Anatomical and Morphological Alterations in Longleaf Pine Needles Resulting from Growth in
Elevated CO2: Interactions with Soil Resource Availability
Author(s): Seth G. Pritchard, Cecilia Mosjidis, Curt M. Peterson, G. Brett Runion, Hugo H.
Rogers
Source: International Journal of Plant Sciences, Vol. 159, No. 6 (November 1998), pp. 10021009
Published by: The University of Chicago Press
Stable URL: http://www.jstor.org/stable/10.1086/314092 .
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Int. J. Plant Sci. 159(6):1002–1009. 1998.
䉷 1998 by The University of Chicago. All rights reserved.
1058-5893/98/5906-0012$03.00
ANATOMICAL AND MORPHOLOGICAL ALTERATIONS IN LONGLEAF PINE NEEDLES
RESULTING FROM GROWTH IN ELEVATED CO2: INTERACTIONS WITH
SOIL RESOURCE AVAILABILITY
Seth G. Pritchard,1,* Cecilia Mosjidis,† Curt M. Peterson,‡ G. Brett Runion,§ and Hugo H. Rogers*
*USDA-ARS National Soil Dynamics Laboratory, Auburn, Alabama 36831, U.S.A.; †Department of Horticulture, Auburn University, Auburn,
Alabama 36849, U.S.A.; ‡Department of Biology, University of Northern Colorado, Greeley, Colorado 80639, U.S.A.; §School of
Forestry, and Alabama Agricultural Experiment Station, Auburn University, Auburn, Alabama 36849, U.S.A.
Studies of anatomical changes in longleaf pine (Pinus palustris Mill.) needles for plants exposed to elevated
atmospheric CO2 may provide insight into the potential influences of global CO2 increases on plant productivity.
Longleaf pine seedlings were grown in open-top field chambers supplied with either ambient (∼365 mmol
mol⫺1) or elevated (∼720 mmol mol⫺1) atmospheric CO2 for 20 mo. Two levels of soil nitrogen (40 and 400
g ha⫺1 yr⫺1) and two soil moisture regimes (⫺0.5 or ⫺1.5 MPa predawn xylem pressure potential) were used
in combination with CO2 treatments. Needle tissue was collected 12 and 20 mo after treatment initiation and
subjected to light and scanning electron microscopy. There was no effect of elevated CO2 on stomatal distribution or the proportion of internal leaf area allocated to a given tissue type at either sampling date. Although
the relationships between vascular, transfusion, mesophyll, and epidermal tissue cross-sectional areas to total
leaf cross-sectional areas appear nonplastic, leaves grown in elevated CO2 with low N availability exhibit
anatomical characteristics suggestive of reduced capacity to assimilate carbon, including decreased mesophyll
cell surface area per unit needle volume (in low-N soil). Significantly greater (8%) needle fascicle volume as
a result of growth in elevated CO2 was observed after 12 mo because of thicker needles. After 20 mo of
exposure, there was a trend indicating smaller fascicle volume (8%) in plants grown with elevated CO2
compared with those grown in ambient conditions, resulting from shorter needles and smaller mesophyll,
vascular tissue, and epidermal cell cross-sectional areas. These results indicate short-term stimulation and longterm inhibition of needle growth in longleaf pine as a result of exposure to elevated CO2 and suggest at the
leaf level that pine species are less responsive to elevated CO2 than are dicotyledons, including other tree
species.
Introduction
increased leaf thickness resulting from the formation of an
extra mesophyll cell layer (Rogers et al. 1983a; Thomas and
Harvey 1983; Mousseau and Enoch 1989; Vu et al. 1989) or
increased leaf cell size (St. Omer and Horvath 1984; Radoglou
and Jarvis 1990a, 1992). Studies on pine species have shown
that needles from plants grown for short durations in elevated
CO2 were thicker because of increases in all cell layers in Pinus
taeda (Rogers et al. 1983b). Thomas and Harvey (1983) reported that P. taeda needles were significantly thicker mainly
as a result of increases in mesophyll tissue. Similarly, Conroy
et al. (1986) reported that growth in elevated CO2 caused an
increase in diameter, length, and surface area of Pinus radiata
needles. However, Pritchard et al. (1997) suggested that
phloem cross-sectional area may be reduced in mature needles
of Pinus palustris following exposure of seedlings to elevated
CO2.
Increased mesophyll and vascular tissue area often reported
may be important determinants of both photosynthetic rates
and assimilate transport capacity. However, examining allometric relationships between tissue and whole-leaf cross-sectional areas, mesophyll cell volumes versus intercellular space
volumes, and changes in cell surface area exposed to intercellular spaces may be of more use in evaluating the effects of
elevated CO2 on leaf function than studies that simply measure
leaf thickness. In one study that examined these relationships,
Variability in leaf features is perhaps the most significant of
plant morphological adaptations to disparate environments
(Esau 1977). The close association between anatomical adaptations and efficiency of physiological processes has been
demonstrated (Ashton and Berlyn 1994). Clearly, alterations
of interactions between structural modifications and physiological function resulting from changing global environments
will either increase or decrease fitness (Lewis 1972). However,
it is not known what direction fitness will be driven by increasing atmospheric CO2 (Gunderson and Wullschleger
1994). Because form is inextricably linked to function, subtle
alterations in leaf anatomy may greatly impact the evolutionary fate of species and the systems of which they are a part.
Many studies have shown that leaf anatomy and morphology are sensitive to atmospheric [CO2]. In a dramatic example,
elevated CO2 caused the conversion in leaves of aquatic plants
from the normal land form into the aquatic form (Bristow
1969). In other species, growth in elevated CO2 has led to
1
Author for correspondence and reprints. USDA-ARS National Soil
Dynamics Laboratory, P.O. Box 3439, Auburn, Alabama 36831-3439,
U.S.A.; E-mail pritcsg@mail.auburn.edu.
Manuscript received April 1998; revised manuscript received June 1998.
1002
PRITCHARD ET AL.—CHANGES IN PINE NEEDLES RESULTING FROM ELEVATED CO 2
Leadley et al. (1987) reported that Glycine max leaves were
thicker when grown in elevated CO2; however, they had less
palisade cell surface area per unit of leaf area. Internal chlorenchyma cell surface area exposed to intercellular spaces is
highly correlated with photosynthesis and water use. Additionally, they reported that a greater percentage of total leaf
cross-sectional area was occupied by vascular tissue in leaves
sampled from plants grown in elevated CO2. Similarly, Pushnik
et al. (1995) observed an increase in the percentage of total
needle cross-sectional area in Pinus ponderosa occupied by
vascular tissue.
The reported enhancement of plant growth, including
changes in leaf size, anatomy, and allometry following atmospheric CO2 enrichment, indicates that continued increases
in atmospheric [CO2] may result in physiologically significant
changes in leaf structure and function. However, resource limitations, such as reduced levels of soil N or drought stress,
may mediate plant response to this atmospheric change. For
example, Conroy et al. (1986) reported for P. radiata that
mesophyll area of needles was increased by elevated CO2 when
P was nonlimiting but was not affected when P was limiting.
The purpose of this study was to determine whether anatomical and allometric changes occur in longleaf pine (P. palustris)
needles as a result of CO2 enrichment under optimal conditions
of soil nitrogen and moisture and also under limiting resources.
Material and Methods
Longleaf pine seedlings were grown in 12 open-top chambers (Rogers et al. 1983a) over a 20-mo period in 1993 and
1994. Plants were maintained in 45-L containers (three per
container) filled with a coarse, sandy medium (pH 5.1) (Mitchell et al. 1995). Ambient air (∼365 mmol CO2 mol⫺1) was
supplied to six open-top chambers while six other chambers
received air enriched with elevated CO2 (∼720 mmol CO2
mol⫺1). Two levels of soil N were applied, using a modification
of the method described by Bazzaz and Miao (1993); N was
applied at concentrations of 400 (high) or 40 (low) g ha⫺1 yr⫺1
(Mitchell et al. 1995). Two water-stress treatments (⫺0.5 or
⫺1.5 MPa xylem pressure potential) were implemented after
seedling initiation (19 wk after planting) and were maintained
by installation of Teflon (5-mil fluorinated ethylpropylene)
rain-exclusion caps on all chambers to eliminate rainfall. Adequately watered plants were maintained between 0 and ⫺0.6
MPa predawn xylem water potential whereas water-stressed
plants were allowed to dry to an average value of ⫺1.3 MPa
before watering. Xylem pressure potentials were determined
with a pressure bomb and correlated with gravimetric measurements in order to maintain both water regimes using total
pot weight loss. Treatments were arranged in a split-plot design
with six replications. Carbon dioxide treatments (main plots)
were randomly assigned to chambers. Nitrogen and waterstress treatments (subplots) were randomly assigned in a
2 # 2 factorial design to a total of 16 pots within each chamber. Pot locations were rerandomized monthly to avoid withinchamber location effects.
Samples for microscopy studies were collected at two harvests (March and November 1994, corresponding to 12 and
20 mo after study initiation). Needles selected for the spring
harvest were chosen from a previously delineated cohort of
1003
needles that were known to have expanded entirely under experimental conditions from buds set just before initiation of
treatments. Needles of longleaf pine abscise in their second
year (Chamberlain 1941); thus, considering the length of this
study, needle ontogeny and development to maturity for needles selected at the second harvest (20 mo) also occurred entirely under imposed experimental conditions. Therefore, needles from the 12-mo harvest were from the same cohort of
needles while the needles from the second (20-mo) harvest may
have represented cohorts from one of several bud flushes.
However, they were clearly second-year needles because of the
presence of secondary needle phloem.
At each harvest, needle pieces (4 mm) were excised from
center portions of needles and were fixed for 2 h in 5% buffered glutaraldehyde (pH 6.8) under aspiration (⫺0.1 to ⫺0.2
MPa) for light microscopy studies. Samples were then trimmed
at both ends to leave a 2-mm piece that was transferred to
fresh fixative for another 2 h. Tissue was aspirated as before,
and the fixative was changed once more after 2 h had elapsed.
Tissue was rinsed three times in phosphate buffer after an
initial 6 h in the three changes of glutaraldehyde solution and
then fixed overnight in 2% osmium tetroxide at 4⬚C. The tissue
was washed twice in buffer and then passed through an ethanol
and propylene oxide dehydration series. Samples were infiltrated and embedded in Spurr’s resin (Spurr 1969). Thick sections were cut (1.5-mm thickness) on a Sorvall JB-4 microtome
and stained for carbohydrates and protein using periodic
acid–Schiff’s reagent and aniline blue black, respectively (Jensen and Fisher 1969).
Stomate distribution was investigated using scanning electron microscopy (SEM). In preparation for SEM, pine needle
segments were fixed with formalin–acetic acid–alcohol (Jensen
1962). After 24 h, tissue was dehydrated in a graded ethanol
series, critical-point dried, mounted onto aluminum stubs, and
gold coated in a sputter-coater before viewing in a Zeiss (Carl
Zeiss, Jena, Germany) SEM.
Stereological methods, as described by Parkhurst (1982),
were used to measure two anatomical variables likely to affect
photosynthesis, including cell surface area per unit of tissue
volume, and percentage of needle area occupied by cells versus
intercellular spaces. The fraction of needle tissue volume occupied by cells versus air space was determined by overlaying
an acetate sheet containing computer-generated dots (2.15 dots
cm⫺2) onto black-and-white photographs (12.5 # 17.5 cm).
The number of dots falling within cells in a given tissue region
(i.e., mesophyll and transfusion tissue) was divided by the
number of dots falling within the whole tissue layer to determine the fraction of a specific tissue area occupied by cells
versus air space. According to Parkhurst (1982), the fraction
of dots within tissue components provides an estimate of the
volumetric fraction of that component. Dots falling outside of
the section were not included.
The second method used an acetate overlay sheet containing
computer-generated random sampling lines (1.56 lines cm⫺2)
rather than points to measure the amount of cell surface area
within a given volume of tissue. Surface area is of physiological
importance in leaf functions such as transpiration and photosynthesis (Parkhurst 1982). The formula used by Parkhurst
(1982) is as follows:
1004
INTERNATIONAL JOURNAL OF PLANT SCIENCES
S/V ⫽ f 7 C/L,
Table 1
where S/V ⫽ surface area/unit volume of tissue (cm2 cm⫺3),
f ⫽ proportionality factor, C ⫽ total number of “cuts” or intersections, and L ⫽ total length of random sampling lines falling within the area of the section being sampled (cm). A proportionality factor (f) equal to 2 was used (for explanation,
see Parkhurst 1982).
Needle tissue areas and perimeters were determined using
an Optimas (Thomas Optical, Columbus, Ga.) image analysis
system interfaced to a Nikon Optiphot brightfield microscope.
Calibrations of each objective were determined using a stage
micrometer. The area of the epidermis included the sclerified
hypodermis. The area of the transfusion tissue included the
endodermis but excluded the veins.
The number of stomates and stomate rows within a 0.629
mm2 area of needle epidermal surface were counted using SEM.
Fascicle volume was obtained applying the formula for the
volume of a cylinder:
Stomatal Distribution on Longleaf Pine Needles Harvested 12 and
20 mo after the Experiment Was Initiated
Volume ⫽ (needle length)(p)(fascicle radius)2 .
Sections were prepared from three to six leaf segments for
each treatment. Analysis was performed by using the General
Linear Models (GLM) procedure of the Statistical Analysis
System (SAS Institute 1985). Error terms appropriate to the
split-plot analysis were used to test the significance of main
effect variables and their interactions. Differences were considered significant at the P ≤ 0.05 level.
Results
Stomate rows
Flat
needle
surface
Variable
Elevated CO2:
High N/well watered .......
High N/water stressed ......
Low N/well watered ........
Low N/water stressed ......
Ambient CO2:
High N/well watered .......
High N/water stressed ......
Low N/well watered ........
Low N/water stressed ......
Significance levels:
CO2 ..........................
N .............................
W .............................
CO2 # N .....................
CO2 # W ....................
N # W .......................
CO2 # N # W ..............
Curved
needle
surface
Stomatal
densitya
12
20
12
20
12
20
5.2
4.3
4.3
4.5
5.2
4.2
4.2
4.0
9.8
8.2
7.5
7.5
9.7
10.0
9.0
8.5
26.2
26.1
25.7
23.3
32.3
25.2
28.8
26.0
4.8
4.8
3.5
5.3
5.3
4.2
3.7
3.8
8.7
7.8
7.3
7.3
10.0
9.7
9.0
8.7
25.9
24.2
25.2
27.1
30.0
27.8
29.3
26.0
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
∗∗
∗
ns
ns
∗
ns
∗
∗
∗∗
ns
ns
ns
ns
Note. High N ⫽ 40 g m yr N; low N ⫽ 4 g m⫺2 yr⫺1 N; elevated CO2 ⫽ 720 mmol mol⫺1; and ambient CO2 ⫽ 360 mmol mol⫺1.
a
Number of stomates/0.629 mm2.
∗
P ≤ 0.05.
∗∗
P ≤ 0.01.
⫺2
⫺1
There was no effect of elevated CO2 on the number of stomate rows on either the curved or flat needle surfaces or stomatal densities on either needle surface at 12- or 20-mo harvests (table 1). However, it is important to note that stomatal
indices, reflective of stomatal patterning during leaf ontogeny
(Radoglou and Jarvis 1990b), could not be calculated in this
study because individual epidermal cells could not be resolved
using SEM.
Stomatal density was not effected by soil N availability at
either harvest date (table 1). However, needles from plants
grown with high N had significantly more rows of stomates
on the curved surface at the 12-mo (P ⫽ 0.05) and 20-mo
(P ⫽ 0.03) harvests, and on the flat surface at the 20-mo harvest (P ! 0.01), resulting from larger needles (table 2).
Although stomatal density was not affected by N or CO2 at
any harvest, needles from seedlings grown with water stress
had reduced numbers of stomata per unit area compared with
needles grown with adequate water (20-mo harvest only;
P ⫽ 0.01). Increased stomatal density was the result of decreased numbers of stomate rows on the flat needle surface
(table 1; P ⫽ 0.03).
bient CO2 (table 2; fig. 1A; P ⫽ 0.05). This was the result of
15% greater cross-sectional area (table 2; fig. 1A; P ⫽ 0.10),
not longer needles (data not shown; P ⫽ 1.00). Increases in
total cross-sectional area were the result of a small (3%) but
significant increase in transfusion tissue area (table 2; P ⫽
0.005) and nonsignificant increases in mesophyll area (17%)
and vascular tissue area (7%). However, after 20 mo of exposure, plants grown in elevated CO2 had an 8% smaller fascicle volume than those grown in ambient CO2 (table 2; fig.
1B; P ⫽ 0.08) resulting from 7% shorter needles (data not
shown; P ⫽ 0.21), 19% less mesophyll cross-sectional area
(table 2; P ⫽ 0.43), 10% less vascular tissue cross-sectional
area (table 2; P ⫽ 0.19), and 19% less epidermal cell area
(table 2; P ⫽ 0.74).
Nitrogen availability had the greatest and most consistent
impact on needle anatomy. Needles from seedlings grown in
low N had a smaller fascicle volume than those grown with
high N availability at both the 12- and 20-mo harvests because
of shorter needles (data not shown; P ! 0.01) and a smaller
cross-sectional area (table 2). Reduced cross-sectional area was
the result of less mesophyll, vascular, and transfusion tissue
cross-sectional area. There were no main treatment effects of
water on any anatomical variable at either harvest.
Needle Anatomy
Needle Allometry and Stereology
After 12 mo of exposure to elevated CO2, longleaf pine
fascicles had an 8% greater volume than those grown in am-
There were no significant main effects of CO2 treatment on
needle allometric relationships (fig. 1; table 3). However, al-
Needle Stomatal Characteristics
PRITCHARD ET AL.—CHANGES IN PINE NEEDLES RESULTING FROM ELEVATED CO 2
1005
Table 2
Anatomical Measurements from Needles Harvested 12 and 20 mo after the Experiment Was Initiated
Crosssectional
area (mm2)
Variable
Elevated CO2:
High N/well watered . . . . . . .
High N/water stressed . . . . . .
Low N/well watered . . . . . . . .
Low N/water stressed . . . . . .
Ambient CO2:
High N/well watered . . . . . . .
High N/water stressed . . . . . .
Low N/well watered . . . . . . . .
Low N/water stressed . . . . . .
Significance levels:
CO2 ..... . . . . . . . . . . . . . . . . . . . . .
N ........ . . . . . . . . . . . . . . . . . . . . .
W ......... . . . . . . . . . . . . . . . . . . . .
CO2 # N . . . . . . . . . . . . . . . . . . . . .
CO2 # W . . . . . . . . . . . . . . . . . . . .
N # W ... . . . . . . . . . . . . . . . . . . . .
CO2 # N # W . . . . . . . . . . . . . .
Note. High N ⫽ 40 g m yr
mol⫺1; and tr ⫽ 0.05 ≤ P ≤ 0.15.
∗
P ≤ 0.05.
∗∗
P ≤ 0.01.
∗∗∗
P ≤ 0.001.
⫺2
⫺1
Fascicle
volume
(mm2)
Epidermal
area (mm2)
Mesophyll
area (mm2)
Transfusion
tissue area
(mm2)
Vascular
area (mm2)
12
20
12
20
12
20
12
20
12
20
12
20
1.14
1.05
0.85
0.85
1.17
1.14
0.93
0.87
1197
1012
567
575
1494
1412
618
937
0.16
0.14
0.12
0.13
0.17
0.17
0.28
0.13
0.63
0.60
0.47
0.46
0.65
0.62
0.42
0.46
0.23
0.21
0.18
0.19
0.22
0.22
0.17
0.18
0.09
0.10
0.06
0.07
0.13
0.13
0.06
0.10
0.99
0.81
0.77
0.77
1.17
1.22
1.08
1.09
1061
841
580
626
1684
1422
791
937
0.16
0.13
0.11
0.11
0.18
0.16
0.27
0.28
0.52
0.45
0.44
0.44
0.63
0.61
0.55
0.53
0.22
0.16
0.16
0.15
0.21
0.22
0.17
0.18
0.09
0.08
0.06
0.07
0.15
0.12
0.09
0.10
tr
tr
ns
ns
ns
ns
ns
ns
tr
ns
ns
ns
ns
ns
tr
ns
tr
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
∗
∗∗∗
∗∗∗
ns
ns
ns
ns
ns
ns
ns
ns
N; low N ⫽ 4 g m
⫺2
∗∗∗
ns
⫺1
yr
∗
ns
ns
ns
ns
ns
∗∗
∗∗
tr
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
∗∗∗
∗∗∗
∗∗∗
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
tr
∗
ns
N; elevated CO2 ⫽ 720 mmol mol ; and ambient CO2 ⫽ 360 mmol
though not statistically significant, there was a 12% reduction
in mesophyll cell surface area per unit area of volume in needles
grown with elevated CO2 compared with ambient CO2 at the
12-mo harvest (table 3, see “Mesophyll S/V”; P ⫽ 0.21). At
the 20-mo harvest, there was a trend for a CO2 # N interaction
for this characteristic; when grown in N-limiting soil, exposure
of seedlings to elevated CO2 resulted in 17% less mesophyll
cell surface area per unit of volume compared with those
grown in ambient CO2 (table 3; P ⫽ 0.14). There was also a
trend for a CO2 # N interaction within the transfusion tissue
at the 12-mo harvest; needles from plants grown in elevated
CO2 had 9% less transfusion tissue cell surface area than those
grown in ambient CO2 when N was limiting. However, when
N was nonlimiting, plants grown in elevated CO2 had 14%
more transfusion cell surface area than those grown in ambient
CO2 (table 3).
The ratio of cell volume to intercellular space volume (table
3) may also have a large effect on dynamics of carbon uptake
and water loss. There were no significant main effects of elevated CO2 on this variable in the transfusion or mesophyll
tissue. However, there were consistent interactive effects of
water with CO2. For the mesophyll tissue region, the nature
of this interaction differed from the 12-mo to the 20-mo harvests. At the 12-mo harvest, the cell area : intercellular space
area ratio (CA : ISA) was higher in elevated compared with
ambient CO2 when plants were well watered (95% vs. 91%).
However CA : ISA was lower in elevated compared with ambient grown plants when subjected to periodic episodes of
water stress (92% vs. 95%). At the 20-mo harvest, CA : ISA
⫺1
was lower in elevated CO2 compared with elevated CO2 when
adequately watered (68% vs. 80%) but was higher in elevated
CO2 than in ambient CO2 when plants were grown with water
stress (78% vs. 59%).
Although it appears that longleaf pine needles may exhibit
plasticity with respect to cellular organization within the mesophyll and transfusion tissue regions (table 3), the proportion
of leaf area allocated to a given tissue type (allometry) appears nonplastic (fig. 1). The proportions of needle crosssectional area occupied by the epidermis, mesophyll, transfusion tissue, and vascular tissue were constant for needles
collected at the same time regardless of treatment-induced
fluctuations in needle size (fig. 1A, B). However, as needles
aged (12 vs. 20 mo), a greater percentage of total crosssectional area was occupied by the vascular tissue and epidermis and a smaller percentage was occupied by the mesophyll and transfusion tissue (fig. 1A, B).
Discussion
It is well established that patterns and rate of development
of stomata are sensitive to environmental conditions (Ticha
1982; Jones 1985; Radoglou and Jarvis 1990b; Boetsch et al.
1996). However, studies that have examined the effects of elevated CO2 on stomatal physical properties have reported
widely variable results. For example, stomatal density has been
reported to increase (Thomas and Harvey 1983), decrease
(Woodward and Bazzaz 1988), or stay the same (Mousseau
and Enoch 1989; Radoglou and Jarvis 1990b, 1992; Estiarte
1006
INTERNATIONAL JOURNAL OF PLANT SCIENCES
Fig. 1 Graphs showing the main treatment effects on needle crosssectional area for the 12-mo (A) and the 20-mo (B) harvests. Bars
(total cross-sectional area in mm2) are subdivided into epidermis (Ep),
mesophyll (Mes), transfusion tissue (T-Tis), and vascular tissue (V);
percentage values presented within each tissue region indicate the percentage of the entire needle cross-sectional area occupied by that tissue
type. HN ⫽ 400 g ha⫺1 yr⫺1 nitrogen; LN ⫽ 40 g ha⫺1 yr⫺1 nitrogen;
WW ⫽ well watered; WS ⫽ water stressed; e CO2 ⫽ elevated CO2
(720 mmol mol⫺1 ); and a CO2 ⫽ ambient CO2 (360 mmol mol⫺1 ).
et al. 1994). Although no studies have experimentally examined the effects of elevated CO2 on stomatal density in a pine
species, Wagner et al. (1996) reported that in fossil leaves of
Pinus flexilis, stomatal density fluctuations correlate with glacial-interglacial CO2 changes. However, results from the current study indicate that stomatal distribution in longleaf pine
is not impacted by growth in elevated CO2.
The increase in fascicle volume at the 12-mo harvest and
decrease in the 20-mo harvest suggest short-term stimulation
and long-term inhibition of needle growth in longleaf pine
resulting from elevated CO2. In other studies on pine species,
Thomas and Harvey (1983) reported that needles were thicker
when grown in elevated CO2 because of a substantial increase
in numbers of mesophyll cells, and Conroy et al. (1986) re-
ported an increase in the diameter, length, and surface area of
individual needles. Conroy et al. (1986) suggested that the
development of a third layer of cells, as suggested by Thomas
and Harvey (1983), may require a longer exposure to elevated
CO2. However, the current study was conducted over 20 mo,
compared with 45 d and 22 wk in the previous studies, and
thus supports the idea that pine needles will ultimately be
similar in size when mature. There is growing evidence in the
literature indicating that in some species, exposure to elevated
CO2 stimulates early growth of leaves, with this effect diminishing over time, ultimately resulting in leaves of similar size
(Pinus taeda, Tolley and Strain 1984; Populus, Radoglou and
Jarvis 1990a; Populus clones, Taylor et al. 1994). Indeed, this
phenomena may reflect photosynthetic acclimation to elevated
atmospheric CO2 concentrations observed to occur over time
in many studies (e.g., Yelle et al. 1989). Gunderson and
Wullschleger (1994) have suggested that alterations in leaf
anatomy of tree species may contribute to the acclimation
phenomena.
Although leaf size is often reported to increase in plants
grown with elevated atmospheric CO2, few studies have examined how more subtle leaf characteristics may be affected,
such as needle volume occupied by cells versus intercellular
spaces, cell surface area exposed to intercellular spaces, and
the proportion of leaf volume occupied by different tissue
types. These characteristics have a large impact on processes
such as photosynthesis and transpiration within the mesophyll
(Parkhurst 1982; Leadley et al. 1987) and may affect movement of water and solutes within the transfusion tissue. The
reductions in mesophyll cell surface area (table 2) and mesophyll cross-sectional area (table 2; 20-mo harvest only) resulting from growth in CO2-enriched atmospheres observed
here provides evidence that longleaf pine may undergo anatomical adaptations that may reduce its capacity to uptake
carbon dioxide (per unit of needle volume) as needles mature.
Similarly, Leadley et al. (1987) reported that, although leaves
of Glycine max were thicker when grown in elevated CO2,
there was less palisade cell surface area per unit of leaf volume.
Clearly, such structural modifications could contribute to
down-regulation in rates of photosynthesis observed to occur
over time in many studies (Amthor 1995).
The longleaf pine seedlings sampled in the current study
exhibited greater rates of photosynthesis when grown in elevated CO2 than when grown in ambient CO2 (G. B. Runion,
unpublished data). Thus, although leaf anatomical characteristics would not intuitively suggest greater photosynthetic capacity based on structural attributes, plants were still able to
fix greater amounts of carbon when grown in elevated CO2.
However, it is important to note that measurements of leaf
photosynthesis were made on needles that were less than 12
mo old (G. B. Runion, unpublished data). In the current study,
anatomy of 12-mo-old needles was more positively affected
by elevated CO2 than were 20-mo-old needles. Furthermore,
greater increases in rates of photosynthesis resulting from
growth in elevated CO2 were observed in plants that were
adequately watered and provided with high N availability
compared with those grown under N and water stress. Moreover, it is important to remember that leaf anatomy reflects
the cumulative effects of treatments over the entire course of
the study (20 mo). Measurements of photosynthetic rates are
PRITCHARD ET AL.—CHANGES IN PINE NEEDLES RESULTING FROM ELEVATED CO 2
1007
Table 3
Stereological Measurements from Needles Harvested 12 and 20 mo
after the Experiment Was Initiated
Variable
Elevated CO2:
High N/well watered . . . . . . .
High N/water stressed . . . . . .
Low N/well watered . . . . . . . .
Low N/water stressed . . . . . .
Ambient CO2:
High N/well watered . . . . . . .
High N/water stressed . . . . . .
Low N/well watered . . . . . . . .
Low N/water stressed . . . . . .
Significance levels:
CO2 . . . . . . . . . . . . . . . . . . . . . . . . . .
N .............................
W .............................
CO2 # N . . . . . . . . . . . . . . . . . . . . .
CO2 # W . . . . . . . . . . . . . . . . . . . .
N # W .......................
CO2 # N # W . . . . . . . . . . . . . .
Transfusion
tissue
cell volume
(% dots)
Mesophyll
S/Va
Transfusion
tissue S/V
Mesophyll
cell volume
(% dots)b
12
20
12
20
12
20
12
20
5.07
4.80
4.78
4.82
5.26
6.00
5.53
5.43
9.01
9.22
8.01
8.73
8.47
7.37
7.61
9.51
94.4
91.9
95.9
92.2
71.0
72.1
65.4
84.1
96.2
95.8
93.5
95.2
67.0
71.0
86.5
65.7
6.29
5.19
5.21
5.15
5.24
5.83
6.52
6.26
8.61
7.44
8.67
8.93
7.36
6.49
7.84
8.08
93.1
95.7
90.1
94.0
88.3
65.3
70.8
52.8
93.9
93.0
94.4
94.7
68.8
79.6
72.8
90.4
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
tr
ns
ns
ns
ns
ns
ns
tr
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
tr
ns
ns
ns
ns
ns
ns
ns
ns
tr
ns
ns
∗
∗
ns
ns
ns
ns
∗∗
ns
∗
Note. High N ⫽ 40 g m⫺2 yr⫺1 N; low N ⫽ 4 g m⫺2 yr⫺1 N; elevated CO2 ⫽ 720 mmol mol⫺1;
and ambient CO2 ⫽ 360 mmol mol⫺1; and tr ⫽ 0.05 ≤ P ≤ 0.15.
a
S/V ⫽ cell surface area per unit of tissue volume (cm2 cm⫺3).
b
% dots ⫽ percentage of area occupied by cells vs. intercellular space.
∗
P ≤ 0.05.
∗∗
P ≤ 0.01.
instantaneous and, in this study, were taken over the course
of a 2-wk period during the active portion of the growing
season, and may not reflect plant function over the course of
longer periods of time. Körner (1991) aptly pointed out that
exclusive use of gas-exchange data to predict plant success has
been overvalued and overrepresented in the literature on plant
response to elevated CO2. Consideration of plant physiological
alterations, structure modifications, and their interactions resulting from growth in elevated CO2 will provide a more holistic concept of how plants will respond to increasing atmospheric CO2 concentrations and will help bridge data collected
at the physiological level to whole-plant and canopy-level processes (Murthy and Dougherty 1997). Future studies on the
effects of elevated CO2 on leaf anatomy should include measurements of photosynthesis and transpiration at the exact time
at which samples for microscopy are collected. Furthermore,
measurements of photosynthesis should be made in different
seasons and also at different times of the day in order to more
closely link structural with functional data.
It has been reported previously that nutrient availability may
mediate the effects of elevated CO2 on pine needle anatomy,
as was observed in the current study. For example, Conroy et
al. (1986) reported for Pinus radiata that the combined crosssectional area of the endodermis, transfusion tissue, and vascular tissue was decreased 14% by exposure to elevated CO2
when P was limiting but was increased 23% by elevated com-
pared with ambient CO2 when P was not limiting. Furthermore, they found that the area of the mesophyll tissue was
38% greater in plants grown in elevated CO2 when P was not
limiting; however, when P was limiting, there was no effect of
CO2 level on mesophyll area.
Other data collected from the longleaf pine seedlings from
which needles were collected for the current study, representing
processes at biochemical, ultrastructural, and whole-plant levels of organization, reflect the interaction of CO2 with N observed here. For example, growth in elevated CO2 increased
above- and belowground biomass in longleaf pine seedlings
grown with high but not low N (Prior et al. 1997b). Needles
from seedlings grown with elevated CO2 and low N exhibited
altered epicuticular wax morphology and decreased wax density compared with plants grown with ambient CO2 and low
N (Prior et al. 1997a). Furthermore, mesophyll chloroplasts
from needles grown with elevated CO2 and low N exhibited
stress symptoms associated with excessive starch accumulation
compared with chloroplasts from plants grown with ambient
CO2 and low N (Pritchard et al. 1997). Increased starch deposition reported by Pritchard et al. (1997) coupled with the
anatomical alterations observed in the current study resulted
in greater specific leaf mass (g m⫺2) in plants grown in elevated
CO2 with low N compared with plants grown in ambient CO2
with low N (Prior et al. 1997a). It is becoming increasingly
evident that pine species are carbon limited (and thus respond
1008
INTERNATIONAL JOURNAL OF PLANT SCIENCES
positively to elevated CO2) only when other resources are provided at optimal levels.
Seemingly contradictory effects of elevated CO2 on leaf anatomy in different seasons of the year as observed in the current
study have been reported previously. For example, Ferris et al.
(1996) reported that in the spring, plants grown in elevated
CO2 had larger leaves as a result of increased cell expansion
and increased epidermal cell density, which resulted in greater
epidermal cell numbers per leaf and increased mesophyll cell
area. However, in the summer, growth in elevated CO2 decreased leaf and cell expansion with reductions in epidermal
cell length and mesophyll cell area. These results, considered
with the findings of the current study, suggest that elevated
CO2 may have differential effects on cell division, cell expansion, and resultant patterns of tissue organization, depending
on resource availability, cell type, and season. Moreover, increased carbon supply as well as altered plant, tissue, and
cellular water potentials would be expected to have independent as well as interactive effects on the cellular and molecular
events controlling cell division, patterning, and expansion. Understanding how these basic processes are regulated, a topic
of immediate interest to developmental plant biologists, will
surely provide a mechanistic basis to explain some of the conflicting reports in the literature concerning CO2 effects on plant
leaf anatomy. Several studies of recent origin have provided
inroads toward understanding the mechanistic basis for plant
structural responses to increased carbon (Francis 1992; Taylor
et al. 1994; Ranasinghe and Taylor 1996; Kinsman et al. 1997)
In conclusion, when exposed to [CO2] predicted for the next
century, needle growth in longleaf pine may be stimulated in
the earlier stages of needle development (12 mo), but, at maturity, needles will be similar in size when soil N availability
is high and slightly smaller when soil N is limiting. Furthermore, although allometric relationships relating different leaf
tissue cross-sectional areas to whole-leaf cross-sectional areas
appear nonplastic, leaves grown in elevated CO2 with low N
availability generally exhibit anatomical characteristics suggestive of reduced capacity to assimilate carbon, such as decreased mesophyll cell surface area per unit needle volume and
decreased mesophyll cross-sectional area (20-mo sampling
date). This lack of a positive long-term response to elevated
atmospheric CO2 contrasts with many studies on dicot species
reporting an increase in final leaf size as a result of elevated
CO2 (Rogers et al. 1983b; Thomas and Harvey 1983; St. Omer
and Horvath 1984; Mousseau and Enoch 1989; Vu et al. 1989;
Kelly et al. 1991) and also with short-term studies on pine
needles reporting increases in size (Rogers et al. 1983b; Thomas and Harvey 1983; Conroy et al. 1986). Since leaf structural adaptations are intimately correlated with efficiency of
physiological processes (Lewis 1972; Jones 1985; Ashton and
Berlyn 1994), lack of a sustained positive response to elevated
CO2 by pine needles may partially explain the differences in
response patterns between pines and broad-leaved species. Pine
species have been shown to be far less responsive to elevated
CO2 than other tree species (Tolley and Strain 1984; Kaushal
et al. 1989; Ceulemans and Mousseau 1994; Gunderson and
Wullschleger 1994). Effects of anatomical leaf adjustments on
long-term tree productivity and, ultimately, the ability of longleaf pine to compete in nature with more responsive forest
species remain to be elucidated.
Acknowledgments
This material is based on research supported by the Southeastern Regional Center of the National Institute for Global
Environmental Change by the U.S. Department of Energy under cooperative agreement DE-FC03-90ER61010, the Experimental Program to Stimulate Competitive Research by the
U.S. Environmental Protection Agency under contract
R821826-01-1, the U.S. Department of Agriculture under contract 93-34208-9058B for effect on water quality, and by Alabama Agricultural Experiment Station project 50-010. Any
opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not
necessarily reflect the views of the U.S. Department of Energy
or the U.S. Environmental Protection Agency. We thank Leigh
Crouse and Regina Burton for technical assistance and S. Prior,
J. Qiu, and M. Davis for critical reviews of this article.
Literature Cited
Amthor JS 1995 Terrestrial higher-plant response to increasing atmospheric [CO2] in relation to the global carbon cycle. Global
Change Biol 1:243–274.
Ashton PMS, GP Berlyn 1994 A comparison of leaf physiology and
anatomy of Quercus (section Erthrobalanus-Fagaceae) species in
different light environments. Am J Bot 81:589–597.
Bazzaz FA, SL Miao 1993 Successional status, seed size, and responses of tree seedlings to CO2, light and nutrients. Ecology 74:
104–112.
Boetsch J, J Chin, M Ling, J Croxdale 1996 Elevated carbon dioxide
affects the patterning of subsidiary cells in Tradescantia stomatal
complexes. J Exp Bot 47:925–931.
Bristow JM 1969 The effects of carbon dioxide on the growth and
development of amphibious plants. Can J Bot 47:1803–1807.
Ceulemans R, M Mousseau 1994 Effects of elevated atmospheric
CO2 on woody plants. New Phytol 127:425–446.
Chamberlain CJ 1941 Gymnosperms structure and function. University of Chicago Press, Chicago.
Conroy J, EWR Barlow, DI Bevege 1986 Response of Pinus radiata
seedlings to carbon dioxide enrichment at different levels of water
and phosphorus: growth, morphology and anatomy. Ann Bot 57:
165–177.
Esau K 1977 Anatomy of seed plants. 2d ed. Wiley, Toronto.
Estiarte M, J Peñuelas, BA Kimball, SB Idso, RL La Morte, PJ Pinter
Jr, GW Wall, RL Garcia 1994 Elevated CO2 effects on stomatal
density of wheat and sour orange trees. J Exp Bot 45:1665–1668.
Ferris R, I Nijs, T Behaeghe, I Impens 1996 Elevated CO2 and temperature have different effects on leaf anatomy of perennial ryegrass
in spring and summer. Ann Bot 78:489–497.
Francis D 1992 The cell cycle in plant development. New Phytol 122:
1–22.
Gunderson CA, SD Wullschleger 1994 Photosynthetic acclimation in
trees to rising atmospheric CO2: a broader perspective. Photosynth
Res 39:369–388.
Jensen WA 1962 Botanical histochemistry. W. H. Freeman, San Francisco. 407 pp.
PRITCHARD ET AL.—CHANGES IN PINE NEEDLES RESULTING FROM ELEVATED CO 2
Jensen WA, DB Fisher 1969 Cotton embryogenesis: the entrance and
discharge of the pollen tube in the embryo sac. Planta 78:158–183.
Jones HG 1985 Adaptive significance of leaf development and structural responses to environment. Pages 155–173 in NR Baker, WJ
Davies, CK Ong, eds. Control of leaf growth. Cambridge University
Press, Cambridge.
Kaushal P, JM Guehl, G Aussenac 1989 Differential growth response
to atmospheric carbon dioxide enrichment in seedlings of Cedrus
atlantica and Pinus nigra ssp. Laricio var. Corsicana. Can J For Res
19:1351–1358.
Kelly DW, PR Hicklenton, EG Reekie 1991 Photosynthetic response
of Geranium to elevated CO2 as affected by leaf age and time of
CO2 exposure. Can J Bot 69:2482–2488.
Kinsman EA, C Lewis, MS Davies, JE Young, D Francis, B Vilhar, HJ
Ougham 1997 Elevated CO2 stimulates cells to divide in grass meristems: a differential effect in two natural populations of Dactylis
glomerata. Plant Cell Environ 20:1309–1316.
Körner CH 1991 Some overlooked plant characteristics as determinates of plant growth: a reconsideration. Funct Ecol 5:162–173.
Leadley PW, JA Reynolds, JF Thomas, JF Reynolds 1987 Effects of
CO2 enrichment on internal surface area in soybeans. Bot Gaz 148:
137–140.
Lewis MC 1972 The physiological significance of variation in leaf
structure. Sci Prog 60:25–51.
Mitchell RJ, GB Runion, SA Prior, HH Rogers, JS Amthor, FP Henning 1995 Effects of nitrogen on Pinus palustris foliar respiratory
responses to elevated atmospheric CO2 concentration. J Exp Bot 46:
1561–1567.
Mousseau M, HZ Enoch 1989 Carbon dioxide enrichment reduces
shoot growth in sweet chestnut seedlings (Castanea sativa Mill.)
Plant Cell Environ 12:927–934.
Murthy R, PM Dougherty 1997 Effect of carbon dioxide, fertilization
and irrigation on loblolly pine branch morphology. Trees 11:
485–493.
Parkhurst DF 1982 Stereological methods for measuring internal leaf
structure variables. Am J Bot 69:31–39.
Prior SA, SG Pritchard, GB Runion, HH Rogers, RJ Mitchell 1997a Influence of atmospheric CO2 enrichment, soil N and
water on needle surface wax formation in Pinus palustris (Pinaceae).
Am J Bot 84:1070–1077.
Prior SA, GB Runion, RJ Mitchell, HH Rogers, JS Amthor 1997b Effects of atmospheric CO2 on longleaf pine: productivity and allocation as influenced by nitrogen and water. Tree Physiol 17:397–405.
Pritchard SG, CM Peterson, SA Prior, HH Rogers 1997 Elevated atmospheric CO2 differentially affects needle chloroplast ultrastructure and phloem anatomy in Pinus palustris: interactions with soil
resource availability. Plant Cell Environ 20:461–471.
1009
Pushnik JC, RS Demaree, JLJ Houpis, WB Flory, SM Bauer, PD Anderson 1995 The effect of elevated CO2 on a Sierra-Nevadan dominant species: Pinus ponderosa. J Biogeogr 22:249–254.
Radoglou KM, PG Jarvis 1990a Effects of CO2 enrichment on four
poplar clones. I. Growth and leaf anatomy. Ann Bot 65:617–626.
——— 1990b Effects of CO2 enrichment on four poplar clones II.
Leaf surface properties. Ann Bot 65:627–632.
——— 1992 The effects of CO2 enrichment and nutrient supply on
growth morphology and anatomy of Phaseolus vulgaris L. seedlings.
Ann Bot 70:245–256.
Ranasinghe S, G Taylor 1996 Mechanism for increased leaf growth
in elevated CO2. J Exp Bot 47:349–358.
Rogers HH, WW Heck, AS Heagle 1983a A field technique for the
study of plant responses to elevated carbon dioxide concentrations.
Air Pollut Control Assoc J 33:42–44.
Rogers HH, JF Thomas, GE Bingham 1983b Response of agronomic
and forest species to elevated atmospheric carbon dioxide. Science
220:428–429.
SAS Institute 1985 SAS user’s guide: statistics. Version 5 ed. SAS Institute, Cary, N.C.
Spurr AR 1969 A low-viscosity epoxy resin embedding medium for
electron microscopy. J Ultrastruct Res 26:31.
St. Omer L, SM Horvath 1984 Developmental changes in anatomy,
morphology and biochemistry of Layia platyglossa exposed to elevated carbon dioxide. Am J Bot 71:693–699.
Taylor G, S Ranasinghe, C Bosac, SDL Gardner, R Ferris 1994 Elevated CO2 and plant growth: cellular mechanisms and
responses of whole plants. J Exp Bot 45:1761–1774.
Thomas JF, CN Harvey 1983 Leaf anatomy of four species grown
under continuous CO2 enrichment. Bot Gaz 144:303–309.
Ticha I 1982 Photosynthetic characteristics during otogenesis of
leaves. 7. Stomata density and sizes. Photosynthetica 16:375–471.
Tolley LC, BR Strain 1984 Effects of CO2 enrichment on growth of
Liquidambar styraciflua and Pinus taeda seedlings under different
irradiance levels. Can J For Res 14:343–350.
Vu JCV, LH Allen Jr, G Bowes 1989 Leaf ultrastructure, carbohydrates and protein of soybeans grown under CO2 enrichment. Environ Exp Bot 29:141–147.
Wagner F, R Below, P De Klerk, DL Dilcher, H Joosten, WM Küreschner, H Visscher 1996 A natural experiment on plant acclimation:
lifetime stomatal frequency response of an individual tree to annual
atmospheric CO2 increase. Proc Natl Acad Sci USA 93:
11705–11708.
Woodward FI, FA Bazzaz 1988 The responses of stomatal density to
CO2 partial pressure. J Exp Bot 39:1771–1781.
Yelle S, RC Beeson, MJ Trudel, A Gosselin 1989 Acclimation of two
tomato species to high atmospheric CO2. Plant Physiol 90:
1465–1472.