Directory UMM :Data Elmu:jurnal:E:Environmental and Experimental Botany:Vol44.Issue1.Aug2000:

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Impacts of urban levels of ozone on

Pinus halepensis

foliage

Costanza Soda

_{, Filippo Bussotti}

_{*, Paolo Grossoni}

_{, Jeremy Barnes}

_,

Bruno Mori

_{, Corrado Tani}

a_{Department of Plant Biology,}_Uni₆_{ersity of Florence,}_{Piazzale delle Cascine} ₂₈_,_I-₅₀₁₄₄ _Firenze, _Italy b_{Air Pollution Laboratory,}_{Department of Agricultural and En}₆_{ironmental Science,}_{Ridley Building,}

The Uni6ersity of Newcastle Upon Tyne,Newcastle NE1 7RU, UK

Received 4 February 2000; received in revised form 10 April 2000; accepted 13 April 2000

Abstract

Between May and September, 1996, seedlings ofPinus halepensiswere placed at a site adjacent to an automated air pollution monitoring station within the urban area of Florence. Additional ‘control’ plants were placed in chambers ventilated with charcoal/Purafil®_{-filtered air. All trees were well watered throughout the whole experimental period.} During the exposure period, ambient levels of sulphur dioxide were very low, whilst the accumulated hourly exposure to ozone above 40 ppb (i.e. AOT40) exceeded 20 000 ppb h−1 _{— peak hourly ozone concentrations rising to levels} above 100 ppb. Trees exposed to ambient levels of air pollution exhibited typical symptoms of ozone damage (chlorotic mottle) on previous year needles toward the end of the summer. Similar symptoms were not observed on equivalent trees exposed to filtered-air, nor were visible symptoms accompanied by insect or pest infestation. Anatomical and ultrastructural observations made on symptomatic needles revealed degeneration in mesophyll cells bordering sub-stomatal cavities and alterations in chloroplast ultrastructure (fat accumulation, starch and tannin pattern modifications). These observations are consistent with the known effects of air pollutants (namely ozone) recorded in the literature. Findings are discussed in relation to the impacts of ozone on P. halepensis in the Mediterranean region. © 2000 Elsevier Science B.V. All rights reserved.

Keywords:Cell ultrastructure; Mediterranean; Sulphur dioxide; Symptomatology; Urban air

www.elsevier.com/locate/envexpbot

1. Introduction

Visible symptoms attributed to the pervasive photochemical air pollutant ozone have been recorded for many years on the needles of several pine species in California and the South-Eastern United States (Miller et al., 1963; Miller and

Millecan, 1971; Evans and Miller, 1972a,b; Miller and Evans, 1974; Fox and Mickler, 1995; Ar-baugh et al., 1998; Chappelka and Samuelson, 1998), as well as in the mountains bordering Mexico City (Hernandez Tejeda and Nieto de Pascual, 1996; Alvarez et al., 1998). In Europe, among coniferous trees, visible symptoms of foliar injury appear to be restricted to a single species,

Aleppo pine (Pinus halepensis), the most common

landscape tree of the Mediterranean littoral. The * Corresponding author.

E-mail address:[email protected] (F. Bussotti).

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question of whether this species is particularly sensitive to ozone remains to be established (see Barnes et al., 1999). However, ambient concentra-tions of the pollutant are known to be particularly high in the Mediterranean region (Gimeno et al., 1994) because of the high summer temperatures and radiations, and the natural distribution of this species is coincident with the highest ground level

ozone concentrations experienced in Europe

(Butkovic et al., 1990; Milla´n et al., 1996). Ac-cording to the UN-ECE standards (Ka¨renlampi and Ska¨rbi, 1996; Fuhrer et al., 1997; Fuhrer and Achermann, 1999), risks for forests are currently evaluated in Europe with the index AOT40 (accu-mulated hourly ozone exposure above 40 ppb threshold). AOT40 values exceeding 10 000 ppb.h are considered dangerous for forest vegetation (Level I Critical Levels). The threshold of 40 ppb was chosen to exclude the non-anthropogenic ozone, but this threshold has been seriously ques-tioned. Sutinen et al. (1990), for example, found that considerably lower ambient ozone concentra-tions (about 30 ppb) caused ultrastructural

alter-ations on Picea abiesneedles.

Typical visible symptoms of ozone injury on the

needles of P. halepensis have been observed in

California (Ka¨renlampi, 1987) and in several parts of Southern Europe (Gimeno et al., 1992; Velis-sariou et al., 1992; Davison et al., 1995; Gimeno et al., 1995; Velissariou et al., 1996). These symp-toms comprise chlorotic mottling that becomes visible towards the end of summer or early winter

(Velissariou et al., 1996). Previous year (C+1)

needles are primarily affected (Ka¨renlampi, 1987; Davison et al., 1995), though current year (C) needles can be affected if trees are exposed to particularly high levels of the pollutant (see Barnes et al. 1999). In the most severe cases, the symptoms degenerate into necrosis and are associ-ated with premature needle loss. The chlorotic mottle observed is considered a specific symptom of ozone injury, since the same symptoms have been reproduced during several controlled or

semi-controlled exposures of P. halepensis to

ozone (Gimeno et al., 1992; Wellburn and Well-burn, 1994; Elvira et al., 1995; Anttonen et al., 1998).

Several ultrastructural studies have been

car-ried-out on symptomatic needles of P. halepensis

in order to characterize the histological changes induced by ozone (Ka¨renlampi, 1986, 1987; Anttonen et al., 1994; Wellburn and Wellburn, 1994). Most of these studies have been performed on plant material exposed to ozone under con-trolled or semi-concon-trolled conditions (Anttonen et al., 1994; Wellburn and Wellburn, 1994), although one study was conducted on trees exposed in the field (Ka¨renlampi, 1986, 1987). Findings have been variable, presumably because of differences in the way in which trees have been exposed to the pollutant (cf. Anttonen et al., 1998) and the fact that several studies have focused on the ef-fects on juvenile needles on young seedlings rather than effects on mature needles of forest trees (Kelly et al., 1995; Kolb et al., 1998).

In this study, we examined (i) the relationship between ambient concentrations of ozone at an urban Mediterranean location and the appearance

of visible symptoms on the needles ofP. halepen

-sis, (ii) the impacts of ambient levels of ozone on needle ultrastructure and (iii) compared the ob-served ultrastructural modifications with those de-scribed in the literature as typical of ozone damage in this species.

2. Materials and methods

2.1. Plant material and sites study

Trees about 150 cm tall and 4 years old of P.

halepensis Mill. (Aleppo pine) field grown in the

regional nursery of Roccastrada (lat. 43°38% _N

long. 11°10% _{E), in a remote (and assumed as}

non-polluted) forest area, were transplanted in March 1996 and placed in individual pots in steam-sterilized soil: peat: Perlite (1:1:1). At the beginning of the experiment (May) trees had

cur-rent year (C) and previous year (C+1) needles.

C+2 needles were being shed and were not

con-sidered. Foliage did not show any biotic or abiotic symptoms.

Between May and September of the same year, eight of these potted trees were placed at a site

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area of Florence (central Italy) where the ground level concentrations of ozone and sulphur dioxide were constantly measured by ARPAT (Regional Agency for Environmental Protection) with state-of-the-art instruments (for further details see Grechi and Bruni, 1998). Air samplers were placed at about the same height as the crowns. The index AOT40 was calculated.

Another eight plants (‘control trees’), were

placed in a indoor chamber about 2×2 m wide,

ventilated with charcoal-Purafil®

-filtered air, next the University of Pisa (lat. 43°41%_{N long. 10°21}%_E).

In these chambers the average daytime tempera-ture tracked external conditions (mean daytime

temperature was 2591°C and mean night-time

temperature was 2091°C; mean daily

photosyn-thetic photon flux density was 530mmol m−2s−1

and mean relative humidity was 8593%, see

Sol-datini et al., 1998). All trees were watered to field-capacity

Fig. 1 shows monthly mean data for temperature and precipitation at the field site over the

experi-mental period. The displayed pattern is usual for the considered period. Fig. 2 provides a summary of daily ground-level sulphur dioxide and ozone concentrations over the same period. Sulphur diox-ide concentrations were generally low over the experimental period. In contrast, monitoring data revealed that 7 h (10:00 – 17:00 h) daily mean ozone concentrations generally exceeded the 40 ppb threshold with peak hourly mean concentrations approaching 100 ppb during six episodes in June and July. These peaks are quite high but not unusual in Mediterranean conditions (see Chalou-lakou et al., 1999). Hourly mean ozone data for each month over the experimental period is shown in Fig. 3. These data revealed that between May and September, mean hourly ozone concentrations exceeded the 40 ppb threshold for about one third of the total exposure time (8 consecutive hours: 10:00 – 18:00 h)-with the highest levels of ozone experienced during July when there were 11 consec-utive h exceeding the 40 ppb threshold. The AOT40

was 20 000 ppb h−1_{between May and September.}

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Fig. 2. Ground-level sulphur dioxide (A) and ozone (B) concentrations at the urban field site during the experimental period. Sulphur dioxide concentrations expressed as the 24 h daily mean (M24) and hourly maximum concentration (MAX). Ozone concentrations expressed as 24 h daily mean (M24), 7 h (10.00 – 17.00 h) daily mean concentration (M7) and maximum hourly concentration (MAX).

2.2. Foliar symptoms and microscopical analyses At the end of the exposure period (end of September), 30 needles pairs of the previous year

needles (i.e. C+1) were collected from each tree,

from the upper and outer part of the crown (120 – 150 cm from the soil), and the extent of visible symptoms (i.e. chorotic mottling) evalu-ated. Additional needles (ten pairs) were sampled from each tree for histological and ultrastructural analyses by means of light and transmission elec-tron microscopy.

In order to assess the extent of visible injury, the percentage of the surface of each needle af-fected by chlorotic mottling was visually esti-mated on each needle, using a 5% proportional classes scale (i.e. a scoring scale based on 5% increments of needle area affected). A chlorotic mottle index (CMI) was calculated based on the mean area of each needle displaying visible injury.

Light microscopy and transmission electron mi-croscopy (TEM) were performed on the needles from ‘control trees’ as well as needles showing visible injury (i.e. chlorotic mottle). In this latter case, we compared the chlorotic areas with the green areas of the same damaged needles. Obser-vations were made on sections from ten needles per tree. Segments measuring about 2 cm in length were removed from the central portion of each needle; these were then examined in primary epifluorescence with a Leitz Dialux 22 microscope employing a 450 – 490 nm wide-band filter. Fur-ther needle segments of the same length were fixed in a 4% solution of formalin, and then kept in a 1% saccharose solution for 24 h. Using a Reichert

Jung freeze-microtome, 30 mm thick transverse

sections were prepared from each needle. These cross-sections were observed in primary fluores-cence at 450 – 490 nm to determine the chlorophyll response (Adams and Lintilhac, 1993); and at

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340 – 380 nm to determine effects on lipids (Adams and Lintilhac, 1993). Then, sections

were stained with either: Neutral Red/Astra

Blue-to highlight cell wall modifications (Jensen, 1962) or Fluoral Yellow 088-to highlight lipid modifications (Brundrett et al., 1991), and sec-tions viewed with the aid of a Zeiss Axioplan microscope. Additional segments measuring 2 – 3 mm were removed from the central portion of each needle, fixed in formalin and then dehy-drated by passing through an increasing ethanol series prior to pre-embedding sections in L.R. White resin and absolute alcohol (1:1). Finally, sections were transferred to pure resin for 24 h and oven-dried at 50°C. Transverse sections (0.5

mm) were cut with a Reichert OM U3

micro-tome, using a glass knife, and subsequently stained with Toluidine Blue 0 to examine overall cell structure (O’Brien and Mc Cully, 1981),

with Schiff’s reagent (PAS) to examine starch content.

Samples destined for observation by (TEM) were prefixed in phosphate buffer (pH 7.2) con-taining 2.5% glutaraldehyde and 4% paraformal-dehyde. After 20 h at 5°C, samples were rinsed

twice (2×10 min) in the same buffer, then

post-fixed (2 h) in 2% osmium tetroxide prepared in the same buffer. Subsequently, samples were de-hydrated in an increasing ethanol series (10 min at each stage of the fixation series). Finally, af-ter two 5 min rinses in propylene oxide (100%), the samples were embedded in resin, according to Spurr’s procedure (Spurr, 1969). A Reichert Ultracut S microtome was used to cut ultra-thin

sections (0.09 mm) with a diamond knife. These

sections were stained with uranyl acetate and lead citrate, and then observed with a Philips EM-300 microscope.

Fig. 3. Hourly mean ground-level ozone concentrations over the course of the day at the urban field site. Data represent monthly averaged hourly concentrations over the experimental period (May – September).

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3. Results

By the end of the experimental period, typical visible symptoms of ozone damage (i.e. chlorotic

mottle) were evident on previous years’ (C+1)

needles of all trees exposed to ambient levels of urban pollution, and correspond to the mottle illustrated by Gimeno et al. (1992) forP.halepen

-sis and Flagler and Chappelka (1995) for Pinus

echinata as typical ozone symptoms. The exten-sion and intensity of mottling varied considerably

between trees: the number of affected C+1

needles ranges from 36 to 74% and the mean chlorotic needle surface ranges between 11.5 and 28.6% (only symptomatic needles considered) and between 4.3 and 6.6% (considering all needles). Chlorotic mottling was not associated with insect damage or fungal disease. On the other hand, no visible symptoms were visible on needles from trees maintained in charcoal-Purafil®_{-filtered air} (i.e. ‘control trees’).

Fig. 4(A – G) illustrates the main findings from

light microscopy. Primary fluorescence was

markedly reduced in symptomatic needles-with effects particularly pronounced around the stom-ata and in the mesophyll cells lining the sub-stom-atal cavity (Fig. 4A, B). Several collapsed mesophyll cells were observed in longitudinal cross-sections prepared from symptomatic needles (Fig. 4C), and there was evidence of lipid (Fig. 4D, E) and starch (Fig. 4F, G) accumulation in mesophyll cells and bundle sheath cells.

Fig. 5(A – F) illustrates some of the ultrastruc-tural modifications observed by TEM in symp-tomatic needles. Fig. 5A and B shows the condition of the chloroplasts. The mesophyll cells of ‘control’ (Fig. 5A) needles possessed large and elongated chloroplasts; the grana’ were well-orga-nized, while plastoglobuli were few and small (arrow). In contrast, their counterparts in symp-tomatic needles (Fig. 5B) were shorter and rounder, and exhibited electron-dense membranes (arrow); plastoglobuli were larger and more abun-dant and appeared not only in the chloroplast, but also in the vacuole. In addition, in symp-tomatic needles the stroma appeared to be granulated.

In the cells that make up the bundle sheath (Fig. 5C, ‘control’, and Fig. 5D, symptomatic needles) a marked difference in the content of lipidic bodies and starch grains was also observed. Both lipids and starch were more abundant in symptomatic needles.

Additional features observed in mesophyll cells are illustrated in Fig. 5(E – F). Fig. 5E shows a section through the mesophyll of a ‘control needle’, while 5F shows an equivalent part of a symptomatic needle. In symptomatic needles, starch grains were visible in the mesophyll cells (Fig. 5F) and the vacuoles contained electron-dense material (possibly tannins) as well as nu-merous lipid bodies (Fig. 5F).

Symptomatic needles (Fig. 6B) also exhibited modifications in phloem structure in comparison with ‘control needles’ (Fig. 6A), cribrum elements appearing to have collapsed and their lumen flat-tened (Fig. 6B). Symptomatic needles also

exhib-ited a greater accumulation of calcium

oxalate-like crystals in epidermal tissue (Fig. 6C, control, and D, symptomatic needle).

The ultrastructural alterations described above occur only in the exposed needles and are associ-ated with chlorotic mottle. In the green areas of the exposed needles they are much less evident or absent.

4. Discussion

The appearance of typical visible symptoms of ozone damage (chlorotic mottle) on the previous

(C+1) year needles’ of field-exposed leaves is

consistent with the reported effects of controlled exposure to ozone (at levels not dissimilar to those recorded in the field in the present study) on P. halepensis. Gimeno et al. (1992) found that an

average concentration of 70 ppb (7 h day−1

) caused mottle within two months of exposure, whereas Wellburn and Wellburn (1994) stressed the role of episodic peaks (up to 120 ppb). Elvira et al. (1995) in an OTC (open top chamber)

experiment (ambient air+ozone 40 ppb)

ob-served chlorotic mottle in the second year of exposure. Finally, Anttonen et al. (1998) found chlorotic mottle after a 5-week ozone treatment

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Fig. 4. Light micrographs of previous year (C+1) needles from trees exposed to ambient air at the field site and equivalent ‘control trees’ maintained in charcoal-Purafil®_{-filtered air (May – September). Primary epi-fluorescence in control (A) and symptomatic (B)} needles observed in transverse sections with blue filter. Arrows indicate weaker red response of chlorophyll in symptomatic needle than in equivalent control needles (bar=100mm). Collapsed cells in the mesophyll of symptomatic needles (stained with neutral red) (C; bar=50 mm). Control (D) and symptomatic (E) needle sections stained with Fluoral Yellow 088. Arrows indicate greater abundance of lipid deposits in symptomatic needle (bar=100mm). Control (F) and symptomatic (G) needle sections stained with Schiff’s reagent. Arrows indicate the greater starch accumulation in the bundle sheath cells of symptomatic needles (bar=100mm).

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Fig. 5. Transmission electron micrographs of previous year (C+1) needles from trees exposed to ambient air at the field site and equivalent ‘control trees’ maintained in charcoal-Purafil®_{-filtered air (May – September). Chloroplast ultrastructure: fewer} plas-toglobuli were evident in control needles (A, arrow) than in their symptomatic counterparts (B), were are more abundant. Arrow indicates the electron-dense membrane (bar=1mm). Bundle sheath cells: numerous lipid bodies and starch grains were visible in symptomatic needles (C) with respect to control needles (D) (bar=5mm). Vacuole appearance: control needles (E) exhibited less accumulation of electron-dense tannins than equivalent symptomatic needles (F) (bar=5mm). L, lipid; PL, plastoglobuli; ST, starch grain; V, vacuole; T, tannins.

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Fig. 6. Transmission electron micrographs of previous year (C+1) needles from trees exposed to ambient air at the field site and equivalent ‘control trees’ maintained in charcoal-Purafil®_{-filtered air (May – September). Phloem in control (A) and symptomatic (B)} needles-latter samples exhibiting the presence of collapsed cells (bar=2mm). Epidermal cells in control (C) and symptomatic needles (D). Arrows indicate greater abundance of calcium oxalate-like crystals in symptomatic needles (bar=5 mm). L, lipid; PL, plastoglobuli; ST, starch grain; V, vacuole; T, tannins.

with 150 ppb (12 h day−1

). Different kinds of symptoms (tip necrosis, reddening and browning

of needles) have been reported as acute SO2or O3

injuries (Flagler, 1998), for very high levels of these pollutants, but they were not found in this survey.

The anatomical observations reported in the present study revealed that chlorotic mottling was associated with the degeneration of mesophyll tissue. Damage appeared primarily in those cells in close proximity to the sub-stomatal cavity-con-sistent with the accepted view that air pollutants

are predominantly absorbed through the stomata (Wolfenden and Mansfield, 1991). It has been reported in the literature that the mesophyll cells of pine needles are especially sensitive to ozone (Evans and Miller, 1972a,b; Miller and Evans, 1974; Evans and Miller, 1975; Evans and Leon-ard, 1991; Evans and Fitzgerald, 1993). Damage to mesophyll was also related to several kinds of pollutants (O3, SO2, NO2), alone or in combina-tion (Fink, 1989; Hasemann and Wild, 1990; Schiffgens-Gruber and Lu¨tz, 1992), to acid rain

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forest decline (Fink, 1988; Vogelmann and Rock, 1988). Hasemann and Wild (1990) described the so-called ‘bone cells’ as a specific symptom of the injury caused by exposure to a variety of air pollutants. The array of modifications observed at the ultrastructural level in symptomatic needles during the present study are consistent with the observations of Moss et al. (1998) on red spruce (P.rubens) needles in ozone polluted regions.

The physiological relevance of some of the ul-trastructural modifications observed in

symp-tomatic needles is particularly worthy of

attention. The change in shape and dimension of the chloroplasts, along with the degeneration of the thylakoids and a marked increase in the size and number of plastoglobuli, are amongst the symptoms most frequently associated with ozone-induced damage (Sutinen et al., 1990; Anttonen et al., 1994; Holopainen et al., 1996). Granulation of the stroma is considered a specific symptom of ozone damage (Sellde´n et al., 1996). On the other hand, swelling of chloroplasts has also been

ob-served as SO2 damage (Wellburn et al., 1972). In

the present study lipid-like bodies were also ob-served in the vacuole of symptomatic needles. This could be interpreted as an ozone-induced premature ageing response, and is consistent with the observations on ozone-treated needles made by Ka¨renlampi (1986, 1987). Symptomatic needles were also observed to exhibit enhanced starch accumulation in the chloroplasts. This is consis-tent with observations made on the bundle sheath of conifer needles (Wellburn and Wellburn, 1994), but contrasts with several authors reports’ of a reduction in the starch content of ozone-treated needles (McQuattie and Schier, 1993; Anttonen and Ka¨renlampi, 1995; Holopainen et al., 1996). In the present study, the observed starch accumu-lation in symptomatic needles was consistent with the observed collapse and resulting inactivation of phloem elements-also observed by Wellburn and

Wellburn (1994) in ozone-treated needles of P.

halepensis. It is possible that this phenomenon is indicative of accelerated needle ageing under the influence of ozone and other pollutants (Schmitt and Ruetze, 1990; Fink, 1991a). Starch pattern and allocation, as well as phloem transport, are known to be affected by pollutants (Wolfenden

and Mansfield, 1991). The alteration of carbon partitioning also causes the unbalance of the shoot – root ratio (Grulke and Balduman, 1999).

Finally, the extracellular accumulation of cal-cium oxalate crystals in the epidermis (cf. Fink, 1991a,b) and changes in the behaviour of tannins in the vacuole (cf. Ka¨renlampi, 1986, 1987) are also features characteristically associated with the modifications induced by ozone (Rosemann et al., 1991; Kangasja¨rvi et al., 1994; Booker et al., 1996).

5. Conclusions

The levels of ozone experienced during the sum-mer months in urban areas of Central Italy are clearly higher than the threshold of toxicity for conifers. As far as SO2 is concerned, the sensitiv-ity of conifers varies considerably and is the ob-ject of discussion. Lichtentaler and Buschmann (1984) proposed a threshold of 50 ppb (in our experimental site the concentration of this pollu-tant was conspollu-tantly lower, Fig. 2), but Darrall (1989) shows how the response varies species by species, and the same happens for the interactive effects of SO2+O3(Darrall, 1989). Dust also may cause ultrastructural and physiological distur-bances (Dixit, 1988; Bacic et al., 1999). Overall, the role of different stress agents cannot be ex-cluded a priori, but in the present survey ozone seems to be the predominant factor.

This pollutant potentially constitutes a hazard to elements of the native woody Mediterranean flora. In addition, Mediterranean vegetation is a natural emission source of isoprenoid compounds (monoterpenes and isoprene) into the atmosphere (Street et al., 1997; Pen˜uelas and Lusia`, 1999). These emissions are a considerable source of reac-tive carbon, and thus may play a crucial role in the formation and persistence greenhouse gas air pollutants, such as carbon monoxide and ozone (Brasseur and Chatfield, 1991; Seufert et al., 1995).

However, several important questions remain unanswered. For example, are visible symptoms a good indicator of effects on tree vitality/ perfor-mance? are dose-effect relationships similar for

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seedling and mature trees and how do they differ between pot-grown (i.e. well-watered, as in the present study) and field-grown (i.e. commonly subjected to severe water deficit during the sum-mer months in the Mediterranean region)? The phytotoxicity of the pollutant is largely governed by the dose of the pollutant taken-up into the foliage, and hence (in the case of ozone) on stomatal conductance (Dixon et al., 1998). It is

well know thatP.halepensisis a drought-avoiding

species that closes its stomates rapidly in response to soil water shortage (Melzack et al., 1995; Schiller and Cohen, 1995). Under field conditions, it is thus possible that the injurious action of the pollutant is reduced during the hottest and driest months (see Barnes et al., 1999).

The anatomical and ultrastructural observa-tions made in the present study are entirely con-sistent with the view that ozone pollution was the predominant cause of the visible damage observed (i.e. chlorotic mottling). Moreover, the type of analysis adopted enables possible confounding ef-fects associated with insect infestation or patho-gen infection to be readily identified (sensu Wenner and Merrill, 1998). The study draws at-tention to the potentially detrimental effects of ambient ozone on a native mediterranean tree in its natural habitat. However, the findings cannot be extrapolated to forest trees (cf. Kolb et al., 1998) and it is quite possible that summer water shortage markedly reduces ‘damage’ under natu-ral conditions (Barnes et al., 1999). Moreover, the appearance of foliar symptoms, and evidence of mesophyll degeneration, should not necessarily be accepted as evidence of damaging effects on the whole tree. For example, Temple and Miller (1994) found that trees with visible ozone lesions on older needles grew as well as those maintained

under controlled conditions. Compensatory

changes in the patterns of growth and carbon allocation (Wellburn and Wellburn, 1994) as well as stimulations in the photosynthetic rate of new foliage (Langebartels et al., 1997; Wellburn et al., 1997) suggest that visible symptoms are important as indicators of a potential risk for the ecosystem, but need to be viewed with considerable scepti-cism when diagnosing ‘damage’ to vegetation (see Davison and Barnes, 1998).

Acknowledgements

The authors are indebted to Prof. Giacomo Lorenzini (University of Pisa) for supervising the culture of ‘control trees’ in filtered air chambers, and ARPAT (Tuscan Regional Agency for Envi-ronmental Protection) for providing access to the relevant air pollution monitoring data.

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needles-latter samples exhibiting the presence of collapsed cells (bar=2mm). Epidermal cells in control (C) and symptomatic needles

(D). Arrows indicate greater abundance of calcium oxalate-like crystals in symptomatic needles (bar=5 mm). L, lipid; PL,

plastoglobuli; ST, starch grain; V, vacuole; T, tannins.

with 150 ppb (12 h day

−1

). Different kinds of

symptoms (tip necrosis, reddening and browning

of needles) have been reported as acute SO2

or O3

injuries (Flagler, 1998), for very high levels of

these pollutants, but they were not found in this

survey.

The anatomical observations reported in the

present study revealed that chlorotic mottling was

associated with the degeneration of mesophyll

tissue. Damage appeared primarily in those cells

in close proximity to the sub-stomatal

cavity-con-sistent with the accepted view that air pollutants

are predominantly absorbed through the stomata

(Wolfenden and Mansfield, 1991). It has been

reported in the literature that the mesophyll cells

of pine needles are especially sensitive to ozone

(Evans and Miller, 1972a,b; Miller and Evans,

1974; Evans and Miller, 1975; Evans and

Leon-ard, 1991; Evans and Fitzgerald, 1993). Damage

to mesophyll was also related to several kinds of

pollutants (O

, SO

, NO

), alone or in

combina-tion (Fink, 1989; Hasemann and Wild, 1990;

Schiffgens-Gruber and Lu¨tz, 1992), to acid rain

treatment (Ba¨ck and Huttunen, 1992) and

/

or to

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forest decline (Fink, 1988; Vogelmann and Rock,

1988). Hasemann and Wild (1990) described the

so-called ‘bone cells’ as a specific symptom of the

injury caused by exposure to a variety of air

pollutants. The array of modifications observed at

the ultrastructural level in symptomatic needles

during the present study are consistent with the

observations of Moss et al. (1998) on red spruce

(P.

rubens

) needles in ozone polluted regions.

The physiological relevance of some of the

ul-trastructural modifications observed in

symp-tomatic

needles

is

particularly

worthy

of

attention. The change in shape and dimension of

the chloroplasts, along with the degeneration of

the thylakoids and a marked increase in the size

and number of plastoglobuli, are amongst the

symptoms most frequently associated with

ozone-induced damage (Sutinen et al., 1990; Anttonen et

al., 1994; Holopainen et al., 1996). Granulation of

the stroma is considered a specific symptom of

ozone damage (Sellde´n et al., 1996). On the other

hand, swelling of chloroplasts has also been

ob-served as SO2

damage (Wellburn et al., 1972). In

the present study lipid-like bodies were also

ob-served in the vacuole of symptomatic needles.

This could be interpreted as an ozone-induced

premature ageing response, and is consistent with

the observations on ozone-treated needles made

by Ka¨renlampi (1986, 1987). Symptomatic needles

were also observed to exhibit enhanced starch

accumulation in the chloroplasts. This is

consis-tent with observations made on the bundle sheath

of conifer needles (Wellburn and Wellburn, 1994),

but contrasts with several authors reports’ of a

reduction in the starch content of ozone-treated

needles (McQuattie and Schier, 1993; Anttonen

and Ka¨renlampi, 1995; Holopainen et al., 1996).

In the present study, the observed starch

accumu-lation in symptomatic needles was consistent with

the observed collapse and resulting inactivation of

phloem elements-also observed by Wellburn and

Wellburn (1994) in ozone-treated needles of

P. halepensis. It is possible that this phenomenon is

indicative of accelerated needle ageing under the

influence of ozone and other pollutants (Schmitt

and Ruetze, 1990; Fink, 1991a). Starch pattern

and allocation, as well as phloem transport, are

known to be affected by pollutants (Wolfenden

and Mansfield, 1991). The alteration of carbon

partitioning also causes the unbalance of the

shoot – root ratio (Grulke and Balduman, 1999).

Finally, the extracellular accumulation of

cal-cium oxalate crystals in the epidermis (cf. Fink,

1991a,b) and changes in the behaviour of tannins

in the vacuole (cf. Ka¨renlampi, 1986, 1987) are

also features characteristically associated with the

modifications induced by ozone (Rosemann et al.,

1991; Kangasja¨rvi et al., 1994; Booker et al.,

1996).

5. Conclusions

The levels of ozone experienced during the

sum-mer months in urban areas of Central Italy are

clearly higher than the threshold of toxicity for

conifers. As far as SO2

is concerned, the

sensitiv-ity of conifers varies considerably and is the

ob-ject of discussion. Lichtentaler and Buschmann

(1984) proposed a threshold of 50 ppb (in our

experimental site the concentration of this

pollu-tant was conspollu-tantly lower, Fig. 2), but Darrall

(1989) shows how the response varies species by

species, and the same happens for the interactive

effects of SO2

+

O3

(Darrall, 1989). Dust also may

cause ultrastructural and physiological

distur-bances (Dixit, 1988; Bacic et al., 1999). Overall,

the role of different stress agents cannot be

ex-cluded a priori, but in the present survey ozone

seems to be the predominant factor.

This pollutant potentially constitutes a hazard

to elements of the native woody Mediterranean

flora. In addition, Mediterranean vegetation is a

natural emission source of isoprenoid compounds

(monoterpenes and isoprene) into the atmosphere

(Street et al., 1997; Pen˜uelas and Lusia`, 1999).

These emissions are a considerable source of

reac-tive carbon, and thus may play a crucial role in

the formation and persistence greenhouse gas air

pollutants, such as carbon monoxide and ozone

(Brasseur and Chatfield, 1991; Seufert et al.,

1995).

However, several important questions remain

unanswered. For example, are visible symptoms a

good indicator of effects on tree vitality

/

perfor-mance? are dose-effect relationships similar for

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seedling and mature trees and how do they differ

between pot-grown (i.e. well-watered, as in the

present study) and field-grown (i.e. commonly

subjected to severe water deficit during the

sum-mer months in the Mediterranean region)? The

phytotoxicity of the pollutant is largely governed

by the dose of the pollutant taken-up into the

foliage, and hence (in the case of ozone) on

stomatal conductance (Dixon et al., 1998). It is

well know that

P. halepensis

is a drought-avoiding

species that closes its stomates rapidly in response

to soil water shortage (Melzack et al., 1995;

Schiller and Cohen, 1995). Under field conditions,

it is thus possible that the injurious action of the

pollutant is reduced during the hottest and driest

months (see Barnes et al., 1999).

The anatomical and ultrastructural

observa-tions made in the present study are entirely

con-sistent with the view that ozone pollution was the

predominant cause of the visible damage observed

(i.e. chlorotic mottling). Moreover, the type of

analysis adopted enables possible confounding

ef-fects associated with insect infestation or

patho-gen infection to be readily identified (sensu

Wenner and Merrill, 1998). The study draws

at-tention to the potentially detrimental effects of

ambient ozone on a native mediterranean tree in

its natural habitat. However, the findings cannot

be extrapolated to forest trees (cf. Kolb et al.,

1998) and it is quite possible that summer water

shortage markedly reduces ‘damage’ under

natu-ral conditions (Barnes et al., 1999). Moreover, the

appearance of foliar symptoms, and evidence of

mesophyll degeneration, should not necessarily be

accepted as evidence of damaging effects on the

whole tree. For example, Temple and Miller

(1994) found that trees with visible ozone lesions

on older needles grew as well as those maintained

under

controlled

conditions.

Compensatory

changes in the patterns of growth and carbon

allocation (Wellburn and Wellburn, 1994) as well

as stimulations in the photosynthetic rate of new

foliage (Langebartels et al., 1997; Wellburn et al.,

1997) suggest that visible symptoms are important

as indicators of a potential risk for the ecosystem,

but need to be viewed with considerable

scepti-cism when diagnosing ‘damage’ to vegetation (see

Davison and Barnes, 1998).

Acknowledgements

The authors are indebted to Prof. Giacomo

Lorenzini (University of Pisa) for supervising the

culture of ‘control trees’ in filtered air chambers,

and ARPAT (Tuscan Regional Agency for

Envi-ronmental Protection) for providing access to the

relevant air pollution monitoring data.

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