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
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