Ozone induced leaf loss and decreased leaf production of European Holly (Ilex aquifolium L.) over multiple seasons

Environmental Pollution 145 (2007) 355e364 www.elsevier.com/locate/envpol

Ozone induced leaf loss and decreased leaf production of European Holly (Ilex aquifolium L.) over multiple seasons Jonathan Ranford*, Kevin Reiling Applied Sciences, Faculty of Health and Sciences, Staffordshire University, College Road, Stoke-on-Trent, Staffordshire ST4 2DE, UK Received 29 July 2005; received in revised form 21 February 2006; accepted 25 February 2006

Ozone significantly alters Ilex aquifolium leaf production and loss over multiple seasons. Abstract European Holly (Ilex aquifolium L.) was used to study the impact of one short (28 day) ozone fumigation episode on leaf production, leaf loss and stomatal conductance ( gs), in order to explore potential longer term effects over 3 growing seasons. Young I. aquifolium plants received an episode of either charcoal-filtered air or charcoal-filtered air with 70 nl l1 O3 added for 7 h d1 over a 28 day period from June 15th 1996, then placed into ambient environment, Stoke-on-Trent, U.K. Data were collected per leaf cohort over the next three growing seasons. Ozone exposure significantly increased leaf loss and stomatal conductance and reduced leaf production over all subsequent seasons. Impact of the initial ozone stress was still detected in leaves that had no direct experimental ozone exposure. This study has shown the potential of ozone to introduce longterm phenological perturbations into ecosystems by influencing productivity over a number of seasons. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Ozone; Ilex aquifolium; Leaf loss; Leaf production; Stomatal conductance

1. Introduction Ozone is a major air pollutant with current and predicted levels (Vingarzan, 2004) being well above concentrations known to elicit significant adverse effects on many aspects of vegetation. Ozone effects on native vegetation (Chappelka and Samuelson, 1998; Davison and Barnes, 1998; Nussbaum et al., 2001; Bermejo et al., 2003; Elvira et al., 2004; Gardner et al., 2005; Oksanen et al., 2005) are well known but the comparative paucity of longer term experimental data (Krupa, 1990; Laurence et al., 1996; Scagel and Andersen, 1997; Oksanen, 2003a,b; Rebbeck et al., 2004) together with existing evidence of substantial genotypic (Reiling and Davison, 1995; Davison and Barnes, 1998; Paludan-Muller et al., 1999; * Corresponding author. Tel.: þ44 782 294 892; fax: þ44 782 294 986. E-mail addresses: [email protected] (J. Ranford), k.reiling@staffs. ac.uk (K. Reiling). 0269-7491/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2006.02.032

Oksanen and Holopainen, 2001) and developmental (Cooley and Manning, 1988; Reiling and Davison, 1994; Black et al., 2000) variation in ozone response indicates our knowledge of potential long-term impact on perennial species or communities is uncertain (Ashmore, 2005). Ozone exposure has been observed to accelerate foliar senescence and abscission (Wiltshire et al., 1993; Pell et al., 1999; Yun and Laurence, 1999) with a consequent impact on whole-plant leaf area (Byres et al., 1992; Stow et al., 1992; Shelburne et al., 1993; Pearson et al., 1996). Effects of ozone on leaf production have also been observed and although variable there is a tendency for reduced biomass production to be reported (Paakkonen et al., 1993) with carry-over effects not unknown (Andersen et al., 1997; Oksanen and Saleem, 1999; Oksanen, 2003a,b; Yonekura et al., 2004). If the reduced biomass leads to a reduction in leaf area this will impact upon photosynthetic ability and may result in a reduction in net carbon gain (Topa et al., 2001).

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The link between exposure and dose (Krupa et al., 1994; Legge et al., 1995; Ashmore and Davison, 1996) and the setting of experimental exposure levels is a long and unresolved issue (Karlsson et al., 2003), for this experiment the concentration of ozone to be used throughout the fumigation period was set to a typical summer ambient level of 70 nl l1. In the UK, hourly mean concentrations of ozone vary between 0 and 150 nl l1, although the typical range is 10 to 30 nl l1. Days which exceed maximal levels of 50 nl l1 imply a photochemical episode and these occur across the UK mainly from April to September and typically range between 30 and 50 days across the country annually (NETGAP, 2001). Studies on tree seedlings have used varying ozone concentrations e.g. 30 nl l1 on Fagus sylvatica L. (Bortier et al., 2000), 80 nl l1 Quercus robur L. (Farage, 1996) and 50/100/150 nl l1 on Picea rubens Sarg. (Waite et al., 1994). Many of these experimental studies are short-term destructive experiments i.e. 4 weeks of ozone fumigation and then plant analysis. The main objective in this study was to investigate the effects over a 3e4 year period in the ambient environment after a relatively short exposure. An advantage to this approach is that the effects of ozone can be observed on several subsequent growing seasons which are particularly relevant to trees that retain their foliage for several years. Whatever the links between exposure and dose the role of gas exchange in controlling the responses of the plant to ozone is very important. A rapid decline in stomatal aperture and hence stomatal conductance is commonly observed in the presence of ozone over a short term exposure (Grantz et al., 1999; Panek and Goldstein, 2001; McAinsh et al., 2002; Panek, 2004; Paoletti and Grulke, 2005) whilst longer term exposure cause stomatal responses to become sluggish (McAinsh et al., 2002). Whether plants with higher intrinsic rates of stomatal conductance are susceptible to ozone injury because of their greater gas exchange rates has not been fully determined. We investigated the effects of a relatively short term ozone exposure at an environmentally realistic level; our objective was to establish any links between that exposure and any long-term carry-over impacts, especially on leaves that had not undergone the experimental exposure. 2. Materials and methods 2.1. Plant growth conditions and fumigation First year I. aquifolium plants obtained from a commercial supplier (Kerry Hill Nurseries, Staffordshire, UK) were potted into square 10 cm pots (John Innes no. 3) and placed in a glasshouse for 28 days in May 1996. Plants were kept fully watered during the glasshouse and experimental period. The plants were then transferred to four controlled environment chambers (AAF Ltd Cramlington, Northumberland, UK), forty in each, and left to acclimatise to chamber conditions for 14 days. All chambers were ventilated with charcoal-filtered air (CFA). The environmental conditions in each of the chambers were uniform and no significant between-chamber variation was detected. The mean temperature in the chambers was 18  C  2  C day, 12  C  2  C night and illumination was provided by metal halide lamps (Siemens Son-T Harrier 250 W unit fitted with Osram Powerstar HQI-T 250 W\D lamp) providing a photon flux density of 550 mmol m2 s1 at plant canopy height over a 16 h photoperiod.

All experimental chambers were ventilated to receive CFA 24 h d1 (<5 nl l1 O3) with a mean airspeed of w1 m s1 at plant height. In addition half of the chambers received a supplement of ozone resulting in a level of 70 nl l1 O3  4 nl l1 O3 from 10.00e17.00 (AOT40 5880) for a period of 28 days. Ozone concentration was measured continuously using a Dasibi R1000 ozone monitor (Dasibi Environmental Corp, Glendale, California, USA). Ozone was generated by an ozonater (Wallace and Tiernan, Tonbridge, Kent UK) from charcoal scrubbed air. To prevent contamination by higher oxides of nitrogen (Brown and Roberts, 1988) the after generation O3-enriched air was passed through water prior to injection into the main chamber inlet. Post fumigation all plants were returned to the glasshouse for 14 days and then placed into ambient conditions (Staffordshire University, Stoke-on-Trent, and Staffordshire, UK) for the remainder of the experiment. Each year the plants were re-potted with John Innes no. 3, in pots 10 cm larger than the previous year, thus an annual supply of nutrients were supplied via the new compost.

2.2. Plant measurements Biomass harvests were conducted prior to placing the plants in ambient conditions in order to ascertain any direct short term impacts. Soil was carefully removed from roots and each seedling was divided into root, stem and foliage. It was noted that root systems of the largest plants were not restricted by pot size. Dry weight (dried at 60  C for at least 48 h until constant weight attained) was then assessed. All I. aquifolium plants were 1st year material at the beginning of the experiment and these leaves have been designated cohort 1. Leaves of subsequent growing seasons were termed cohort 2, cohort 3 and cohort 4 respectively. In Ilex leaf age can be readily distinguished by their position between annual nodes on the branch (Brewer and Gaston, 2002). Experimental plants were randomly selected from a homogenous parent supply and then randomly selected for the treatment regimes. Leaf counts were recorded monthly over 50 months in order to assess ongoing loss and production. The main production of leaves in Ilex is in a spring flush between April and July. Monthly leaf production was assessed for the cohorts 2, 3, and 4 over these months. Stomatal conductance was recorded monthly using an AP4 portable porometer (Delta-T Devices, Cambridge, Cambridgeshire, UK) at a standardised time (14:00 h). Fully expanded leaves were randomly selected per plant for each cohort and conductance recorded. Different cohorts were measured on subsequent days.

2.3. Statistics Statistical Analyses were performed using the SPSS statistical package (SPSS Inc., Chicago, Illinois, USA). The experiments consisted of randomized block design with the main factor ‘treatment’ for leaf loss, leaf production, gs and plant biomass. The treatment effect of CFA plus O3 and CFA were tested with Repeated Measures (RM) ANOVA for all measurements excluding plant biomass in which instance ANOVA was applied. In addition, pair-wise comparisons within treatments (monthly means of CFA plus ozone and CFA) were conducted using t-tests. Statistical significance in all tests was set at the 0.05 probability level (P  0.05).

3. Results 3.1. Ambient ozone exposure and climatological data Daily mean (24 h) and daily maximum ambient ozone concentrations at ambient site conditions are summarized in Fig. 1 for the experimental period. The initial sections of the trace in the figures indicate the level and duration of the experimental fumigation thus highlighting the relevance to typical daily mean and maximum ozone concentrations. The mean daily ambient ozone concentration ranged between 7 and 126 ppb with most summer ozone concentrations ranging between 40

J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 355e364

a

357

200 180 160

Ozone ppb

140 120 100 80 60 40 20 0

Jun Aug Oct Dec Feb Apr May Jul Sep Nov Jan Mar Apr Jun Aug Oct Dec Mar May Jun Aug Oct Dec Jan Mar May Jul Sep Oct Dec 96 97 98 99 00

Date

b

200 180 160

Ozone ppb

140 120 100 80 60 40 20 0

Jun Aug Oct Dec Feb Apr May Jul Sep Nov Jan Mar Apr Jun Aug Oct Dec Mar May Jun Aug Oct Dec Jan Mar May Jul Sep Oct Dec 96 97 98 99 00

Date

Fig. 1. a. Mean daily (24 h) ambient ozone concentration during the experimental period, at the experimental area, including the initial ozone fumigation exposure (June 15theJuly 12th,open symbols) and the charcoal-filtered air control (closed symbols). Source e Air Quality Archive. b. Maximum daily ambient ozone concentration during the experimental period, at the experimental area, including the initial ozone fumigation exposure (June 15theJuly 12th, open symbols) and the charcoal-filtered air control (closed symbols). Source e Air Quality Archive.

and 80 ppb. The maximum daily ozone concentration ranged between 4 and 194 with a typical summer range between 60 and 90 ppb. The AOT40 and climate data for the experimental period at the ambient site are summarized in Table 1. Interestingly AOT40s in April and May are considerably higher than the other months. This coincides with the main leaf production in I. aquifolium. The year following fumigation (1997), had the maximum temperature and sunlight hours over the experimental period. The first winter after fumigation also exposed the plants to more air frost days (67) than following years. The following 3 had broadly similar values for the various climate measurements over the experimental period. 3.2. Plant Biomass Following the initial fumigation the dry mass (harvested 44 days after the beginning of the experiment) of the roots

and shoots were both significantly decreased by the ozone treatment (Table 2). Compared with the controls, this equated to w16% reduction in dry mass for both roots and shoots, however root:shoot ratios were unaffected. 3.3. Leaf Loss Fig. 2 shows the percentage leaf loss by cohort over the experimental period. RM ANOVA was applied to all cohort data and leaf loss was found to be significantly different (Table 3). Cohort 1 (Fig. 2a) is the only cohort actually to receive a controlled ozone episode. There is a marked impact of the treatment on leaf loss with ozone treated plants showing a marked significant early loss, with w50% (Table 4) of their leaves shed by February compared to April for the control plants. This period of loss coincides with a high number of

J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 355e364

358

Table 1 Summary of AOT40a and climateb data covering the experimental period, Stoke-on-Trent, Staffordshire Date

June-96c Julyc August September October November December

AOT40

3360 4671 2435 1094 1385 498 1834

Temperature  C mean max.

mean min.

22 21.3 21.1 17.8 14.7 9.1 5.2

12 10.2 11.3 8.3 7.4 1.6 0.4

Air frost (d)

n/a 0 0 0 0 15 17

Precip. (mm)

n/a 23.6 47.2 13.7 69.8 65.8 42.3

Table 1 (continued ) Date

Sunshine (h)

n/a 226.8 174.4 133.2 86.6 91.8 51.0

June July August September October November December

12,047 4491 3186 1467 2226 2785 2964

Total

72,972

a

Total January-97 February March April May June July August September October November December Total January-98 February March April May June July August September October November December Total January-99 February March April May June July August September October November December

15,277 3241 2321 2007 2314 5623 2998 2695 5069 656 331 268 1392

4.9 10.0 11.8 13.1 16.2 17.5 21.3 23.4 17.6 13.6 11.2 8.2

1.2 3.0 3.7 4.0 5.7 9.5 10.8 13.0 9.4 4.9 5.1 2.2

28,915 1764 1291 1928 3723 4290 1186 612 1064 1387 1824 412 1042

7.3 10.9 11.3 10.9 17.3 17.7 19.1 20.2 17.9 13.5 9.1 8.3

1.6 3.5 4.4 3.1 7.7 10.1 10.9 10.4 10.2 6.5 1.8 0.2

20,523 2969 690 2657 5136 6856 3932 3283 2978 1411 244 453 1666

Total

32,275

January-00 February March April May

6031 5172 2743 10,263 19,597

8.5 8.2 10.4 13.5 16.7 18.1 22.5 19.9 19.6 14.3 10.6 7.8

8.0 9.5 10.9 11.5 16.5

1.1 1.3 3.2 4.8 8.4 8.9 11.7 11.1 10.0 6.3 4.1 1.1

1.4 1.9 2.5 3.2 6.1

AOT40

32

262.4

763.8

20 5 3 7 2 0 0 0 0 9 6 10

10.4 43.0 24.2 21.9 87.0 96.9 40.6 91.0 21.0 58.8 68.3 61.1

33.1 80.0 119.6 137.9 232.9 123.0 237.4 195.7 138.4 117.8 39.3 31.8

62

624.2

1486.9

11 6 5 8 0 0 0 0 0 1 11 14

81.4 16.4 64.4 87.8 12.8 72.6 35.3 40.4 77.3 106.8 41.2 45.8

46.5 78.2 88.1 139.2 205.4 145.9 159.9 193.9 112.2 95.6 72.2 38.2

56

682.2

1375.3

11 10 3 5 0 0 0 0 0 1 3 10

90.7 39.6 47.2 39.8 54.1 64.7 14.0 128.1 125.3 80.8 34.2 79.8

59.9 62.9 100.0 159.7 147.4 211.8 242.9 153.2 178.5 116.4 66.1 44.8

43

798.3

1543.6

13 7 11 7 0

23.0 55.1 15.8 114.7 43.6

59.8 102.2 120.8 146.5 211.2

b

Temperature  C mean max.

mean min.

18.8 19.4 20.6 18.3 13.6 9.7 7.9

10.1 10.6 10.8 10.3 5.8 2.8 2.4

Air frost (d)

Precip. (mm)

Sunshine (h)

0 0 0 0 0 7 8

45.5 72.6 91.1 101.7 114.1 111.6 77.1

161.1 174.6 168.2 119.0 93.6 53.4 52.7

53

865.9

1463.1

Air Quality Archive, 2006 (primary data). The Met Office, 2006.

days with frost (Table 1). A continuing significant difference, following a similar pattern, was also apparent the following year with more leaves lost over the winter period by the ozone treated plants. Total leaf loss of this cohort occurred by June compared with September in the control plants. Cohort 2 (Fig. 2b) leaves follow a similar pattern to those of cohort 1 with a greater loss of leaves over the winter periods from plants receiving the short extra ozone fumigation. In comparison to the first cohort data both treatments did however take longer to reach 50% leaf loss, September and November for ozone and control respectively. Data from Cohort 3 (Fig. 2c), although showing a similar overall trend as the previous cohorts, with earlier reductions apparent for the ozone treated plants, gives no statistically significant differences in the monthly data. 3.4. Leaf production Prior to fumigation, leaf production was measured in all plants to provide starting data and to check the random selection procedure. Leaf production data reflects plant response to the ozone treatment as none of the leaves measured were subjected to experimental ozone fumigation. Again, a RM ANOVA was applied to all cohort data and leaf production was found to be significantly different (Table 3). The cohort 2 (Cohort 2 to maintain consistency with leaf loss labelling but actually the first cohort of leaf production post-fumigation) data (Fig. 3a) shows that ozone treated plants produce significantly fewer leaves equating to an overall reduction of >40% compared with control plants. As with leaf loss, ozone had an impact on the phenology of Ilex with main leaf production occurring a month later. Table 2 Mean dry weight for root and shoot after ozone fumigation (70 nl1 28 d 7 h d) on I. aquifolium 44 days post initial fumigation

Roots Shoots Root:Shoot ratio

Control (g)

O3 (g)

161 215 0.749

135a 179a 0.754

a Indicates significant (P < 0.05) differences between O3 and fumigated means.

J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 355e364

a

Table 3 Summary of Repeated Measures ANOVA on the various plant measurements investigating the effects of ozone (70 nl1 28 d 7 h d1, June 1996) on I. aquifolium compared with the control (charcoal-filtered air)

100 90 80

Leaf Loss (%)

359

70 60 50 40

Measurement

F-value

Significance

Leaf loss Leaf production Stomatal conductance

165.57 247.97 35.00

P < 0.05 P < 0.05 P < 0.05

30 20 10 0

O N D Jan F M A M J '96 '97

J A S O N D Jan F M A M J '98

J A S

Month

b

100 90

Leaf Loss (%)

80 70 60 50

3.5. Stomatal conductance

40 30 20 10 0

D Jan F M A M J '97 '98

J

A S O N D Jan F M A M J '99

J

A S O

Month

c

100 90 80

Leaf Loss (%)

The cohort 4 data (Fig. 3c) still displays a significant reduction in monthly leaf production although the production trend is less divergent for the treatments than in previous cohorts. Ozone treated plants still produced w10% less compared with control and have subsequently reached the same level of leaf production as the cohort 2 control plants. Thus both leaf loss and production data have shown the ability of ozone to introduce long term phenological changes in plant material.

70 60 50 40 30 20 10 0

Jan F M A M J '99

J

A S O N D Jan F M A M J '00

J

Prior to fumigation, gs was assessed to give a baseline for future cohort assessments and also to verify the random selection process of the experimental plants. There was no significant difference in gs between the plants prior to treatment. RM ANOVA was applied to all cohort data and gs were found to be significantly different (Table 3) between the treatments. Mean monthly plots for gs for cohort 1 are presented in Fig. 4. The ozone treatment causes an initial increase of >50 mmol m2 s1 in gs. This increase was still apparent but more pronounced (>100 mmol m2 s1) in these leaves the following summer when compared with the control plants. The increase in gs is thus continuous for the following two years from the initial fumigation. The only significant stomatal differences were found in leaves that actually received the additional ozone treatment. Leaves produced under ambient conditions were not different with respect to gs.

A S O N

Month

Fig. 2. a. Mean cumulative monthly leaf loss of I. aquifolium, cohort 1 (1996). Ozone fumigated (70 nl1 28 d 7 h d1 June 1996) open symbols or maintained in charcoal-filtered air (CFA) closed symbols. Significant differences from CFA are denoted star, P < 0.05. b. Mean cumulative monthly leaf loss of I. aquifolium, cohort 2 (1997). Ozone fumigated (70 nl1 28 d 7 h d1 June 1996) open symbols or maintained in charcoal-filtered air (CFA) closed symbols. Significant differences from CFA are denoted star, P < 0.05; 2 stars, P < 0.01. c. Mean cumulative monthly leaf loss of I. aquifolium, cohort 3 (1998). Ozone fumigated (70 nl1 28 d 7 h d1 June 1996) open symbols or maintained in charcoal-filtered air (CFA) closed symbols.

Cohort 3 (Fig. 3b) shows a similar trend to cohort 2 with all monthly results being significantly different. In this cohort ozone treated plants produced >30% less leaves than the control. Although both treatments show an increase in total leaf number compared with cohort 2 the ozone treated plants still produced less leaves than the cohort 2 control plants did in the preceding year.

4. Discussion After fumigation, both the ozone and control treatment experienced the same ambient ozone and climatological conditions. Whilst this approach does potentially input a large range of differing climate variables it is important to realise Table 4 Summary table exemplifying the effect of ozone fumigation (70 nl1 28 d 7 h d1 June 1996) on leaf loss and production on I. aquifolium Number of months to lose 50% of leaves for:

CFA

CFA þO3

Cohort 1 Cohort 2 Cohort 3 Mean leaf production (n)

10 11 11 379

14 13 11 272

CFA e charcoal-filtered air.

J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 355e364

360 140

Stomatal Conductance (mmol m-2 s-1)

Cumulative Leaf Production (n)

a

120 100 80 60 40 20 0

April

May

June

July

Month

Cumulative Leaf Production (n)

b

200 150 100 50 0 Jun J A S O N D Jan F M A M J J A S O N D Jan F M A M J J '96 '97 '98

Fig. 4. Mean stomatal conductance of I. aquifolium, cohort 1 (1996). Ozone fumigated (70 nl1 28 d 7 h d1 June 1996) open symbols or maintained in charcoal-filtered air (CFA) closed symbols. Data were collected monthly at 14:00 h. Significant differences from CFA are denoted star, P < 0.05; 2 stars, P < 0.01.

120 100 80 60 40 20

April

May

June

July

June

July

Month

Cumulative Leaf Production (n)

250

Month

140

0

c

300

140 120 100 80 60 40 20 0

April

May

Month

Fig. 3. a. Mean cumulative monthly leaf production of I. aquifolium, cohort 2 (1997). Ozone fumigated (70 nl1 28 d 7 h d1 June 1996) open symbols or maintained in charcoal-filtered air (CFA) closed symbols. Significant differences from CFA are denoted star, P < 0.05; 2 stars, P < 0.01. b. Mean cumulative monthly leaf production of I. aquifolium, cohort 3 (1998). Ozone fumigated (70 nl1 28 d 7 h d1 June 1996) open symbols or maintained in charcoal-filtered air (CFA) closed symbols. Significant differences from CFA are denoted star, P < 0.05; 2 stars, P < 0.01. c. Mean cumulative monthly leaf production of I. aquifolium, cohort 4 (1999). Ozone fumigated (70 nl1 28 d 7 h d1 June 1996) open symbols or maintained in charcoal-filtered air (CFA) closed symbols. Significant differences from CFA are denoted star, P < 0.05; 2 stars, P < 0.01.

that the only difference between the plants was the initial treatment. Thus we can study the differential impacts of that treatment without trying to model every potential climatalogical variable. The reported early leaf loss and decreased production on I. aquifolium agrees with much of the published literature e.g.

premature leaf loss (Byres et al., 1992; Pell et al., 1999; Martin et al., 2001; Schreuder et al., 2001; Nunn et al., 2005) and impaired leaf production (Paakkonen et al., 1993, 1997; Schreuder et al., 2001). A study with a similar experimental regime to this work demonstrated that ozone exposure during the growing season induced early leaf fall, late bud break, a reduction in the number of leaves per bud in the following spring and a reduction in leaf non-structural carbohydrates on Fagus crenata (Yonekura et al., 2004). Similarly Populus species have shown to have lower photosynthetic biomass in ozone treatments due to production of fewer new leaves and premature leaf abscission (Schreuder et al., 2001). No change in leaf production and slower leaf abscission rates has been observed with ozone exposure on young Fraxinus excelsior (Wiltshire et al., 1996) while Betula pendula clones showed increased shoot:root ratio and enhanced leaf production (Yamaji et al., 2003). It was noted that the initial major loss of leaves was coincidental with the onset of frost. Ambient frost exposure has frequently demonstrated increased damage and sensitivity of plants previously exposed to ozone (Barnes and Davison, 1988; Waite et al., 1994; Foot et al., 1997; Skarby et al., 1998; Oksanen et al., 2005). The timing of leaf loss witnessed in this experiment may be an indication of increased frost sensitivity in a species whose distribution and habitat preference already show sensitivity to low temperature. This study demonstrates that the impact of ozone on leaf production is not only significant in the first year but persists at a statistically significant level over the following two years. This carry-over effect is not unique and has been observed in several species of trees (Pandey and Agrawal, 1994; Bortier et al., 2000; Oksanen, 2003a,b; Yonekura et al., 2004). In this study the decreased leaf production is not only compounded over several successive growing seasons but is likely to affect the photosynthetically competitive advantage often associated with evergreens and winter/spring leaf retention. The data exhibits a tentative link between differential leaf production and ambient sunshine levels, however this maybe an

J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 355e364

overtly speculative conclusion as AOT40 and sunshine also correlate. In the UK, the highest AOT40 values occur in April and May which corresponds with spring growth. While some of the impact on leaf production appears to be due to direct impact on the plant it is interesting to note that the differences in gs appears to be confined to leaves actually receiving the treatment. Leaves treated with ozone in the initial fumigation (cohort 1) display significantly increased gs compared with the control plants. Subsequent leaf cohorts for both treatments received ambient ozone (Table 1) and did not show any significant differences in gs. It is thus likely that ozone is exerting impact on productivity via at least two different routes, one, the direct effect of ozone causing dysfunction to leaf gas exchange which can induce leaf abscission (Faeth et al., 1981), resulting in further reduced photosynthesis (Sanderman, 1996; Matyssek and Innes, 1999) and secondly, by altering leaf number and retention at the whole plant level. Changes in carbon assimilation as reflected in reduced plant biomass may also be disrupting partitioning (Andersen, 2003) thus altering whole-plant nutrient balance and leading to an impact on leaf production in the following spring (Millard and Thomson, 1989). These effects can be cumulative over several seasons in perennial plants and could ultimately affect the composition of plant communities where the ecosystem is dominated by perennials (Wiltshire et al., 1996). In addition tree species, like I. aquifolium, that have evolved a slow rate of leaf fall are generally less efficient at recovering nitrogen from senescing leaves (Arco et al., 1991). The accelerated leaf fall as witnessed in this study may thus also have impact on nitrogen cycling within the habitat. Shifts in carbon allocation are the predominant way in which plants compensate for stress (Pell et al., 1994). Any decrease in carbon assimilation will have a cumulative yearly effect in perennial plants and therefore subsequent seasonal ozone exposure will have an increasing effect (Wiltshire et al., 1996). Decreased root growth has been reported the following spring after ozone exposure even in the absence of additional ozone (Andersen and Rygiewicz, 1991; Anderson et al., 1997). This was attributed to a decrease in stored reserves and premature leaf loss the previous autumn. Decreased allocation to the roots after ozone exposure occurs rapidly (Andersen and Rygiewicz, 1991; Gorissen et al., 1991; Andersen and Rygiewicz, 1995; Oksanen and Rousi, 2001) with the reduction in biomass occurring within one growing season (Andersen and Rygiewicz, 1995). It has been demonstrated that a higher proportion of photosynthate remained in the foliage and branches of P. strobus L. and less was exported to the bole and the roots after exposure to ozone (McLaughlin et al., 1982). There is a large variation in plant responses to ozone between species and even between genotypes of the same species (Mansfield, 1998) although it has been reported that the relationship between stomatal conductance and foliar injury is species specific (Zhang et al., 2001). In most instances ozone is known to decrease stomatal conductance in plants either as an injury or defence mechanism (Paakkonen et al., 1998; Minnocci et al., 1999; Gunthardt-Goerg et al., 2000; Oksanen

361

and Holopainen, 2001; Zhang et al., 2001; Gerosa et al., 2003; Oksanen, 2003b). This experiment has shown I. aquifolium to possess an atypical response in that gs increased on fumigation. Although Taylor et al. (1986) have reported similar higher stomatal conductance in P. rubens seedlings exposed to moderate ozone levels, while (Hassan et al., 1994) observed increased conductance in Raphanus sativus L. An increase in stomatal conductance and loss of stomatal control has also been reported with prolonged exposure to ozone (Keller and Hasler, 1987; Skarby et al., 1987). A similar study found that water loss could not be controlled, resulting in reduced water use efficiency (Maurer and Matyssek, 1997). The higher gs may indicate either damage to the guard cells (Frey et al., 1996) or a plant mediated response to the stress (Pearson et al., 1996). An increase in conductance may reflect an increase in carbon flux in the plant for metabolic products that are involved with defence and repair mechanisms, although a reduction of photosynthesis may occur due to the effects of ozone. Additional water loss may exacerbate potential ozone and soil moisture interactions (Davison and Barnes, 1998; Bungener et al., 1999; Broadmeadow and Jackson, 2000; Nussbaum et al., 2000; Grulke et al., 2002). Higher stomatal conductance and therefore increased water loss over periods of freezing when water availability is low may have an additional stress effect. It is likely that the increased water loss played an important part is the increased winter leaf loss, winter survival being partially a drought phenomenon. The timing of leaf loss and production poses obvious problems for the plant and potentially the ecosystem and also in this particular species it is probably of major importance for the associated leaf miner, Phytomza ilicis. Pupation and leaf drop are closely linked (James and Pritchard, 1988). If the pupa is formed before leaf drop then the pupa will continue to pupate but if in its larval stage, earlier leaf abscission as in this experiment will increase the mortality of the leaf miner (Ranford, 2006). There are a variety of responses that seem to reflect differences in experimental methodology (Manning, 2005) especially in relation to the type of environmental chamber and the age of the tree species (Chappelka and Samuelson, 1998; Skarby et al., 1998; Manning, 2005). In recognition of these experimental differences it may be concluded that with the impairment of leaf production and early leaf abscission on I. aquifolium, relative fitness of young I. aquifolium saplings are detrimentally affected by ozone but similar impacts may not be experienced by the mature tree. 5. Conclusions This study thus highlights the problems of scaling results from leaf to whole plant and also reinforces the care needed when extrapolating results obtained via material exposed at one developmental stage. This study has shown the potential of ozone to introduce long-term phenological perturbations into ecosystems by influencing productivity over a number of seasons. Leaf loss was significantly increased up to two years and leaf production up to three year post-fumigation.

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The significant loss of leaves prior to the main part of the growing season will result in a decrease of photosynthate production that will be further impacted upon by the reduction and timing of leaf production in the ozone fumigated plants. The increase in stomatal conductance will also have serious impacts for evergreen broad-leaved plants which are exposed to summer and winter desiccation stress.

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