Leaf structural responses to recent and projected changes in atmospheric [CO2] and temperature affect leaf function in Eucalyptus sideroxylon.

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Leaf structural responses to pre-industrial, current
and elevated atmospheric [CO2] and temperature affect
leaf function in Eucalyptus sideroxylon
Renee A. SmithA, James D. LewisA,B,C, Oula GhannoumAand David T. TissueA
AUniversity of Western Sydney, Hawkesbury Institute for the Environment, Richmond, NSW 2753, Australia.
BFordham University, Louis Calder Center - Biological Field Station and Department of Biological Sciences,
Armonk, NY 10504, USA.
CCorresponding author. Email: jdlewis@fordham.edu
Abstract.
in climate change research is the role of changes in leaf structure in photosynthetic responses to temperature and
atmospheric CO2concentration ([CO2]), ranging from pre-industrial to future levels. We examined the interactive
effects of [CO2] (290, 400 and 650mLL–1) and temperature (ambient, ambient +4?C) on leaf structural and chemical
traits that regulate photosynthesis in Eucalyptus sideroxylon A.Cunn. ex Woolls. Rising [CO2] from pre-industrial
to elevated levels increased light-saturated net photosynthetic rates (Asat), but reduced photosynthetic capacity (Amax).
ChangesinleafNperunit area(Narea)andthenumberofpalisade layersaccountedfor56and14%ofthe variation inAmax,
respectively, associated with changes in leaf mass per area. Elevated temperature increased stomatal frequency, but did not
affect Amax. Further, rising [CO2] and temperature generally did not interactively affect leaf structure or function. These
results suggest that leaf Nareaand the number of palisade layers are the key chemical and structural factors regulating
photosynthetic capacity of E. sideroxylon under rising [CO2], whereas the lack of photosynthetic responses to elevated
temperature may reflect the limited effect of temperature on leaf structure and chemistry.
Leaf structure and chemistry both play critical roles in regulating photosynthesis. Yet, a key unresolved issue
Additional keywords: leaf anatomy, leaf physiology, nitrogen, photosynthesis, pre-industrial [CO2], stomata,
temperature.
Received 28 May 2011, accepted 16 January 2012, published online 20 March 2012
Introduction
Rising atmospheric CO2 concentrations ([CO2]) have a
significant effect on global climate by increasing temperature.
Over the past 200 years, atmospheric [CO2] has risen from
280 to 390mLL–1(Sage and Coleman 2001; Körner 2006).
Models project [CO2] to reach 600mLL–1within this century,
accompanied by a 0.3–3.4?C rise in mean air temperature for
Australia (Hennessy et al. 2007). Forests may play a key role in
ameliorating rises in [CO2] and temperature because trees
account for ~70% of terrestrial primary production (Field et al.
1998; Atwell et al. 2007) and sequester a sizeable fraction of
CO2released to the atmosphere (Melillo et al. 1993; Schimel
etal.2001;Norbyetal.2005).Consequently,itiscrucialthatwe
understand how the capacity of trees to absorb CO2is influenced
by a changing climate. Although tree responses to elevated
[CO2] have been well documented (Saxe et al. 1998; Norby
et al. 1999; Ainsworth and Long 2005), less is known about
responses to past rises in [CO2] (Lewis et al. 2010) or the
interactive effects of [CO2] and temperature (Ghannoum et al.
2010a).
Akeyunresolvedissueintheresponseofcarbonsequestration
by trees to climate change is the role of leaf anatomy. Leaf
structure is closely linked to the function and growth of the
whole plant and is sensitive to environmental conditions such
as temperature (Klich 2000; Niinemets et al. 2009). Growth
[CO2] may also affect leaf structure, but few general trends
have emerged. Leaf thickness has been reported to increase
(Pritchard et al. 1999) and decrease (Rengifo et al. 2002) with
rising [CO2]. Growth in elevated [CO2] is also reported to
increase (Thomas and Harvey 1983) and decrease mesophyll
cell layer thickness (Oksanen et al. 2005). The effect of elevated
temperature similarly varies (Saxe et al. 2001) and elevated
temperature sometimes enhances (Zha et al. 2001; Vu 2005)
or does not enhance (Bannayan et al. 2009) the response to
elevated [CO2]. There have been few studies of the effects of
pre-industrial [CO2] on leaf anatomy; however, leaf mass per
area (LMA) has been shown to increase with rising [CO2] from
glacial or pre-industrial levels (Tissue et al. 1995; Ghannoum
et al. 2010a; Tissue and Lewis 2010). Studies involving current
and elevated [CO2] also report an increase in LMA, which
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suggests leaves become more dense or thicker with increasing
[CO2] (Pritchard et al. 1999).
Changes in LMA in response to climate change may
significantly alter leaf function and plant growth, as LMA is
closely related to photosynthetic rates (Reich et al. 1998;
Wright et al. 2005) and relative growth rates in many species
(Atkin et al. 1999; Wilson et al. 1999; Wright and Westoby
2000). LMA reflects both leaf thickness and density, but these
two traits differ in their relationship to leaf function. Increases
in thickness reflect anatomical changes, such as increases in
the size and number of layers of palisade cells (Pereira and
Kozlowski 1976; James et al. 1999), whereas increases in
density also reflect changes in leaf chemistry, including
increases in soluble protein and starch per unit leaf area and
changes in cell wall content (Dijkstra and Lambers 1989).
Increased protein per unit leaf area is associated with increased
photosynthetic rates (Field and Mooney 1986; Evans 1989), but
increased starch accumulation may reduce photosynthetic
rates through source:sink limitation (Herold 1980; Lewis
et al. 2002b). As a result, to understand how the response of
LMA to climate change may affect photosynthesis, it is critical
to consider the relationship between LMA and both leaf
thickness and chemistry.
Changes in leaf anatomy may also affect photosynthetic
responses to climate change by altering CO2diffusion from
the atmosphere to the site of photosynthesis (Guerfel et al.
2009). CO2diffusion is primarily regulated by stomates and
reductions in stomatal frequency with rising [CO2] reduce
CO2diffusion (Woodward et al. 2002). Conversely, growth
in pre-industrial [CO2] may increase stomatal frequency,
reducing the resistance to CO2diffusion, but increasing water
loss by transpiration (Ward and Strain 1997). Mesophyll
packing also alters gas diffusion (Evans 1995), by altering
the total surface area of mesophyll cells exposed to
intercellular air space (James et al. 1999; Marchi et al. 2008;
Evans et al. 2009).
Inrelatedstudies,wehaveassessedtheeffectsofrising[CO2]
and temperature on growth (Ghannoum et al. 2010a),
photosynthesis (Ghannoum et al. 2010b), light energy
partitioning (Logan et al. 2010) and nocturnal stomatal
conductance during drought (Zeppel et al. 2011) of Eucalyptus
sideroxylon A.Cunn. ex Woolls. Ghannoum et al. (2010a)
observed that elevated temperature increased the stimulatory
effect of rising [CO2] on E. sideroxylon, but temperature
did not affect photosynthetic responses to rising [CO2].
Upregulation of electron transport capacity (Jmax) was a key
factor drivingtheobserved
(Ghannoum et al. 2010b). Upregulation of Jmaxin response to
climate change is uncommon, but Ghannoum et al. (2010b)
lacked a mechanistic explanation for the response because
they did not determine whether the change was the result of
anatomical or chemical changes. Our objective in this study
was to extend our previous research by identifying the
structural (e.g. number of palisade layers, stomatal frequency)
and chemical (e.g. leaf [N]) changes that may have regulated
the photosynthetic response of E. sideroxylon to rising [CO2]
and temperature. This study was conducted on plants grown
separately from Ghannoum et al. (2010a, 2010b), but under
similar environmental conditions. The studies primarily differ
photosyntheticresponses
in that plants in this study were grown for ~210 days, compared
with~140daysinGhannoumetal.(2010a,2010b).Wefocussed
on Eucalyptus because few studies have examined structural
and functional responses of Eucalyptus to climate change
(Conroy 1992; Roden and Ball 1996; Roden et al. 1999),
despite Eucalyptus being an iconic genus with ecological
importance inAustraliaand
worldwide. We selected E. sideroxylon because it represents
an ecologically important eucalypt with slower growing, more
drought tolerant traits than eucalypts which have been more
commonly studied.
commercial importance
Materials and methods
Growth conditions
Detailed experimental set-up is described by Ghannoum et al.
(2010a). Briefly, 9kg of air-dried loamy-sand field soil was
added to 10-L PVC pots, which were transferred to six
adjacent, naturally-lit and temperature controlled glasshouse
compartments. Three compartments were programmed to
simulate the local ambient temperature (Richmond, NSW) and
the remaining three compartments were maintained at ambient
+4?C. Average temperatures for the ambient and elevated
temperature treatments were 26/18 and 30/22?C (day/night)
respectively. Within each temperature treatment, compartments
were maintained at pre-industrial (280mLL–1target), current
(400mLL–1target) or elevated (640mLL–1target) [CO2].
Atmospheric [CO2] was controlled and monitored as described
by Ghannoum et al. (2010a). Mean daytime [CO2] during
the experiment for the pre-industrial, current and elevated
treatments was 290, 400 and 650mLL–1respectively. RH,
monitored by Tinytag data loggers (TinyView, Gemini Data
Loggers LTD, Chichester, UK), averaged 57% during the
study and did not differ among [CO2] and temperature
treatments. As a result, vapour pressure deficit (VPD) was
higher in the elevated compared with the ambient temperature
treatment (1.8 vs 1.4kPa, on average). Maximum mid-day
photosynthetically active radiation (PAR), measured at a
nearby (1km away) weather station, was 2360mmolm–2s–1.
Across the study,peak
1250mmolm–2s–1. The glasshouse structure attenuated direct
sunlight by ~10–15%.
midday PAR averaged
Plant growth
Seeds of red ironbark (Eucalyptus sideroxylon A.Cunn. ex.
Woolls) were obtained from Ensis (Australian Tree Seed
Centre, ACT) and germinated at ambient [CO2] in plastic
greenhouses. Four weeks after germination, seedlings were
transplanted by planting one seedling into the middle of each
prepared pot. Pots were irrigated every 2–3 days as needed.
Pots were irrigated on three occasions (30, 120 and 135 days
after planting (DAP)) with a commercial fertiliser (General
Purpose, Thrive Professional, Yates, Sydney, NSW) at a
concentration of 0.2g NL–1(N:P:K:S:Fe:Mn:B; 25:4.1:
17.3:1.6:0.06:0.003:0.022%). Pots were routinely moved
within the glasshouse compartments during the experimental
period. Five pots from each treatment were randomly selected
for this study.
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Internal leaf anatomy
Duplicate 1.0?0.5cm sections were collected, after 7 months
of growth, on opposite sides of the midvein in the mid region
of the lamina; one leaf was used per pot. The leaf tissue was
immediately fixed in double aldehyde fixative containing 0.01g
of Triton-X, washed in three changes of 0.1M phosphate buffer
(pH 6.9) and then dehydrated in an ethanol series before being
embedded in LR White Resin (ProSci Tech, Townsville, Qld).
Transverse sections 2mm thick were stained with 0.1%
toluidine blue. Sections were photographed using a JenOptik
C14 digital camera attached to an Olympus compound light
microscope (Olympus BX60, Center Valley, PA). Five images
were recorded at various points along the leaf section. Image-
Pro Plus (ver. 5.1; Media Cybernetics Inc., Bethesda, MD) was
used to analyse leaf and epidermal thickness, palisade cell size,
number of palisade layers and the contributions of cell layers
and intercellular air space to leaf thickness and density. Percent
palisade and percent intercellular airspace were calculated as
the fraction of leaf structure between the epidermal layers
occupiedbypalisadecellsandintercellularairspacerespectively.
Leaf mass per area (LMA) was measured in conjunction
with gas-exchange measurements. A minimum of 40 leaf
disks from 8–10 recently fully expanded leaves per plant were
collected using a 0.2cm2leaf corer. The disks were oven-
dried at 70?C for 48h and weighed for dry mass. LMA was
calculated as leaf dry mass/area (gm–2).
Surface leaf anatomy
Two 1-cm2sections per plant were taken from the mid-lamina
regionofrecentlyfullyexpandedmatureleavesafter7monthsof
growth. Epidermal peels were made using a method adapted
from Jain (1976). The epidermal peels were stained in 0.5%
aqueous safranin overnight at room temperature, washed with
distilled water and mounted in 40% glycerol. Three images
were taken on the upper and lower surface of each epidermal
peelusingaJenOptikC14digitalcameraattachedtoacompound
light microscope (Olympus BX60). The counting function in
Image-Pro Plus was used to determine the number of stomata
and epidermal cells per field of view. Stomatal index (SI) was
calculated as: SI (%)=(SF/(SF+EF))?100, where SF is
stomatal frequency mm–2and EF is epidermal cell frequency
mm–2.
Leaf gas-exchange measurements
Net photosynthesis at saturating light (Asat) and near-saturating
CO2(Amax), stomatal conductance (gs), the ratio of intercellular
to ambient [CO2] (Ci/Ca) and water-use efficiency (WUE) were
measured, after 7 months of growth, on one attached, recently
fully expanded leaf per plant in the top-third of the plant, using a
portable open gas-exchange system (Li-6400XT, Li-Cor,
Lincoln, NE). Asatand Amaxmeasurements were made on the
same leaf at saturating light (1800mmolm–2s–1) with a target
VPD of 1.4kPa. Asatwas measured at target growth [CO2]
(280, 400 or 640mLL–1) and midday growth temperature (26
or 30?C). Amax was measured at 1200mLL–1[CO2] and
26?C. Each leaf was allowed to stabilise before measurements
were taken.
Leaf chemistry
The leaf used for gas-exchange measurements was immediately
harvested from the plant and snap frozen in liquid nitrogen
for analysis of leaf [N]. Leaves were freeze-dried for 24h,
ground to a fine dust, then analysed for N concentration
using a CHN analyser (LECO TruSpec, LECO Corporation,
St Joseph, MI).
Three leaves per plant were harvested on a sunny day during
the gas-exchange campaign, between 1100 and 1300hours,
for carbohydrate and cell wall content analysis. Leaf samples
were snap frozen in liquid nitrogen and stored in a ?85?C
freezer. Beforeanalysis, subsamples
for 24h, then ground to a fine dust. Soluble sugars were
assayed using a modified anthrone method as described in
Ebell (1969) and total starch was enzymatically assayed on the
residual pellet using a Megazyme total starch kit (Megazyme
were freeze-dried
0
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LMA (g m–2)
Leaf thickness (µm)
Leaf density (g cm–3)
Fig. 1.
at three atmospheric [CO2] (290, 400 or 650mLL–1) and two air
temperatures (ambient (*) or ambient+4?C (*)). Values represent means
?s.e.
Leaf structural characteristics of Eucalyptus sideroxylon grown
Leaf anatomy affects photosynthetic responses
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International Ireland Ltd, Wicklow, Ireland); total non-structural
carbohydrate (TNC) was calculated as the sum of soluble sugar
and starch.
Cell wall content was determined as described by Harrison
et al. (2009). About 10mg of ground, freeze-dried leaf material
was vortexed in 1.5mL of buffer (50mM tricine, pH 8.1)
containing 1% PVP40 (Sigma-Aldrich, St Louis, MO), then
centrifuged at 12000g for 5min. The supernatant was
removed and the pellet re-suspended in 1.5mL of buffer
without PVP containing 1% sodium dodecyl sulfate (Sigma-
Aldrich). The tube was vortexed and incubated in a 90?C
water bath for 5min, then centrifuged at 12000g for 5min.
This step was repeated. The pellet was then washed twice with
0.2M KOH, twice with distilled water and twice with ethanol,
mixing well and centrifuging at 12000g for 5min at each step.
The pellet was oven-dried at 70?C and the dry mass was
assumed to represent the leaf structural biomass.
Four leaf disks from recently fully expanded leaves were
collected from each plant, in conjunction with samples
collected for carbohydrate analyses, for chlorophyll and
soluble protein content. Leaf disks were snap frozen in liquid
nitrogen and stored in a ?85?C freezer until extraction. Disks
were extracted in 1.49mL of buffer (0.077g 5mM DTT, 1g
10% glycerol, 100mL 1mM MgCl2, 40mL 0.5M EDTA, 20mL
0.5M EGTA, 120mL aminocaproic acid, 80mL benzamidine,
made up to 10mL with 50mM HEPES (pH 8.0)), 0.1g 1%
PVP and 10mL 10mM PMSF in a pre-cooled mortar. Total
chlorophyll content was determined on subsamples of the
extract using an acetone-extract colourimetric method, as
described by Porra et al. (1989). The remaining extract was
centrifuged for 2min and soluble protein content was
determined on aliquots of the supernatant using a Coomassie
Plus kit (VWR International, Brisbane, Qld).
Data analyses
The main and interactive effects of growth [CO2] and
temperature on leaf anatomy, chemistry and gas exchange
were tested using two-way analysis of variance (Statistica,
StatSoft Inc., Tulsa, OK). Data were tested for normality and
Table 1.
on leaf structural and functional parameters of Eucalyptus sideroxylon grown at two temperatures
and three [CO2] (see ‘Materials and methods’)
Significance levels are: n.s., not significant (P>0.05); *, P<0.05; **, P<0.01; ***, P<0.001. TNC, total
non-structural carbohydrate
Summary of the two-way ANOVA results for the effects of temperature and [CO2]
VariableSignificance level
CO2
Temperature
Temperature?CO2
Leaf anatomy
LMA (gm–2)
Density (gcm–3)
Leaf thickness (mm)
Mesophyll thickness (mm)
Epidermal thickness (mm)
No. of palisade layers
Palisade cell length (mm)
Palisade cell width (mm)
Intercellular air space (%)
Palisade (%)
Cell wall content (%)
*
*
***
**
*
**
**
***
n.s.
*
**
**
n.s.
n.s.
n.s.
n.s.
n.s.
**
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
***
n.s.
*
*
n.s.
Leaf surface anatomy
Epidermal cell frequency (no.permm–2)
Stomatal frequency (no.permm–2)
Stomatal index (%)
n.s.
*
*
***
***
**
n.s.
n.s.
*
Leaf gas exchange
Amax(mmolm–2s–1)
Asat(mmolm–2s–1)
gs(molm–2s–1)
Ci/Ca
n.s.
**
n.s.
n.s.
***
***
**
n.s.
n.s.
n.s.
n.s.
n.s.
Leaf biochemistry
Leaf Nmass(mgg–1)
Leaf Narea(gm–2)
Soluble protein (gm–2)
Chl a+b (gm–2)
Chl a/b
Total soluble sugars (g glucose equivalents m–2)
Starch (g glucose equivalents m–2)
TNC (g glucose equivalents m–2)
n.s.
n.s.
*
*
n.s.
n.s.
***
**
***
**
**
**
n.s.
***
***
***
n.s.
n.s.
*
n.s.
n.s.
n.s.
**
*
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homogeneity of variances; extreme outliers were removed
from the datasets before proceeding with the statistical
analysis. Where [CO2] and temperature interactions were
significant, pair-wise comparison of treatment means was done
using a Newman-Kewls post-hoc test (Statistica, StatSoft Inc.).
Relationships between leaf gas exchange and leaf structure
and chemistry were analysed using linear regression analysis
(MicrocolOriginver.6.0,MicrocolSoftware,Inc.,Northampton,
MA). In all analyses, test results were considered significant if
P?0.05.
Results
Leaf internal anatomy
LMA increased ~35% between pre-industrial and elevated
[CO2] (Fig. 1; Table 1). Growth in elevated temperature
decreased LMA by 10% compared with ambient temperature.
Although the effect of rising [CO2] appeared to vary between
temperature treatments, the interaction was not significant
(Table 1). Effects of elevated [CO2] on LMA were associated
with increases in leaf thickness and density, whereas elevated
temperature increased leaf density, but did not affect leaf
thickness (Fig. 1; Table 1). As with LMA, although the effect
of rising [CO2] appeared to differ between temperature
treatments, the interactions were not significant (Table 1).
Mesophyll thickness increased with rising [CO2], but did
not vary with growth temperature (Fig. 2; Table 1). Further,
although both rising [CO2] and elevated temperature affected
the size and distribution of palisade cells, the effects differed
betweenthesefactors(Fig.2).Rising[CO2]enhancedthenumber
of palisade layers; on average, one extra layer of palisade cells
was observed at elevated compared with pre-industrial [CO2].
Palisadecellwidthincreasedwithrising[CO2],whereaspalisade
cell length was not affected by growth [CO2]. In contrast,
increasing temperature reduced palisade cell length by an
average of 12%, but did not affect the number of layers or the
widthofpalisadecells.Rising[CO2]alsoincreasedthefractionof
internal leaf space occupied by palisade cells and reduced that
occupied by intercellular air space (Fig. 2). In contrast, elevated
temperature increased the fraction of intercellular air space and
reduced the fraction of internal leaf space occupied by palisade
cells (Fig. 2).
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# Palisade layers
% Palisade
Palisade cell length (µm)
% Intercellular air space
Growth [CO2] (µL L–1)
Palisade cell width (µm)
Fig. 2.
400 or 650mLL–1) and two air temperatures (ambient (*) or ambient+4?C (*)). Values represent
means ?s.e.
Mesophyll characteristics of Eucalyptus sideroxylon grown at three atmospheric [CO2] (290,
Leaf anatomy affects photosynthetic responses
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Leaf surface anatomy
Epidermal thickness increased and epidermal cell frequency
decreased with rising [CO2], but temperature did not affect
epidermal thickness or cell frequency (Fig. 3; Table 1). The
effects of elevated [CO2] on stomatal frequency and stomatal
index differed between temperature treatments. Stomatal
frequency and stomatal index decreased with increasing [CO2]
between pre-industrial and current [CO2] in both temperature
treatments. However, stomatal frequency and stomatal index
also decreased with rising [CO2] between current and elevated
[CO2] in the elevated temperature treatment, but both increased
with increasing [CO2] between current and elevated [CO2]
in the ambient temperature treatment. As a result, although
elevatedtemperatureincreased
stomatal index at pre-industrial and current [CO2], elevated
temperature decreased stomatal frequency and stomatal index
in elevated [CO2]
stomatalfrequencyand
Leaf gas exchange
Asat increased with rising [CO2] and elevated temperature
(Fig. 4; Table 1), but the increase was larger between current
and elevated [CO2] (33% increase) than between pre-industrial
and current [CO2] (7% increase). The comparatively smaller
effect in the transition from pre-industrial and current [CO2]
reflected a 27% decrease in Amaxin the transition from pre-
industrial to current [CO2]; no change was observed from
current to elevated [CO2] (Table 2). Amax did not differ
between temperature treatments (Table 1).
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Epidermal thickness (µm)
Stomatal freq. (# mm–2)
Epidermal cell freq. (# mm–2)
Growth [CO2] (µL L–1)
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b
Stomatal index (%)
a
Fig. 3.
sideroxylongrown at three atmospheric [CO2] (290, 400 or 650mLL–1) and two air temperatures (ambient(*) or
ambient+4?C (*)). Values represent means ?s.e. There was a significant interaction between [CO2] and
temperature treatments on stomatal index only; superscripts in (d) indicate significant differences between
treatments.
(a) Epidermal thickness, (b) stomatal frequency, (c) cell frequency and (d) stomatal index of Eucalyptus
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gs (mol m–2 s–1)
Growth [CO2] (µL L–1)
(a)
(b)
Fig. 4.
and temperature (Asat) and (b) stomatal conductance (gs) of Eucalyptus
sideroxylon grown at three atmospheric [CO2] (290, 400 or 650mLL–1)
and two air temperatures (ambient (*) or ambient +4?C (*)). Values
represent means ?s.e.
(a) Light-saturated photosynthetic rates measured at growth [CO2]
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Results showed that gs decreased with rising [CO2] but
did not vary with growth temperature (Fig. 4; Table 1). Ci/Ca
was not affected by either growth [CO2] or temperature
(Table 2). Overall, leaf gas exchange was affected more by
growth [CO2] than temperature (Table 1). Temperature and
[CO2] had no interactive effects on leaf gas-exchange
parameters (Table 1).
Leaf chemistry
Leaf N per unit dry mass (Nmass) decreased 45% with
rising [CO2], but did not vary with growth temperature
(Tables 1, 2). Similar responses to rising [CO2] and
temperature were observed when leaf N was expressed on an
area or structural dry mass basis. Although growth temperature
did not affect the response of leaf N per unit area (Narea) to
rising [CO2], the response of soluble protein content to rising
[CO2] differed between growth temperatures. At ambient
temperature, soluble protein decreased by 28% in the
transition from pre-industrial to current [CO2], but did not
differ between current and elevated [CO2]. At elevated
temperature, soluble protein did not differ among [CO2]
treatments. Elevated temperature reduced soluble protein in
pre-industrial [CO2], but did not affect soluble protein in
current or elevated [CO2]. Total chlorophyll decreased with
rising [CO2] and increased with temperature. Neither growth
[CO2]nortemperaturehadaneffectonthechlorophylla/bratioor
on cell wall content.
Soluble sugars nearly doubled between pre-industrial and
elevated [CO2], but the effect of rising [CO2] on starch
differed between temperature treatments (Tables 1, 2). At
ambient temperature, starch nearly doubled between current
and elevated [CO2], but there was no effect between pre-
industrial and current [CO2] (Tables 1, 2). At elevated
temperature, starch increased by 48% between pre-industrial
and current [CO2] and 40% between current and elevated
[CO2]. Elevated temperature decreased starch ~50% across
[CO2] treatments. As a result of the combined treatment
effects on soluble sugars and starch, TNC doubled with rising
[CO2] anddeclined ~20%
(Tables 1, 2). Overall, growth [CO2] had a greater effect on
leaf chemical properties than high temperature (Table 1).
withelevated temperature
Relationships between leaf structure and function
To assess those factors that may have regulated Amax, we
examined relationships between Amax and structural and
chemical variables (LMA, leaf and mesophyll thickness,
number of palisade layers, fraction of intercellular air space,
and leaf Narea, starch, soluble sugars, TNC and soluble
Table 2.Leaf biochemistry traits of Eucalyptus sideroxylon grown at two temperatures and three [CO2]
(see ‘Materials and methods’)
Values represent means ?s.e. For those leaf traits where there was a significant interaction between [CO2] and
temperature treatments, different letters indicate significance between treatments. In all cases n=5. TNC, total
non-structural carbohydrate
VariableTemperature[CO2] (mlL–1)
400290 650
Leaf anatomy
Cell wall content (%)Ambient
High
30±1
30±1
30±1
30±1
32±1
28±1
Leaf gas exchange
Ambient
High
Ambient
High
Amax(mmol m–2s–1) 32.0±0.1
32.9±1.8
0.64±0.04
0.68±0.04
24.4±1.7
23.5±1.1
0.66±0.03
0.59±0.04
23.2±1.9
26.2±1.1
0.59±0.03
0.60±0.03
Ci/Ca
Leaf biochemistry
Ambient
High
Ambient
High
Ambient
High
Ambient
High
Ambient
High
Ambient
High
Ambient
High
Ambient
High
Leaf Nmass(mgg–1)20.9±0.8
24.5±2.8
2.5±0.1
2.9±0.3
8.8±0.4b
5.5±0.8a
0.55±0.06
0.62±0.04
2.5±0.0
2.4±0.0
9.6±1.2
10.2±1.7
6.0±0.9a
3.1±0.6a
15.5±1.3a
13.8±1.6a
14.1±1.8
15.9±2.8
1.9±0.1
1.9±0.3
6.3±1.0a
5.6±0.5a
0.37±0.02
0.45±0.07
2.4±0.1
2.2±0.1
13.8±2.5
15.5±1.6
7.2±0.3a
4.6±0.5a
21.9±3.2b
20.1±1.8ab
11.7±1.2
13.5±1.1
2.1±0.2
2.0±0.1
4.7±0.6a
4.8±0.2a
0.37±0.05
0.51±0.01
2.4±0.2
2.4±0.4
19.9±2.2
17.9±1.8
14.3±1.1b
6.4±0.5a
35.6±2.5c
24.4±2.0b
Leaf Narea(gm–2)
Soluble protein (gm–2)
Chl a+b (gm–2)
Chl a/b
Total soluble sugars
(g glucose equivalents m–2)
Starch (g glucose equivalents m–2)
TNC (g glucose equivalents m–2)
Leaf anatomy affects photosynthetic responses
Functional Plant Biology
291

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proteins). Amaxincreased with increasing fraction of intercellular
air space and leaf Narea, whereas Amaxdecreased with increasing
LMA, leaf and mesophyll thickness, number of palisade layers
and starch, soluble sugar and TNC per unit area (P<0.05 in all
cases).ThestrongestrelationshipswerebetweenAmaxandLMA,
number of palisade layers, leaf Nareaand TNC per unit area
(Fig. 5). Amaxdidnotvary with leafsoluble proteins perunit area
(P=0.186).
To assess those factors that may have regulated leaf density,
we examined the relationships between leaf density and
structural (fraction of palisade cells) and chemical (leaf starch,
soluble sugars, TNC) variables that may have influenced leaf
density. Leaf density increased with leaf starch and the fraction
of palisade cells (Fig. 6). Leaf starch per unit volume accounted
for 41% of the variation in leaf density, while fraction of
palisade cells accounted for 29% of the variation in leaf density.
Discussion
Rising [CO2] affected Asat directly and through effects on
photosynthetic capacity (Amax). Rising [CO2] reduced Amaxby
reducing leaf Nareabut increased the number of palisade layers.
Leaf Nareaaccounted for 56% of the variation in Amaxand the
number of palisade layers accounted for 14% of the variation,
respectively. Reduction in Nareawas associated with increased
leaf mass per area (LMA), which, in turn, was associated with
increased starch and number of palisade layers. Changes in
these three factors were associated with linear reductions in
Amax between pre-industrial and elevated [CO2], suggesting
consistent patterns in the effects of these factors on Amax
across CO2 concentrations. In contrast, although elevated
temperature increased stomatal frequency, it did not affect
Amax, indicating acclimation to growth temperature. Further,
there generally were no interactions between rising [CO2] and
temperature on leaf structure or function. These results indicate
leaf Nareaand the number of palisade layers, associated with
changes in LMA, were the key chemical and anatomical factors
regulating photosynthetic responses of E. sideroxylon to rising
[CO2], whereas the lack of photosynthetic responses to elevated
temperature may reflect both acclimation of photosynthesis and
the limited effect of elevated temperature on leaf anatomy.
Relationships between leaf structure and function
In C3 plants, long-term exposure to rising [CO2] often is
associated with reductions in Amax, reducing the relative Asat
response to [CO2] (Tissue and Oechel 1987; Ainsworth and
Rogers 2007), as we observed in this study. These reductions
may result from a wide range of structural and physiological
changes, including changes in leaf thickness and reductions in
total Rubisco activity. Further, these changes may be tied to
changes in leaf chemistry, including reductions in leaf Narea
45
40
35
30
25
20
15
10
5
0
45
40
35
30
25
20
15
10
5
0
1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2
Leaf [N] (g m–2)
LMA (g m–2) # Palisade layers
TNC (g Glu eq. m–2 s–1)
5101520 253035 4045
80
Amax = 39.6 – 0.1 *LMA
r2 = 0.23
p < 0.01
Amax = 65.6 – 6.8 *#layers
r2 = 0.14
p < 0.05
Amax = 34.5 – 0.4 *[TNC]
r2 = 0.31
p < 0.01
Amax = 9.6 – 8.1 *[N]
r2 = 0.56
p < 0.01
Amax (µmol m–2 s–1)
100
(a)
(b)
(c)
(d)
120 140160 1802005.0 5.25.45.65.86.0 6.2
Fig. 5.
layers,(c)leafNareaand(d)totalnon-structuralcarbohydrates.Symbolsrepresentgrowth[CO2];290mLL–1(!),400mLL–1(*)
and 650mLL–1(~), with open symbols representing ambient temperature and closed symbols representing ambient
+4?C. There were five replicates per treatment and each data point represents a single observation. Data were fitted using a
linear regression (solid line). The equation of the linear fit, adjusted R2value and its significance are shown.
The relationships of Eucalyptus sideroxylon photosynthetic capacity (Amax) with (a) LMA, (b) number of palisade
292
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R. A. Smith et al.

Page 9

and increased carbohydrate concentrations (Griffin et al. 2001;
Lewis et al. 2004; Ainsworth and Rogers 2007). Our results
suggest that variation in Amaxwas primarily driven by changes
in LMA and associated changes in leaf chemistry and the
number of palisade layers. These changes, in turn, partially
reflected changes in allocation of resources within the leaf.
Changes in leaf Narea had the largest effect on Amax,
accounting for 56% of the variation in Amax. The effect of leaf
Nareaon Amaxmay have been in part through effects on soluble
protein content per unit area, which exhibited similar patterns
of response to rising [CO2] and temperature as leaf Narea. In C3
leaves, Rubisco constitutes ~25% of leaf N content and up to
50% of the soluble protein fraction (Evans 1983; Conroy 1992).
As a result, soluble protein content and Amaxclosely vary with
leaf Narea(Field and Mooney 1986; Evans 1989); reductions in
leaf Narea and soluble protein content with rising [CO2]
were an indication that Rubisco content was reduced (Tissue
et al. 1993). Reductions in Amaxalso occurred in conjunction
withincreasedTNC, suggesting
downregulation may have occurred at least in part due to a
that photosynthetic
source:sink imbalance. Downregulation of Amaxin response
to carbohydrate accumulation has been observed in a wide
range of studies (Thomas and Strain 1991; Lewis et al. 2002b;
Ainsworth and Long 2005).
The close relationships between Amaxand leaf Narea, soluble
proteins and TNC per unit area at least partially account for the
differential responses of Amaxto pre-industrial versus elevated
[CO2]. Amax, leaf Narea, and soluble proteins decreased while
carbohydrates increased with rising [CO2]. The differential
effects on leaf chemistry of pre-industrial [CO2] compared
with elevated [CO2] were partially a function of changes in
leaf structure, as has been observed in previous studies (e.g.
PoorterandEvans1998;Reichetal.1998).Leafstructure,which
is often correlated with LMA, affects processes such as N
allocation and concentration (Field and Mooney 1986).
Accordingly, there often is a strong correlation between LMA
and photosynthesis–nitrogen relationships (Reich et al. 1998;
Wright et al. 2005). For instance, species with high LMA
generally have higher Amaxand Nareathan species with low
LMA (Poorter and Evans 1998; Sefton et al. 2002). In this
study, E. sideroxylon with high-LMA leaves had lower Amax
than that observed in the low-LMA leaves; low leaf Nareaand
high LMA together generated low Amax. This result highlights
the role of N in determining Amaxand in regulating responses to
rising [CO2] from pre-industrial to elevated [CO2]
Higher TNC at elevated [CO2] was associated with the
production of an additional palisade cell layer, as has been
observed in other studies (Thomas and Harvey 1983; Pritchard
et al. 1999). Increased numbers of palisade cell layers generally
increaseAmax(Linetal.2001).However,increasesinthenumber
of palisade cell layers may reduce Amaxby reducing leaf Narea
througheffectsonLMA.Increasesinpalisadelayernumbermay
also affect processes such as CO2diffusion (James et al. 1999;
Marchi et al. 2008; Evans et al. 2009). Here, we observed with
rising [CO2] an increase in the fraction of internal leaf surface
occupied by palisade cells and a reduction in the fraction
occupied by intercellular air space. Reductions in the
intercellular air space have been associated with reduced
mesophyll conductance of CO2, which would offset the
stimulatory effect of an additional palisade layer on Amax.
Likewise, increasing the fraction of intercellular air space at
pre-industrial [CO2] facilitates plant compensation for low
[CO2] by reducing the resistance to [CO2] diffusion.
Accordingly, rising [CO2] may have reduced Asatthrough
effects on both mesophyll and stomatal conductance. Stomates
are the primary factor regulating CO2diffusion into the leaf and
reductions in stomatal frequency with rising [CO2] reduce CO2
diffusion into the leaf (Woodward et al. 2002). Consistent with
this expectation as well as empirical data (Medlyn et al. 2001;
Lewisetal.2002a;AinsworthandLong2005),gsdecreasedwith
rising [CO2]. Growth in pre-industrial [CO2] often is associated
with comparatively high stomatal frequency, reducing the
resistance to CO2diffusion and offsetting the reduction in the
driving gradient for CO2diffusion into the leaf (Ward and Strain
1997). The observed changes in stomatal frequency likely
reflected effects of rising [CO2] on epidermal cell production
rather than on stomatal initiation. Rising [CO2] had a small
negative effect on stomatal index, which suggests that changes
in stomatal frequency were largely proportional to changes in
Starch (g Glu eq. cm–3)
% Palisade
D = 0.1 + 0.006 *[%p]
r2 = 0.29
p < 0.01
D = 0.4 + 3.5 *[Starch]
r2 = 0.41
p < 0.001
Leaf density (g cm–3)
Fig. 6.
starch and fraction palisade cells. Symbols represent growth [CO2];
290mLL–1(!), 400mLL–1(*) and 650mLL–1(~), with open symbols
representing ambient temperature and closed symbols representing
ambient +4?C. There were five replicates per treatment and each data point
represents a single observation. Data were fitted using a linear regression
(solid line). The equation of the linear fit, adjusted R2value and its
significance are shown.
The relationships of Eucalyptus sideroxylon leaf density with leaf
Leaf anatomy affects photosynthetic responses
Functional Plant Biology
293

Page 10

epidermal cell frequency (stomatal index is the ratio of the
number of epidermal cells to stomata). Hence, rising [CO2] is
unlikely to have affected stomatal initiation but instead
stimulated epidermal cell production and expansion.
We noted that the response of Asatto rising [CO2] was
independent of temperature. During short-term exposure, the
response of Asatto rising [CO2] is predicted to increase with
increasing temperature because of differences in the temperature
responses of photosynthesis and photorespiration (Berry and
Bjorkman 1980; Sage and Kubien 2007). However, responses
to long-term exposure to elevated [CO2] and temperature
depend on the extent to which photosynthesis acclimates to the
new conditions. In our study, the strong thermal acclimation
of Amaxprecluded temperature?[CO2] interactions. The lack
of a temperature effect on Amaxindicates that photosynthesis
underwent partial thermal acclimation in response to growth
temperature(Ghannoum
et
acclimation to high temperature is commonly observed in a
wide variety of plants (Berry and Bjorkman 1980; Cowling
and Sage 1998), although not always (Lewis et al. 2001). The
lack of a temperature effect on Amaxfurther indicates that the
anatomical changes associated with elevated temperature,
including the decrease in leaf density, decreased palisade cell
length and percent palisade cells, were not sufficient to alter
photosynthetic processes.
al.2010b).Photosynthetic
Conclusions
Rising [CO2] increased Asatthrough direct effects, but indirectly
affectedAsatthroughreductionsinAmax.ReductionsinAmaxwith
rising [CO2] were driven by reductions in leaf Nareaand an
increase in the number of palisade layers, which accounted for
56 and 14%, respectively, of the variation in Amax. Reductions in
Nareawere associated with increased LMA, which, in turn, was
associated with increased starch and numbers of palisade
layers. Amax responded linearly to changes in these traits,
suggesting consistent effects of these traits on Amax across
[CO2] levels. Elevated temperature was associated with
increases in stomatal frequency, but did not significantly affect
Amax. Further, as has been found in a wide range of tree species
(Lewis et al. 1999; Wang et al. 2003; Allen and Vu 2009;
Ghannoum et al. 2010b), the effects of rising [CO2] and
elevated temperature generally were additive, rather than
interactive.Our resultssuggest
photosynthesis in E. sideroxylon primarily through effects on
leaf Narea, stomatal frequency and the number of palisade layers,
while the lack of effect of elevated temperature on Amax
indicated photosynthetic acclimation to elevated temperature
and that structural changes associated with rising temperature
were not extensive enough to alter Amax.
rising[CO2]affects
Acknowledgements
We thank Renee Attard, Liz Kabanoff, Dr Markus Loew, Dr Kaushal
Tewari, Linda Westmoreland and Roslyn Woodfield for their assistance
with the field and laboratory work. This study was supported by Discovery
Project Number DP0879531 of the Australian Research Council (DTT),
University of Western Sydney International Research Initiatives Scheme
(Grant No. 71846; JDL) and Fordham University (JDL). This is
contribution number 254 from the Louis Calder Center and Biological
Station, Fordham University.
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