Annals of Botany 78 : 489–497, 1996
Elevated CO2 and Temperature have Different Effects on Leaf Anatomy of
Perennial Ryegrass in Spring and Summer
R A C H E L F E R R IS*‡, I. N I J S*, T. B E H A E G HE† and I. I M P E N S*
* Department of Biology, UniŠersity of Antwerp, UniŠersiteitsplein 1, B-2610 Wilrijk, and † Faculty of Agricultural
and Applied Biological Sciences, UniŠersity of Ghent, Coupure Links 653, B-9000 Ghent, Belgium
Received : 27 November 1995
Accepted : 1 May 1996
Mature second leaves of Lolium perenne L. cv. Vigor, were sampled in a spring and summer regrowth period. Effects
of CO enrichment and increased air temperature on stomatal density, stomatal index, guard cell length, epidermal
#
cell density, epidermal cell length and mesophyll cell area were examined for different positions on the leaf and seasons
of growth.
Leaf stomatal density was smaller in spring but greater in summer in elevated CO and higher in both seasons in
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elevated temperature and in elevated CO ¬temperature relative to the respective controls. In spring, leaf stomatal
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index was reduced in elevated CO but in summer it varied with position on the leaf. In elevated temperature, stomatal
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index in both seasons was lower at the tip}middle of the leaf but slightly higher at the base. In elevated
CO ¬temperature, stomatal index varied with position on the leaf and between seasons. Leaf epidermal cell density
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was higher in all treatments relative to controls except in elevated CO (spring) and elevated CO ¬temperature
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(summer), it was reduced at the leaf base. In all treatments, stomatal density and epidermal cell density declined from
leaf tip to base, whilst guard cell length showed an inverse relationship, increasing towards the base. Leaf epidermal
cell length and mesophyll cell area increased in elevated CO in spring and decreased in summer. In elevated
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CO ¬temperature leaf epidermal cell length remained unaltered in spring compared to the control but decreased in
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summer. Stomatal conductance was lower in all treatments except in summer in elevated CO it was higher than in
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the ambient CO .
#
These contrasting responses in anatomy to elevated CO and temperature provide information that might account
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for differences in seasonal leaf area development observed in L. perenne under the same conditions.
# 1996 Annals of Botany Company
Key words : Lolium perenne, perennial ryegrass, elevated CO and temperature, stomatal density, stomatal index, cell
#
size.
INTRODUCTION
In temperate western Europe, perennial ryegrasses are the
main component of lowland pasture production, being
grown under conditions of high nitrogen input and intensive
management. Since atmospheric CO concentration is
#
increasing with a predicted rise to 600 µmol mol−" by the
second half of the next century and summer temperatures
are also expected to increase across Europe (Houghton,
Jenkins and Ephraums, 1992), pasture management could
be affected.
The response of stomatal density (SD, the number of
stomata per unit area) and stomatal index (SI, the
proportion of stomata in relation to total number of
epidermal plus stomatal cells) to elevated CO in a wide
#
range of species has been reviewed (Beerling, Putland and
Woodward, 1995). Some studies have shown no effect of
CO on SD, SI or both in plants including Lolium perenne
#
(Ryle and Stanley, 1992) ; Populus species (Radoglou and
Jarvis, 1990 b, 1992), Castanea satiŠa (Moussea and Enoch,
‡ For correspondence at : Plant Environment Laboratory, Department of Agriculture, The University of Reading, Cutbush Lane,
Shinfield, Reading, RG2 9AD UK.
0305-7364}96}100489­09 $18.00}0
1989) Triticum aestiŠum and Citrus aurantium (Estiarte et al.,
1994). Others have shown a reduction in SD only, for
example in 14 herbarium tree species (Penuelas and
Matamala, 1990) or mixed responses in SD and SI of four
perennial herbs (Ferris, 1994 ; Ferris and Taylor, 1994).
Relatively few studies have focused on the response of SD
and SI to elevated temperature and CO (Beerling and
#
Chaloner, 1993 ; Morgan et al., 1994). Stomatal density can
vary across the leaf and can be affected by other environmental variables such as light, shade, humidity and drought
(Ticha, 1982 ; Smith, Weyers and Berry, 1989 ; Ferris, 1991).
Leaf SD is an important ecophysiological parameter that
affects gas exchange. It has been suggested that declining
stomatal numbers over the last century (Woodward, 1987,
1993 ; Tyree and Alexander, 1993) might lead to greater
instantaneous water use efficiency which is usually defined
as the ratio of assimilation to transpiration (Ferris, 1994 ;
Ferris and Taylor, 1995) and that stomatal conductance
may change as a result of differences in stomatal aperture,
geometry or density (Meidner and Mansfield, 1968 ;
Berryman, Eamus and Duff, 1994 ; Clifford et al., 1995 ;
Pearson, Davies and Mansfield, 1995).
Woodward (1987) showed that changes in SD were often
paralleled by changes in the SI, indicating that the effect of
# 1996 Annals of Botany Company
490
Ferris et al.—CO and Temperature Effects on Leaf Anatomy
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CO was on stomatal initiation, rather than a result of
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changes in the rates of leaf cell expansion. Stomata arise
through differential divisions in the protoderm which
becomes secondarily meristematic, forming guard mother
cells. The governing mechanisms are not clearly understood,
but the placement of division planes in parent cells (Palevitz,
1981) leads to the formation of stomata. A more recent
study of variegated leaves suggested that leaf structure may
be important in determining the SD}SI responses to elevated
CO (Beerling and Woodward, 1995).
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We have observed that spring and summer leaves of L.
perenne differ in appearance even under elevated CO and
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temperature conditions. This experiment investigated effects
of elevated CO and temperature on the developmental
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anatomy of leaves of L. perenne by (1) examining leaf SD,
SI, epidermal cell density and cell size of different cell types
in second fully expanded leaves which had grown during a
spring and summer regrowth period ; and (2) by examining
any interactions with position on leaf and season of sampling
which might confound the CO and temperature effects on
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these characteristics.
MATERIAL AND METHODS
Plant growth and exposure to eleŠated CO and
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temperature
Lolium perenne L. cv. Vigor seeds (caryopses) were planted
at a density of 6 g m−# in steamed loamy soil in 3±85 l plastic
containers (34±0 cm deep, 11±8 cm diameter) on 11 Oct.
1994. Pots were fertilized with N, P and K : 10 g N m−# (as
NH NO ), 15 g P m−# (as P O ), and 15 g K m−# (as K O).
# &
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%
$
They remained in the field during the following winter at the
Faculty of Agriculture field site, University of Ghent,
Belgium (50°59« N, 3°49« E). In Mar. 1995, 45 containers
were placed in each of four transparent sunlit growthchambers. This hybrid design is in between an open-top
chamber and a closed conditioned greenhouse (described
in detail in Nijs, Impens and Behaeghe, 1995). Each
‘ greenhouse ’ was covered with 180 µm thick transparent
polythene ; containing 14 % vinyl acetate for reinforcement which transmitted appprox. 80 % of the UV-B
(280–320 nm) in natural sunlight as well as most of the UVA wavelengths. Average photosynthetically active radiation
(PAR) in the chambers was about 17 % lower than field
measurements with no significant differences observed
between chambers. A 75 cm high strip of plastic gauze
covered the lateral walls from the ground up (6±3 m length)
to facilitate heat removal. Each unit was fed with filtered
temperature-controlled outside air to the exposure table via
a dust filter (fibre-glass filled filter, EFC, Herk-de-Stad,
Belgium), a cooling system with separate condenser and an
evaporator of 4±3 kW, and through an electrical resistance
heating grid (6 kW). The turbine ventilator (2±2 kW)
supplied air at 4000 m$ h−", which was reduced to
2000 m$ h−" through partial obstruction of the air inlet.
Inside each greenhouse, the containers were placed on a
‘ table ’ (2±75 m¬1±45 m). The table was an aluminium
cabinet designed to convert the horizontal air stream,
produced by the air-conditioning unit, into a homogeneous
upward flow of well-mixed air to the plants. A perforated
plate and horizontal and vertical deflectors distributed the
vertical air stream uniformly, producing the necessary
homogeneity of windspeed over the surface of the exposure
table to minimize environmental gradients. Windspeed was
0±9 m s−" in all chambers (fluctuating between 0±8 and
1±0 m s−") and spatially uniform over the 4±5 m# area of the
exposure table. Transparent acrylic shields around the table
minimized the influx of ‘ false ’ turbulent air into the air
column. Elevated CO concentrations were maintained by
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the injection of a constant flow of pure CO in the air#
conditioning cabinet. Pots were rotated on the table twice a
week. Treatments were as follows. Control : field conditions
of CO concentration and air temperature continuously
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tracked ; Temp : increased air temperature at 4 °C above
ambient ; CO : elevated CO concentration at 700 µmol
#
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CO mol−" air ; CO ¬temp : a combination of elevated
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temperature and CO .
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On 6 Apr. 1995, stands were cut back to 3 cm height
(stubble layer left intact) and swards were then cut on 15
May, 16 Jun., 14 Jul. and 17 Aug. Pots were arranged to
form a closed canopy and irrigated each day with a trickle
system (one drip tube per container) which maintained field
capacity. After clipping, the harvested material was removed
and the remaining 3-cm high stubble layer was refertilized
with a standard NPK mixture. Ample nutrients were
supplied after each cut with 13±0 g N m−#, 3±18 g P m−# and
10±6 g K m−# on 15 May (generative period) and 9±0 g N m−#,
2±2 g P m−# and 7±35 g K m−# after all other cuts.
The microclimate in the chambers was measured at
10 min intervals and registered with a time-controlled
datalogger DL2 (Delta-T, Burwell). Environmental conditions and sensors were : air temperature—copperconstantan thermocouples (Honeywell, Washington, USA) ;
relative humidity (RH)—macro-polymer humidity sensors
[(RH-8) General Eastern, Watertown, Maine, USA] ; photosynthetically active radiation—quantum sensors with gallium arsenide photodiode (Pontailler, 1990) ; CO
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concentration—absolute infrared CO analyser SBA-1
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OEM (PP-Systems, Herts., UK). Relative humidity (RH)
and photosynthetically active radiation (PAR) were comparable with outside values. The RH was slightly reduced
compared to field values (8 % on average). There was no
additional illumination so the stands were cultivated under
normal day length and seasonal light fluctuations with
decreased absolute irradiance. Average PAR in the field
over a 16 h day (n ¯ 1254) was 415 µmol m−# s−" in spring
and 639 µmol m−# s−" in summer. The average day-time CO
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concentrations (³s.d.) were 371³22 µmol mol−" in the
−
ambient CO treatments and 701±3³72 µmol mol " in the
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elevated CO treatments (n ¯ 2221). At night CO concen#
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trations rose by approximately 20 % due to respiration.
During the measurement period the average difference in air
temperature between the ambient treatments and the
treatments with increased air temperature was 3±77 °C (n ¯
6580) in spring and 4±10 °C (n ¯ 5064) in summer compared
to a target value of 4 °C. Average (³s.d.) air temperatures
were spring : 13±2³4.4, 16±8³3±8, 13±0³4±0, 17±1³4±0 °C
and summer : 21±4³4±8, 25±1³5±1, 21±7³5±4, 25±9³5±1 °C
in control, temperature, CO , and CO ¬temperature,
#
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respectively.
Ferris et al.—CO and Temperature Effects on Leaf Anatomy
#
Stomatal and epidermal cell counts and preparation of
epidermal replicas
Most of the stomata in L. perenne are located linearly
along the flanks of the longitudinal grooves of the adaxial
leaf surface. Adaxial stomatal anatomy was examined in ten
fully expanded second leaves sampled after 35 d of growth
following cutting. Leaf length and width (top, middle, and
base of leaf lamina) were measured and leaf area calculated
from length¬average width. A coefficient of determination
of 0±98 was obtained for regression of length¬width on leaf
area measured with a photoelectronic planimeter (Nijs,
unpubl. res.). Leaves were gently washed in a 1 % solution
of Teepol. When dry a thin layer of clear nail varnish was
painted onto both surfaces of each leaf blade and left for
20 min. A strip of transparent sticky tape (‘ Sellotape ’) was
placed over the dried varnish at the base, middle and tip of
the leaf blade, and pressure applied to obtain an imprint.
The sellotape with varnish imprint was peeled from the leaf
and placed onto a glass microscope slide. Replicas were
examined under the light microscope to obtain the SD and
SI for each leaf. The number of stomata and other epidermal
cells were counted from three half fields of view (area
0±0868 mm#) from each of ten individual leaf surface replicas
(30 microscopic half fields per treatment) at ¬400 magnification. An eyepiece graticule, precalibrated with a stage
micrometer, was used to calculate the area of the half field
of view. The number of stomata per half field was converted
to stomata mm−# and the ‘ stomatal index ’ (SI) was
calculated which relates the number of stomata per unit area
(SD) to the number of epidermal cells per unit area (ECD)
where SI ¯ [SD}(ECD­SD)]¬100, (Salisbury, 1927). Stomata and epidermal cell density per leaf (not shown) was
calculated by multiplying mean leaf SD and ECD averaged
from measurements at each position, with each leaf area.
The length of three epidermal cells (ECL) from the
corresponding abaxial surface (middle) of ten expanded
leaves per treatment (one per pot), was measured at ¬400
magnification.
491
during the early afternoon of clear warm days in early June
and mid-August. An automatic diffusion porometer (Mark
II, Delta-T Devices Ltd., Cambridge, UK) measured gs at
the CO and temperature conditions in which plants were
#
grown and under saturating irradiance. Mean (³s.d.)
afternoon PAR in spring was 931³289 µmol m−# s−" and
summer PAR was 940³270 µmol m−# s−" and ambient RH
(approx. 58 %). Tests showed 300 µmol m−# s−" was
sufficient to reach 95 % stomatal opening (Nijs, Impens and
Behaeghe, 1989). Precautions taken to ensure reliable values
included rapid measurements, frequent calibration, and
shading the porometer between readings to ensure similar
leaf and cup temperatures (Turner, 1991).
Statistical analysis
Using Minitab, data were analysed with an ANOVA
(mixed model) to test any significance effects of CO ,
#
temperature, position on leaf, time and interactions. Leaves
and microscopic fields of view were included as random
factors in the models but were not significant unless stated.
Data of transverse sections were similarly analysed omitting
position on leaf. Data were tested for normality using
normal probability plots and homogeneity of variance
(Sokal and Rohlf, 1981). No transformations were
necessary.
RESULTS
ANOVAs (Table 1) show significant CO ¬temperature
#
¬position¬time interactions on leaf SD, SI, and ECD
(P ! 0±001). In elevated CO , SD was lower and higher
#
in spring and summer leaves, respectively relative to the
controls. In elevated temperature and CO ¬temperature,
#
SD in both seasons was higher relative to the controls (Fig.
1). The four significant main effects (Table 1) show a high
percentage of the variation in SD was due to position on leaf
(36±7 %) and time of sampling (17±6 %). Overall SD declined
from tip to base and from spring to summer irrespective of
Preparation of transŠerse sections
Measurements of stomatal conductance
Stomatal conductance (gs) was measured at the leaf tip
and base of 10–15 fully expanded second leaves per treatment
200
Stomatal density (mm–2)
Leaf sections (10 mm) from the centre of second fully
mature leaves were placed in fixative [formalin : glacial
acetic : 70 % ethanol (1 : 1 : 18, v}v)]. Using sharp razor
blades and polystyrene blocks, transverse sections were
hand cut, placed on clean microscope slides and covered
with cover slips. At ¬400 magnification, the thickness of
the leaves and epidermal cells was measured. A camera
lucida attached to a light microscope projected cell images
onto graph paper covered with acetate sheets. Images of five
mesophyll cells from eight leaves per treatment were traced
in a field of view (196¬196 µm) at ¬400 magnification
(n ¯ 40). After magnification correction, average cell areas
in cross sections were calculated : cell area ¯ (weight of
paper cells}weight of paper field)¬area of field.
Spring
Summer
150
100
50
0
Tip
Middle
Base
Tip Middle
Position on leaf
Base
F. 1. The effects of either ambient CO (*) or 700 µmol mol−" CO
#
#
(9) and elevated temperature (­4 °C) on leaf adaxial stomatal density
of mature spring and summer grass of Lolium perenne. The symbols for
the temperature treatments are : elevated temperature (8), elevated
CO ¬temperature (+). Each point is the mean of 30 measurements.
#
For statistical analysis, see Table 1.
685
719
1
1
2
1
1
2
1
2
1
2
2
1
2
2
2
d.f.
***
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***
ns
**
***
**
***
***
**
***
*
*
***
23±6
1±08
9±25
36±7
17±6
0±11
0±34
4±97
0±38
0±73
2±90
0±38
0±45
0±27
0±29
0±58
% of
sums of
squares
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*
***
***
***
***
ns
***
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ns
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Stomatal
index
58±7
1±63
2±28
7±07
0±49
6±65
3±94
7±92
1±47
0±09
1±86
2±13
0±21
2±64
1±51
1±22
% of
sums of
squares
ns
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*
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Epidermal
cell
density†
*, P % 0±05 ; **, P % 0±01 ; ***, P % 0±001. n ¯ at least 10 leaves per treatment. d.f. ¯ degrees of freedom.
† Anova revealed significant (P % 0±001) variation between leaves sampled.
CO
#
Temperature
Position
Time
CO ¬temperature
#
CO ¬position
#
CO ¬time
#
Temperature¬position
Temperature¬time
Position¬time
CO ¬temperature¬position
#
CO ¬temperature¬time
#
CO ¬position¬time
#
Temperature¬position¬time
CO ¬temperature¬position
#
¬time
Error
Total
Environmental factors
Stomatal
density
18±8
0±03
6±69
27±7
29±0
1±83
1±76
0±12
1±12
1±92
8±60
0±94
0±32
0±45
0±36
0±75
% of
sums of
squares
Plant characteristics
ns
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ns
ns
***
**
***
ns
*
***
ns
**
ns
Guard cell
length
29±1
0±063
14±8
14±3
26±4
0±14
0±017
7±26
0±45
0±74
0±11
0±28
5±53
0±016
0±50
0±083
% of sums
of squares
118
133
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
d.f.
T     1. Summarised analysis of Šariance table for Figures 1–4 and 6 showing percentage sums of squares
ns
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***
ns
**
***
***
**
*
**
ns
***
*
Stomatal
conductance
19±78
0±63
11±3
44±4
3±40
2±03
0±39
1±33
6±07
2±09
1±47
0±77
1±30
0±09
4±24
0±73
% of
sums of
squares
492
Ferris et al.—CO and Temperature Effects on Leaf Anatomy
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Ferris et al.—CO and Temperature Effects on Leaf Anatomy
#
40
Spring
Summer
Middle
Base
Tip Middle
Position on leaf
Guard cell length (µm)
Stomatal index (%)
35
30
25
20
15
10
5
0
Tip
Base
Epidermal cell density (mm–2)
F. 2. The effects of either ambient CO (*) or 700 µmol mol−" CO
#
#
(9) and elevated temperature (­4 °C) on leaf adaxial stomatal index
(%) of mature spring and summer grass of Lolium perenne. The
symbols for the temperature treatments are : elevated temperature (8),
elevated CO ¬temperature (+). Each point is the mean of 30
#
measurements. For statistical analysis, see Table 1.
400
Spring
350
60
55
50
45
40
35
30
25
20
15
10
5
0
Tip
Spring
Summer
Middle
Base
Tip Middle
Position on leaf
T     2. AŠerage total leaf areas (mm#) of L. perenne
sampled from each treatment
Summer
Mature
spring leaves
250
200
100
50
Middle
Base
Tip Middle
Position on leaf
Base
F. 3. The effects of either ambient CO (*) or 700 µmol mol−" CO
#
#
(9) and elevated temperature (­4 °C) on leaf epidermal cell density of
mature spring and summer grass of Lolium perenne. The symbols for
the temperature treatments are : elevated temperature (8), elevated
CO ¬temperature (+). Each point is the mean of 30 measurements.
#
For statistical analysis, see Table 1.
treatments. Figure 2 shows that with CO enrichment, leaf
#
SI was lower in spring and declined from tip to base but in
summer SI was only lower at the tip, a higher SI was
recorded towards the base relative to each control. Although
highly significant the CO ¬time and CO ¬time¬position
#
#
effect accounted for only 7±92 % and 2±64 % of the variation
respectively. In contrast, in elevated temperature, SI in both
seasons was lower at the tip}middle of the leaf and higher
at the base relative to the controls. In elevated
CO ¬temperature, SI in spring was smaller at the tip (with
#
little differences in other positions) relative to the controls,
whilst in summer it was greater at the middle}base of the
leaf relative to respective controls. In elevated
CO ¬temperature there was some variation in SI over the
#
leaf and overall SI was slightly higher in summer as shown
in Fig. 2.
In elevated CO , elevated temperature and both com#
binations, ECD was higher in spring and summer leaves
relative to ambient conditions and decreased from the tip to
the base of the leaf (Fig. 3). Of the three highly significant
Mature
summer leaves
CO concentration (µmol mol−")
360
700
360
700
Ambient temperature
Elevated temperature
CO
#
Temp
Time
CO ¬temp
#
CO ¬time
#
Temp¬time
CO ¬temp¬time
418
350
751
400
514
425
437
371
#
150
Tip
Base
F. 4. The effects of either ambient CO (*) or 700 µmol mol−" CO
#
#
(9) and elevated temperature (­4 °C) on leaf adaxial guard cell length
of mature spring and summer grass of Lolium perenne. The symbols for
the temperature treatments are : elevated temperature (8), elevated
CO ¬temperature (+). Each point is the mean of 30 measurements.
#
For statistical analysis, see Table 1.
300
0
493
#
**
***
ns
**
***
**
**
**, P % 0±01 ; ***, P %0±001. n ¯ 10 leaves per treatment.
main effects (P % 0±001, Table 1) a high percentage of the
variation in ECD was due to position on leaf (27±7 %), time
of sampling (29±0 %) and the interaction (8±6 %). Figure 3
shows overall ECD was higher, particularly at the leaf tip,
in spring leaves relative to summer leaves irrespective of the
CO and temperature treatments (Fig. 3). Figure 4 shows in
#
elevated CO , leaf GCL was overall larger in spring and
#
smaller in summer with 7±3 % of the total sums of squares
partitioned to this CO ¬time effect (P % 0±001, Table 1). In
#
elevated temperature and in CO ¬temperature, GCL of
#
spring and summer leaves were smaller relative to the
controls and GCL increased from the tip to the base of the
leaf. A high percentage of variation in GCL was due to the
significant (P % 0±001) main effects of temperature (14±8 %),
position (14±3 %) and time (26±4 %, Table 1). GCL was
overall larger in CO ¬temperature in summer leaves as
#
compared to spring leaves (Table 1 and Fig. 4).
Spring leaves of L. perenne were larger in elevated CO ,
#
whilst in summer they were smaller in contrast to controls
(Table 2). Leaves were smaller in elevated temperature and
CO ¬temperature in spring and summer relative to each
#
494
Ferris et al.—CO and Temperature Effects on Leaf Anatomy
#
T     3. The influence of CO and temperature on the leaf anatomy of fully expanded second leaŠes of L. perenne
#
Approximate
concentration of
CO (µmol mol−")
#
Spring leaves
Ambient temperature
Elevated temperature
Summer leaves
Ambient temperature
Elevated temperature
CO
#
Temp
Time
CO ¬temp
#
CO ¬time
#
Temp¬time
CO ¬temp¬time
Thickness of
lower layer
of epidermal
cells (µm)
Thickness (µm) of
leaf (abaxial
surface to top
of ridge)
Thickness (µm) of
leaf (abaxial
surface to top
of furrow)
Area of
mesophyll
cells (µm#)†‡
360
700
360
700
360
700
360
700
37
35
51
49
271
265
323
315
136
125
161
150
587
535
836
797
46
42
37
39
336
324
300
315
168
154
147
153
861
590
565
471
#
**
ns
ns
ns
***
ns
ns
**
ns
***
ns
***
ns
ns
ns
ns
**
ns
***
ns
ns
ns
ns
***
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***
**
Epidermal cell length ( µm)
Mean values from eight leaves are shown. An ANOVA with randomised block† (leaves) was used to test the difference between cell means for
each treatment. *, P % 0±05 ; **, P % 0±01 ; ns, not significant.
‡ ANOVA showed no significant variation between leaves (n ¯ 40 cells).
1000
900
800
700
600
500
400
300
200
100
0
Control
CO2 × temp
Temp
CO2
Treatment
DISCUSSION
ANOVA
CO2
Temp
Time
CO2 × temp
CO2 × time
Temp × time
CO2 × temp × time
significant CO ¬temperature¬time interaction on ECL
#
which was similar in size in spring but reduced in summer
relative to each respective control. In spring, in elevated
CO , epidermal cells were longer, but in summer they were
#
shorter than controls. Figure 6 shows leaf gs was lower in
elevated CO (spring only), temperature and in combined
#
treatments over the whole leaf and overall gs declined from
tip to base irrespective of season. In contrast, in summer
leaves gs was slightly higher in elevated CO relative to the
#
control leaves. Table 1 shows three highly significant main
effects (P % 0±001), with position on leaf accounting for
44±36 % of the total sums of squares.
ns
***
ns
ns
***
ns
***
F. 5. The effects of either ambient CO or 700 µmol mol−" CO and
#
#
elevated temperature (­4 °C) on leaf abaxial epidermal cell length of
mature spring (*) and summer grass (+) of Lolium perenne. Each
point is the mean of 30 measurements. Asterisks identify significant
treatment effects from the ANOVA. *, P % 0±05 ; **, P % 0±01 ;
***, P % 0±001.
respective control (Table 2 ; Ferris et al., 1996). Table 3
shows a significant CO ¬time interaction on the thickness
#
of the leaf, abaxial layer of epidermal cells and MCA : all
increased in spring but decreased in summer leaves relative
to controls. In high temperature, MCA of spring and
summer leaves were smaller whilst in combination with
elevated CO , MCA was larger in spring leaves but smaller
#
in summer leaves relative to controls. Figure 5 shows a
The data presented here provide new information on the
responses of both stomatal and other cell types to elevated
CO and temperature over different parts of the leaf during
#
two seasons of development. The inclusion of cutting
treatments has shown seasonal differences in the studied
responses in L. perenne. In this study SD and ECD were
reduced by warmer summer temperatures. Beerling and
Chaloner (1995) found for Quercus robur leaves, formed in
warmer summer temperatures, that SD and SI were reduced
compared to their spring counterparts. Our study shows
that any change in the SI was paralleled by a similar change
in SD or ECD although the direction and magnitude of the
change in SD and ECD differed beween treatments and with
time (Figs 1, 2 and 3). In elevated CO in spring, leaf size, SD
#
and ECD increased whilst SI decreased, suggesting CO
#
reduced stomatal initiation in the meristem. In contrast, in
summer, leaf size decreased and SI varied : reduced at the
tip, unchanged in the middle and increased at the base. CO
#
had different effects on stomatal initiation which were
confounded by season of growth and position on the leaf.
Both increases and decreases in SD in elevated CO and
#
other environmental variables (e.g. drought, temperature)
s )
400
–2 –1
500
Stomatal conductance (mmol m
Ferris et al.—CO and Temperature Effects on Leaf Anatomy
#
Spring
450
Summer
350
300
250
200
150
100
50
0
Tip
Base
Tip
Position on leaf
Base
ANOVA
CO2
temperature
position
time
CO2 × temperature
CO2 × position
CO2 × time
temperature × position
temperature × time
position × time
CO2 × temperature × position
CO2 × temperature × time
CO2 × position × time
temperature × position × time
CO2 × temperature × position × time
ns
***
***
***
***
ns
**
***
***
**
*
**
ns
***
*
F. 6. The effects of either ambient CO (*) or 700 µmol mol−" CO
#
#
(9) and elevated temperature (­4 °C) on leaf adaxial stomatal
conductance (gs) of fully expanded spring and summer grass of Lolium
perenne. Symbols for the temperature treatments are : elevated
temperature (8), elevated CO ¬temperature (+). Each point is the
#
mean of 10–15 measurements. Statistical analysis and symbols as for
Figure 5.
have been observed (Ticha, 1982 ; Beerling and Chaloner,
1993 ; Ferris and Taylor, 1994 ; Clifford et al., 1995), all
owing to changes in the degrees of cell expansion. In our
study, variation in the SI over the leaf is because ECD and
SD did not change in parallel. If CO and}or temperature
#
affects cell differentiation in the meristem and stomatal
initiation, SD will alter without ECD changing in parallel
and SI will be affected.
In elevated temperature and CO ¬temperature SI was
#
lower at the tip in spring and higher at the leaf base in
summer. Whilst in spring, SI at the leaf base and summer SI
at the tip remained insensitive thus stomatal initiation
remained unchanged over part of the leaf as this index is
independent of leaf cell expansion : the increase in SD was a
result of increased stomatal initiation parallel with increased
epidermal cell divisions (Figs 1, 2 and 3). A similar response
was observed on both leaf surfaces of Lotus corniculatus in
elevated CO (Ferris and Taylor, 1994). Ceulemans, Van
#
Praet and Jiang (1995) found for two poplar clones, reduced
adaxial SD and abaxial SD and SI in elevated CO in
#
495
expanding leaves of the upper portion of the plant, but no
effect on mature leaves on the middle}lower portion of the
plant. They concluded that interactions with leaf age and}or
position often confounded the CO effect. One might
#
hypothesize from our study and the study of Ceulemans et
al. (1995) that with these interactions and subtle differences,
gas exchange and instantaneous water use efficiency might
vary but (depending on root growth) predicting the
ecophysiological response of the whole plant is difficult.
Morgan et al. (1994) found no temperature or elevated CO
#
effects on SD in two steppe grasses whilst Knapp et al.
(1994) found SD in Andropogon gerardii and SalŠia pitcheri
decreased and increased respectively in elevated CO .
#
Woodward (1987) showed SD in three temperate trees and
a herbaceous species was reduced by 67 % after 3 weeks at
340 µmol mol−" CO relative to lower preindustrial concen#
trations. Differences in the light environment within our
swards might be an interacting factor since light (Schoch,
Zinsou and Sibi, 1980), leaf age or position (Ticha, 1982)
interactions have been documented.
Variation in stomatal production in elevated CO and
#
temperature is not surprising since cellular development in
the epidermis can vary : Sachs, Novoplansky and Kagan
(1993) suggested epidermal patterning may occur during
rather than preceding stomatal development. How SD is
controlled during leaf growth is still unknown. Giles and
Shehata (1984) found cell division and elongation in Zea
mays were related to the plastochron index and guard
mother cell differentiation was linked to the potassium
pump. Friend and Woodward (1990) hypothesized that
SD}SI responses were under the mechanistic control of a
substance such as ATP but this has been questioned.
Recently, Beerling and Woodward (1995) studied stomatal
features of five variegated species and suggested that leaf
structure was important in assessing the sensitivity response
to CO .
#
Reduced SD in elevated CO (spring only) was mirrored
#
by a decline in gs. This is the general response of gs to
elevated CO with decreases of 10–60 % (Oberbauer, Strain
#
and Fetcher, 1985 ; Eamus, 1991 ; Ryle, Powell and Tewson,
1992 ; Ferris and Taylor, 1995) often found but exceptions
to this response have occurred (Eamus and Jarvis, 1989 ;
Conroy, Barlow and Bevege, 1986). Paoletti, Gellini and
Manes (1993) suggested that the main factor reducing gs and
improving instantaneous water use efficiency at elevated
CO is not the reduction in SD or pore size but stomatal
#
opening. In contrast, Berryman et al. (1994) showed a longterm irreversible decline in gs was a result of decreased SD
in Maranthes corymbosa and they concluded that the supply
of abscisic acid and calcium were not limiting responses of
gs. In summer, there was a negative effect of CO , on leaf
#
growth (Ferris et al., 1996 ; Table 2) and SD increased
slightly at the tip, but more at the base whilst gs also
increased slightly at the leaf tip (Fig. 6). Similar gs responses
occurred in the morning but the magnitude of the response
declined (data not shown). Examples of increased gs to
elevated CO have been reported with increased plant
#
hormones such as indole-3-yl acetic acid or cytokinins
suggested as factors increasing gs (Eamus, 1991). Overall,
leaf GCL decreased in elevated CO , suggesting pore length
#
496
Ferris et al.—CO and Temperature Effects on Leaf Anatomy
#
was smaller : the increase in pores per unit area or wider
pore opening might explain the increase in gs. In elevated
temperature and CO ¬temperature, SD increased and gs
#
and GCL were reduced relative to each respective control in
both seasons. Reduced gs was probably the result of
stomatal closure or perhaps reduced pore size or both.
Morgan et al. (1994) found in a C grass, Pascopyrum
$
smithii that a combination of high temperature and enriched
CO resulted in closed, Ci-unresponsive stomates (where Ci
#
is leaf intercellular CO concentration). Developmental
#
plasticity may affect both short and long-term vegetational
water use efficiency. Water use efficiency might be enhanced
by lower SD (Tyree and Alexander, 1993) as the number of
open pathways to water vapour diffusion, from parastomatal regions is decreased. However, assimilation is less
sensitive to SD since an additional diffusional resistance to
CO uptake by chloroplasts resides in the liquid and
#
membrane diffusion pathways (Tyree and Yianoulis, 1980).
An overall reduction of SD in summer might be a water
saving mechanism whilst treatment differences in SD suggest
instantaneous water use efficiency might be greater in spring
leaves in elevated CO , but unaltered or decreased in
#
summer and in the other treatments. Two earlier independent studies of L. perenne in elevated CO alone
#
suggested water use efficiency increased in spring (Nijs et al.,
1989) but decreased slightly in summer (Nijs, Impens and
Behaeghe (1988). However, Ferris and Taylor (1995) found
that for two herbs that differed in SD, leaf area and rooting
depths in elevated CO , instantaneous water use efficiency
#
increased irrespective of SD.
The area of spring leaves increased with CO enrichment
#
due to increased cell expansion (Fig. 5) and, over parts of
the leaf, increased ECD (Fig. 3) resulting in greater
epidermal cell numbers per leaf (not shown) : a response also
observed in L. corniculatus (Ferris and Taylor, 1994) and in
poplar (Ceulemans et al., 1995). In contrast, there was a
negative effect of CO on leaf and cell expansion in summer
#
with reductions in ECL and MCA (Fig. 5, Tables 2 and 3 ;
Ferris et al., 1996) and no effect on epidermal cell numbers
per leaf (not shown). In high temperature, both ECL and
MCA (middle of leaf) decreased but less so in summer than
spring but in both cases ECD increased (Fig. 3). But, in
elevated CO ¬temperature, leaves were larger in spring and
#
MCA increased but ECL remained unaltered, whilst in
summer, reduced leaf growth was linked with reduced ECL
and MCA relative to those in ambient conditions (Tables 2
and 3 and Fig. 5 ; Ferris et al., 1996). This suggests that
elevated CO ¬temperature has a different effect on cell
#
division and expansion (Arkebauer and Norman, 1995)
depending on cell type and season. In contrast to our
results, non-seasonal studies of Ryle and Stanley (1992) and
Radoglou and Jarvis (1990a) found no effect of CO
#
enrichment alone on ECD, stoma and ECL in L. perenne or
leaf cell numbers per mm# in four poplar clones, respectively.
Gardner, Taylor and Bosac (1995) found no effect of CO
#
alone on mature leaf cell size in one poplar clone even
though leaves were larger and concluded increased area was
possibly due to a concomitant effect on cell production. In
our study, SD was inversely correlated with GCL : a
response observed previously for species grown in elevated
CO (Ferris, 1994) and in manipulated environmental
#
conditions (Ferris, 1991) illustrating that treatment effects
on cell expansion also affect the stomatal complex.
This study shows different effects of elevated CO and
#
temperature on stomatal and cell characteristics of L.
perenne with some of the variation explained by confounding
effects of season of sampling and position on leaf. However,
one can reasonably conclude that CO and temperature
#
effects on cell size and}or cell differentiation account for
some differences in leaf area development, ECD, SD, and SI
responses in spring and summer leaves.
A C K N O W L E D G E M E N TS
Rachel Ferris thanks the Federal Office for Scientific,
Technical & Cultural Affairs (Brussels) for a Postdoctoral
Fellowship under the Impulse Programme for Global
Change. Ivan Nijs is indebted to the Belgian National
Science Foundation for a Postdoctoral Fellowship. We
thank Kurt Schamp, Jan Bogaert and Bart van de Weghe
for technical assistance.
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