Journal of Experimental Botany, Vol. 57, No. 2, pp. 373–380, 2006
Phenotypic Plasticity and the Changing Environment Special Issue
doi:10.1093/jxb/eri278 Advance Access publication 19 September, 2005
Stomatal development in new leaves is related to the
stomatal conductance of mature leaves in poplar
(Populus trichocarpa3P. deltoides)
Shin-Ichi Miyazawa1,*, Nigel J. Livingston1 and David H. Turpin2
1
Centre for Forest Biology, Department of Biology, University of Victoria, PO Box 3020, Victoria,
BC V8W3N5, Canada
2
Department of Biology, University of Victoria, PO Box 3020, Victoria, BC V8W3N5, Canada
Received 27 May 2005; Accepted 3 August 2005
Abstract
In general, stomatal density (SD) decreases when
plants are grown at high CO2 concentrations. Recent
studies suggest that signals produced from mature
leaves regulate the SD of expanding leaves. To determine the underlying driver of these signals in poplar
(Populus trichocarpa3P. deltoides) saplings, a cuvette
system was used whereby the environment around
mature (lower) leaves could be controlled independently of that around developing (upper) leaves. A
series of experiments were performed in which the CO2
concentration, vapour pressure deficit (D), and irradiance (Q) around the lower leaves were varied while the
(ambient) conditions around the upper leaves were
unchanged. The overall objective was to break the
nexus between leaf stomatal conductance and transpiration and photosynthesis rates of lower leaves and
determine which, if any, of these parameters regulate
stomatal development in the upper expanding leaves.
SD, stomatal index (SI), and epidermal cell density (ED)
were measured on the adaxial and abaxial surfaces of
fully expanded upper leaves. SD and SI decreased with
increasing lower leaf CO2 concentration (150–780 ppm)
at both ambient (1.3–1.6 kPa) and low (0.7–1.0 kPa) D.
SD and SI at low D were generally higher than at
ambient D. By contrast, ED was relatively insensitive
to both vapour pressure and CO2 concentration. When
lower leaves were shaded, upper leaf SD, SI, and ED
decreased but did not change with varying CO2 concentration. These results suggest that epidermal cell
development and stomatal development are regulated
by different physiological mechanisms. SI of the upper
leaves was positively and highly correlated (r2 >0.84)
with the stomatal conductance of the lower leaves
independent of their net photosynthesis and transpiration rates, suggesting that the stomatal conductance of
mature leaves has a regulatory effect on the stomatal
development of expanding leaves.
Key words: CO2 concentration, leaf development, photosynthesis, poplar, transpiration, stomata, stomatal conductance,
stomatal density, stomatal index, vapour pressure deficit.
Introduction
Plants commonly respond to increased atmospheric CO2 by
adjusting their uptake of CO2 and their water loss. These
adjustments are brought about by changes in stomatal
aperture and/or stomatal density (SD). In response to CO2
enrichment, some species decrease their SD (Woodward
and Kelly, 1995). However, there are also reports that
leaves grown at high CO2 concentrations have a similar or
higher SD compared with those grown at lower CO2
concentrations (Woodward and Kelly, 1995; Woodward
et al., 2002). For example, in poplars such as Populus
trichocarpa, P. deltoides, and P. trichocarpa3P. deltoides,
SD of leaves grown at 700 and 350 ppm CO2 did not differ
(Radoglou and Jarvis, 1990). On the other hand, in P.
trichocarpa3P. deltoides, when grown at 700 ppm CO2,
the SD of young leaves on an upper portion of the plant
decreased significantly, but the SD of mature leaves on the
lower portion did not change (Ceulemans et al., 1995).
The response of SD to high CO2 concentrations probably
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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374 Miyazawa et al.
depends on species, leaf age, and plant growth conditions
(Ceulemans et al., 1995; Woodward et al., 2002).
Recent studies suggest that growing leaves are not fully
autonomous with respect to their stomatal development.
For example, in Arabidopsis thaliana, the SD of new leaves
grown under ambient CO2 (360 ppm) concentrations decreased by 20–30% (relative to control plants grown at
ambient CO2) when mature leaves were concurrently
exposed to twice the CO2 concentration (Lake et al.,
2001). This indicates that mature leaves might play a pivotal role in the stomatal development of new leaves through
the generation of systemic signals. However, the nature
of these signals is not known and it is unclear how such
signals are elicited.
There are several possible sources of systemic signals;
leaves exposed to high CO2 typically increase their rates of
carbohydrate production. Under these conditions, carbohydrate accumulations are known to regulate the expression
of photosynthetic genes such as ribulose-1,5-bisphosphate
carboxylase/oxygenase (sugar sensing mechanism; Sheen,
1990; Moore et al., 1999; Sheen et al., 1999). Tobacco
plants with overproduction of cytokinins (CK) have 1.26
times higher SD than control plants (Wang et al., 1997).
The promoter of the cytokinin oxidase/dehydrogenase gene
(AtKCX6), which encodes enzymes catalysing CK breakdown, is active in developing stomatal guard cells (Werner
et al., 2003). The application of abscisic acid (ABA) to
emerging leaves increases their SD in tomato (Bradford
et al., 1983) and in Tradescantia virginiana (Franks and
Farquhar, 2001). These results suggest that plant hormones
such as CK and ABA might be involved in the regulation
of stomatal development. High CO2 treatments generally
induce stomatal closure and hence reduce transpiration rates
(Kramer and Boyer, 1995). This might affect the delivery of CK and ABA that are produced in the roots and
transported to leaves via the transpiration stream (Davies
and Zhang, 1991; Pons and Bergkotte, 1996; Pons et al.,
2001).
Experiments have demonstrated that the transpiration
rates of individual leaves are not independent of the whole
plant transpiration rate, so that, for example, when a portion
of a plant’s foliage is shaded or excised, the transpiration
rates of the remaining leaves tend to increase (Meinzer and
Grantz, 1991; Pepin et al., 2002). This suggests that the
transpiration rates of mature leaves could influence the
delivery rates of hormones from the roots to the expanding
leaves. It is also possible that hydraulic signals could elicit
the release of hormones or signalling cascades in the leaves
(Fuchs and Livingston, 1996). Carbohydrate production
and/or transpiration might, therefore, be involved in the
systemic control of stomatal development.
In cases where leaves are well ventilated and the
boundary layer conductance is very large and does not
vary, leaf transpiration rate per unit leaf area (E) can be
expressed as:
E = gs ðwi wa Þ
where gs is stomatal conductance to water vapour, wi is the
mole fraction of water in the intercellular spaces of the leaf,
and wa is the mole fraction of water in the surrounding air.
Thus wi–wa, the vapour pressure difference between leaf
and air (D), is the driving force for evaporation. In general,
gs decreases with increasing D (Kramer and Boyer, 1995).
Net photosynthesis (Pn) can be expressed as the product
of stomatal conductance to CO2 (0.625 gs) and the gradient
in CO2 concentration between the leaf intercellular spaces
and atmosphere. Hence, Pn and E are inextricably linked
through gs.
A cuvette system was used whereby the environment
around the mature (lower) leaves of clonal poplar (P.
trichocarpa3P. deltoides) saplings could be manipulated
independently of the environment around their developing
(upper) leaves. In addition, shade cloth was used to reduce
the irradiance received by lower leaves while the upper
leaves remained fully illuminated. A series of manipulative
treatments was carried out whereby, for example, Pn could
be increased while, at the same time, gs could either be
decreased or increased, or held relatively constant. Our
hypothesis was that, by breaking the nexus between E, Pn,
and gs, the underlying driver of signals sent from mature to
developing leaves could be isolated.
Materials and methods
Plant growth conditions and treatments
Hybrid poplar plants H 11-11 (Populus trichocarpa3Populus
deltoides) were propagated from greenwood cuttings in peat
(Terra-Lite Redi-Earth, WR Grace, Ajax, ON, Canada) in 2.4 l
plastic pots. Each plant had a single main stem. Plants were kept in
a growth cabinet (Conviron, Winnipeg, MB, Canada) under 16-h
days at 20–22 8C. Night temperatures were 17–18 8C. Plants were
supplied with 120 ml of water and 100 ml of nutrient solution
containing 0.1 g l1 20-20-20 Plant-Prod complete fertilizer (Plant
Products, Brampton, ON, Canada) every other day. Otherwise,
plants were supplied with 220 ml of water. Photosynthetically active
photon flux (Q) measured at the top of the plants ranged from 190
to 240 lmol m2 s1. When plants were 16–17 cm high, they were
transferred to a cuvette system housed in another growth cabinet
where the treatments outlined below were imposed. The irrigation
and fertilization levels, growth temperature, and light conditions
were unchanged.
A cuvette system (Fig. 1) that accommodates three plants was used
to control the CO2 concentration, vapour pressure, and temperature of
the air surrounding the mature leaves. This system, made from
Plexiglas, 90 cm (L)330.5 cm (W)322 cm (H), is based on the design
described by Livingston et al. (1994). The air within the cuvette is
circulated at 0.070 m3 s1 by means of two 12 V DC fans
(CFA128025MS, Circuit-Test, Burnaby, BC, Canada). CO2 and
vapour pressure in the cuvette are measured with an infrared gas
analyser (LI-820, Li-Cor Inc., Lincoln, NE, USA). Cuvette air is
pumped through a soda lime column when cuvette CO2 concentration
exceeds a given set point. Conversely, cylinder CO2 (5000 ppm CO2
in air) is injected into the cuvette through a solenoid valve to balance
that taken up by the plants. Cuvette air is circulated through desiccant
Systemic responses of stomatal density in poplar
375
Fig. 1. Schematic diagram of the cuvette system (not to scale). The upper young leaves of each of the three plants protrude through small, sealable holes
out of the top of the cuvette.
columns (approximately 4 kg of oven-dried CaSO4) with two 12 V
DC fans (D601T, Micronel, Vista, CA, USA) when the cuvette
vapour pressure exceeds a specified vapour pressure. Desiccant was
changed every day or every other day. In addition, air is continuously
passed through a water-cooled heat exchanger using an additional
12 V DC fan to provide temperature control.
The upper 2–4 young leaves (lamina length; 19–97 mm) of each of
the three plants protrude through small, sealable holes out of the top
of the cuvette. In the experiments these leaves were exposed to air
with a CO2 concentration of 350–380 ppm and a vapour pressure
deficit of 1.3–1.7 kPa during the illumination period. The remaining
(mature) leaves were exposed to a range of treatments during the
illumination period; three different CO2 treatments were imposed:
low CO2 (150 ppm), ambient CO2 (380 ppm), and high CO2 (780
ppm) at two different D: low D (0.7–1.0 kPa) and ambient D (1.3–1.6
kPa). In addition, shade treatments were imposed by covering the top
of the cuvette with black cloth. Shade reduced Q, measured at the
middle of the cuvette, from 130–150 to 39–50 lmol m2 s1. In
the shade treatments, D in the cuvette was held at 0.7–1.0 kPa and
the CO2 concentration varied.
Measurements were taken only on those upper leaves with a lamina
length of less than 45 mm when treatments were started. Lamina
length and width of upper leaves were measured every day or every
other day. The upper leaves took 10–15 d to expand fully. Lamina
area was calculated from the equation, lamina area=0.6623lamina
length3lamina width (r2=0.995, P <0.001), which was obtained from
17 leaves. For each treatment, measurements were carried out on 4–6
leaves that were obtained from 2–5 plants.
When upper leaves were fully expanded, an approximately 2 cm2
area in the middle sections of leaf adaxial and abaxial lamina surfaces
was painted with nail varnish which when dry was peeled from the
surface and mounted on slides. 10–15 microscopic fields of each
epidermis impression were randomly selected. Micrographs of the
epidermis impressions were taken with a digital camera linked to
a light microscope to determine the numbers of stomata and epidermal cells. Stomatal index was calculated as the value of stomatal
density divided by the sum of stomatal density and epidermal
cell density.
Changes in stomatal number with leaf area expansion
Following the methodology described by Geisler et al. (2000), dental
resin impressions were used to obtain changes in stomatal number
with leaf area expansion. The dental resin mould was filled with nail
varnish to create a cast that was examined by a light microscope.
Lamina length, lamina width, and stomatal density were measured
every other day during leaf development. Five microscopic fields of
each epidermal cell impression were randomly selected, and light
micrographs were taken with a digital camera. Stomata and stomatal
precursor cells (meristemoids and guard mother cells) were counted
on the micrographs. Lamina area was calculated using the same
equation as described above. Stomatal number per leaf was calculated
as the value of stomatal density multiplied by the lamina area. Two
leaves were used for each set of measurements.
Gas exchange
Gas exchange measurements were taken on mature fully expanded
leaves with a portable infrared gas analyser (LI-6400, Li-Cor Inc.,
Lincoln, NE, USA). Measurements were made on plants in a growth
chamber before they were enclosed in the three-plant cuvette system
and exposed to the treatments described above. The environmental
conditions in the LI-6400 cuvette were adjusted to match those
that would be imposed during the treatments. Changes in net
photosynthesis rates, transpiration rates, and stomatal conductance
were continuously monitored after the leaves were exposed to the
treatment conditions. It took approximately 30–60 min for the gas
exchange parameters to reach their steady-state values. Three leaves
were used for measurements.
Preliminary experiments (data not shown) were conducted in
single whole-plant cuvettes in which the environment in the bottom
portion of a plant could be independently and precisely controlled
and gas exchange continuously measured (Pepin et al., 2002). It
was established that plant transpiration rates, stomatal conductance,
and net photosynthesis rates did not change markedly over 10 d of
treatments and that values of gas exchange parameters obtained with
the portable gas analyser reflected those of plants exposed to the same
conditions in the three-plant cuvette system.
376 Miyazawa et al.
Pn (µmol m-2 s-1)
Figure 2 shows the response of Pn, E, and gs of lower
mature leaves to changes in CO2 concentration, D, and light
intensity. At both low and ambient D, gs and E decreased,
and Pn increased with increasing CO2. Because of the
limited effect of D on gs (and hence Pn) increases in E were
almost in direct proportion to increases in D. When leaves
were shaded Pn, gs and E decreased.
Stomatal number per leaf increased rapidly with leaf area
expansion, reaching a maximum value by the time the leaf
area had reached approximately half its final value (Fig. 3).
Stomatal density decreased as the leaf area increased
without the concurrent change in stomatal number (data
not shown).
When lower leaves were shaded, the lamina area of fully
expanded (unshaded) upper leaves was generally higher
than that of leaves in unshaded treatments regardless of
the CO2 treatment imposed on lower leaves (Fig. 4). The
largest area was in those plants subjected to the highest CO2
concentrations. Conversely, in unshaded treatments, the
lamina area of upper leaves did not significantly change
with increasing CO2 concentration at the lower leaves.
There was not a significant difference in the lamina area
between ambient and low D treatments.
The stomatal density and stomatal index of adaxial surfaces were lower than those of abaxial surfaces (Fig. 5).
Conversely, the epidermal cell density of adaxial surfaces
was significantly higher than that of abaxial surfaces. The
changes in stomatal density, epidermal cell density, and
stomatal index with the treatments were similar between
the two epidermal surfaces.
The stomatal density of upper leaves decreased when
the lower leaves were grown at elevated CO2, at ambient
and low D (Fig. 5). The stomatal density of upper foliage
also decreased significantly when the lower foliage was
shaded. At low D, the stomatal density of the upper leaves
at 780 ppm CO2 was significantly lower than that at 380
ppm CO2. By contrast, at ambient D, there was no significant difference in stomatal density between the 380 and
780 ppm CO2 treatments. When the lower leaves were
shaded, the upper leaf stomatal density was insensitive to
changes in the CO2 concentration at the lower leaves.
In general, changes in stomatal index in response to
CO2 and D treatments mirrored those of stomatal density
(Fig. 5). At both low D and shade, the epidermal cell
density of upper leaves did not change when lower leaves
were exposed to elevated CO2. Conversely, at ambient
D, the epidermal cell density of upper leaves increased
5
4
3
2
1
0
2.5
E (mmol m-2 s-1)
Results
6
2.0
1.5
1.0
0.5
0.0
0.20
gs (mol m-2 s-1)
Statistical analysis
Means were compared with the Tukey–Kramer multiple comparison
test using SPSS (SPSS Inc., Chicago, Ill., USA). Correlation
coefficients of linear regressions were calculated using Origin 6.1J
(OriginLab Corporation, Northampton, MA, USA).
0.15
0.10
0.05
0.00
150
380
780
CO2 concentration (ppm)
Fig. 2. Net photosynthesis rates (Pn), transpiration rates (E), and
stomatal conductance (gs), expressed per unit leaf area, of mature leaves
under various CO2 concentrations, vapour pressure deficits (D), and light
regimes in poplar (Populus trichocarpa3P. deltoides) saplings. Low D
(open circles); D=0.7–1.0 kPa and photosynthetically active photon flux
(Q)=130–150 lmol m2 s1. Ambient D (open squares); D=1.3–1.6 kPa
and Q=130–150 lmol m2 s1. Shade (closed circles); D=0.7–1.0 kPa
and Q=39–50 lmol m2 s1. Each value represents the mean 6standard
deviation (n=3).
slightly when the lower leaves were exposed to either high
or low CO2.
The relationships between the stomatal index of the
upper leaves and Pn, E, and gs measured in the lower leaves
are shown in Fig. 6. Both Pn and E were poorly correlated
with stomatal index in adaxial and abaxial surfaces (coefficient of variation, r2=0.004–0.41). By contrast, stomatal
60
300
50
250
Leaf area (cm2)
c
c
40
200
c
c
30
150
b
100
20
10
50
a
0
0
2
4
6
8
10
12
14
Stomatal number per leaf ( x 103)
Systemic responses of stomatal density in poplar
0
Time (d)
Fig. 3. Developmental changes in lamina area (open circles) and
stomatal number per leaf (closed circles) in poplar (Populus
trichocarpa3P. deltoides) saplings. Stomatal number per leaf was
calculated as the value of stomatal density multiplied by the lamina
area. Each value with bars represents the mean 6standard error (n=10).
Different letters next to the symbols indicate significant differences between the treatments at P <0.05. Two leaves were used for measurements.
100
b
80
Leaf area (cm2)
ab
ab
60
ab
a
40
a
a
a
a
20
0
150
380
780
CO2 concentration (ppm)
Fig. 4. Lamina area of fully expanded upper leaves in relation to
differing vapour pressure deficit (D), CO2 concentration, and light
conditions imposed on lower leaves in poplar (Populus trichocarpa3P.
deltoides) saplings. Low D treatment (open circles); D=0.7–1.0 kPa and
photosynthetically active photon flux (Q)=130–150 lmol m2 s1.
Ambient D treatment (open squares); D=1.3–1.6 kPa and Q=130–150
lmol m2 s1. Shade treatment (closed circles); D=0.7–1.0 kPa and
Q=39–50 lmol m2 s1. The upper leaves were grown under constant
conditions throughout the experiments. Each value represents the mean
6standard deviation (n=4–6). Different letters next to the symbols
indicate significant differences between the treatments at P <0.05.
index has a very strong positive relationship with gs in both
adaxial and abaxial surfaces (r2 >0.84). There was poor
correspondence between the internal CO2 concentration of
mature leaves and stomatal index (r2 <0.50, data not
377
shown). This suggests that any delivery of CO2 from
mature to developing leaves via xylem sap exerted little or
no influence on stomatal development.
Discussion
Both stomatal density and index generally decrease with
decreasing light intensity (Tichá, 1985). In this study, shade
treatments imposed on lower mature leaves significantly
decreased the stomatal density and the stomatal index of
the upper developing leaves (Fig. 5). Similar results have
been reported for Vigna sinensis L. (Schoch et al., 1980),
Arabidopsis thaliana (Lake et al., 2001), and tobacco
(Thomas et al., 2003). These results suggest that signals
triggered by changes in irradiance are transmitted from
mature to young leaves and regulate stomatal development
(Lake et al., 2001; Yano and Terashima, 2001; Thomas
et al., 2003). These results indicate that the effect of
changing CO2 concentration (at the mature leaves) on
stomatal development in young leaves is not independent
of the light regime (Fig. 5), suggesting an interdependence
of the respective signalling mechanisms.
Shading of mature leaves brought about a significant
reduction in the epidermal cell density of new (unshaded)
leaves (Fig. 5). A similar result has been reported in tobacco
where the epidermal cell density of young leaves was
reduced when mature leaves were grown at light intensities
lower than those provided to young developing leaves
(Thomas et al., 2003). Lake et al. (2002) indicated that, in
Arabidopsis, the epidermal cell density of upper new leaves
was insensitive to the elevated CO2 on lower mature leaves.
By contrast, in these experiments, upper leaf epidermal
cell density was sensitive to the CO2 concentration at the
lower leaves when the same leaves were illuminated and
held at ambient D.
This study’s results suggest that changes in epidermal
cell density are unrelated to changes in carbohydrate
production and/or transpiration rates of mature leaves since
the large changes in net photosynthesis and transpiration
rates in response to changes in CO2 supply or evaporative
demand did not have a corresponding effect on the
epidermal cell number of the developing leaves (Figs 2,
5). The systemic changes in epidermal cell density of upper
young leaves in response to changes in CO2 concentration,
vapour pressure, and light intensity (at lower mature leaves)
were different from those in stomatal index (Fig. 5). This
suggests that stomatal differentiation is regulated by
a different physiological mechanism from that which
determines epidermal cell division and/or expansion.
Some previous papers indicate that in Populus
trichocarpa3P. deltoides stomatal density significantly decreases when they are grown at elevated CO2 concentration
(Ceulemans et al., 1994, 1995), but there are other reports
that stomatal density does not change in response to the
378 Miyazawa et al.
Adaxial
Abaxial
160
80
a
Epidermal cell density (mm-2)
Stomatal density (mm-2)
70
140
a
c
a
ab
b
60
120
b
b
abc
a
bcd
c
100
50
d
d
d
d
d
40
80
30
60
1200
800
a
d
a
a
a
ab
ab
ab
ab
ab
1000
b
bc
b
c
600
c
c
c
c
c
800
400
6.5
15.5
a
Stomatal index (%)
a
15.0
6.0
ac
ab
c
ab
14.5
5.5
ab
b
b
5.0
b
14.0
ab
b
ab
b
b
4.5
b
13.5
b
b
13.0
4.0
150
380
780
150
380
780
CO2 concentration (ppm)
Fig. 5. Stomatal density, epidermal cell density, and stomatal index of upper leaves in relation to differing vapour pressure deficit (D), CO2
concentration, and light conditions imposed on lower leaves in poplar (Populus trichocarpa3P. deltoides) saplings. Stomatal index is calculated as:
stomatal density/(stomatal density+epidermal cell density). Low D treatment (open circles); D=0.7–1.0 kPa and photosynthetically active photon flux
(Q)=130–150 lmol m2 s1. Ambient D treatment (open squares); D=1.3–1.6 kPa and Q=130–150 lmol m2 s1. Shade treatment (closed circles);
D=0.7–1.0 kPa and Q=39–50 lmol m2 s1. The upper leaves were grown under constant conditions throughout the experiments. Each value represents
the mean 6standard error (n=60). Different letters next to the symbols indicate significant differences between the treatments at P <0.01.
elevated CO2 concentration (Radoglou and Jarvis, 1990).
This inconsistency might be due to differences in growth
light intensity and/or humidity in these studies because, as
these results show, the systemic responses of stomatal and
epidermal cell development are affected by these variables
(Fig. 5).
As shown in Fig. 3, the stomatal number was determined
before leaf area reached half its maximum value. This
Systemic responses of stomatal density in poplar
379
Adaxial
7.0
r = -0.03 P = 0.94
r = 0.96 P < 0.01
r = 0.64 P = 0.06
6.5
6.0
5.5
5.0
Stomatal index (%)
4.5
4.0
0
1
2
4
3
Pn (µmol
m-2
5
0
s-1)
1
2
E (mmol m
-2
0.2
0.1
0.0
s-1)
gs (mol
m-2
0.3
s-1)
Abaxial
16.0
r = -0.07 P = 0.86
r = 0.92 P < 0.01
r = 0.57 P = 0.10
15.5
15.0
14.5
14.0
13.5
13.0
0
1
2
4
3
Pn (µmol
m-2
s-1)
5
0
1
2
E (mmol
m-2
s-1)
0.2
0.1
0.0
gs (mol
m-2
0.3
s-1)
Fig. 6. Relationships between stomatal index of the upper new leaves and net photosynthesis rate (Pn), transpiration rate (E), and stomatal conductance
(gs) of the lower mature leaves in poplar (Populus trichocarpa3P. deltoides) saplings. Pn, E, and gs are all expressed per unit leaf area. Stomatal index is
calculated as: stomatal density/(stomatal density+epidermal cell density). Low D (open circles); D=0.7–1.0 kPa and photosynthetically active photon
flux (Q)=130–150 lmol m2 s1. Ambient D (open squares); D=1.3–1.6 kPa and Q=130–150 lmol m2 s1. Shade (closed circles); D=0.7–1.0 kPa and
Q=39–50 lmol m2 s1. Correlation coefficients (r) and the levels of significance (P) are shown on the panels.
suggests that signals produced from mature leaves play
a significant role in stomatal differentiation of developing
leaves only when they are at their early stages of expansion.
The relatively poor correlation between Pn and E and
stomatal index (r2 <0.41) suggests that neither carbohydrate
production nor transpiration is strongly associated with the
systemic control of stomatal development. Beerling and
Woodward (1995) argued that local carbohydrate concentrations in the leaf might not influence the systemic control
of stomatal development because there is a normal reduction in stomatal density in variegated leaves when
they are exposed to high CO2 concentrations. The results
support this argument. E of mature leaves was not correlated with stomatal index of new leaves (Fig. 6). The
transpiration stream delivers plant hormones such as
cytokinins, and plays an important role in leaf biochemical acclimations to changes in light intensity (Pons and
Bergkotte, 1996; Pons et al., 2001). However, this theory
does not account for any mechanisms that alter the concentrations and compositions of plant hormones in the transpiration stream. The very poor correlation between E of
mature leaves and the stomatal index of new leaves would
not necessarily rule out the involvement of plant hormones
in the systemic control of stomatal development.
In contrast to Pn and E, there was a consistent relation
between gs and stomatal index (Fig. 6). The results suggest
that the production of long-distance signals, hormonal or
otherwise, that regulate stomatal development in new
leaves is directly related to changes in the stomatal conductance of the mature leaves. Any manipulation of the
CO2, light, and humidity regimes that brings about an
increase in mature leaf stomatal conductance, regardless
of the effects on Pn and E, will promote stomatal differentiation in developing leaves.
Acknowledgements
We thank Ichiro Terashima, David Whitehead, and Charles Warren
for their comments on an earlier draft of this manuscript, and Ivan
Petrovic and Peter Ward for their work on constructing the cuvette
systems. We thank Peter Constabel, Ian Major, and Brad Binges
for providing poplar cuttings. S-IM would like to thank Yoshiro
380 Miyazawa et al.
and Emiko Arakawa for their hospitality during his stay in Canada.
We thank Michiru Miyazawa for helping with epidermal cell
analyses. Funding (NJL and DHT) for this research was provided,
in part, by the Natural Sciences and Engineering Research Council
of Canada.
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