New Phytol. (1996), 132, 135-143
T h e effects of ozone and nutrient supply
on stomatal response in birch (Betula
pendula) leaves as determined by digital
image-analysis and X-ray microanalysis
BY B E A T FREYX,C H R I S T O P H S C H E I D E G G E R , M A D E L E I N E S.
G U N T H A R D T - G O E R G A N D RAINER MATYSSEKT
Swiss Fedeval Institute fov Fovest, S n o w and Landscape Reseavch, Ziivchevstvasse 111,
CH-8903 Bivmensdovf, Switaevland
(Received 19 June 1995;accepted 12 Septembev 1995)
SUMMARY
C u t t i n g s o f Betula pendula R o t h were g r o w n i n field fumigation c h a m b e r s t h r o u g h o u t o n e growing season i n
filtered air w i t h < 3 nl 1-I 0 , ( c o n t r o l ; C ) or d a y l n i g h t = 90140 nl 1-I 0 , ( o z o n e f u m i g a t i o n ; 0,).Plants w e r e
watered w i t h either l o w (0.005 O h ; L F ) or high-concentrated (0.05 ",b; H F ) fertilizer solution. Discs b e t w e e n
second-order veins i n t h e central portion o f t h e leaves w e r e excised and i m m e d i a t e l y cryofixed i n liquid nitrogen
for low-temperature scanning electron-microscopy ( L T S E M ) at 1000 h o u r s and 1400 hours. Stomatal w i d t h , area
and density were measured b y digital image-analysis. X - r a y c o u n t s o f potassium ( K ) and calcium ( C a ) ions w e r e
determined b y m e a n s o f energy-dispersive X - r a y microanalysis i n guard and subsidiary cells. Accurate and fast
m e a s u r e m e n t s o f stomatal apertures b y image analysis w e r e possible i n b i r c h leaves, because t h e darkness o f t h e
stomatal pore contrasts w i t h t h e brightness o f t h e guard cells and t h e cuticular ledges. Regression analysis showed
a close relationship b e t w e e n t h e stomatal w i d t h and t h e pore area (s = 0,938, P < 0.01). At all harvest t i m e s , t h e
stomatal pores were significantly narrowed i n t h e h i g h fertilization control treatment ( C / H F v s . C / L F ) , and i n t h e
ozone treatment at 1400 h o u r s ( O , / H F v s . O , / L F ) . I n addition t o t h i s fertilization e f f e c t , o z o n e had also narrowed
t h e stomatal pores ( O , / H F v s . C / H F at 1400 h o u r s , O , / L F v s . C / L F at all harvest t i m e s ) . I n addition t o these
functional e f f e c t s , morphological e f f e c t s (individual leaf area, stomatal d e n s i t y ) w e r e d e t e r m i n e d . Single-leaf area
was increased b y h i g h fertilization, w i t h a t e n d e n c y t o decrease w i t h 0 , fumigation. T h e stomatal density i n
intercostal fields was increased b y 0 , b u t decreased b y h i g h fertilization. Stomatal widening w a s accompanied b y
increased K c o u n t rates i n t h e guard cells, i n contrast t o constant K values i n t h e subsidiary cells, irrespective o f
t h e fumigation or fertilization regimes. C a l c i u m c o u n t s i n t h e guard cells w e r e similar t o t h o s e i n t h e subsidiary
cells, and w e r e i n d e p e n d e n t o f t h e aperture w i d t h . I n samples w i t h established o z o n e i n j u r y , t h e K / C a ratio i n
collapsed guard cells increased compared w i t h turgid guard cells irrespective o f t h e pore aperture. Collapsed
subsidiary cells o n l y differed f r o m turgid subsidiary cells w h e n t h e guard cells had also collapsed and t h u s closed
t h e pore.
K e y w o r d s : Betula pendula, o z o n e , stomatal aperture, X - r a y microanalysis, low-temperature scanning electronmicroscopy.
INTRODUCTION
Leaf ontogeny is controlled by the species-dependent
response to environmental conditions including air
pollution (Sestac, 1985; Gunthardt-Goerg et al.,
1993 a). During leaf formation, these conditions
influence the final leaf size and differentiation (such
as tissue structure and stomatal density), and later
control leaf gas-exchange (Hinckley & Braatne, 1994)
*
T o xvhorn correspondence should be addressed.
Current address: Lehrstuhl Forstbotanik, LudwigMaximilians-Universitat Munchen, Hohenbachernstr. 22, D85354 Freising, Germany.
t
by regulating stomatal apertures through volume
changes in the guard cells. T h e stomatal movements
are mediated by the accumulation and release of ions
(Kf, balanced by C1- and malate from starch) to and
from the vacuoles of the guard cells (MacRobbie,
1981 ; Hite & Outlaw, 1994).
Tropospheric ozone ( 0 , ) concentrations have been
increasing with industrial activity and have reached
phytotoxic levels (Ashmore, 1994). Stomata respond
to ozone depending on the growth conditions
(Darrall, 1989). Many reports have shown that ozone
induces at least partial stomatal closure (Unsworth &
Black, 1981 ; Scheidegger et al., 1991), whereas
136
B . Frey and others
increased aperture could also be observed in newly
formed young leaves in trees with established injury
(Schmutz et al., 1995). Phytotoxicity, or detoxification and acclimation mechanisms might lead
finally to changes in ultrastructure, morphology and
biomass (Matyssek et al., 1992 ; Gunthardt-Goerg et
al., 1993 a ; Paakkonen et al., 1993).
I n birch leaves (Betula pendula Roth), stomatal
conductance determines the uptake of 0, into the
inner air space of the leaf. T h e natural status of
individual stomatal apertures can be analysed by
low-temperature scanning electron-microscopy
( L T S E M ) . Cryotechniques have to be used, because
not only chemical fixatives, but also critical-point
drying or freeze-drying cause considerable changes
in the width of stomatal apertures (Van Gardingen,
Jeffree & Grace, 1989; Scheidegger et al., 1991).
Samples rapidly immobilized and stabilized by
c?yofixation retain most of their water in a frozenhydrated state after partial freeze-drying (Read &
Jeffree, 1991) so that cell volume stays unaffected.
Stomatal behaviour inferred from gas exchange
measurements is integrated over the entire functioning area of the leaf, and subsumes morphological
characteristics such as stomatal size and number,
and different stomatal apertures in the case of
'patchiness ' and cell injury (Beyschlag et al., 1994).
Gas exchange measurements demand structural
knowledge of the leaf surface, to ensure that the
latter contains a representative number of stomata.
Stomatal apertures are generally measured with a
ruler after the photograph has been taken using a
scanning electron-microscope (Shiraishi, Hashimoto
& Kuraishi, 1978; Scheidegger et al., 1991).
This study describes the influence of ozone and
fertilizer supply on morphological aspects (singleleaf area, stomatal density) and stomatal behaviour
of birch leaves grown in fumigation chambers in the
field. Stomatal aperture was investigated using
L T S E M and digital image-processing (Van
Gardingen et al., 1989). First, the reliability of the
data from image analysis was compared with
conventional methods (measured by a ruler) and
then we applied this technique to evaluate responses
of stomatal apertures to ozone and fertilization.
Potassium and calcium composition in guard and
their subsidiary cells which are known to be
important for stomatal regulation (Hite & Outlaw,
1994) were obtained by energy-dispersive X-ray
microanalysis (EDX).
M A T E R I A L S AND NIETHODS
Plants and fumigation
Cuttings of a birch clone (Betula pendula Roth) were
grown in 10 1pots with sand and transferred to a field
fumigation installation (Landolt, Pfenninger &
Luthy-Krause, 1989; Matyssek et al., 1991) before
leaf flushing. Plants (80 plants, one per pot, eight per
fumigation chamber, without competition) were
maintained in the fumigation chambers from April
through to October 1993 and were separated into
groups of four different treatments, namely: control
(C, charcoal-filtered air < 3 n1 1-I O,), filtered air
with added 0, (0700-21 00121 00-0700 hours =
90140 nl1-I O,), and each with low fertilization ( L F ,
0.005 :/, fertilizer solution; Hauert Nahrsalz, T y p A,
Bern, Switzerland), or high fertilization ( H F ,
0.05 94). Nutrient concentrations of the high nutrient
supply fertilizer were as follows: macroelements
(mn4): 6.5 N ; 0.9 P ; 0.2 S ; 2.0 K ; 0.2 Ca; 0.2 M g ;
microelements ( ~ N :I ) 11.0 F e ; 5.6 M n ; 3.4 C u ;
1.9 Z n ; 0.6 M o ; 9.2 B. Ozone was produced from
pure oxygen by an ozone generator (Fischer Mod.
502). T h e concentrations were measured with an
ozone monitor (Monitor Labs Mod. 8810). O n clear
sunny days, a shading roof limited the photon flux
density to a maximum of c. 600 pmol m-2 s-' to
prevent overheating in the open-top chambers; the
roof was not employed under overcast or cloudy
conditions, nor at dawn and dusk.
Sampling
Individual leaf growth rate, age and phenology
(including visible injury symptoms) were recorded
along the main stem axis. Leaf samples taken from
the four treatments were homogeneous in terms of
formation date and duration of their expansion
growth, because these parameters influence leaf
differentiation and in particular stomatal density
(Gunthardt-Goerg et al., 1993a). Discs (diameter
8 mm, between 2nd-order veins) were excised from
each half of the central lamina for light and scanning
electron-microscopy (sampling see Table 1). Stomatal density was determined by light microscopy
( L M ) in leaf discs excised into methanol (bleaching
and conservation) and stained with J J K solution
(2 g KJ and 1 g J in 100 ml distilled water). Stomata
were counted within a counting net at 10 randomlychosen disc positions of 0.3 mm2 area, each situated
in intercostal fields. For S E M investigations, leaves
of similar age and without shading were selected and
sampled simultaneously in the control and the 0,
treatments. Leaf discs were mounted on aluminium
stubs using a cryo-adhesive (MethocelB, Fluka) and
immediately frozen in liquid nitrogen. Sampling
proceeded from leaves that were 35 ( C I L F , O,/LF)
and 41 ( C I H F , O,/HF) to 69 ( L F ) and 79 ( H F ) days
old. Leaf area was determined with a Delta-T area
meter M K 2 .
Low-temperature scanning electron-microscopy
(LTSEM)
A Philips 51 5 microscope (Philips, T h e Netherlands)
equipped with a SEN1 cryo unit (SCU 020,
Bal-Tec, Balzers, Liechtenstein) was used for the
preparation and microscopical analysis of the frozen-
Ozone and fertilization effects on stomatal behaviour
Table 1. Sampling
Measured
parameter
Individual leaf
area
Stomatal
density ( L M )
Stomatal
aperture ,(SENI)
*
Number
o f trees
Number
Per
o f leaves
t r e a t m e n t per tree
D a t e and
time (h)
o f harvest
5
2
12 A u g .
5
5
12 A u g .
1"
5
1"
3
1"
3
14 S e p .
1400 h o u r s
15 S e p .
1000 h o u r s
17 S e p .
1400 h o u r s
Harvest conditions,
t e m p e r a t u r e (OC)
and relative
humidity ( O A )
o f harvest
Cloudy
18.8, 52
Clear s u n n y
17.2, 57
Clear s u n n y
18.8, 57
T o t a l n u m b e r o f stomata measured
In
micrographs
Image
analysis
EDX
232
196
64
276
216
64
287
168
64
D i f f e r e n t trees i n each harvest
hydrated specimens. Cryo-preserved samples were
transferred to the cold stage in the preparation
chamber of the SEM. Surface ice crystals were
removed by sublimation in the preparation chamber
for 10 min at -80 OC under high vacuum (P <
2 x 10-4 Pa) (Scheidegger et al., 1991). T h e specimen
was sputter-coated with platinum (to give good
electrical conductivity) in an argon atmosphere after
raising the pressure to 2.2 Pa. T h e coating thickness
was 15 nm, measured by a quartz thin-film monitor.
After coating, the specimen was transferred from the
preparation chamber to the cold stage of the S E M by
means of a transfer rod under high vacuum, after
opening the transfer valve. T h e coated specimen was
analysed in the S E M at a temperature below
- 120 OC with an acceleration voltage of 12 kV.
T h e stomata were viewed without a tilt, normal to
the leaf surface and photographed at a standard
instrument magnification of x 300. Photographic
images randomly selected from five areas on the leaf
disc were recorded on T M X film (Kodak) and
recording times were 32 ms per line for a total
exposure of 2000 lines with 9-22 stomata per
300 x 400 p m viewing area ( = 0.12 mm2).
based on selected image grey-level ranges. That
resulted in a binary image (stomatal aperture white
and background black). Measurements of the stomatal aperture were obtained after the binary image of
the detected pore had been optimized and dust had
been removed.
Binary filter operations were performed on the
recorded images with a single erosion step to remove
pixel noise, an operation to fill holes within objects
and finally a dilatation operation was applied to
compensate for the erosion steps before the stomatal
aperture was measured. After these operations, we
normally obtained a satisfactory separation between
background leaf matrix and stomatal aperture.
T h e processed images were used for evaluating the
pore length, the pore width (perpendicular to the
pore length), the pore area, the percentage area
occupied by stomatal aperture within the viewed
area (300 x 300 pm), the aspect ratio, and the perimeter of each stoma. T h e total number of apertures
within an image was counted using the convention of
excluding parts of stomata overlapping the margins
of the image and was converted to stomata per mm2.
T h e data from all leaves (with five replicates per leaf)
per harvest and treatment were combined.
Image analysis
The video signal from the S E M was directed to the
image analyser connected to a work station with
24 M b RAM memory and a 730 M b hard disk. One
image was built by averaging eight single frames
with a resolution of 5 12 horizontal x 5 12 vertical
pixels (256 possible grey values per pixel). T h e
recorded images were processed with the interactive
image-analysis package Voyagera (Noran Instruments, USA). T h e digital image analysis consisted of
grey-scale image-processing, thresholding and the
binary image-processing (Omasa Sr. Onoe, 1984).
The threshold to distinguish between the stomatal
aperture and the leaf matrix in the image was
determined by an interactive image-segmentation
Energy-dispersive X-ray microanalysis
Elemental analysis of selected cells was performed in
the S E M equipped with a TracormNorthern energydispersive X-ray analysis system. Electron-induced
X-rays were detected by a Pioneerm Si(Li) lightelement analytical detector (30 mm2 Microtrace)
with a take-off angle of 15'. T h e microscope was
operated at an acceleration voltage of 18 kV with a
beam current of 80 pA and a working distance of
12 mm. T h e specimen was sputter-coated with
chromium instead of platinum. T h e coating thickness was 3 nm.
Microanalysis was carried out in the centre of
guard cells and their subsidary cells. A 2 p m x 2 p m
138
B. Frey and others
Figure 1. SEM image analysis: open stomata from the 1000 hour harvest; C / L F treatment, ( a ) grey image;
(b) binary image as used to determine the dimensions of the aperture (bar = 100 p m ) .
Figure 2. SEM micrographs from the C / L F (a) with turgid guard cells (arrow) and turgld subsidiary cells
(arrowhead), and the O , / L F (b) regimes (harvest 1000 hours). Note the collapsed guard cells (long arrow) and
collapsed subsidiary cells (arrowhead) in the leaf with established 0,-injury symptoms. Only a few guard cells
(short arrow) and subsidiary cells (double arrowhead) are turgid (bar = 100 pm).
area was scanned with a maximum magnification of
x 10000. At least eight stomata1 complexes per leaf
containing closed, narrowly open and wide open
stomata (when available) were analysed. All spectra
were acquired for 120 s (live time) and a dead time of
20 0/,. Peaks of characteristic elements were detected
in the X-ray energy range of 0-10 keV. Spectra were
processed to determine net counts using the Voyager
software package including an automatic peak identification and a standardless analysis quantitation. T h e
resulting X-ray counts corrected for background are
semi-quantitative measures and were not converted
O z o n e and fer~tilizationeffects on stolnatal behaviozir
15 September 1000 hours
40
30
20
10
0
C/LF
03/LF
CIHF
O,/HF
14 September 1400 hours
17 September 1400 hours
Treatment
Figure 3. Betula pendz~la:(a) length (0)
a nd aperture ( m ) of guard cells measured in SERtI micrographs and
(b) length (0)
and width ( m ) of stomata1 pore measured by image analysis. Leaves were harvested at different
harvest times (sampling Table 1). Treatments were: C, filtered air control; O,, fumigated with ozone; L F , low
fertilization; H F , high fertilization. Bars show mean values with one standard error from all measured leaves
per treatment. + denotes a fertilization effect, namely C / H F vs. C / L F or O , / H F vs. O , / L F , * denotes an 0,
effect, namely O , / L F vs. C / L F or O , / H F vs. C / H F tested by a one-way ANOVA, L S D 99
to concentrations because of the problems of
obtaining fully quantitative results from bulk-frozen
hydrated samples (Van Steveninck & Van
Steveninck, 1991). Net counts of K and Ca and the
mean ratios of K to Ca were given.
Statistics
Linear regression was performed with the Statview
4 . 0 , analysis of variance with the Statgraphics Plus
for Windows@ 1 . 0 program. Data were tested
140
B. F r e y and others
by a one-way analysis of variance (ANOVA).
Least significant differences ( L S D ) were calculated
(P < 0.01) for all significant F-ratios.
RESULTS
T h e processed steps of the digital image-analysis led
to a binary image of the stomatal openings, which
corresponded well with the original grey-scale image
(Fig. 1). T h e grey values of the stomatal aperture
were generally less than 80 and the difference from the
background was in the range 175-21 5. T h e total time
of processing of one single image was c . 10 m i n ; i.e.
image capture (2 min), and image and data analysis
(8 min), whereas measurements from S E M micrographs by a ruler took longer ( 1 0 m i n for each
picture).
Data of the stomatal aperture from different leaves
(see sampling, 'Materials and Methods '), harvested
from the same treatment and at the same time, did
not differ significantly, and therefore were combined.
However, in leaves with established ozone injury
where single or groups of epidermal cells had
collapsed, the stomatal aperture within a leaf varied
considerably (Fig. 2). When measured in micrographs, such zones of established ozone injury were
excluded.
O n the whole, the pore width was similar in both
methods of measurement (in micrographs, Fig. 3 a ,
or in digital images, Fig. 3 b, dark bars). Pore length
(Fig. 3 b, white bars), however, could not be related to
the guard cell length (Fig. 3 a ) as determined from
micrographs, since the first is a measure of the length
of the open pore and the latter of the whole size of the
guard cell. Guard cell length was significantly
decreased by high fertilization (Fig. 3 a , white bars).
At all harvest times, the stomatal pores (Fig. 3) were
significantly narrowed in the control regime under
"0
2
4
6
8
10
12
14
16
Pore width (,urn)
Figure 4. Relationship between pore area ( p m 2 ) and pore
width (pm) (data from image analysis, all three harvests,
2 2 . 3 ~(v2= 0.9, P < 0.0001).
n = 580): y = -29.9
+
/
80
80
100
120
140
Stomata per mm2
160
180
Figure 5. T h e effect of ozone and nutrient supply on
stomatal density (stomata per mm2) in leaves of Betula
pendula as determined by light microscopy ( L M ) and
image analysis (IA). LM and IA values are plotted along
the x and y axes, respectively. Each treatment is identified
with a separate symbol: (0)
C / H F , (0)
C / L F , (a)
O , / H F , (m) O , / L F (key as in Fig. 3). T h e horizontal and
vertical bars indicate the standard errors of the parameter
means for LR/I (n = 1120) and IA (n = 250). T h e dashed
line represents the 1 : 1 relationship.
denotes a
fertilization effect, namely C / H F vs. C / L F or O , / H F vs.
O,/LF. * denotes an 0, effect, namely O , / L F vs. C / L F or
O , / H F vs. C / H F tested by a one-way ANOVA, L S D
99 96.
+
high fertilization ( C / H F vs. C / L F ) . Ozone resulted
in an additional narrowing ( 0 3 / H F vs. C / H F at
1400 hours, 0 3 / L F vs. C / L F all harvest times).
Within the ozone regimes the high fertilization had
narrowed the pores at the 1400 hours harvests
( 0 3 / H F vs. 0 3 / L F ) . At the 1000 hours harvest, the
pores in the 0 3 / L F regimes were decreased more
than those in the 0 3 / H F regime (Fig. 3).
Additional parameters, namely individual pore
area, percentage area of pores within a S E M image,
aspect ratio, and pore perimeter were easily obtained
by image analysis (data not shown). At all harvest
times, there was a close relationship between stomatal width and stomatal area. Figure 4 shows the good
linear fit between the two parameters over all
sampling times ( r = 0.938, P < 0.01). T h e stomatal
density (stomata per m m 2 ) determined by L M was
similar to the value derived from the scanning view
(image analysis), especially in the C / H F and the
O , / L F treatments (Fig. 5) taking into account the
different area viewed and the different sampling
(harvest time and number of samples).
Leaf differentiation parameters were affected by
nutrient supply and ozone. T h e stomatal density was
increased by 0, but decreased by high fertilization
(Fig. 5 ) . Single-leaf area was increased by high
fertilization with no significant effect of 0, exposure
Ozone and fertilization effects on stomata1 behaviour
141
irrespective of the fumigation or fertilization regimes
(data not shown). Calcium, unlike potassium, did
not show any regular change between guard and
subsidiary cells in open stomata. I n samples with
established ozone injury, high K and K/Ca ratios
were detected in guard cells in the zones of collapsing
epidermis (Table 2), independent of the aperture
width, indicating a changed ion balance in collapsed
guard cells. Collapsed subsidiary cells only differed
from turgid subsidiary cells when the guard cells had
also collapsed and thus closed the pore.
DISCUSSION
0
C/LF
03/LF
C/H F
03/HF
Treatment
Figwre 6. T h e effect of ozone and nutrient supply on
single-leaf area of Betula pendz~la(10 leaves per treatment,
selected for similar age and phenology). Vertical bars
denotes a
indicate means with one standard error.
fertilization effect, namely C / H F vs. C / L F or 0 3 / H F vs.
0 3 / L F tested by a one-way ANOVA, L S D 99 9,. Key as
in Figure 3.
+
(Fig. 6). A negative linear correlation of the single
leaf area with the stomatal density (correlation
coefficient -0.78, r2 = 61.2) and the length of the
guard cells (correlation coefficient -0.65, r2 = 41.7)
was found. Differentiation parameters such as
stomatal density or single leaf area did not correlate
with stomatal pore aperture.
Microanalytical study of potassium and calcium in
stomatal guard and subsidiary cells
X-ray spectra showed that K and Ca were at levels
significantly above background when probed
through the cuticles. Other ions such as Mg, P, S
and C1 were at the limit of probe detection and in
most instances not even detectable. Stomatal widening was accompanied,by increased K counting-rates
in turgid guard cells (5-10-fold in the open state as
compared with the partly closed state), in contrast
to constant K values in the subsidiary cells (Table 2)
T h e present observations using L T S E M showed a
decreased stomata1 width and pore area under ozone
compared with that under filtered air at similar
external conditions, and thus confirmed earlier S E M
investigations (Scheidegger et al., 1991 ; GunthardtGoerg, Matyssek & Keller, 1993 b) and gas exchange
measurements (Matyssek et al., 1991). Stomatal
aperture depends on the plant genotype and is
regulated by many internal and external factors
(Zeiger, Farquhar & Cowan, 1987; Furuliawa, Park
& Fujinuma, 1990). Stomatal narrowing under or
after exposure to ozone has been reported for many
plants (for review see Darrall, 1989) and was a result
of a disturbance of the leaf function (Matyssek et al.,
1991).
I n addition to the strong influence of ozone, a
significant effect of nutrient supply on stomatal
width was found. Subtle balances, including hormonal, water, carbon and nutrient relations, exist
within plants (Natr, 1992). Mineral nutrients not
only determine the structural components of cells,
but also participate in individual photosynthetic
reactions. Gas-exchange measurements confirmed
that the transpiration rate in leaves of similar age
from the C / L F regime was increased over that in
leaves under the C / H F regime (S. Maurer, pers.
comm.).
Ozone and nutrient supply not only influence the
stomatal aperture, but also independently determine
leaf differentiation during formation (see also
Matyssek et al., 1991; Gunthardt-Goerg et al.,
Table 2. Net counts of K and Ca and elemental ratios ( K I C a ) of selected turgid and collapsed, guard and
subsidiary cells (Fig. Zb) of birch leaves (harvest 14 Sep. 1400 hours; regime : O,/LF) measured by EDX
analysis (mean _+
;n = 12)
Potassium
Pore
Cell state
Open
Open
Turgid
Collapsed
Closed
Closed
Turgid
Collapsed
Calcium
K/Ca
Guard
cell
Subsidiary
cell
Guard
cell
Subsidiary
cell
Guard
cell
+
+
377 k 242
1060 627
206 k 64
2482 631
414+99
503 49
245 60
393+120
638 f209
902+458
379 f63
377+110
6.0 2.7
18.6k2.8
1.6 k 0.3
4.1 1.3
2366 634
9342 1666
383 f88
1603 f558
+
+
+
+
+
+
Subsidiary
cell
+
+
0.7 0.5
1.1 f0.3
0.6 0.3
7.4 k 4.4
142
B. Frey and others
1993a; Paakkonen et al., 1993; Paakkonen &
Holopainen, 1995). Both factors influenced the
stomatal density. Increased stomatal density in birch
seems to be a consistent effect related to ozone
exposure and has also been reported by Matyssek et al.
(1991), Gunthardt-Goerg et al. (1993 a) and
Paakkonen et al. (1993). T h e increased stomatal
density was apparently overridden by the narrowing
stomatal apertures. I n contrast to the stomatal
density, leaf area and guard cell length were only
determined by fertilization. Therefore, neither leaf
size nor stomatal density was correlated with guard
cell size. Once grown, the leaves regulate their
stomatal conductance by the pore width. T h e
resulting stomatal conductance increases with the
stohatal density and aperture (Hinckley & Braatne,
1994, von Willert, Matyssek & Herppich, 1995).
~ T S E Mcombined with digital image-analysis
provides accurate measurements of the stomatal
aperture in birch leaves quicker than do conventional
measurements with a ruler from S E M micrographs.
Although the methods of measurement were carried
out by different persons and included different
subjective bias, the results were very similar. This
indicates that measurements of pore width with
conventional methods (Scheidegger et al., 1991)
have been accurate. Standardized sampling of birch
leaves resulted in relatively homogeneous states of
stomatal aperture per treatment. With increasing
ozone dose, however, cell injury started within single
groups of mesophyll cells. Epidermal cells declined
later and only collapsed when the mesophyll cells
beneath had been collapsed before. Guard cells
collapsed only when subsidiary cells had collapsed
after a transient widely opened state (GunthardtGoerg et al., 1 9 9 3 ~ ) .Therefore, in leaves with
necrotic spots such epidermal zones showed widely
opened stomatal pores at the border between collapsed necrotic tissue with collapsed and closed
stomata, and stomata in different states of injury, or
healthy turgid cells. Such zones had an extremely
large variance of the stomatal aperture (the leaf
O,/LF, Fig. 2). Because stomata in such zones were
out of regulation, they were treated separately.
Similar states have been reported in field beans after
exposure to 175 nl l-I SO, (Black & Black, 1979), in
birch after exposure to 40 nl 1-I SO, +40 nl 1-I NO,
(Wright, 1988), and in radish after exposure to
80 nl1-I 0, (Hassan, Ashmore & Bell, 1994).
T h e E D X of frozen-hydrated samples confirmed
the importance of vacuolar K increase with guardcell swelling and thus with active stomatal widening,
at constant K values in the subsidiary cells (irrespective of fumigation or nutrient supply, data
not shown). These results were consistent with those
obtained by X-ray microanalysis (Humble &
Raschke, 1971 ; Garrec et al., 1983). High K counts
and K/Ca ratios were detected in ozone-injured
subsidiary cells and adjacent guard cells in the zones
of collapsing epidermis. Interpretation of these
results remains speculative and net counts of K have
to be considered with caution, because they are not
converted to concentrations. By forming elemental
ratios of the microanalytical data, the effects of
uneven surface topography on X-ray collection can
be partly eliminated. Changes in membrane permeability (indicated by 'leakiness' of cells) have
often been observed in ozone-treated tissues, and
have been interpreted as evidence of a direct attack
on the plasma membrane (Heath, 1987; Mansfield &
Pearson, 1993). Heath & Frederick (1979) showed
that ozone increased membrane permeability to
potassium ions. Additionally, mass loss, particularly
water loss in cells, can increase X-ray signals
(Zierold, 1982). I n an earlier study GiinthardtGoerg et al. (1993 a) reported that with increasing
ozone-dose, epidermal cells showed shrinkage of the
mucilaginous layer, leading to decreased epidermal
width in connection with injury symptoms and water
loss. Therefore, we suggest that water loss and
decompartmentation in deteriorating cells might
have increased the semiquantitative E D X counts.
ACKNOWLEDGEMENTS
W e gratefully acknowledge t h e technical assistance o f C .
R h i n e r . W e t h a n k S . M a u r e r , U . B i i h l m a n n and A.
Burkhart for t e n d i n g t h e plants. W e are grateful t o D r W .
L a n d o l t and P . Bleuler, w h o p r o g r a m m e d , installed and
operated t h e fumigation. T h e s t u d y w a s partly financed
t h r o u g h t h e ' E U R E K A 447 E U R O S I L V A ' p r o g r a m m e
o f t h e S w i s s ' B u n d e s a m t fiir B i l d u n g u n d W i s s e n s c h a f t ' .
W e also t h a n k NI. J . Sieber for editing t h e English t e x t .
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