Effect of elevated CO2 on the stomatal distribution and leaf physiology of Alnus glutinosa

RESEARCH New Phytol. (2000), 145, 511–521

Effect of elevated CO#

on the stomatal

distribution and leaf physiology of

Alnus glutinosa

I. POOLE†, T. LAWSON*, J. D. B. WEYERS J. A. RAVEN

Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK

Received 29 July 1999; accepted 17 November 1999

Variation in stomatal development and physiology of mature leaves from Alnus glutinosa plants grown under

reference (current ambient, 360 µmol mol−" CO#) and double ambient (720 µmol mol−" CO

#) carbon dioxide (CO

#)

mole fractions is assessed in terms of relative plant growth, stomatal characters (i.e. stomatal index and density)

and leaf photosynthetic characters. This is the first study to consider the effects of elevated CO#concentration on

the distribution of stomata and epidermal cells across the whole leaf and to try to ascertain the cause of intraleaf

variation. In general, a doubling of the atmospheric CO#concentration enhanced plant growth and significantly

increased stomatal index. However, there was no significant change in relative stomatal density. Under elevated

CO#concentration there was a significant decrease in stomatal conductance and an increase in assimilation rate.

However, no significant differences were found for the maximum rate of carboxylation (Vcmax

) and the light

saturated rate of electron transport (Jmax

) between the control and elevated CO#

treatment.

Key words: Alnus, stomatal index, stomatal density, carbon dioxide, conductance, photosynthesis, variation.

The impact of atmospheric CO#

on the changing

climate of the Earth has attracted considerable

attention. Since the beginning of atmospheric moni-

toring at Mauna Loa (Hawaii) in 1958 the CO#

concentration has risen from a mean of c. 320 µmol

mol−" CO#

to an average present-day level of 360

µmol mol−" and is forecast to continue increasing

to approx. 550 µmol mol−" by the middle of the

twentyfirst century (Houghton et al., 1992). How-

ever, the mole fraction of CO#has been much lower

in the recent past ; during the previous interglacial

period (c. 130000 yr BP), the CO#concentration was

approx. 280 µmol mol−" CO#

but during the last

glacial period the maximum dropped to 180–190

µmol mol−" only to increase to 280 µmol mol−" once

more at the onset of the present interglacial (Petit et

al., 1999). This change in CO#

is expected to have

far-reaching effects on the environment such that a

general indication in terms of plant response is

fundamental for predictive analyses.

Plants are known to respond both anatomically

(Woodward, 1987; Beerling & Chaloner, 1994;

*Author for correspondence (present address) :Department of

Biology, John Tabor Laboratories, University of Essex, Wivenhoe

Park, Colchester CO4 3SQ, UK (tel 44 1206 873306; fax 44

1206 873416; e-mail tlawson!essex.ac.uk). †Present address :

School of Earth Sciences, University of Leeds, Leeds LS2 9JT,

UK.

Beerling et al., 1998) and physiologically (Long et

al., 1996; Mulholland et al., 1997) to changes in the

CO#concentrations. Much work has focused on the

changes in leaf anatomy and, in particular, stomatal

characters. However, stomatal density and index are

known to vary interspecifically and have been used as

one of many features in the identification of leaf

material (Masterson, 1994; Mishra, 1997). There-

fore, any response made by a plant to environmental

changes has to be related to a designated reference

point for that particular species.

Variations in anatomical stomatal characters have

been recognized for a number of years within and

between leaves of the same plant (Salisbury, 1927;

Shearman & Beard, 1972; Lugg & Sinclair, 1979;

Ticha' , 1982; Sola! rova! & Posps) ilova! , 1983; Weyers &

Meidner, 1990; Poole et al. 1996; Weyers & Lawson,

1997; Weyers et al., 1997). Differences have also

been found among species and cultivars (Salisbury,

1927; Meidner & Mansfield, 1968; Jones, 1977;

Apel & Peisker, 1995). An extensive review by Ticha'(1982) collated data on within-leaf stomatal character

variation and ontogenic gradients. Stomatal

characters are affected by a number of growth

conditions including light intensity (Brown &

Rosenberg, 1970; Gay & Hurd, 1975; Wild & Wolf,

1980), water availability (Gindel, 1969), nutrient

availability (Hsaio & Fischer, 1975), CO#

concen-

trations (Woodward, 1987; Woodward & Bazzaz,

512 RESEARCH I. Poole et al.

1988; Beerling & Chaloner, 1992; Ferris & Taylor,

1994; Woodward & Kelly, 1995) and leaf age (Davis

et al., 1977).

Although there have been numerous reports on

variations in stomatal characters with different

environments, these have relied on relatively few

samples which might not take into account the

natural trends that exist across a leaf surface. Even

so, some workers inherently recognize natural vari-

ation by standardizing the location on the leaf where

measurements of stomatal density and index are

made. However this does not necessarily take into

account the possibility of the differential effects of

treatments on this standard location and on other

areas of the leaf. Such intrinsic variation in stomatal

density, for example in Commelina communis, has

been illustrated using 2-D contour maps by Smith et

al. (1989). Weyers et al. (1997) used a similar method

to show changes in stomatal density between watered

and unwatered C. communis leaves and Poole et al.

(1996) mapped the variation in stomatal density and

index in the hypostomatous sun and shade leaves of

Alnus glutinosa.

Intrinsic stomatal variation as a response to

external differences over a leaf surface could be

attributed to uneven guard cell differentiation

(differentiation hypothesis) or uneven expansion

(expansion hypothesis) of the epidermal cells after

differentiation (cf. Beerling & Chaloner, 1993; Ferris

& Taylor, 1994; Poole et al., 1996). However, such

spacing could also be a result of the combined effects

of both uneven differentiation and expansion (Poole

et al., 1996).

Numerous studies over the past decade have

investigated the effects of CO#

enrichment on

photosynthesis and related variables. Many studies

have shown that exposure of plant species to elevated

CO#increases net photosynthesis (Long et al., 1993,

1996; Gifford & Seemann, 1996), reduces photo-

respiration (Gifford et al., 1985) and increases above-

and}or below-ground biomass (Kimball, 1983;

Kimball et al., 1995). Long term exposure to elevated

levels of CO#

has also been shown to induce

acclimatory reductions in photosynthesis and photo-

synthetic capacity (Sage et al., 1989). The reduction

in photosynthetic capacity has been shown to be

attributed to a decrease in the content and activity of

Rubisco (Sage et al., 1989). It is believed that this

decrease in Rubisco is a feedback response to an

increase in carbohydrate content within the leaf,

resulting from the accumulation of starch (Long &

Drake, 1992). Most of these studies have been

carried out on crop species in which a sink–source

relationship might play a primary role in photo-

synthetic acclimation due to a balance between

carbon gain from the environment and its con-

sumption in sinks (Farrar, 1996). A number of

deciduous tree species have been the subject of

elevated CO#

studies and most have shown an

increase, to different degrees, in above-ground

biomass (Strain, 1987; Norby, 1989; Eamus &

Jarvis, 1989; Ceulemans & Mosseau, 1994;

Ceulemans et al., 1996). However, other studies have

reported little difference in growth rate of tree

species with elevated CO#

(El Kohen et al., 1993;

Norby et al., 1999).

Photosynthesis is dependent on stomata for its

supply of CO#

which is conducted into the leaf

through the stomatal aperture. Therefore conduc-

tance is affected primarily by stomatal aperture but

also by the number of stomata per unit leaf area (i.e.

stomatal density; Weyers & Lawson, 1997). Changes

in stomatal aperture size and density thus play

influential roles in stomatal conductance and hence

photosynthetic rate (Wong et al., 1979). Stomatal

conductance has been reported to decrease under

conditions of elevated CO#

(Kellomaki & Wang,

1997; Mullholland et al., 1997; Morison, 1998)

through a decrease in aperture size, which continues

to maintain the CO#

concentration within the leaf

but at the same time reduces water loss. Therefore

stomatal conductance plays a significant role in water

use efficiency (Mansfield et al., 1990; Long, 1994).

The aim of the research presented here was to

quantify any changes in growth parameters and

stomatal numbers in A. glutinosa with elevated CO#,

and to monitor consequential effects of such a change

in CO#

concentration on stomatal conductance and

photosynthetic performance.

Plant material

Before sowing, Alnus glutinosa (L.) Gaertn. seeds

were kept at 4°C for 2 wk to break dormancy. Seeds

weresownin3.35-lpotscontainingLevington’smulti-

purpose growing compost (Fisons Ltd, Ipswich,

UK). One pot was placed into each of four glass

growth chambers measuring 0.75¬0.45¬0.61 m

(0.21 m$), under natural light in a glasshouse.

Supplementary lighting was supplied to both am-

bient and elevated treatments from 08.00–23.00

hours by high pressure sodium bulbs giving a

minimum PPFD of 300–350 µmol photons m−# s−" at

pot height. A 15 h day length was selected to

maintain a seasonal average for the duration of the

experiment. The temperature within the chambers

was maintained at 15°C at night and rarely exceeded

35°C during the day. On days when the temperature

did exceed 35°C the supplementary lighting was

turned off. The base of each chamber was lined with

capillary matting to ensure the plants received a

regular water supply. Two control chambers were

supplied with ambient air (360 µmol mol−" CO#) and

the remaining treatment chambers were supplied

with ambient air supplemented with CO#to maintain

the mole fraction at a constant 720 µmol mol−" CO#.

RESEARCH Alnus and elevated CO#

513

Table 1. Mean growth of the nine plants of Alnus glutinosa within each cabinet at the whole-plant level

illustrating differences when grown at ambient (360 µmol mol−" CO#) and elevated CO

#(720 µmol mol−" CO

#)

mole fractions

CO#

treatment

(µmol mol−" CO#)

Mean height

(mm)

Mean leaf area

of plant (mm#)

No. of lateral

branches on

main axis

Mean total no.

of leaves on

main axis

Mean specific

leaf area

(mm# g−" d. wt)

360 277 (³22) 2734 (³183) 9 (³1±3) 13 (³0±6) 2760 (³38)

720 307 (³16) 3211 (³135) 19 (³2±5) 15 (³0±6) 2376 (³81)

Values are means³SE.

Outside air was supplied to the chambers at a rate of

1.56 m$ h−" and was pre-warmed by passing through

the piping system within the glasshouse. This

maintained a relatively constant CO#

mole fraction

of 360 µmol mol−" CO#, whilst additional CO

#was

supplied through small diameter pipes at a constant

pressure to enrich the atmosphere to 720 µmol mol−"

CO#. To ensure these pressures were constant CO

#

mole fractions were monitored regularly (for further

details, see Godber, 1997).

When the seedlings were 30–40 mm high, four

seedlings were transplanted into each 1.45-l pot and

nine such pots were placed, randomly arranged, on

individual plant trays and returned to their re-

spective chamber. The plants were watered daily and

fed with the nutrient-rich Hoagland’s solution

(Hoagland & Arnon, 1938) three times per week.

Plants and CO#treatments were rotated twice weekly

to average the effects of any unknown differences

between the cabinets which might have resulted in

experimental bias. The pots within each of the

chambers were then moved accordingly and

positioned randomly within the chamber to

minimize possible within-chamber effects. When the

seedlings were 70–80 mm high the three smallest

plants were removed thus leaving the strongest and

healthiest plant in each pot. The internodal distance,

length and breadth of each leaf and the total number

of leaves of each plant was recorded every 2 d.

Stomatal measurements

At maturity (maturation was taken as being complete

when there was no change in leaf area based on

length and width), negative silicone rubber

impressions of abaxial leaf surfaces were taken from

the hypostomatous leaf at insertion point 9 of each

plant using Xantopren VL Plus dental impression

material (Beyer Dental, Leverkusen, Germany)

according to the method of Weyers & Johansen

(1985). Leaves at insertion point 9 were selected

because they were present on all plants, unfolded

fully at maturity and had a large leaf surface area for

physiological measurements and from which a

representative number of sites could be studied

microscopically. The impressions were divided into

10¬10 mm sampling squares from which positive

impressions were made with clear nail varnish spread

onto microscope slides. Stomatal and epidermal cell

counts were made following the systematic sampling

strategy outlined by Poole & Ku$ rschner (1999) using

a microscope linked to a television monitor via a

video camera as described by Poole et al. (1996). The

stomatal density and index values were calculated

(n.b. A. glutinosa has no subsidiary cells to com-

plicate stomatal index determinations) and mapped

using the bilinear interpolation option within the

Unimap 2000 program (UNIRAS Ltd, Slough,

UK; see Lawson, 1997 for details). The x and y

spatial co-ordinates represent the centre of each

sample site whilst the z value represents the site

mean for stomatal and cell characters as appropriate.

However, it should be noted that 2-D colour maps

should be interpreted with care (Lawson & Weyers,

1999) as the maps involve interpolation based on an

arbitrary algorithm. Nevertheless previous studies

have shown extensive validation of the use of such a

program and indicated that excellent reproduction

patterns based on mathematical models could be

obtained using a similar sampling strategy (Lawson,

1997). Data were tested to confirm normality and

parametric statistical analyses were undertaken as

indicated.

Gas exchange measurements

Using a Combined Infrared Gas Analysis system

(CIRAS-PP Systems, Hitchin, UK) stomatal con-

ductance values were measured directly under light

saturating conditions at the relevant ambient CO#

concentration for that plant once the plant had

stabilized to the cuvette conditions. Photosynthetic

characters of at least three of the leaves at insertion

point 9 from the plants grown at the different CO#

concentrations were determined from the PPFD}net

CO#

assimilation rate (A) and A}internal carbon

dioxide partial pressure (Ci) curves (Parsons et al.,

1997). The PPFD}A curves were carried out on

plants at their respective chamber CO#mole fraction

(i.e. 360 µmol mol−" CO#

or 720 µmol mol−" CO#)

with neutral density filters after ensuring that

stomatal conductance was not limiting. Reverse

PPFD}A curves were also carried out at 720 µmol

mol−" CO#

on plants grown at 360 µmol mol−" CO#


(a) (b)

(c) (d)

5 cm

Stomatal index

>13.0

12.5–13.0

12.0–12.5

11.5–12.0

11.0–11.5

10.5–11.0

10.0–10.5

9.5–10.0

9.0–9.5

<9.0

Stomatal density

>340

330–340

320–330

310–320

300–310

290–300

280–290

270–280

260–270

250–260

240–250

<240

Fig. 1. Maps of stomatal index (%) and stomatal density (stomata mm−#) over the abaxial surface of Alnusglutinosa leaves at insertion point 9 from the base. (a) Stomatal index at 720 µmol mol−" CO

#, (b) stomatal density

at 720 µmol mol−" CO#, (c) stomatal index at 360 µmol mol−" CO

#, (d) stomatal density at 360 µmol mol−" CO

#.


515

and vice versa. The A}Cianalyses were carried out

on four plants at an incident PPFD of 2028 µmol

photons m−# s−" with the internal CO#pressure taken

as the value calculated by the algorithm provided

within the CIRAS. The relative humidity was 66%

(water vapour pressure 2.3 kPa, temperature 26.6°C)

for 360 µmol mol−" CO#

and 69% (water vapour

pressure 2.1 kPa, temperature 23.4°C) for 720 µmol

mol−" CO#. Photosynthetic capacities were deter-

mined as the highest value recorded and photo-

synthetic efficiencies determined from the gradient

of linear regressions for data values close to A ¯ 0.

Other photosynthetic parameters were determined

using the Photosyn Assistant computer program

(Dundee Scientific, Dundee, UK). Differences in

conductance and photosynthetic parameters between

the plants grown at the control and elevated CO#

concentrations were determined using a two tailed

t-test (Minitab Inc., State College, PA, USA).

At the end of the experiment (approx. 6 months

after sowing) all nine plants were harvested and final

plant height, total leaf area (using an area meter,

Delta-T Devices, Cambridge, UK), number of

lateral branches and leaves of every plant were

evaluated. Leaf dry mass was obtained after drying

the leaves in an oven at 80°C for 24 h.

Morphological and anatomical differences

Alnus glutinosa plants grown in the elevated CO#

concentration showed an overall general increase in

growth of the measured parameters relative to those

grown at ambient CO#(Table 1). There was a signi-

ficant increase in the number of branches (P !0.01)

and a decrease in specific leaf area (P !0.01)

with elevated CO#, but no significant increase in

plant height, number of leaves and absolute leaf area.

This supports the general observation that an

increase in atmospheric CO#concentration enhances

overall plant growth (McKee et al., 1995; Long et

al., 1996; Mulholland et al., 1998). The higher

specific leaf area exhibited by the plants grown at

360 µmol mol−" CO#compared with those grown at

720 µmol mol−" CO#suggests that the former plants

might have thinner leaves. However, other studies

have shown that differences in leaf morphology, such

as leaf thickness, with elevated CO#(Madsen, 1973;

Ho, 1977) could be attributed to photosynthetic

acclimation and an accumulation of starch and}or an

increase in the number of palisade cells (Arp, 1991;

Hofstra & Hesketh, 1975; Thomas & Harvey, 1983;

Acock & Pasternak, 1986).

Over each representative leaf, approx. 2000 sto-

mata were counted although the number of sample

sites varied as a result of the difference in leaf areas.

The data are represented two-dimensionally in

Fig. 1, which shows the general variation in intra-

leaf stomatal characters over abaxial leaf surfaces

fromplants grown at bothCO#concentrations. There

was no consistent pattern to either stomatal density

or index between the leaves although the patterns of

stomatal density and index over the leaf grown at 360

µmol mol−" CO#show broad similarities (Fig. 1a,b).

There was a significantly lower number of epidermal

cells on leaves grown at 720 µmol mol−" CO#

than

those grown at 360 µmol mol−" CO#

(P !0.001;

Table 2). The stomatal densities for the leaves grown

at 360 and 720 µmol mol−" CO#were not significantly

different (P ¯ 0.66) and had similar ranges in value

(i.e. 240–340 stomata mm−# ; Fig. 1) which agrees

with the findings of Ceulemans et al. (1995) working

on Populus. The mean stomatal index of leaves

grown at 720 µmol mol−" CO#

was significantly

higher than those grown at 360 µmol mol−" CO#

(P !0.001) and the range of the index values in-

creased with increasing CO#

concentration (Fig. 1).

The observations of stomatal density and index de-

scribed here for A. glutinosa are similar to those

found by Boetsch et al. (1996) for Tradescantia

where an increase in stomatal index but no change in

stomatal density with elevated CO#

was described.

The ratio of stomatal density between the leaves

grown at the two different CO#

concentrations was

approx. 1 : 1 whereas the epidermal cell density

decreased by c. 13% and stomatal index increased

by c. 11% with elevated CO#

(Table 2). The leaves

grown at 360 and 720 µmol mol−" CO#

both had

lower coefficients of variation in stomatal index when

comparedwith the respective stomatal density values.

Therefore we conclude that the increase in stomatal

index found in leaves grown at 720 µmol mol−" CO#,

compared with those grown at 360 µmol mol−" CO#,

appears to be largely because of a decrease in density

of epidermal cells.

Further analysis at the site level allowed a deeper

investigation into the causes of variability. Spearman

Rank Correlation coefficients were used to determine

the relationship between stomatal density, stomatal

index and epidermal cell density (Fig. 2). The

correlations between stomatal density and stomatal

index for both treatments were significant (Fig. 2a).

A significant negative correlation was found between

epidermal cell density and stomatal index for the

leaves grown at 720 µmol mol−" CO#

(Fig. 2b),

whereas no correlation was found between epidermal

cell density and stomatal index for the leaves grown

at 360 µmol mol−" CO#

(Fig. 2b). A significant

positive correlation was also found between epi-

dermal cell density and stomatal density (Fig. 2c) for

both the leaves grown at 360 and 720 µmol mol−"

CO#

(Fig. 2c). These results suggest that the

differentiation of the guard cells (illustrated by the

correlation between stomatal density and stomatal

index) and possibly epidermal cells (illustrated by

the correlation between epidermal cells and stomatal


Table 2. Comparison of stomatal characters between two representative leaves of Alnus

glutinosa from insertion number 9 from one plant grown under 360 µmol mol−" CO#

and another plant grown

under 720 µmol mol−" CO#

CO#

treatment

Character

360

(µmol mol−" CO#)

720

(µmol mol−" CO#)

720:360 (%)

(µmol mol−" CO#)

No. sites sampled 30 40 133

Leaf area (mm−#) 2260 3770 167

Stomatal density

Mean no. pores (mm−#) 291 287 99

Median 294 292

Standard deviation 63±0 40±9Coefficient of variation (%) 10±5 14±3

Epidermal cell density

Mean no. cells (mm−#) 2518 2193 87

Median 2217 2495

Standard deviation 151.3 307.7

Coefficient of variation (%) 6.0 14.0

Stomatal Index

Mean (%) 10±3 11±4 111

Median 11±3 10±3Standard deviation 0±84 1±21

Coefficient of variation (%) 8.2 10.6

Differences between mean values of epidermal cell density and stomatal index were significant at P !0±001. Differences

between mean values of stomatal density were not significant.

density) were the cause of differences in stomatal

index over the leaf surface of plants grown at 360

µmol mol−" CO#. However, although the same

correlations were observed for the plants grown at

720 µmol mol−" CO#, there was an additional

correlation observed between epidermal cell density

and stomatal index. This suggests that epidermal cell

expansion was also a contributory factor to the

variability in stomatal index possibly through a

mixed differentiation–expansion mechanism. How-

ever, this is the subject of ongoing research and

therefore will not be discussed further here.

Physiological differences

At least three replicates of all photosynthetic analyses

were carried out to assess the impact of increasing

CO#on the stomatal conductance and assimilation of

A. glutinosa in relation to current ambient CO#

concentration.

Stomatal conductance values, taken at saturated

PPFD (Fig 3a), in plants grown at elevated CO#

levels revealed a significant decrease (i.e. by 25%,

P !0.05) compared with those plants grown at 360

µmol mol−" CO#

(Fig. 3a). Since stomatal density

was unchanged by an elevated CO#concentration (as

already described), this decrease in stomatal con-

ductance could only have arisen from reduced

stomatal apertures. This therefore suggests that A.

glutinosa adjusts stomatal conductance to the chang-

ing environment through stomatal aperture rather

than stomatal numbers.

However, the maximum assimilation rate at satu-

rating irradiance and CO#(A

max) depended upon the

CO#concentration under which the plant was grown.

A significant increase in maximum assimilation, by

c. 5 µmol CO#m−# s−" (P !0.1), was observed in the

plants grown under 720 µmol mol−" CO#, which

agrees with a number of other species grown under

elevated CO#concentrations (e.g. Long et al., 1996;

Norby et al., 1999). There were no significant

differences found in Vcmax

, Jmax

and triose phosphate

utilization (TPU) rates calculated from the A}Ci

curves (Table 3). This is contrary to a number of

observations made on other tree species (e.g.

Gunderson & Wullschleger, 1994) where a decrease

in Vcmax

was reported in plants grown under elevated

CO#

concentrations.

The relationship between A and PPFD observed

by constructing a PPFD}A curve at the CO#

concentration at which the plants were grown (Fig.

3b) showed there was a significant increase (P !0.05)

in assimilation rate as expected with plants grown at

the elevated CO#

concentration. Analysis of the

PPFD}A curves revealed an increased PPFD satu-

rated maximum assimilation rate of saturating

irradiance (Asat

) by 10 µmol CO#

m−# s−" with a

doubling of the CO#

concentration (Table 3). The

quantum efficiency was significantly lower (i.e. by

66%, P !0.05) in plants grown at 360 than those

grown at 720 µmol mol−" CO#

(Table 3).

PPFD}A curves carried out at CO#

levels of 360

µmol mol−" CO#on plants grown at 720 µmol mol−"

CO#revealed a significantly lower (P !0.05) PPFD


517

3000

2500

2000

1500

1000

500

00 100 200 300 400 500

Stomatal density (cells mm–2)

(c)

Ep

ider

mal

cel

l den

sity

(ce

lls m

m–2

)

12

10

8

6

4

2

00 500 1000 1500 2000 2500

Epidermal cell density (cells mm–2)

(b)

20

15

10

5

00 100 200 300 400 500

Stomatal density (cells mm–2)

(a)S

tom

atal

ind

ex (

%)

3000

14

Sto

mat

al in

dex

(%

)

Fig. 2. Correlations between stomatal and epidermal cell

characters of two Alnus glutinosa leaves grown at ambient

and elevated CO#concentrations. (a) Correlations between

stomatal density and stomatal index at 360 µmol mol−" CO#

(r# ¯ 0.829, P !0.001) and 720 µmol mol−" CO#

(r# ¯0.441, P !0.001) ; (b) correlations between epidermal cell

density and stomatal index at 360 µmol mol−" CO#

(r# ¯0.069, P"0.05) and 720 µmol mol−" CO

#(r# ¯ 0.407,

P !0.001) ; (c) correlations between stomatal density

and epidermal cell density at 360 µmol mol−" CO#

(r# ¯0.494, P !0.001) and 720 µmol mol−" CO

#r# ¯ 0.428, P

!0.001). Open circles represent Values from one repre-

sentative leaf at insertion number 9 grown at 720 µmol

mol−" CO#

(open circles) and at 360 µmol mol−" CO#

(closed circles).

saturated rate of photosynthesis (c. 12 µmol CO#m−#

s−") when compared with PPFD}A curves carried

out at 720 µmol mol−" CO#

(Table 3). However,

although this rate was lower it was not significantly

different (P ¯ 0.36) from the rate obtained from the

plants grown, and measured, at 360 µmol mol−" CO#

(Table 3). Possible acclimation of photosynthesis

was further studied using PPFD}A analysis per-

formed at 720 µmol mol−" CO#

on plants grown at

25

20

15

10

5

0

0 500 1000 1500 2000 2500PPFD (µmol photons m–2 s–1)

(b)

A (

µmo

l CO

2 m

–2 s

–1)

500

400

300

200

100

0360 720

CO2 mole fraction (µmol mol–1)

(a)

Gs

(mm

ol m

–2 s

–1)

Fig. 3. The effect of CO#

concentration on stomatal

conductance (Gs) and PPFD-dependent rates of photo-

synthesis on three leaves of Alnus glutinosa grown under

360 µmol mol−" CO#

(black bar, closed circles) and 720

µmol mol−" CO#(white bar, open circles). (a) Influence of

growth CO#

concentration on mean leaf conductance

measurement taken at saturating PPFD, (b) influence of

PPFD on net assimilation rate (A) (see text for details).

360 µmol mol−" CO#(Table 3). The PPFD saturated

rate of photosynthesis was significantly increased (by

c. 6 µmol CO#

m−# s−", P !0.05) when compared

with the same plants measured at 360 µmol mol−"

CO#. Even though these plants showed an increase in

PPFD saturated rate of photosynthesis, it was still

significantly less (P !0.05) than the rate shown by

the plants grown and measured at 720 µmol mol−"

CO#. Yelle et al. (1989) observed similar responses

and suggested that the decrease in photosynthetic

capacity of plants grown at elevated CO#was caused

by a corresponding decrease in Rubisco activity.

This pattern of acclimation would explain why many

studies have found that plants grown under elevated

CO#

show a decrease in photosynthesis, relative to

control plants, when measured at 360 µmol mol−"

CO#but not when measured at the elevated growth

concentration (Long et al., 1996).

Alnus glutinosa responds to a doubling of atmos-

pheric CO#by a significant (P !0.1) increase in the

PPFD and CO#

saturated Amax

(Table 3). This

agrees with the findings of, for example, Long et al.


(1996) and appears to be due to a lower PPFD

compensation point (as would be expected with

elevated CO#), in conjunction with an increase in

TPU (which indicates an availability of inorganic P

for the Calvin cycle (Sharkey, 1985)) and not an

increase in the quantity and}or activity of Rubisco.

An increase in either quantity or efficiency of

Rubisco (¯ carboxylation conductance) would have

been revealed by an increase in Vcmax

and a greater

carboxylation efficiency (e.g. Harley et al., 1992) but

no significant difference was seen between control

and treatment in this study.

In summary, this study shows the variation in

stomatal characters with elevated CO#. This revealed

a c. 1.6-fold and c. 1.4-fold difference in stomatal

density and stomatal index, respectively, over the

mature leaf as represented in Fig. 1. Interestingly,

there was no difference in mean stomatal density

between plants grown at ambient and elevated CO#.

However, there was a significant increase in mean

stomatal index with elevated CO#.

In addition, this

native British tree species shows a general increase in

growth and photosynthetic parameters together with

a decrease in stomatal conductance when grown at

elevated CO#

concentration and this suggests an

acclimatory effect shown by a change in the PPFD

saturated rate of assimilation when carried out at the

reversed CO#

concentration (i.e. 360 µmol mol−"

CO#). This acclimatory effect is positive rather than

a downregulation of photosynthesis that has been

seen in studies on other species (e.g. Long & Drake,

1992).

This is the first study to report the effects of elevated

CO#

on leaf stomatal counts across whole leaves in

conjunction with studies on photosynthetic capa-

bilities and growth characters of A. glutinosa. The

results agree with other published work docu-

menting variation in growth (Mulholland et al.,

1998), photosynthetic rates (Harley et al., 1992;

Bryant et al., 1998), stomatal characters (Woodward

& Bazzaz, 1988; Woodward & Kelly, 1995; Wagner

et al., 1996) and epidermal cells (Ku$ rschner et al.,

1998) with changing CO#

concentration in species

other than A. glutinosa.

Significant differences between control plants

grown at a CO#

mole fraction of 360 µmol mol−"

CO#

and treatment plants grown at a CO#

mole

fraction of 720 µmol mol−" CO#

were found for

number of branches and specific leaf area. This

supports the observation that an increase in CO#

concentration enhances plant growth (Long, 1994).

No consistent pattern in stomatal characters across

leaves of A. glutinosa grown at the different CO#

concentrations was observed which accords with

Poole et al. (1996) where the same phenomenon in

leaves of the same species grown under sun and

Table

3.E

ffec

tsofel

evate

dC

O#in

rela

tion

toam

bie

ntC

O#on

photo

synth

etic

chara

cter

sofatle

ast

thre

ele

aves

ofA

lnus

glu

tinosa

atin

sert

ion

num

ber

9asdet

erm

ined

from

net

CO

#ass

imilation

rate

}inte

rnalC

O#

part

ialpre

ssure

(A}C

i)and

PP

FD

}Acu

rves

(see

text

and

Fig

.3

for

det

ails)

A}C

i

CO

#tr

eatm

ent

( µm

olm

ol−

"C

O#)

PPF

Dand

CO

#sa

tura

ted

Am

ax

(µm

olC

O#

m−#

s−")

Vcm

ax

(µm

olC

O#

m−#

s−")

J max

(µm

olC

O#

m−#

s−")

TPU

(µm

olC

O#

m−#

s−")

Carb

oxyla

tion

effi

cie

ncy

(µm

olC

O#

m−#

s−")

Com

pensa

tion

poin

t

(µm

olm

ol−

"C

O#)

360

18±9

32±3

87±5

6±9

0±0

54

9±2

9

720

24±1

28±0

88±5

8±7

0±0

49

8±3

5

t-te

stP

!0±1

ns

ns

ns

ns

P!

0±1

PPF

D}A

Quantu

meffi

cie

ncy

PPF

Dcom

pensa

tion

PPF

Dsa

tura

ted

Asat(µ

molC

O#

m−#

s−")

CO

#tr

eatm

ent

PPF

Dsa

tura

ted

Asat

(µm

olC

O#

µm

ol−

"poin

t(µ

molphoto

ns

(µm

olm

ol−

"C

O#)

(µm

olC

O#

m−#

s−")

photo

ns)

m−#

s−")

Measu

red

at

360

Measu

red

at

720

t-te

st

360

11±5

30±0

37

19±9

811±5

317±0

6P

!0±0

5

720

22±5

50±0

64

17±5

29±4

22±5

3P

!0±0

5

t-te

stP

!0±0

5P

!0±0

5ns

ns

P!

0±0

5

Ele

vate

dC

O#,720

µm

olm

ol−

"C

O#;am

bie

nt

CO

#,360

µm

olm

ol−

"C

O#.

Vcm

ax,m

axim

um

rate

of

carb

oxyla

tion

;J m

ax,light

satu

rate

dra

teof

ele

ctr

on

transp

ort

;T

PU

,ra

teof

trio

sephosp

hate

utilization.

Revers

ed

CO

#m

ole

fraction

PPF

D}A

curv

esw

ere

carr

ied

outat720

µm

olm

ol−

"C

O#on

pla

nts

gro

wn

at360

µm

olm

ol−

"C

O#and

at360

µm

olm

ol−

"C

O#on

pla

nts

gro

wn

at720

µm

ol

mol−

"C

O#

and

statist

ically

com

pare

dw

ith

the

curv

es

carr

ied

out

under

the

concentr

ation

at

whic

hth

epla

nts

were

gro

wn.ns,

not

signifi

cant.


519

shade conditions is illustrated. Such natural variation

emphasizes the need to adopt a consistent sampling

protocol that takes into account the intrinsic vari-

ation in stomatal characters over a leaf lamina. This

would allow a reference point to be established for

the species under study and with which any

treatment effects can be compared (Poole &

Ku$ rschner, 1999). Intra-leaf stomatal variation in A.

glutinosa leaves grown at the control CO#

con-

centration appeared to be due to differentiation of

guard cells (differentiation hypothesis) whereas

intra-leaf stomatal variation at the treatment CO#

concentration can be attributed to cell differentiation

and epidermal cell expansion (the combined differen-

tiation and expansion hypothesis). No statistical

difference was found for stomatal densities between

leaves grown under the control and treatment CO#

concentration, a phenomenon also observed on

mature leaves by, for example, Radoglou & Jarvis

(1990 a,b) and Ceulemans et al. (1995). Statistically

significant differences were obtained for stomatal

index values between leaves grown under the control

and treatment CO#concentrations. The interactions

of the environmental parameters, leaf age and

position, significantly confound the CO#

treatment

effect on stomatal and epidermal cell densities, which

might contribute to the problem of stomatal density

reduction under elevated atmospheric CO#and thus

illustrate the need for similar studies to cite stomatal

index values rather than solely stomatal density

values.

Significant changes were found in stomatal con-

ductance and assimilation rate between plants grown

at the control and treatment CO#

concentrations,

which agrees with the findings of, for example, Long

et al. (1996), Mulholland et al. (1997) and Kellomaki

& Wang (1997). Since stomatal density was un-

changed by elevated CO#concentration this suggests

the change in conductance might have risen from a

change in stomatal aperture rather than stomatal

numbers. Surprisingly no significant differences

were found in Vcmax

, Jmax

and TPU rates.

In trying to determine responses of plants to

increased atmospheric CO#

concentrations an ex-

perimental single-step CO#enrichment on seedlings

of long-lived plants might not be totally represen-

tative of responses to global increases of 0.1–0.2 µmol

mol−" CO#

yr−" (Gunderson & Wullschleger, 1994;

Ko$ rner, 1995). Therefore it is suggested that future

work be directed towards longer term experimental

studies, such as those conducted under free air CO#

enrichment (FACE; Miglietta et al., 1998) or open

top chamber experiments (Ceulemans et al., 1997;

Mulholland et al. 1997). Furthermore the results

presented here suggest that more work needs to be

carried out on plant responses at the cellular level to

elevated CO#. This could be investigated with

respect to: responses of tree species in terms of

possible differences between seedlings and the

mature plant, responses of non-arboreal species

compared with trees, and response in terms of carry-

over between generations (Case et al., 1998). Studies

to determine the effect of elevated atmospheric CO#

concentrations, via epidermal cell expansion and}or

guard cell differentiation on stomatal variation, will

then provide a greater understanding of plant

adaptational response to our changing environment.

This research was funded by a grant from the Misses

Durham Bequest, which is gratefully acknowledged. The

authors wish to thank Dr R. Parsons for help in setting up

the environmental chambers and Dr J. Morison for useful

discussions. T.L. was supported by the UK BBSRC.

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Documents

Effect of elevated CO2 on the stomatal distribution and leaf physiology of Alnus glutinosa