Reduced soil water availability did not protect two competing grassland species from the negative effects of increasing background ozone

Environmental Pollution 165 (2012) 91e99

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Reduced soil water availability did not protect two competing grassland species from the negative effects of increasing background ozone Serena Wagg a, b, *, Gina Mills a, Felicity Hayes a, Sally Wilkinson b, David Cooper a, William J. Davies b a b

Centre for Ecology and Hydrology, Environment Centre Wales, Deiniol Road, Bangor, Gwynedd LL57 2UW, UK Lancaster Environment Centre, University of Lancaster, Bailrigg, Lancaster LA1 4YQ, UK

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 June 2011 Received in revised form 10 February 2012 Accepted 12 February 2012

Two common (semi-) natural temperate grassland species, Dactylis glomerata and Ranunculus acris, were grown in competition and exposed to two watering regimes: well-watered (WW, 20e40% v/v) and reduced-watered (RW, 7.5e20% v/v) in combination with eight ozone treatments ranging from preindustrial to predicted 2100 background levels. For both species there was a significant increase in leaf damage with increasing background ozone concentration. RW had no protective effect against increasing levels of ozone-induced senescence/injury. In high ozone, based on measurements of stomatal conductance, we propose that ozone influx into the leaves was not prevented in the RW treatment, in D. glomerata because stomata were a) more widely open than those in less polluted plants and b) were less responsive to drought. Total seasonal above ground biomass was not significantly altered by increased ozone; however, ozone significantly reduced root biomass in both species to differing amounts depending on watering regime. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Background ozone Carbon allocation Climate change Drought Senescence Stomatal conductance Soil moisture deficit Roots

1. Introduction Although ozone is a naturally occurring gas in the troposphere, background concentrations have been steadily increasing since the industrial revolution due to anthropogenic activities, with a 0.5e2.0% increase per annum being reported over the last 3 decades (Vingarzan, 2004). This upward trend is predicted to continue (Sitch et al., 2007; Vingarzan, 2004) with mean ozone concentrations increasing by 20e25% by 2050 and by 40e60% by 2100 based on the A2 scenario from the Special Report on Emission Scenarios (SRES) (IPCC, 2007). At present, current peak ozone concentrations across much of Europe are already at levels high enough to cause visible and physiological changes to more sensitive crop and (semi-) natural vegetation species (Ashmore, 2005; Bassin et al., 2004; Mills et al., 2011). Further increases in ambient ozone concentrations will, therefore, be of considerable environmental importance. Ozone inputs from the free troposphere (and stratosphere) are significantly greater in upland areas due to increased mixing ratios of air masses (Vingarzan, 2004). Thus, rural upland areas tend to have higher background ozone levels than more low-

* Corresponding author. E-mail address: [email protected] (S. Wagg). 0269-7491/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2012.02.010

lying regions (Coyle et al., 2002); this potentially puts these habitats, which include (semi-) natural grasslands, at increased risk. In Europe, grasslands cover over 50% of the land area and are important sinks for ozone deposition (Ashmore et al., 2007). It is, therefore, important to understand the adverse effects of ozone on these habitats as it has already been shown that ozone impacts may relate to shifts in species diversity, functional types and dominant vegetation types (Bergmann et al.,1999; Krupa et al., 2001; Ren et al., 2007) as well as changes in the productivity of single species (Bungener et al., 1999b; Davison and Barnes, 1998; Hayes et al., 2007). At present, grasslands have the potential to sequester 240 g C m
2 y
1 (Soussana et al., 2007). Changes in primary productivity and C sequestration and storage that may arise due to the adverse effects of ozone, especially on sink strength of roots (Andersen, 2003; Cooley and Manning, 1987; Fiscus et al., 2005; Hunt et al., 1987), would, therefore, indirectly increase carbon dioxide levels in the atmosphere, thereby, contributing to global warming (Sitch et al., 2007) and as such, further impact on grassland ecosystems. Ozone causes oxidative stress in plants and has various negative physiological effects, with leaf injury (necrotic lesions and/or stippling) and/or accelerated foliar senescence the more obvious visual symptoms (Castagna and Ranieri, 2009; Mészáros et al., 2009; Mills et al., 2007, 2011; Rai and Agrawal, 2008). In addition to visual foliar injury, altered stomatal conductance (Davison and Barnes, 1998;

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S. Wagg et al. / Environmental Pollution 165 (2012) 91e99

Paoletti, 2005; Wilkinson and Davies, 2009, 2010; Wittig et al., 2007), reduced photosynthetic capacity (Pell et al., 1997), decreased biomass productivity (Davison and Barnes, 1998; Hayes et al., 2007) and alteration of assimilate partitioning (Cooley and Manning, 1987; Davison and Barnes, 1998) are also reported symptoms of ozone stress. However, fluctuations in environmental conditions often mean that plant responses to ozone are dynamic (Bassin et al., 2007) because stomatal conductance (gs) is strongly influenced by abiotic factors, such as, photosynthetically active radiation (PAR), air vapour pressure deficit (VPD) and soil moisture deficit (SMD) with the response to these modifiers being speciesspecific. Increased SMD has previously been reported to have a protective effect against the adverse effects of ozone pollution by reducing gs (Heggestad et al., 1988; Manes et al., 2001; Nussbaum et al., 2000; Pell et al., 1993; Schaub et al., 2003). However, other reports document either no change in gs or injury with enhanced ozone under drought conditions (Bungener et al., 1999a,b), or that stomata do not close in response to increased SMD, thereby continuing to allow influx of ozone into the plant (Hayes et al., in press; Maier-Maercker, 1989; Pearson and Mansfield, 1993; Wilkinson and Davies, 2009) with an associated increase in leaf injury. Such alterations in gs primarily depend on the magnitude of change in SMD (Hayes et al., in press; Heggestad et al., 1988). Further, under drought stress, photosynthesis can be reduced and plants detoxification and defence performance impaired, under these circumstances drought can actually increase overall oxidative stress (Bussotti, 2008; Tausz et al., 2007). Paoletti (2005) describes “sluggish” stomatal response in A. unedo leaves to changes in PAR after plants grown in high ozone for 90 days and recent work by Wilkinson and Davies (2009, 2010) documents that ozone stress may cause disruption to abscisic acid (ABA) mediated stomatal closure due to the antagonistic effect of ozone-induced ethylene at the guard cell membrane. Other studies show that enhanced ozone may lead to alterations in the permeability of the guard cell plasma membrane leading to a loss of cell turgor (Manes et al., 2001). The consequence of the above effects may result in reduced stomatal sensitivity to environmental stresses potentially leading to increased transpiration and loss of shoot water balance (Bussotti, 2008; Mills et al., 2009; Wilkinson and Davies, 2009) in addition to the continued flux of ozone to the plant interior. The aims of this study were to: 1) investigate whether prolonged reduction in water availability provides protection against, or modifies, the adverse effects of ozone toxicity in two commonly competing temperate grassland species, Dactylis glomerata and Ranunculus acris; 2) determine whether any protective effect found is associated with stomatal closure; 3) quantify biomass partitioning patterns and investigate whether there is a shift in balance between two competing species in relation to increasing background ozone.

Table 1 Mean daily maximum, and mean ozone concentrations after 8, 13 and 20 weeks ozone exposure. Values for the mean ozone concentration are the mean followed by the 1st and 3rd quartile values in brackets. Ozone Mean daily Mean hourly treatment maximum ozone (ppb) ozone, ppb after 8 weeks exposure

Mean hourly ozone (ppb) after 13 weeks exposure

Mean hourly ozone (ppb) after 20 weeks exposure

AA
20 AA AA þ 12 AA þ 24 AA þ 36 AA þ 48 AA þ 60 AA þ 72

16.7 34.8 46.5 53.4 67.3 77.5 93.1 94.7

16.2 33.9 44.1 50.7 62 72.6 88.9 89.5

20.8 41.6 53.8 62.4 80.6 89.1 108.4 110.7

16.5 34.5 45.5 52.6 67.9 73.9 90.1 89.2

(12.6, (31.6, (42.9, (50.3, (66.6, (72.8, (87.9, (89.2,

20.6) 40.3) 52.6) 61) 80.1) 86) 108.3) 107.9)

(12.9, (31.5, (43.3, (51.4, (63.9, (74.8, (90.5, (93.2,

20.1) 40) 52.8) 61.7) 79.7) 88.7) 108.8) 111.1)

(12.2, (30.7, (41.6, (47.7, (54.3, (69.9, (87.9, (86.5,

20.2) 40.5) 53.1) 62.5) 78.7) 88.6) 108.4) 111.5)

watered (RW) treatments at 7.5e20% v/v soil water content as measured using a hand-held theta probe (HH2 Delta T, Cambridge, UK). 2.2. Ozone exposure The experiment exposed 96 communities of Dactylis glomerata and Ranunculus acris to an ozone range of between 16 ppbe90 ppb within 8 hemispherical dome-shaped greenhouses (“solardomes”, 2 m tall, and 3 m diameter) at the CEH Bangor Air Pollution Facility, North Wales, UK. Ozone supplied to the solardomes was produced from concentrated atmospheric oxygen (Workhorse 8, Dryden Aqua, UK) using a G11 ozone generator (Dryden Aqua) and supplied to each of the domes via polytetrafluoroethylene (PTFE) tubing. Ozone concentrations within each of the domes were measured from above the canopy for 5 min in every 30 min using two ozone analysers (Model API 400A) of matched calibration. The ozone concentration in one solardome was measured continuously with a Thermoelectron (Model 49C, Reading, UK) ozone analyser and used for feedback control. LABVIEW software (version 7) controlled the quantity of ozone injected into the airstreams for any of the given domes, via mass flow controllers; if ozone concentrations exceeded targets by 20 ppb, an automatic cut-off solenoid was triggered to prevent over dosing. The eight ozone treatments were based on a weekly profile measured at the Marchlyn Mawr monitoring site, Snowdonia, North Wales, UK (altitude 610 m; grid reference SH613619). The ozone range for this period was 30e67 ppb giving a mean weekly ozone concentration of 43.3 ppb. Superimposed on the base line data (þ0 ppb, ambient air (AA)) were six incremental increases (AA þ 12, AA þ 24, AA þ 36, AA þ 48, AA þ 60 and AA þ 72 ppb) and one decrease (AA-20 ppb) in ozone concentration giving a mean range of between 16 ppb (pre-industrial) to 90 ppb (simulated post-2100) over the growing season (Table 1). 2.3. Biomass measurements

2. Materials and methods

For each community the above ground biomass was harvested to 7 cm above the soil surface during week 13 (12th August 2008), simulating a hay meadow cut (hereafter referred to as the mid-season cutback), and then allowed to re-grow for a further 7 weeks. After the 20-week ozone exposure, a two-stage final harvest was carried out on all community pots at 7 cm cutting height, then to soil level, to determine total seasonal above ground biomass per species (combined mid-season cutback, destructive harvest and stubble cut to soil level). Below ground biomass was determined for four of the treatments, (AA, AA þ 12, AA þ 48 and AA þ 60 ppb) by carefully washing a preweighed vertical subsection of each pot, containing the same number and combination of plants, and representing approximately 15% of the soil volume; this was subsequently multiplied up to give total root biomass per pot. D. glomerata and R. acris roots were separated by species as they were easily distinguishable. Biomass was oven dried at 65  C to a constant weight and presented as dry weight (d. wt).

2.1. Two-species communities

2.4. Visual assessment of senescence/ozone injury

Two-species communities of Dactylis glomerata and Ranunculus acris, both raised from plug plants (British Wildflower Plants, Norfolk, UK), were established 5 weeks prior to the start of ozone exposure in 14 litre pots containing Levington organic mix topsoil inoculated with 200 ml slurry per pot of sieved soil from an upland conservation meadow, High Keenley Fell, Northumberland (grid reference NY 7922 5586, 360 m a.s.l.). Within each community three clumps of D. glomerata and four R. acris were arranged in a pattern that was repeated for each pot. Community pots were randomly assigned to one of the eight ozone treatments and to either a well or reduced-watered regime, with 6 communities per ozone/watering treatment combination. Prior to the onset of ozone exposure, communities were placed in the solardomes and supplied with charcoal-filtered air only for 5 days to allow for acclimatization. The community pots were watered by hand 2e3 times per week depending on weather conditions to maintain soil water content of the wellwatered treatments at 20e40% v/v soil water content (WW) and the reduced-

Visual assessments of portions of senesced/ozone injured leaves were carried out the day prior to ozone exposure and then in weeks 1, 4, 8, 12 & 20. For each pot assessment sub-samples of 40e70 leaves (D. glomerata) or 10e15 leaves (R. acris) were randomly selected (these leaves were representative of the age classes present in the whole pot) and any exhibiting >10% injury/senescence were recorded as ozone damaged. For R. acris there was no segregation into senescence and ozone injury as these were difficult to distinguish. For D. glomerata ozone caused a yellowing of the leaf blade (chlorophyll bleaching) which is described here as senescence. Percentage injured/senesced leaves were calculated from leaf counts for each species per pot. 2.5. Stomatal conductance measurements Stomatal conductance (gs) measurements were made using a Delta-T AP4 porometer (Cambridge, UK) in selected weeks during the 20-week ozone exposure.

S. Wagg et al. / Environmental Pollution 165 (2012) 91e99 Stomatal conductance measurements were made on days with similar climatic conditions and in the data reported here temperatures within the solardomes ranged from between 23.4(0.15) to 30.5(0.2)  C and PAR ranged from between 342.3(23.9) to 1304.9(78.4) mmol m
2 s
1 (Table 2). All measurements were made for upper canopy fully expanded leaves of both species and were made on leaves that showed 10% injury/senescence. 2.6. Statistical analysis The data were analysed using the R statistical package. The experimental design was analysed as a split plot with ozone concentration as the main-plot (between domes) treatment and water regime as the split-plot (within domes) treatment. The split plot structure was accommodated within a linear mixed effects (lme) model analysis. The stratum effects used in an analysis of variance for split plots is equivalent to using random effects to represent strata, but it avoids possible implied negative variance components. For leaf count senescence data which are integral, a generalized lme model was fitted with binomial link function. For other variables, a simple lme model was used. In all analysis any dome effect was treated as random, with ozone and treatment as fixed effects. In the absence of true replication of ozone treatment, it is assumed in the analysis that any lack of smoothness in a quadratic response is due to dome effects not associated with ozone concentration. On this assumption, linear and quadratic effects of ozone treatment have been extracted for testing against any remaining between dome variability.

3. Results 3.1. Ozone exposure and climatic conditions The mean daily maximum ozone concentrations ranged from 20.8 to 110.7 ppb; seasonal mean ozone concentrations ranged from 16.2 to 89.5 ppb over the 20-week exposure period (although these values were slightly higher over the first 8 and 13 weeks of ozone exposure) (Table 1). Minimum and maximum temperatures for the hourly intervals that stomatal conductance (gs) measurements were made in weeks 3 and 9 ranged between 23.4(0.15) to 30.5(0.2)  C, and minimum and maximum PAR ranged from 342.3(23.9) to 1304.9(78.4) mmol m
2 s
1 over the same period. Average soil moisture ranged from 8.2 to 14.3 (% v/v) in the RW treatment and from 21 to 28 (% v/v) in the WW treatment. Within measurement week ranges were similar (Table 2) allowing comparisons to be made. 3.2. Effects of ozone and drought on stomatal conductance Initial (week 3) analysis revealed that plants grown under reduced soil water availability showed reduced gs in comparison to plants grown under WW conditions for both D. glomerata and R. acris (Fig. 1A and C, p < 0.001 and 0.001 respectively). However, after 9 weeks ozone exposure, stomata in plants that experienced reduced soil water availability showed similar conductance to that for plants that were grown under the WW conditions in ozone

93

concentrations of AA þ 36 and above for both species, although the ozone/water treatment interaction was not statistically significant (Fig. 1B and D). Statistical analysis of the five ozone treatments used in week 9, for D. glomerata, revealed that there was a significant increase in stomatal conductance with increasing ozone in the RW treated plants (r2 ¼ 0.93; p ¼ 0.001) with loss of stomatal response to reduced soil moisture deficient (SMD) being more apparent in the highest ozone treatment. Thus, the percentage reduction in gs by the RW treatment declined as the ozone concentration increased, such that gs was barely reduced in response to RW at high ozone. For the WW plants there was also a significant ozone effect with gs increasing as ozone concentration increased (r2 ¼ 0.61; p ¼ 0.01). However, analysis of data for R. acris revealed that although there was an indication of an ozone effect in the RW treatment, this was not statistically significant. 3.3. Effects of ozone on above-ground biomass By the mid-season cutback, the foliage and stems of D. glomerata increased from 18.02 g (1.04) to 27.68 g (1.57) and from 28.21 g (2.29) to 36.56 g (1.34) in the RW and WW treatments respectively when AA was compared to AA þ 72 ppb; these differences showed a significant ozone effect (Fig. 2A, p ¼ 0.014). There was also a highly significant water treatment effect (p ¼ 0.001), with shoot weights increasing by between 18% and 35% in the RW and WW treatments respectively in the highest ozone profiles in comparison to the AA treatment. There was no ozone by water treatment interaction. Analysis of the above-ground biomass at the final harvest revealed that the increase in biomass with increasing ozone seen at the mid-season cutback was no longer present and there was no significant ozone effect. Nevertheless, the water treatment effect remained highly significant (Fig. 2B, p ¼ <0.001). For R. acris there was no significant ozone effect on above ground biomass at the mid-season cut back or the final harvest, however, the above-ground biomass was significantly greater in the WW treatment compared to the RW treatment (Fig. 3A, p ¼ 0.001). Further, at both the mid-season cut back and the final harvest R. acris was out competed by D. glomerata; average biomass over the 20 week exposure for R. acris was 2.41 g and 4.06 g in the RW and WW treatments respectively, whereas, for D. glomerata it was 20.85 g and 28.34 g respectively. 3.4. Effects of ozone on below-ground biomass Statistical analysis for below ground biomass in D. glomerata showed a significant ozone effect (p ¼ 0.049) with reductions in both the RW and WW treatments of between 37.5% and 50%

Table 2 Hourly mean (+/
SE) temperature, PAR and relative humidity within the solardomes at the time gs measurements were taken during weeks 3 and 9 of the ozone exposure. The mean (+/
SE) soil moisture content, measured for each pot, at the time of gs measurements are also provided. Ozone treatment

Hourly mean (+/
SE) Temp ( C)

PAR (mmol m
2 s
1)

Relative humidity (%)

Soil moisture %v/v) RW

WW

Week 3 AA AA + 12 AA + 36 AA + 72

25.6 (0.2) 23.6 (0.2) 26.1(0.2) 24.4(0.1)

1304.9 (78.4) 981.3(75.7) 1063.1(126.8) 1267.1 (95.6)

39.7 (0.6) 40.2 (0.7) 45.5 (0.7) 37.3(0.5)

14.3 7.5 10 12.1

(2.5) (0.8) (2.5) (2.4)

24.1 22.1 25.8 23.6

( 0.9) (2.4) (1.3) (2.5)

Week 9 AA AA + 24 AA + 36 AA + 48 AA + 60

27.1 23.6 24 20.8 21

1051.6 959.1 697 441.6 435.1

54.5 65.8 64.5 74.2 74.3

8.9 9.5 11.9 8.2 11

(1.9) (1.5) (1.4) (1.5) (0.9)

21.6 25.8 28.2 26.2 23.2

(3.4) (1.7) (0.8) (1.3) (3.3)

(0.2) (0.6) (0.6) (0.1) (0.1)

(130.8) (137.1) (98.4) (40) (42.5)

(0.6) (1.8) (2.1) (0.5) (0.4)

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S. Wagg et al. / Environmental Pollution 165 (2012) 91e99

Fig. 1. Stomatal conductance of well-watered (black bars) and reduced-watered (grey bars) treatments in relation to ozone treatment after exposure to ozone for (A) 3 weeks and (B) 9 weeks in Dactylis glomerata and (C) 3 weeks and (D) 9 weeks in R. acris (n ¼ 6). **, * and (*) indicate significant differences at p < 0.001, p < 0.05 and p < 0.1 respectively for ozone (O3), water treatment (T) and their interaction (O3  T).

respectively in the highest ozone treatment in comparison to the AA treatment (Fig. 4A). There was also a significant water treatment effect (p ¼ 0.013), with RW increasing overall root biomass, but no significant difference in the slope of the response to ozone between the two watering treatments. Statistical analysis revealed there was no significant ozone effect on root weight in R. acris but a significant water treatment effect (Fig. 4B, p ¼ 0.012). The outcome of below ground competition between R. acris and D. glomerata was very different in the two watering treatments. In the WW treatment there was an increase in the ratio between R. acris and D. glomerata roots with increasing ozone, by contrast, under RW conditions there was a decrease in the ratio (Fig. 4C).

There was a significant water treatment effect (p ¼ 0.001) and a significant interaction between soil water treatment and enhanced ozone (p ¼ 0.05). 3.5. Foliar damage caused by ozone D. glomerata and R. acris showed an increase in foliar damage with increasing ozone concentrations over the exposure period. In R. acris, ozone induced foliar injury consisted of interveinal, upper surface stippling followed by chlorosis, necrosis and abscission. Older, mature leaves were more affected than younger ones. In D. glomerata ozone significantly increased senescence, with some plants from the highest ozone treatment having >60% of the leaves

S. Wagg et al. / Environmental Pollution 165 (2012) 91e99

45

O3: p = 0.014

A

9

O3: n.s. T: p< 0.001 O3xT: n.s.

40

T: p< 0.001

8

35

O3xT: n.s.

7

30

25 20 15

d. wt.shoot, g

d.wt shoot, g

A

6 5 4 3

10

2

5

1 0

0 0

20

40

60

80

100

0

120

20

40 35 d.wt shoot, g

B

60

80

100

120

9

O3: n.s.

T: p< 0.001

8

T: p< 0.001

O3xT: n.s.

7

O3xT: n.s.

O3: n.s.

30 25 20 15

d.wt. shoot, g

45

40

mean seasonal ozone (ppb)

mean seasonal ozone (ppb)

B

95

6 5 4 3 2

10

1

5

0

0 0

20

40

60

80

100

120

0

affected by ozone during the 20 week exposure. In most cases in D. glomerata, senesced leaves were retained by the plant. Figs. 5 and 6 show the percentage senesced/injured leaves (a) 8 weeks after the start of the exposure and (b) 7 weeks after the mid-season cutback allowing a comparison of ozone effects on leaves that had developed over a similar timescale in ozone. Enhanced senescence/injury was observed in both D. glomerata and R. acris as early as 7 days after the onset of the ozone exposure (data not shown). By week 8 of the exposure, D. glomerata grown in the higher ozone profiles showed a highly significant increase in the proportion of leaves that were senesced compared to those grown in the ambient air treatment (AA) in both the RW and WW treatments respectively (Fig. 5A). Significant increases were also observed in R. acris by week 8 (Fig. 6A). Following the mid-season cutback, foliar re-growth in both D. glomerata and R. acris showed lower amounts of ozone damage despite exposure to ozone for a similar number of weeks (7) to the pre-cutback assessment (8 weeks). The numbers of senesced/ injured leaves in the highest ozone exposures, when the midseason cut back was compared to the destructive harvest, were reduced by an average of 28.5% in D. glomerata and 33.5% in R. acris. Despite these reductions there was still a significant increase in injury/senescence with increasing ozone (p ¼ <0.001; Figs. 5B and 6B). Furthermore, it was noticeable that prior to the mid-season cutback there was a significant water treatment/ozone interaction

40

60

80

100

120

mean seasonal ozone (ppb)

mean seasonal ozone (ppb) Fig. 2. Effect of increasing seasonal mean ozone on above-ground biomass in Dactylis glomerata at A) the mid-season cutback and B) the final harvest for the well-watered ) and reduced-watered (B and ---) treatments (n ¼ 6  standard error). (- and The significance of treatments is shown for ozone (O3), water treatment (T) and their interaction (O3  T).

20

Fig. 3. Effect of increasing seasonal mean ozone on above-ground biomass in Ranunculus acris at A) the mid-season cutback and B) the final harvest for the well-watered ) and reduced-watered (B and ---) treatments (n ¼ 6  standard error). (- and The significance of treatments is shown for ozone (O3), water treatment (T) and their interaction (O3  T).

with higher levels of injury/senescence in the RW than WW treatments at lower ozone concentrations for both species. 4. Discussion This research considers the combined effects of reduced soil water availability and increasing ozone, as is predicted for the UK and Europe in the coming decades, in two typical component species of many upland mesotrophic grasslands, and highlights the increased vulnerability of R. acris to these combined stresses, resulting in reduced below-ground carbon allocation. In this study we have also shown that R. acris and D. glomerata were less able to close their stomata in response to reduced soil water availability when exposed to enhanced background ozone. Such loss of stomatal response to SMD occurred at ozone concentrations comparable to current summer maxima in the UK. Furthermore, reduced-watering had no protective effect against ozone related foliar damage in either D. glomerata or R. acris in high ozone concentrations. These findings are not consistent with effects seen in some other species at higher ozone concentrations. For example, Pell et al. (1993) and Schaub et al. (2003) report reduced gs and/or reduced foliar injury in, for example, Prunus serotina, Fraxinus americana, and Raphanus sativus. Here we report that the observed increase in foliar senescence in both the RW and WW treatment in

96

A

S. Wagg et al. / Environmental Pollution 165 (2012) 91e99

A

180 r2 0.48

160

% senescence

root d.wt, g

140 120 100 80 60

O3: p= 0.049

40

T: p= 0.013

20

O3xT: n.s.

r2 0.49

O3: p< 0.001

80

T: p = 0.007

70

O3xT: p= 0.003

60 50 30

20

40

60

80

100

0

120

0

30

B

20

2

r 0.14

% senescence

root d. wt, g

20

40

60

80

100

120

mean seasonal ozone (ppb)

25

15 O3: n.s 10

T:p= 0.012 O3xT: p

5

r2 0.36

=0.08 0

20

40

60

90

O3: p< 0.001

80

T: p = 0.03

70

O3xT: n.s.

60 50

r2 = 0.87

40 30

r2= 0.73

20

0 80

100

120

mean seasonal ozone (ppb)

10 0 0

C 0.25

20

40

60

80

100

120

mean seasonal ozone (ppb)

0.2

ratio

r2 = 0.78

10 mean seasonal ozone (ppb)

B

r2 = 0.6

40 20

0 0

90

Fig. 5. Relationship between increasing seasonal mean ozone concentrations and senescence in D. glomerata for A) week 8 and B) week 20 in the well-watered (- and ) and reduce-watered (B and ---) treatments (n ¼ 6  standard error). The significance of treatments is shown for ozone (O3), water treatment (T) and their interaction (O3  T).

r2 0.35

0.15 0.1 O3: n.s 0.05

T: p=0.001 O3xT: p= 0.05

r2 0.14

0 0

20

40

60

80

100

120

mean seasonal ozone (ppb) Fig. 4. The effects of increasing seasonal mean ozone on below ground biomass in A) Dactylis glomerata, B) Ranunculus acris, and C) the ratio of R. acris to D. glomerata root ) and reduce-watered (B and ---) treatments biomass for the well-watered (- and over the 20 week experimental period (n ¼ 4  standard error). The significance of treatments is shown for ozone (O3), water treatment (T) and their interaction (O3  T).

D. glomerata may, in part, be due to the increase in gs with enhanced ozone which permits a greater influx of ozone into the plant, as originally suggested in Wilkinson and Davies (2009, 2010). However, this explanation is inappropriate for R. acris, where there was no significant ozone effect on gs in either the RW or WW treatment. Reductions in the amount of injury/senescence after the mid-season cut back for both species, suggests that leaves that had emerged and expanded in high ozone concentrations may have developed some resilience to ozone toxicity; the mechanisms for this are unclear, but it could be due to an acclimation processes

(Bussotti, 2008) and/or an increase in the plant’s antioxidant capacity (Tausz et al., 2007). Whilst ozone-induced stomatal closure is commonly observed in many species, it is apparent from the literature that stomatal response to ozone is species-specific (see above), and that closure may be less apparent particularly when combined with other stresses such as drying soil (Hayes et al., in press). For D. glomerata we report a significant increase in gs with increasing ozone under both WW and RW treatments, with stomata in the RW treatment being as open as those in the WW treatment in ozone concentrations of AA þ 36 ppb by week 9 of the exposure. Further, although there was no significant increase in gs with increasing ozone in R. acris there was, as with D. glomerata, a loss of stomatal response to SMD in the higher ozone concentrations. Such loss of stomatal sensitivity may be the result of complex interactions between phytohormones (Bussotti, 2008), for example, Tanaka et al. (2005) demonstrated that Arabidopsis plants supplied with exogenous ethylene in drought stress conditions shown delayed stomatal closure due to an ethylene-depended inhibition of the ABA (the “drought hormone”) signalling pathway. Further, recent work by Wilkinson and Davies (2009) is the first to report that ethylene is involved in an ozone-mediated loss of stomatal response to SMD in 30-leaf-stage Leontodon hispidus plants. The authors showed that by inhibiting ethylene formation, via pre-treatment of plants with 1-methylcyclopropene (1-MCP), stomatal response to soil drying was restored; they attributed this to the antagonistic effect of

S. Wagg et al. / Environmental Pollution 165 (2012) 91e99

% injury

A

90

O3: p= 0.005

80

T: p = 0.04

70

O3xT: p= 0.004

60 r2 = 0.27

50 40 30 20

r2 = 0.01

10 0 0

20

40

60

80

100

120

mean seasonal ozone (ppb)

B

90

O3: p< 0.001

80 70 % injury

r2 = 0.68

T: p = 0.05 O3xT: n.s.

60 50

r2 = 0.62

40 30

20 10 0 0

20

40

60

80

100

120

mean seasonal ozone (ppb) Fig. 6. Relationship between increasing seasonal mean ozone concentration and injury ) and reducein R. acris for A) week 8 and B) week 20 in the well-watered (- and watered (B and ---) treatments (n ¼ 6  standard error). The significance of treatments is shown for ozone (O3), water treatment (T) and their interaction (O3  T).

ethylene on the ABA signalling pathway, as described by Tanaka et al. (2005). In order to confirm that ethylene may play a role in inhibiting ABA response in either D. glomerata or R. acris further work would be required. Reductions, or no change, in above-ground biomass is a frequently reported response of plants to enhanced ozone (Bungener et al., 1999b; Cooley and Manning, 1987; Davison and Barnes, 1998; Hayes et al., 2007). Here we observed an initial transitory increase in above-ground biomass with enhanced ozone in both the RW and WW treatments in D. glomerata by the midseason cutback, followed by a loss of response by the final harvest. We propose that this is likely to be due to a number of factors: 1) Because the stomata were more widely open in high ozone there could have been an increased carbon and water/ nutrient flux into the plant across, respectively, the stomata and the root system (Wilkinson and Davies, 2010); 2). Enhanced growth may be a result of hormesis, where initial-phase stress shows a stimulation, followed by a decrease, in growth (Calabrese and Blain, 2009); 3) Reduced carbon partitioning to the roots, resulting in more assimilate availability for above ground parts or conversely, that the prolonged effect of ozone on below-ground biomass resulting in reduced root stock would mean less nutrient resource available for sustained shoot growth. 4) An ozone-induced reduction in carbon assimilation and/or assimilate diversion to activate antioxidant defence (i.e. increased production of proteins) thereby reducing C availability to both shoots and roots (Nunn et al.,

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2005; Overmyer et al., 2008; Wilkinson et al., in press). Further research is needed to elucidate the mechanism(s) involved. Moreover, ozone commonly reduces assimilate partitioning to below-ground sinks (Cooley and Manning, 1987; Reiling and Davison, 1995; Warwick and Taylor, 1995; Zouzoulas et al., 2009). Conversely, increased SMD is often a root growth promoter as a result of increased synthesis of ABA in the root ozone (refs in Wilkinson and Davies, 2010). In this study we found that although ozone induced a marked and linear decrease in root biomass with increasing ozone for D. glomerata, plants grown under RW conditions had significantly greater root biomass than plants grown under WW conditions. Further, although there was no significant below ground effect under non-water-stress conditions in R. acris, there was an indication of a significant interaction between reduced water availability and increasing ozone. That ozone stress causes preferential partitioning of assimilates to shoots rather than roots may be due to decreased carbon assimilation, as outlined above. Other explanations include; increased metabolic costs for detoxification and repair processes; decreased phloem loading (Andersen, 2003; Grantz et al., 2006) as well as increased ethylene formation (ethylene being a root growth inhibitor) as suggested by Wilkinson and Davies (2010). There is also evidence that ozone injury to lower, mature leaves, as frequently observed in this study, can indirectly limit root growth, because it is these leaves that act as the main source of photosynthates for root development (Andersen, 2003; Cooley and Manning, 1987; Grantz, 2003). Whatever the cause, reduced remobilization of photosynthates from smaller root systems further compromises the competitiveness of more ozone-sensitive species (Tingey et al., 2002) and hence, although R. acris showed no significant above-ground effects to enhanced ozone, it was increasingly outcompeted by D. glomerata under RW conditions below ground. Recent work by Wedlich et al. (in press), demonstrated that Ranunculus species are significantly out competed by grasses and legumes at ozone concentrations of 50e65 ppb during a 3 year free air ozone fumigation (FAOE). Ashmore and Ainsworth (1995) have also reported a linear decrease in forb species (F. rubra, T. repens) with increasing ozone in the absence of water stress. In the current study the significant interaction between increased SMD and enhanced ozone on the ratio of R. acris to D. glomerata root biomass indicates a shift in species balance below-ground. Under RW conditions there was a decrease in R. acris roots with increasing ozone, but the opposite occurred under WW conditions. As the incidence of drought events is likely to increase with global warming, then it is possible that more sensitive forb species may be adversely affected by the combined stresses of ozone and increased SMD and possibly lost from grassland communities. Changes in root ratios between R. acris and D. glomerata may alter the dynamics of competition as well as leading to changes in over wintering and subsequent root growth over time, as described in Andersen (2003). 5. Conclusion In this study we have shown that in two common grassland species, D. glomerata and R. acris, enhanced ozone at levels likely to be seen in the next few decades, caused stomata to be less responsive to soil moisture deficit and remain more open. Such loss of stomatal response to soil drying can lead to an increased efflux of water out of the plant and impact on the soileplant water balance (Bussotti, 2008). Further, more open stomata, as reported here in D. glomerata, but also observed in other plant species (Grulke et al., 2007; Wilkinson and Davies, 2009, 2010), is likely to lead to an increase in influx of ozone through the stomata. In this study increased influx of ozone into the plant in D. glomerata, in the

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highest ozone treatments may have caused the exponential increase in injury/senescence seen before the mid-season cut back (with no protective effect of reduced watering) which in the longerterm may have resulted in reduced below-ground carbon allocation due to the increased need of photosynthates for detoxification and repair processes above-ground. The significant interaction between soil water availability and increasing ozone concentrations on the below-ground biomass ratio between R. acris and D. glomerata implies that in an increasingly drier and ozone rich environment, forb species may become more susceptible to the detrimental effects of concomitant environmental stresses (Wedlich et al., in press). Further work is required to understand the mechanisms of ozone injury and the responses of different species to the combined stresses of ozone and reduced soil water availability, particularly at the field scale/ecosystem level. Acknowledgements We wish to thank Aled Williams for design and maintenance of the ozone exposure system and NERC for funding the PhD studentship of S. Wagg and for supporting the running costs of the solardomes. References Andersen, C.P., 2003. Sourceesink balance and carbon allocation below ground in plants exposed to ozone. New Phytologist 157, 213e228. Ashmore, M.R., 2005. Assessing the future global impacts of ozone on vegetation. Plant Cell and Environment 28, 949e964. Ashmore, M.R., Ainsworth, N., 1995. The effects of ozone and cutting on the species composition of artificial Grassland Communities. Functional Ecology 9, 708e712. Ashmore, M.R., Büker, P., Emberson, L.D., Terry, A.C., Toet, S., 2007. Modelling stomatal ozone flux and deposition to grassland communities across Europe. Environmental Pollution 146, 659e670. Bassin, S., Kolliker, R., Cretton, C., Bertossa, M., Widmer, F., Bungener, P., Fuhrer, E., 2004. Intra-specific variability of ozone sensitivity in Centaurea jacea L., a potential bioindicator for elevated ozone concentrations. Environmental Pollution 131, 1e12. Bassin, S., Volk, M., Fuhrer, J., 2007. Factors affecting the ozone sensitivity of temperate European grasslands: an overview. Environmental Pollution 146, 678e691. Bergmann, E., Bender, J., Weigel, H.J., 1999. Ozone threshold doses and exposureresponse relationships for the development of ozone injury symptoms in wild plant species. New Phytologist 144, 423e435. Bungener, P., Balls, G., Nussbaum, S., Geissmann, M., Grub, A., Fuhrer, J., 1999a. Leaf injury characteristics of grassland species exposed to ozone in relation to soil moisture condition and vapour pressure deficit. New Phytologist 142, 271e282. Bungener, P., Nussbaum, S., Grub, A., Fuhrer, J., 1999b. Growth response of grassland species to ozone in relation to soil moisture condition and plant strategy. New Phytologist 142, 283e293. Bussotti, F., 2008. Functional leaf traits, plant communities and acclimation processes in relation to oxidative stress in trees: a critical overview. Global Change Biology 14, 2727e2739. Calabrese, E.J., Blain, R.B., 2009. Hormesis and plant biology. Environmental Pollution 157, 42e48. Castagna, A., Ranieri, A., 2009. Detoxification and repair process of ozone injury: from O3 uptake to gene expression adjustment. Environmental Pollution 157, 1461e1469. Cooley, D.R., Manning, W.J., 1987. The impact of ozone on assimilate partitioning in plants: a review. Environmental Pollution 47, 95e113. Coyle, M., Smith, R.I., Stedman, J.R., Weston, K.J., Fowler, D., 2002. Quantifying the spatial distribution of surface ozone concentration in the UK. Atmospheric Environment 36, 1013e1024. Davison, A.W., Barnes, J.D., 1998. Effects of ozone on wild plants. New Phytologist 139, 135e151. Fiscus, E.L., Booker, F.L., Burkey, K.O., 2005. Crop responses to ozone: uptake, modes of action, carbon assimilation and partitioning. Plant Cell and Environment 28, 997e1011. Grantz, D.A., 2003. Ozone impacts on cotton: towards an integrated mechanism. Environmental Pollution 126, 331e344. Grantz, D.A., Gunn, S., Vu, H.B., 2006. O-3 impacts on plant development: a metaanalysis of root/shoot allocation and growth. Plant Cell and Environment 29, 1193e1209. Grulke, N.E., Paoletti, E., Heath, R.L., 2007. Comparison of calculated and measured foliar O3 flux in crop and forest species. Environmental Pollution 146, 640e647.

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