijel, 1999; Embersson et al., 2000). However, it must be noted that the Finnish ecotypes tended to have even higher stomatal conductance values, but had fewer light brown flecks, than the Swiss ones. It is also known that long days allow O3 uptake for many hours, and because of the short nights, plants have only limited time over which to recuperate from the oxidative stress (De Temmermann et al., 2002). Additionally, differences

in the nutrient status of the growing media may have effects on the O3 sensitivity of plants (Davison and Barnes, 1998). Visible injuries caused by O3 are not always specific but can be manifested as general reddening, chlorosis and premature senescence (Davison and Barnes, 1998; Bergmann et al., 1999). In the present study, only general reddening was recorded, which has previously been reported as a common non-specific O3 response by members of the Asteraceae family, including C. jacea (Bungener et al., 1999a; Schenk, 2003; Manning and Godzik, 2004). The purple anthocyanin coloration normally becomes visible when the chlorophyll content of the leaves decreases during senescence, which could also be seen in the present study. The role of anthocyanins in leaves is still under debate, but according to one hypothesis, anthocyanins may mitigate the oxidative stress caused by biotic or abiotic stresses (Lee and Gould, 2002; Neill et al., 2002), including those produced from O3 (Sharma et al., 1996). In the present study, it can be assumed that anthocyanin production was a sign of O3 -induced senescence, as it has been reported to be in wild strawberry, Fragaria vesca L. (Manninen et al., 2003). Compared to the Finnish plants, the Swiss populations were faster in their overall development and exhibited higher proportions of leaves with purple pigmentation, also in treatments with ambient O3 . Despite the distinct visible injuries, there were relatively few other treatment effects, which have previously been reported by Mortensen and Nilsen (1992). Of the few O3 effects, the increase in stomatal conductance is opposite to the previous findings on C. jacea (Nussbaum et al., 2000) and other species (e.g. Power and Ashmore, 2002). A similar initial increase (50%) and subsequent progressive reduction in stomatal conductance under elevated O3 have, however, been observed in Mentha aquatica (Power and Ashmore, 2002). The observed decrease in leaf number due to O3 is in accordance with a study on strawberry (Fragaria × ananassa Duch.) (Keutgen et al., 2005), but not with the study by Pearson et al. (1996). Broadly speaking, the types reacted differently to elevated O3 , depending on the measured parameter, which is in agreement with a study on three contrasting populations of Plantago major L. (Pearson et al., 1996). Visible injuries were not well translatable into reductions of biomass or reproductive success, which has previously been observed on other species (e.g. Pleijel and Danielsson, 1997; Davison and Barnes, 1998). One likely explanation may be the low O3 concentrations and the lack of peak O3 concentrations in the present study (Bergmann et al., 1999; Power and Ashmore, 2002). It is also possible that the visible injuries may have correlated with below-ground growth reductions (Power and Ashmore, 2002), but this cannot be verified as we did not determine the root dry weight. The lack of growth reductions does, however, not hinder the use of C. jacea as a bioindicator in mapping the occurrence of detrimental O3 concentrations.

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4.2. OTC and carbon dioxide effects OTCs had a positive effect on the total dry weights and relative growth rates, which is in good agreement with the earlier studies (e.g. Sanders et al., 1991; Danielsson et al., 1999). Temperature was slightly higher and relative air humidity lower in the OTCs compared to the AA plots. The AOT40 index and global radiation were also somewhat reduced in the OTCs. The Finnish ecotypes benefited more from the OTC conditions compared to their Swiss counterparts, which is in agreement with the observations of Danielsson et al. (1999) on Phleum genotypes originating from areas with different summer temperatures. When elevated CO2 was given alone, there were very few treatment effects. The increases in net photosynthesis caused by CO2 were in accordance with previous studies (e.g. Nowak et al., 2004; Ainsworth and Long, 2005). Increased leaf numbers on herbaceous plants exposed to CO2 have been reported previously by Pritchard et al. (1999). The lack of CO2 -mediated responses may be due to several factors including the relatively low levels of CO2 enrichment and nutrients, and relatively small pots compared to other studies (e.g. Mulholland et al., 1997a; Poorter, 1998; Poorter and Perez-Soba, 2001; Marissink et al., 2002). It is also possible that growth responses were manifested below-ground, as previous studies have indicated that elevated CO2 increases root biomass relative to shoot biomass (Rogers et al., 1994), especially when nutrients are limited (Bazzaz, 1990). 4.3. Ozone*carbon dioxide effects As O3 -specific visible injuries were the most consistent O3 effect, the role of CO2 was also most apparent in this parameter. It is interesting that, although elevated CO2 did not modify the specific visible O3 injuries in terms of the proportion of injured individuals, it decreased their extent (% of injured leaves) in the Swiss ecotypes. The Finnish ecotypes, however, tended to develop more specific visible injuries in the NF + O3 + CO2 treatment than following exposure to NF + O3 . Similar results have been presented by Kull et al. (1996), who found that the photosynthesis of an O3 tolerant aspen clone decreased more when O3 and CO2 were given in combination compared to O3 given alone. It seems that, in the case of chronic visible injuries, the capacity of elevated CO2 to ameliorate the harmful effects of O3 depends on the ecotype, and amelioration was here only seen in the most O3 -sensitive ecotype (S). This is in agreement with a study on two Trifolium repens L. clones (Heagle et al., 1993), but opposite to a study by Dickinson et al. (2001), who studied two trembling aspen clones and noticed that the addition of CO2 to O3 exposure only counteracted the negative impact of O3 in the tolerant clone. An opposite trend was recorded for non-specific reddening. When exposed to NF + O3 + CO2 treatment, the Finnish ecotypes hardly showed any reddening, whereas the individuals of the Swiss ecotypes exhibited extensive reddening. It may be speculated that CO2 delayed

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the O3 -related senescence in the Finnish ecotypes. It is possible that the amelioration was dependent on the used concentrations, since Heagle et al. (1993) reported that CO2 did not protect clover (Trifolium pratense L.) against the effects of O3 at concentrations lower than the highest (710 ppm CO2 ) used. Johnson et al. (1996) found similar responses when studying timothy (Phleum pratense L.) and alfalfa (Medicago sativa L.). 4.4. Of the differences in ecotype sensitivity At the end of the study, the Swiss ecotypes had larger percentages of injured leaves than the Finnish ones. This is in accordance with a recent review on northern European field layer plant species, which indicated that most of the studied species proved to be quite tolerant of O3 (Timonen et al., 2004). In addition to the differences in the development of visible injuries, there seemed to be extensive variation among the different ecotypes. On the whole, the Swiss ecotypes resembled each other more than the Finnish ones, and vice versa. The ozone sensitivity of C. jacea did not seem to be related to previous O3 exposure, as noted by Bassin et al. (2004). The correlations between the percentage of injured leaves and plant growth variables revealed that O3 sensitivity correlated positively with plant dry weight and, to some extent, with the relative growth rate, as also observed amongst others by Danielsson et al. (1999) on Phleum genotypes. However, most determinant factors were related to reproductive development. The individuals with early flowering, the highest stem dry weight, the longest stems and the most numerous flower stems were most severely injured at the end of the study. This confirms the observations by Bassin et al. (2004) that flowering is associated with high sensitivity in C. jacea types, and this could be attributed to changes in resource allocation or changes in physical and micro-meteorological conditions. Similar O3 sensitivity at the onset of flowering has been observed in Arabidopsis thaliana L., and it was explained by the plant’s lowered capacity to detoxify oxidative stress in the leaves (Ye et al., 2000). The high relative growth rates and faster (phenological) development of the Swiss ecotypes explain their earlier senescence manifested as a higher percentage of leaves with purple pigmentation and lower SPAD values. According to Schenk (2003), the O3 sensitivity of C. jacea types is related to early senescence, as antioxidants decrease along with age, making senescing plants more susceptible to stress factors, such as air pollutants (Pell et al., 1997; Larcher, 2003). The Finnish ecotypes required a longer time to shift into the reproductive stage, which is in accordance with the observations on the Norwegian type in the study of Bassin et al. (2004). Bassin et al. (2004) suspected that the comparatively short photoperiod at study site in Southern Switzerland might have inhibited the development of the inflorescence. However, the Finnish plants in the present study were grown at latitudes natural to them, and they were still slower in their reproductive development than the Swiss types. Solely on the basis of the present

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study it cannot be ruled out that the Swiss plants were not more sensitive to stress because they were not were growing in a climate to which they were not adapted, but cognizance of results of Bassin et al. (2004) it seems that, regardless of the experimental site, Central and Northern European (Norwegian and Finnish) plants have a type-specific response to O3 , the former being more and the latter less responsive. It is also noteworthy that the Swiss ecotypes had been previously classified as resistant and sensitive, and selected on the basis of their ability to express visible injuries. The highest conductance values were measured from the Finnish types that showed the least visible injuries. This is contrary to the previous studies (e.g. Nebel and Fuhrer, 1994), which have suggested that high stomatal conductance could be related to increased visible O3 injury. According to the present study other growth variables may have more significant role in determining the O3 sensitivity of C. jacea types, but due to only few measurement occasions the results cannot be generalized. However, Timonen et al. (2004) pointed out that Oxalis acetosella L. has low stomatal conductance (K¨orner, 1994), but shows visible injuries at low levels of O3 (Nygaard, 1994). Franzaring et al. (2000) and Postiglione et al. (2000) suggested that species with large, thin leaves seem to be more susceptible to O3 than species with more compact leaves, as they have a higher surface-to-volume ratio. Supporting this, the Swiss ecotypes had large and thin leaves, and the Finnish ones small compact leaves. Plant O3 sensitivity has also been linked to other leaf characteristics such as cell wall thickness, high stomatal density and greater leaf weight (Davison and Barnes, 1998; Ferdinand et al., 2000). Due to the various statistical analyses including several ecotypes, treatments and growth variables in the present study, the factors making ecotypes sensitive should, however, be interpreted with caution.

5. Conclusions The study verifies the sensitivity of C. jacea, as all four ecotypes developed significant and extensive visible O3 injuries, even though the AOT40 level was relatively low (approximately 5000 ppb h). High responsiveness at such low concentrations is most likely to be due to the high relative air humidity and long day length at the study site. Although all individuals showed O3 -specific visible injuries (light brown flecks) at the end of the study, the extent of injuries varied between the different ecotypes. The extent of O3 -specific visible injuries (injured leaves per individual) was associated with early flowering, abundant dry matter production and high stem dry weight. In regard to specific visible injuries, the capacity of CO2 to ameliorate the harmful effects of O3 depended on the ecotype. In the present study, significant amelioration only took place in the most O3 -sensitive ecotype. In addition, the least sensitive ecotypes exhibited more O3 -specific injuries when O3 and CO2 were given in combination. Apart from distinct visible injuries, there were rela-

tively few other treatment effects. The most obvious effects were caused by the OTCs, which had a positive effect on dry matter production, especially in the Finnish ecotypes. The results of this experiment confirm the previous results showing that C. jacea is not a particularly good bioindicator for intact vegetation due to the large intraspecific differences in O3 tolerance. In addition, when using natural plant populations as bioindicators for O3 , the phenological state of the individual plants should be recorded.

Acknowledgements This study was financially supported by funds from Helsinki University Environmental Research Centre and the University of Helsinki. We are grateful to Mr. Peter Huhtala and Mr. Oiva Hakala for their contribution to the development and the maintenance of fumigations throughout the study. The staff of MTT/MPY is thanked for all their assistance. We thank Ms. Satu N¨aykki from H¨ameen maaseutukeskus for borrowing the SPAD-instrument and Dr. Timo Salmi from the Finnish Meteorological Institute for providing the AOT40 data of the Finnish EMEP stations. The comments of two anonymous reviewers on an earlier version of the manuscript are very much appreciated. The English language was revised by Ms. Sirkka-Liisa Leinonen.

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