Journal of Experimental Botany, Vol. 45, No. 280, pp. 1665-1668, November 1994
Journal of
Experimental
Botany
SHORT COMMUNICATION
Elevated CO2 effects on stomatal density of wheat and
sour orange trees
M. Estiarte 1 , J. Penuelas1-3, B.A. Kimball2, S.B. Idso2, R.L. LaMorte 2 , P.J. Pinter2 Jr, G.W. Wall 2
and R.L. Garcia2
1
Received 19 May 1994; Accepted 15 August 1994
Abstract
No significant differences were found in stomatal
densities or stomatal indices of wheat or sour orange
trees grown at high CO2 concentrations in two different CO2 enrichment systems (Free-Air CO 2 Enrichment
for wheat and Open-Top Chambers for orange trees).
These results are in accordance with most of the previous results obtained in short-term experimental studies which suggest that plants do not acclimate to
increasing CO2 concentration by changing stomatal
density within a single generation.
Key words: Triticum aestivum, Citrus aurantium, stomatal
density, elevated atmospheric CO2.
Introduction
Stomata regulate the rates of carbon uptake and water
loss (Friend and Woodward, 1990). The light regime is
the most important determinant of stomatal density, but
atmospheric CO2 concentrations have also been reported
as secondary determinants (Woodward, 1987). In many
cases, variations in stomatal density under different environmental conditions are a consequence of variations in
the final size of epidermal cells and not of variations in
the stomatal index (number of stomata related to total
epidermal cell number) (Ticha, 1982).
Several papers have reported changes in stomatal parameters of fossil and herbarium plant material and correlated them to changes in atmospheric CO2 concentration.
Fossil tree leaves from the last 10 million years (Van der
' To whom correspondence should be addressed. Fax: +34 3 581 13 12.
O Oxford University Press 1994
Burg et al., 1993) and from the last 140000 years (Beerling
et ai, 1993) and herbarium leaves from the last three
centuries (Woodward, 1987; Penuelas and Matamala,
1990; Van der Burg et ai, 1993) have shown a pattern of
stomatal density inversely correlated with atmospheric
CO2 concentrations. However, there are also reports
showing no significant differences in herbaceous plants
from the last 100 years (KSrner, 1988). The results
obtained with plants experimentally grown under lower
levels than current CO2 atmospheric concentrations have
also shown an increase in stomatal numbers (Woodward
and Bazzaz, 1988). In greenhouse conditions, a lower
stomatal density of leaves under elevated CO2 has also
been reported (Madsen, 1973). It is suspected that such
changes are caused by a reduction in the initiation of
stomata as atmospheric CO2 increases.
Typically, however, plants grown under artificially
CO2-enriched atmospheres do not show this pattern
of stomatal reduction as CO2 increases (Thomas and
Harvey, 1983; Kelly et al., 1991; Woodward and Bazzaz,
1988; Radoglou and Jarvis, 1990, 1992). It is not clear,
then, whether the higher atmospheric CO2 concentrations
foreseen for the next decades will produce changes in
stomatal numbers. New insights on short-term versus
long-term responses, phenotypic acclimation versus genotypic adaptation and higher-than-present versus lowerthan-present atmospheric CO2 concentrations are needed.
We provide data on stomata of plants grown under
two different CO2 enrichment conditions: wheat plants in
an open-field with Free-Air CO2 Enrichment (FACE)
and sour orange trees in Open-Top Chambers (OTC).
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CREAF and Centre de Referenda d'Estudis Ambientals, Universitat Autdnoma, Facultat de Ciencies,
08193 Bellaterra, Barcelona, Spain
2
US Water Conservation Laboratory, Agricultural Research Service, US Department of Agriculture,
4331 E. Broadway, Phoenix, AZ 85040, USA
1666
Estiarte et al.
Our aim was to study stomatal numbers under higherthan-present atmospheric CO2 concentrations. A secondary aim was to compare the CO2 response in FACE and
OTC in order to assess possible disturbing effects of
chambers on stomatal numbers.
Materials and methods
No statistical differences (ANOVA) in wheat stomatal
density or stomatal index were found among the four
CO2-irrigation treatments, either in the adaxial or in the
abaxial epidermis (Fig. la, b).
However, plants grown under the dry treatments tended
to have lower stomatal densities in adaxial epidermis and
higher in abaxial epidermis than those from the wet
treatments. Plants from the FACE treatments had slightly
higher stomatal densities in adaxial epidermis and lower
in abaxial epidermis than those from control treatments.
In any case, FACE treatments had more influence on the
dry treatments than on the wet treatments (Fig. la).
Similar to wheat, orange tree leaves did not show any
clear effect of different concentrations of atmospheric
CO2 on stomatal density (Fig. lc).
There were no significant changes in the number of
stomata or in the ratio of the number of stomata to the
total number of epidermal cells in either the orange tree
or the wheat studies (Fig. lb, d).
Orang*
I
I ADAHAL EPIDERMIS
I V \ 1 ABAXIAL EPIDERMIS
Wheat
I
I ADABAL EPIDERMIS
f\~\| ABAXIAL EPIDERMIS
CD
CW
tr*«
(c)
(b)
CD
FW
(4)
V
//A
E
Fig. 1. Stomatal densities (±SE) (a, c) and stomatal indices (±SE) (b,
d) of wheat flag leaves (a, b) and sour orange tree leaves (c. d) under
FACE (F, 550 ^mol mol"1), control (C, ~360^mol mol"'), ambient
(A, -360^mol mol"1), and enriched (E, ambient + 300 ^mol mol"1)
CO2 concentrations and well-watered (W, wet) and dry (D) irrigation
regimes. Wheat results are from abaxial and adaxial surfaces of leaves
from the FACE experiment, while orange tree results are from abaxial
surface of leaves from the OTC experiment.
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A wheat crop {Triticum aestivum L. cv. Yecora rojo, a hard red
spring wheat) was grown in flat beds in an open field at
Maricopa, Arizona, USA. The soil was Trix clay loam (fineloamy, mixed (calcareous) hyperthermic Typic Torrifluvent).
The wheat was planted on 15 December 1992. It emerged on
1 January 1993, and was harvested during the third week of
May 1993. It was sown in rows spaced 25 cm apart at a
population of 130 plants per m2. The crop was irrigated using
a subsurface drip system with tubes spaced 0.5 m apart at a
depth of about 0.2 m. Emitters were spaced every 0.3 m along
each tube.
Similar to previous cotton experiments (Mauney et al, 1994),
20 m-diameter plots of the wheat were subjected to 550 ^mol
mol"1 of CO2 from a free-air CO2 enrichment (FACE) system.
Unlike the cotton experiments, however, the CO2 enrichment
was supplied 24 h per day from emergence until harvest. There
were also control plots at ambient CO2 concentrations (about
370 fimol mol"1 during daytime). Four replicates of both FACE
and control treatments were included.
The wheat plots were split into semi-circular subplots, half
of which were well-watered using the drip irrigation system by
frequent replacement of the water lost by potential evapotranspiration (wet treatment) and half of which received half as
much water as the wet plots (dry treatment). Thus, there were
a total of 16 plots (two concentrations of CO2, two rates of
water supply, four replicates).
Eight sour orange {Citrus aurantium L.) trees rooted in the
ground have been grown from the seedling stage in clearplastic-wall open-top chambers since 1987 (Idso and Kimball,
1992). Half of the trees have been exposed to air enriched with
CO2 by 300 ^mol mol"1 above ambient (350-380 ^mol mol"1
during the daytime and much higher at night). Water and
nutrients have been supplied at ample rates. The trees in the
CO2-enriched atmosphere have exhibited growth rates nearly
triple those of the trees at ambient CO2.
Fully developed flag leaves of wheat were collected on day
125 (5 May), when the wheat was nearing maturity. Three
leaves per plot were collected, resulting in 12 leaves per
treatment. The central part of each leaf was bathed in chloral
hydrate in order to loosen the epidermis (without changing cell
sizes) as described previously (Pefiuelas and Matamala, 1990).
The epidermis from each leaf was mounted on a slide and
photographed with the help of a stereoscope under x 75
magnification. Two fields per slide were randomly selected and
photographed.
Fully developed and exposed leaves from the south side of
each sour orange tree were sampled on 3 December 1992. Two
leaves per tree were sampled yielding eight leaves per plant
treatment. Varnish prints of the middle leaf surface were
collected and photographs were taken under x 160 magnification with the help of a microscope.
Stomatal density was calculated on a leaf surface basis and
stomatal index was calculated as: number of stomata/(number
of stomata + number of epidermal cells) (Salisbury, 1927).
Results and discussion
CO2 effects on stomatal density
Acknowledgements
We gratefully acknowledge financial support from CICYT and
INIA (Spain) grants AGR90-0458 and SC94-011 to JP and a
MEC (Spain) fellowship to ME, from USDA-ARS, and from
the U.S. Dept. of Energy, Atmospheric and Climate Research,
Office of Energy Research under Interagency Agreement no.
DE-AIOS-88ER-69014. We appreciate the help of David Akey,
the technical support of R. Seay and S. Johnson and the
comments on the manuscript by Dr S.R. Malone and Dr
E. Bautista. We also acknowledge and appreciate the
co-operation of the staff of the University of Arizona Mancopa
Agricultural Center.
References
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1993. Stomatal density responds to the glacial cycle of
environmental change. Proceedings of the Royal Society of
London, Series B 251, 133-8.
Friend AD, Woodward M. 1990. Evolutionary and ecophysiological responses of mountain plants to the growing season
environment. Advances in Ecological Research 20, 59-124.
Gaudillere JP, Mousseau M. 1989. Short-term effect of CO,
enrichment on leaf development and gas exchange of young
poplars (Populus euramericana cv. 1 124). Acta Oecologica/
Oecologia Plantarum 10, 95-105.
Grace J, Russell G. 1977. The effect of wind on grasses. 111.
Influence of continuous drought or wind on anatomy and
water relations in Festuca arundmacea Schreb. Journal of
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Idso SB, KimbaM BA. 1992. Above-ground inventory of sour
orange trees exposed to different atmospheric CO, concentrations for three full years. Agricultural and Forest Meteorology
60, 145-51.
Kelly DW, Hicklenton PR, Reekie EG. 1991. Photosynthetic
response of geranium to elevated CO 2 as affected by leaf age
and time of CO 2 exposure. Canadian Journal of Botany
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Kimball BA, Mauney JR, Radin JW, Nakayama FS, Idso SB,
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water relations, and physiology of plants grown under
optimal and limiting levels of water and nitrogen. In:
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Komer C. 1988. Does global increase of CO 2 alter stomatal
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KSrner C, Arnone JA. 1992. Responses to elevated carbon
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Madsen E. 1973. Effect of CO 2 concentration on the morphological, histological and cytological changes in tomato plants.
Acta Agricultura Scandinavica 23, 241-6.
Mauney JR, Kimball BA, Pinter Jr PJ, LaMorte R, Lewin KF,
Nagy J, Hendrey GR. 1994. Growth and yield of cotton in
response to a free-air carbon dioxide enrichment (FACE)
environment. Agricultural
and Forest Meteorology
(in press).
Oberbauer SF, Strain BR, Fetcher N. 1985. Effect of CO 2 enrichment on seedling physiology and growth of two tropical
tree species. Physiologia Plantarum 65, 352-6.
Penuelas J, Matamala R. 1990. Changes in N and S leaf
content, stomatal density and specific leaf area of 14 plant
species during the last three centuries of CO 2 increase. Journal
of Experimental Botany 230, 1119-24.
Radoglou KM, Jarvis PG. 1990. Effects of CO 2 enrichment on
four poplar clones. II. Leaf surface properties. Annals of
Botany 65, 627-32.
Radoglou KM, Jarvis PG. 1992. The effects of CO 2 enrichment
and nutrient supply on growth morphology and anatomy of
Phaseolus vulgaris L. seedlings. Annals of Botany 70, 245-56.
Retuerto R, Woodward Fl. 1993. The influences of increased
CO 2 and water supply on growth, biomass allocation and
Downloaded from http://jxb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 12, 2016
Our results are in accordance with several other sets of
experimental data from plants grown in artificially CO 2 enriched atmospheres. Although some studies (Oberbauer
et al, 1985) have found a decrease in stomatal density
and others have found some increase (Kimball et al,
1986; Gaudillere and Mousseau, 1989), almost all the
reports of experiments on plants growing in artificially
elevated CO 2 have shown no response or a very small
decrease in stomatal density (Thomas and Harvey, 1983;
Oberbauer et al, 1985; Woodward and Bazzaz, 1988;
Radoglou and Jarvis, 1990, 1992; Kelly et al, 1991;
Korner and Arnone, 1992).
Other variables such as humidity, wind, temperature
or irradiance that are modified in experimental conditions
in OTC's and greenhouses could explain some of the
conflicting results in the literature. Water availability
affects stomatal density. Increased humidity in chambers
could reduce stomatal density by increasing epidermal
cell expansion (Ticha, 1982). Wind speed has also been
shown to increase the number of stomata per unit area
as a consequence of a reduction in leaf expansion (Grace
and Russell, 1977). A reduction in stomatal density has
been reported at low wind speeds in chambers as CO 2
concentration increases, while high wind speeds increase stomatal density as CO 2 increases (Retuerto and
Woodward, 1993). These experimental disturbances were
avoided in our FACE study. Nevertheless, we still did
not find any significant differences in stomatal numbers.
Our results are also in accordance with the prediction
that stomatal response to CO 2 concentrations would be
non-linear and that any changes are likely to be smaller
in the range from the current ambient CO 2 concentration
(350 ^mol mol ~') to higher concentrations (e.g. 700 jimo\
mol" 1 ), than from the current to lower concentrations
(225/xmol mol" 1 ) (Woodward and Bazzaz, 1988).
The absence of differences in stomatal density and in
stomatal index supports the hypothesis that there are no
direct effects of CO 2 enrichment on the initiation of
stomata during ontogenesis or in epidermal cell expansion
at a later stage (Radoglou and Jarvis, 1990, 1992), i.e.
that there is no short-term acclimation of the plants within
a single generation. However, these experiments did not
test the genetic component of long-term plant adaptation
to changes in CO 2 concentration that have been suggested
by the observed changes in stomatal numbers from fossil
and herbarium plant material (Woodward, 1987; Pefluelas
and Matamala, 1990; Beerling et al, 1993).
1667
1674
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