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 for P. 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. 4A – 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. 5A – 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. 5E – 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
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 = 100 mm. 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 = 100 mm. 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 = 100 mm.
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 = 1 mm. Bundle sheath cells: numerous lipid bodies and starch grains were visible in symptomatic needles C with respect to control needles D bar = 5 mm. Vacuole appearance: control needles E exhibited less
accumulation of electron-dense tannins than equivalent symptomatic needles F bar = 5 mm. L, lipid; PL, plastoglobuli; ST, starch grain; V, vacuole; T, tannins.
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 = 2 mm. 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 SO
2
or O
3
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
3
, SO
2
, NO
2
, 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 andor to
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 SO
2
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
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