Biologia, Bratislava, 61/1: 85—90, 2006
Section Botany
DOI: 10.2478/s11756-006-0012-1
Tropospheric ozone-induced structural changes in leaf mesophyll
cell walls in grapevine plants
Nikola Ljubešić1 & Mihaela Britvec2
1
Ruder Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia; tel.: ++385-1-4680238, fax: ++385-1-4561177, e-mail:
[email protected]
2
Faculty of Agriculture, University of Zagreb, Svetošimunska cesta 25, 10000 Zagreb, Croatia; tel.: ++385-1-2393838, fax:
++385-1-2315300, e-mail: [email protected]
Abstract: The structural changes in leaves of grapevine plants (Vitis vinifera L.) exposed to different ozone concentrations
were investigated. Ozone fumigations were performed in open-top chambers at four different ozone levels (charcoal-filtered
air (F), ambient air (N), ambient air + 25 mm3 m−3 ozone (O-25) and ambient air + 50 mm3 m−3 ozone (O-50)).
The leaves of plants from chambers with increased ozone concentrations (O-25 and O-50) were significantly thicker than the
controls (F), owing to increased thickness of the mesophyll layer. Observing O-50 leaves, it was found that the mesophyll
cell wall displayed structural changes. In some places cell wall thickness increased up to 1 µm. We found callose deposits
on the inner side of the cell walls of mesophyll cells. These data are in accord with the concept that the mesophyll cell wall
acts as a barrier against the penetration of tropospheric ozone into the cells.
Key words: callose, cell wall, leaf structure, grapevine, tropospheric ozone
Introduction
Tropospheric (i.e. ground-level) ozone (O3 ) is one of
the most ubiquitous and damaging phytotoxic air pollutant. Current ground level concentrations of ozone
(25–45 ppb) are known to have adverse effects on the
vitality of agricultural crops, forest trees and natural
vegetation, and have been predicted to increase globally by 1 to 2% per year (Olszyk et al., 2001). Although the build-up of tropospheric O3 is typical of
urban areas with high vehicle densities, tropospheric
ozone is now known to be transported long distances
to non-urban, rural, and other remote areas so that
many of the world’s most productive agricultural regions are currently exposed to harmful concentrations
of O3 (Chameides & Lodge, 1994).
The response to tropospheric ozone includes the
collapse of the protoplast (plasmolysis), the pile-up
of cytoplasmic content, and the disintegration of cells
(Pell & Weissberger, 1976; Tiedemann & Herrmann, 1992). Such a collapse of individual cells caused
by ozone is associated with an increase in the intercellular air space of the leaf, as well as with water loss
(Landolt et al., 1994). In mesophyll cells, seriously
damaged chloroplast membranes, plasmalemmae, and
tonoplasts as a result of ozone exposure have been found
(Rufner et al., 1975; Sanders et al., 1992). Swelling
of thylakoid membranes leading to the breakdown of
c 2006 Institute of Botany, Slovak Academy of Sciences
chloroplast integrity was shown to be the result of
ozone exposure (Crang & McQuattie, 1986). In addition, ozone caused an increase in the number and size
of plastoglobules in beech chloroplasts (Mikkelsen &
Heide-Jørgensen, 1996; Britvec et al., 2001). This
was considered to be a result of premature ageing of
leaves exposed to ozone.
Ozone is a highly reactive molecule that may lead
to the oxidation of numerous biological components
and metabolites present in cells. Ozone induces formation of reactive oxygen species (ROS), including hydrogen peroxide (H2 O2 ), superoxide (O−
2 ), hydroxyl
radicals (OH. ) and singlet oxygen in plants (Pellinen et al., 1999; Pellinen et al., 2002; Pasqualini
et al., 2002). Troposheric ozone, as a strong oxidant, is considered to react first with oxidizable constituents in the apoplast. The apoplastic matrix is the
first compartment of a mesophyll cell through which
the pollutant has to pass before reaching the symplastic components. Ozone dissolves in the aqueous
matrix which overlays the surface of the cells lining
the sub-stomatal cavity (the apoplast). The dissolution chemistry of O3 in the region of the cell wall
is far from fully understood. Of significance is also
the presence of thicker cell walls of mesophyll cells,
which has been reported for some species (Gravano
et al., 2003). Due to the high reactivity of ozone,
it is still a matter of debate how deeply it diffuses
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86
N. Ljubešić & M. Britvec
into the mesophyll cells (Günthardt-Goerg et al.,
1996).
Here we investigate the structural changes of
grapevine leaves exposed to ambient and increased
ozone concentrations in open-top chambers. The hypothesis tested in this study is that there is a correlation between increased troposheric ozone concentration
and leaf morphology and anatomy, with emphasis on
the cell wall structure.
Flourescence microscopy
Flourescence microscopy was performed on the materials
prepared for elecron microscopy. Semithin sections of leaves
embedded in Araldite (2 µm) were cut using an ultramicrotome and mounted on coverslips. The resin from epoxyembedded tisssue was removed with a freshly prepared
mixture of sodium methoxide, benzene and methyl alcohol (MAYOR et al. 1961). Subsequently, the sections were
stained with aniline blue and screened for callose using an
epiflourescence microscope (Zeiss Axiovert 35, filter set 01,
G365, FT395, LP420) (O’BRIEN & MCCULLY 1981).
Material and methods
Electron microscopy
Leaf samples for electron microscopy were prepared by standard methods (fixation in 1% glutaraldehyde and postfixation in 1% osmium tetroxide) and embedded in Araldite.
Ultrathin sections (60–100 nm thick) were made using a
glass knife (ultramicrotome MT 6000-XL, RMC, Inc., USA),
stained with uranyl acetate and lead citrate, and studied
and photographed with a transmission electron microscope
(EM 10A – Zeiss, Germany).
Plant material
Grapevine plants (Vitis vinifera L. cv. Welschriesling)
were pre-cultivated in 65-L pots (one plant per pot). The
grapevines were planted in compost-based plant growth substrate (Frux Einheitserde ED 63). The plants were irrigated
as required to avoid drought stress and to allow for maximum ozone uptake rates. Pesticides were applied as necessary to avoid the following pests/diseases: Plasmopara
viticola, Oidium tuckeri, Botrytis cinerea, and Panonychus
ulmi. The following agents were used: diazinone (Basudin,
Ciba-Geigy AG, Basel, Germany), brompropylate (Neoron,
Ciba-Geigy AG, Basel, Germany), and paraffinic oil (Paramag, AFA GmbH, Erbach, Germany). In the spring of their
third fertile year, before bud break, 11 plants were placed
in each of eight open-top chambers.
Ozone exposure system
Cylindrical open-Top-Chambers, OTC, are permanently set
up at the experimental site of the Austrian Research Center Seibersdorf, located 35 km to the south-east of Vienna.
The experimental set-up included two chambers per each
ozone treatment: with charcoal filters (F), with ambient air
(N), with ambient air supplemented with 25 mm3 m−3 of
ozone (O-25), and with ambient air supplemented with 50
mm3 m−3 of ozone (O-50). Ozone was generated from pure
oxygen by an ozone generator (Fischer, Model 502, Meckenheim/Bonn, Germany), and added for 7 hours per day
(from 9 a.m. to 4 p.m.) 5 days a week. The treatment period lasted from mid May to the end of September. The
details are described in BRITVEC (1998) and BRITVEC et
al. (2001).
For microscopic investigation two leaves per plant were
taken at the end of the treatment period. These were 8th
and 10th node leaves, counted from the base of this year’s
growth. Interveinal regions (discs) from each leaf were excised for light and electron microscopy.
Light microscopy and morphometry
For light microscopy, hand made sections or semithin sections of Araldite embedded tissue stained with toluidine
blue were used. The thickness of the leaf, upper epidermis,
palisade mesophyll, spongy mesophyll, and lower epidermis
were measured in cross-sections, at 440 × magnification,
with an ocular scale standardized by a stage micrometer.
The observed data were statistically analysed using ANOVA
(SAS Institute Inc., SAS/STAT User’s Guide, Version 6,
Fourth Edition, Volume 1, Cary, NC: SAS Institute Inc.,
1989, p. 943).
Results
Light microscopy and morphometry
Table 1 summarizes the effect of ozone treatment on the
thickness of whole leaves, upper and lower epidermis,
palisade and spongy mesophyll. The leaves of plants
from chamber with increased ozone concentration (O25 and O-50) were significantly thicker than the controls (F), i. e. about 5.23% for O-25, and about 8.17%
for O-50. The dimensions of the cells in the upper and
the lower epidermis did not show significant changes.
However, the thickness of mesophyll layer was increased
(Tab. 1) due to an increase in the size of palisade and
spongy mesophyll cells. Morphometric measurements of
cell areas and cell diameters parallel and perpedicular
to the surface of the leaf corroborated the above findings (data not shown).
Flourescence microscopy
Examination of semithin sections revealed local thickening of the mesophyll cell walls (Fig. 1A). Staining
with aniline blue confirmed the presence of callose deposits (Fig. 1B). Such deposits were only visible on cells
of the spongy mesophyll and usually adjacent to intercellular spaces.
Electron microscopy
The plants grown in filtered (F) and ambient air (N)
had mesophyll cells with typical thin cell walls. The
thickness of about 0.2 µm was constant around the
whole cell (Fig. 2A).
The treatment with ambient air with addition of
25 mm3 m−3 ozone (O-25) did not substantially affect
the cell wall (Fig. 2B). Only, in some places the cell
wall showed slight thickening. In these sites, callose deposits are beginning to be formed (Fig. 2B, arrow).
The other cell structures also exhibited minor changes.
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Tropospheric ozone-induced structural changes
87
Table 1. Thickness of whole grapevine leaves and their individual cell layers (in µm). F – plants grown in open-top chambers with
charcoal-filtered air, N – plants grown in open-top chambers with ambient air, O-25 – plants grown in open-top chambers with ambient
air + 25 mm3 m−3 ozone, O-50 – plants grown in open-top chambers with ambient air + 50 mm3 m−3 ozone, n = 44, mean ± SE.
Leaf and its
histological
components
Whole leaf
Upper epidermis
Palisade mesophyll
Spongy mesophyll
Lower epidermis
Ozone treatment
F
146.48
17.00
46.84
67.41
15.00
±
±
±
±
±
N
2.34
0.37
1.11
1.81
0.52
148.50
17.82
47.73
67.30
15.43
±
±
±
±
±
O-25
2.08
0.34
1.13
1.41
0.37
154.14
17.82
52.09
70.02
14.00
±
±
±
±
±
O-50
1.66
0.37
0.86
1.48
0.36
158.45
18.43
54.64
70.95
14.34
±
±
±
±
±
2.29
0.39
1.36
1.56
0.48
This layer was constantly about 0.2 µm thick. However,
the deposits of callose were only very rarely present
along the whole cell wall and their thickness varied
from zero to nearly one µm. Also, the callose layer
was less homogenous, in comparison with the cellulose layer. The callose fibrils were irregularly arranged.
In the midle of these structures electron opaque inclusions of various size and shape were present (Fig. 2CD, arrowheads). The electron opaque core was enclosed
by an electron-translucent area. Very often the cells
with thickened walls were plasmolysed and the protoplasts were regularly detached from the walls (Fig. 2D).
Sometimes, in the mesophyll cell layer of leaves treated
with 50 mm3 m−3 ozone (O-50) electron-dense deposits
appeared in the intercellular spaces. This is possibly
pectin which forms small exudates near the cell walls
or large electron opaque accumulations between cells
(Fig. 2D).
Discussion
Fig. 1. Leaf cross-sections of grapevine plants grown in opentop chambers with ambient air and the addition of 50 mm3 m−3
ozone (O-50). Arrows in the light micrograph (A) point to areas
of increased thickness of the mesophyll cell wall. The flourescence
micrograph (B) shows callose deposits on the thickened cell wall
(arrows). The markers in all micrographs represent 10 µm.
In the chloroplasts, the thylakoid system was well preserved, but partially swollen, and the size and number
of plastoglobules was increased (Fig. 2B).
The leaves grown at increased (ambient + 50
mm3 m−3 ) ozone concentration (O-50) showed more explicit structural changes in the cell wall. Local wall
thickening, as measured in the cross sections, reached as
much as 1 µm. At the inner side of the cell wall there
were deposits for which flourescence microscopy suggested that the chemical nature of this matter was callose (Figs 2C-D). The cellulose layer was homogeneous
and contained regularly arranged fibers of cellulose.
Increased ozone concentrations (O-25, O-50) resulted in
significantly increased thickness of total leaf, palisade
and spongy layers. We interpret these changes as an
indication of the sensitivity of Vitis vinifera to tropospheric ozone which resulted in morphological response.
This is in accordance with previous studies, in which
O3 sensitivity has been correlated with leaf thickening
(Crang & McQuattie, 1986; Bennett et al., 1992;
Pääkkönen et al., 1995; Bussotti et al., 1998; Oksanen et al., 2001).
Under elevated ozone concentrations we found a
good preservation of the thylakoid membranes whereas
there was an increase in the number and size of plastoglobules. This is in accordance to the work of Miyake
et al. (1989), Violini et al. (1992), and Mikkelsen &
Heide-Jørgensen (1996) who found out that ozone
caused an increase in the number and the size of plastoglobules in the chloroplasts of radish, common bean,
and beech. They interpreted these changes as signs of
premature ageing caused by ozone. Ljubešić (1968,
1969) also described an increase in the number and size
of plastoglobules as a sign of leaf ageing in Nicotiana
tabacum and Chlorophytum comosum grown in ambient air. He interpreted a larger number of plastoglobUnauthenticated
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88
N. Ljubešić & M. Britvec
Fig. 2. Transmission electron micrographs of cross-sections of mesophyll cells of grapevine plants. A. Plants grown in open-top chambers
with charcoal-filtered air (F). B. Plants grown in open-top chambers with ambient air + 25 mm3 m−3 ozone (O-25), the arrow points
to an area of begining callose formation. C – D. Plants grown in open-top chambers with ambient air + 50 mm3 m−3 ozone (O-50).
Arrow heads – electron opaque cores surrounded by electron-transluscent areas, c – cellulose cell wall, ca – callose deposits, p – putative
pectin exudates. The markers in all micrographs represent 0.5 µm.
ules as a sign of changed physiological activity. There
is the possibility of partial plastoglobule reduction by
repeated greening of old and yellow leaves.
Of particular interest in the O-50 – leaves (ambient air and the addition of 50 mm3 m−3 of ozone) was
the observation of significantly thickened cell walls of
mesophyll cells. In some places the cell wall reached
a thickness as large as 1 µm. Previous studies have
reported that ozone may injure plants by increasing
the thickness of the mesophyll cell wall (Pääkkönen
et al., 1995; Günthardt-Goerg, 1996; GünthardtGoerg et al., 1997; Polle et al., 2000). In addition to
confirming these reports, we found deposits of callose
on the inner side of the cell walls of mesophyll cells.
Their thickness was not constant – from zero to nearly
one µm. This apparent accumulation of callose along
the walls of mesophyll cells of grapevine has, to our
knowledge, not been described yet. Callose is a polymer of β-1,3-linked glucose that is deposited between
the plasma membrane and the cell wall in response to
stress (Schmohl et al., 2000).
The callose deposition is in agreement with the
pattern of foliar ozone injury as described by Gravano et al. (2003). The injured cells of Ailanthus altissima leaves were separated from the healthy ones by
a layer of callose. The authors concluded that cell walls,
in addition to acting as mechanical barriers against the
spread of ozone into the protoplast, also provides important role in physiological protection because its aqueous
matrix contains many compounds which can act as antioxidants (and may thus prevent ozone-induced damage), including ascorbate, dehydroascorbate, as well as
several enzymes, such as peroxidases and glutathioneS-tranferase (Moldau, 1998; Moldau et al., 1998;
Ranieri et al., 2000; Turcsányi et al., 2000; Burkey
& Eason, 2002; Burkey et al., 2003). This is in accord
with the previous estimates that a typical cellulose layer
of 0.1–0.5 µm (as found in our study) is able to detoxify
about half of the ozone entering the cell suface, whereas,
in thicker callose layers, detoxification reactions of the
above type may result in more complete decomposition
of O3 (Moldau et al., 1997).
Furthermore, we sometimes found electron dense
deposits in the intercellular spaces in the mesophyll
of grapevines leaves treated with 50 mm3 m−3 ozone.
This was possibly pectin which forms small exudates
near the cell walls or large electron opaque accumulations between cells. These pectinaceous projections
of mesophyll cell walls are also regarded as signs of
activated stress defence mechanisms (GünthardtUnauthenticated
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Tropospheric ozone-induced structural changes
Goerg, 1996; Günthardt-Goerg et al., 2000; Pääkkönen et al., 1998; Polle et al., 2000).
In conclusion, in grapevine plants, the accumulation of callose increasing the overall thickness of the
cell walls, is involved in the adaptation of the mesophyll cells to tropospheric ozone.
Acknowledgements
This work was partly supported by the Austrian Federal
Ministry for Science and Transport (FTSP-Programm). The
authors gratefully acknowledge the Austrian Research Center Seibersdorf for their support providing the plant materials and Dr. Volker MAGNUS (Ruder Bošković Institute,
Zagreb) for help in preparing the manuscript.
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Received Jan. 3, 2005
Accepted Nov. 10, 2005
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