Journal of Experimental Botany, Vol. 60, No. 8, pp. 2303–2314, 2009
doi:10.1093/jxb/erp021 Advance Access publication 13 March, 2009
RESEARCH PAPER
Influence of leaf dry mass per area, CO2, and irradiance on
mesophyll conductance in sclerophylls
Foteini Hassiotou1,*, Martha Ludwig2, Michael Renton1,3, Erik J. Veneklaas1 and John R. Evans4
1
School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley
WA 6009, Australia
2
School of Biomedical, Biomolecular and Chemical Sciences, Faculty of Life and Physical Sciences, The University of Western Australia,
35 Stirling Highway, Crawley WA 6009, Australia
3
Agricultural Landscapes, CSIRO Sustainable Ecosystems, Floreat, WA 6014, Australia
4
Environmental Biology Group, Research School of Biological Sciences, Australian National University, Canberra, ACT 2601, Australia
Received 19 November 2008; Revised 14 January 2009; Accepted 16 January 2009
Abstract
Leaf photosynthesis (A) is limited by mesophyll conductance (gm), which is influenced by both leaf structure and the
environment. Previous studies have indicated that the upper bound for gm declines as leaf dry mass per area (LMA,
an indicator of leaf structure) increases, extrapolating to zero at a LMA of about 240 g m22. No data exist on gm and
its response to the environment for species with LMA values higher than 220 g m22. In this study, laboratory
measurements of leaf gas exchange and in vivo chlorophyll a fluorescence were used concurrently to derive
estimates of gm in seven species of the Australian sclerophyllous genus Banksia covering a wide range of LMA (130–
480 g m22). Irradiance and CO2 were varied during those measurements to gauge the extent of environmental effects
on gm. A significant decrease of gm with increasing LMA was found. gm declined by 35–60% in response to
increasing atmospheric CO2 concentrations at high irradiance, with a more variable response (0–60%) observed at
low irradiance, where gm was, on average, 22% lower than at high irradiance at ambient CO2 concentrations.
Despite considerable variation in A and LMA between the Banksia species, the CO2 concentrations in the
intercellular air spaces (Ci, 26265 mmol mol21) and in the chloroplasts (Cc, 12764 mmol mol21) were remarkably
stable.
Key words: Banksia, CO2, leaf anatomy, leaf internal conductance, light intensity, photosynthesis, sclerophylly.
Introduction
Photosynthesis requires diffusion of CO2 into the leaf. The
diffusion pathway has several components. Turbulent diffusion outside the leaf carries CO2 towards the leaf surface
where it passes through a laminar boundary layer. Entry into
the leaf is restricted by stomatal pores, which are generally
situated on the leaf surface, but are sometimes below the leaf
surface in crypts. From the substomatal cavity, CO2 then
diffuses through the intercellular air spaces to mesophyll cell
walls where it dissolves into the liquid phase. CO2 diffuses
through the cell walls and cytosol and into the chloroplast
where it combines with RuBP (ribulose-1,5-bisphosphate)
and enters the photosynthetic carbon reduction cycle.
* To whom correspondence should be addressed. [email protected]
Abbreviations: A, net CO2 assimilation rate per unit leaf area; Ca, ambient CO2 concentration; Ci, intercellular CO2 concentration; Cc, chloroplastic CO2
concentration; gm, mesophyll conductance; gs, stomatal conductance; gias, conductance to CO2 diffusion through the mesophyll intercellular air spaces; gliq,
conductance to CO2 diffusion through the liquid phase, including cell walls, plasma membrane, cytosol, chloroplast envelope, and stroma; Sc, chloroplast surface
area exposed to the intercellular air spaces; UCO2 , gas exchange-based quantum yield [mol e– (mol incident quanta)1]; UPSII, photochemical efficiency of
Photosystem II; J, rate of electron transport; Jf, rate of electron transport calculated from chlorophyll fluorescence; JCO2 , rate of electron transport obtained from gas
exchange; Vc, maximum rate of RuBP (ribulose-1;5-bisphosphate) carboxylation; C*, photo-compensation point in the absence of mitochondrial respiration; Rd,
respiration in the light; PPFD, photosynthetic photon flux density; a, leaf absorptance; b, fraction of quanta absorbed by Photosystem II; LMA, leaf dry mass per
area.
ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2304 | Hassiotou et al.
Until recently, research focused on the resistance caused
by stomata (Parkhurst, 1994; Evans and von Caemmerer,
1996; Morison and Lawson, 2007; Warren, 2007). For
convenience, conductance through the mesophyll (gm) had
been assumed to be infinite prior to techniques being
developed to measure gm. However, now that methodology
to measure gm is more widely available, increasing attention
is being paid to understanding leaf internal diffusion, as it
actually accounts for more than 40% of the decrease in CO2
concentration between the atmosphere and the sites of
carboxylation (Warren, 2007; Flexas et al., 2008).
The structure of the mesophyll can be a major determinant of gm, either through its gaseous component in
the intercellular air spaces (gias) or through its liquid
component from the cell walls to the chloroplasts (gliq).
Mesophyll porosity and thickness can affect gias (Parkhurst,
1994; Evans and von Caemmerer, 1996), which is dependent
on the length, diameter, and tortuosity of the diffusion
pathway, i.e. it is determined by the depth of the mesophyll layer, the position of stomata relative to mesophyll
cells, and the size, shape, and packing of mesophyll cells.
Morison et al. (2005) computed the vertical gias of 56 species
surveyed by Slaton and Smith (2002), using the diffusion
coefficient of CO2 in still air, mesophyll porosity, and leaf
thickness, and found a range of gias values of 0.1–1.9 mol
m2 s1. Parkhurst and Mott (1990) used helox (air where
nitrogen has been replaced by helium) to measure the
resistance of the gaseous path to diffusion, and found that
gias accounted for 10–60% of gm, being more important in
hypostomatous leaves. On the other hand, Genty et al.
(1998) have shown that gias contributes only a small proportion to the total drawdown in the concentration of CO2
in the mesophyll, and that gliq is the main determinant of
gm, which has also been supported by other investigators
(von Caemmerer and Evans, 1991; Aalto and Juurola, 2002;
Sharkey et al., 2007; Warren, 2007). Leaf anatomical traits
such as mesophyll cell surface area and chloroplast surface
area exposed to intercellular air spaces (Evans et al., 1994;
Evans and Loreto, 2000), chloroplast rearrangements
(Sharkey et al., 1991; Tholen et al., 2007), and cell wall
thickness (Terashima et al., 2006) can all impact on gliq.
Nobel et al. (1975) argued that cell walls impose a major
resistance to photosynthesis, which suggests that sclerophylls
(hard leaves) with thick cell walls may have lower gm.
Interestingly, in the early stages of gm research, von
Caemmerer and Evans (1991) and Lloyd et al. (1992) found
that sclerophylls, such as Eucalyptus blakelyi, Macadamia
integrifolia, Citrus limon, and Citrus paradisi, had gm values
between 0.1–0.25 mol m2 s1, which overlapped with the
range observed for thin and soft leaves of tobacco and bean
with similar rates of photosynthesis. Loreto et al. (1992)
found that seven sclerophylls had low values of gm and low
rates of photosynthesis, but the relationship between photosynthesis and gm did not vary between sclerophyllous plants
and mesophytes. Leaf dry mass per area (LMA) is a leaf
morphological trait that has often been used as an indicator
of sclerophylly (Gratani and Varone, 2006) and a recent
review by Flexas et al. (2008) examined the relationship
between LMA and gm, summarizing data from 17 studies.
While low-LMA mesophytic species present a wide range of
gm values, leaf structure appears to limit gm strongly in
evergreen species with high LMA (Flexas et al., 2008). It is
evident that gm decreases with increasing LMA, extrapolating
to zero at a LMA of 240 g m2. However, since LMA in
sclerophyllous plants can be much higher than 240 g m2,
there is a need to extend the range of measurements.
During the last decade, it has been shown that leaf
structure is not the only determinant of gm. Interestingly, gm
seems to respond to environmental factors such as soil
water availability, salinity, and temperature (Warren, 2007;
Flexas et al., 2008). Bernacchi et al. (2002) argued that the
temperature response coefficient (Q10) of approximately 2.2
for gm that they found for tobacco leaves suggests a role for
proteins in the path of CO2 diffusion. Current research is
focusing on two proteins, carbonic anhydrase (CA) and
aquaporins. CA has been shown to have a modest effect on
gm (Price et al., 1994; Williams et al., 1996). However,
Gillon and Yakir, (2000) suggested that the role of CA in
gm regulation may be more important when gm is low, such
as in sclerophyllous species. Moreover, research on aquaporins, initially using inhibitors, then antisense and overexpression genotypes, suggests that certain aquaporins
located in the plasma and chloroplast membranes are
permeable to CO2 and are involved in the regulation of gm
(Terashima and Ono, 2002; Üehlein et al., 2003, 2008;
Hanba et al., 2004; Flexas et al., 2006; Miyazawa et al.,
2008). The findings of Flexas et al. (2007b) that gm
responded rapidly to changes in atmospheric CO2 concentration in six species and that gm was reduced in tobacco
when measured under low irradiance show that part of the
resistance pathway can be reversibly altered within minutes,
which further supports an active regulation of gm.
Given that LMA values of Banksia species are between
130 g m2 and 500 g m2 (F Hassiotou, unpublished data),
this genus provides an excellent model to examine the effect
of leaf structure as indicated through LMA on gm as well as
to extend the current knowledge on gm for species at the
high-LMA end. In this context, seven Banksia species
covering a wide range of LMA were selected and mesophyll
conductance was estimated from A–Ci curves at two
irradiances using combined gas exchange and chlorophyll
fluorescence measurements.
Materials and methods
Plant material and growth conditions
Three to five-year-old plants of seven Banksia species that
covered a wide range of LMA were used (Table 1). The
experiment was done in two phases: (i) in September–
October 2007 in Canberra, eastern Australia (Southern
Hemisphere spring), measurements were carried out on
three species (Banksia integrifolia L.f., Banksia serrata L.f.,
and Banksia paludosa R.Br.), using plants purchased from
a local nursery. Upon purchase, the plants were repotted
Mesophyll conductance in sclerophylls | 2305
Table 1. Leaf dry mass per area (LMA) of the Banksia species
examined.
Species
LMA (g m2)
B.
B.
B.
B.
B.
B.
B.
134612.5
17163.6
21761.4
23863.6
26262.6
26968.2
47868.2
serrata
integrifolia
attenuata
solandri
paludosa
repens
elderiana
Values are averages 6standard errors of three replicates per species.
into 10 L pots containing a mixture of grey sand and potting
mix and grown for 2 months prior to the measurements in
a greenhouse (25/20 oC day/night); and (ii) in November–
December 2007 in Perth, Western Australia (Southern
Hemisphere spring to summer), measurements were done
on four species (Banksia solandri R.Br., Banksia elderiana
F.Muell. & Tate, Banksia attenuata R.Br., and Banksia
repens Labill.), using plants in 10 L pots containing river
sand and potting mix. In 2002, seeds of the above four
Banksia species were germinated and plants were grown
outdoors until about three weeks before the measurements,
when they were transferred to a controlled-temperature
greenhouse (23/18 oC day/night).
Gas exchange and chlorophyll a fluorescence
Combined gas exchange and chlorophyll a fluorescence
measurements (the so-called ‘fluorescence method’; Evans
and von Caemmerer, 1996) were carried out using the
youngest fully expanded leaves. Three leaves were measured
from each species, each leaf originating from a different
plant, with the exception of B. integrifolia and B. paludosa,
where three leaves of the same plant were used. For each
leaf, two sets of measurements were taken: (i) the CO2
response of gas exchange and chlorophyll fluorescence
measured simultaneously, at ambient O2 concentration and
at two irradiances (500 and 1500 lmol quanta m2 s1);
and (ii) the CO2 response of gas exchange and chlorophyll
fluorescence measured simultaneously, at 2% O2 and at two
irradiances (500 and 1500 lmol quanta m2 s1). At the end
of the measurements, all leaves were collected, leaf area
(using a leaf area meter, LI-300A, Li-Cor, Lincoln, NE,
USA), and leaf dry mass (after drying at 70 oC for 3 d) were
measured, and leaf dry mass per area (LMA) was calculated. For (i) and (ii), a LI-6400 open gas exchange system
was used with an integrated 2 cm2 chamber fluorometer (LI6400-40, Li-Cor, Lincoln, NE, USA). The photochemical
efficiency of Photosystem II (UPSII) was determined by
measuring steady-state fluorescence, Fs, followed by a multiple saturating pulse of 7000 lmol quanta m2 s1 (Fm# )
according to Genty et al. (1989):
UPSII ¼
Fm# Fs
Fm#
ð1Þ
Before the measurements were taken, CO2 and H2O
values were zeroed and fresh drierite (and soda lime if
necessary) was used. Leaf temperature was between 21–23
o
C, except for B. integrifolia, where the leaf temperature
was 18.7–19.4 oC. Leaf-to-air vapour pressure difference
(VPD) was kept between 1.2–1.4 kPa; this range had no
effect on stomatal conductance (gs). Leaves were kept in the
gas exchange chamber at high irradiance (1500 lmol quanta
m2 s1) and low ambient CO2 concentration (Ca) (100
lmol mol1) for at least 30 min before the start of the
simultaneous measurement of chlorophyll fluorescence and
the response of net leaf photosynthesis (A) to the intercellular CO2 concentration (Ci) (A–Ci response), ensuring
stomata were fully open and steady-state was reached. The
CO2 response of photosynthesis and chlorophyll fluorescence measurements were started at a Ca of 380 lmol
mol1, then the Ca was decreased to 50 or 100 lmol mol1
and subsequently increased at 50 lmol mol1 intervals to
800 lmol mol1. At each Ca, 2–4 measurements were
recorded, at least 1 min apart. This was sufficient time for
all leaf parameters to stabilize after the pulse of CO2.
Between different Ca values, gas exchange and chlorophyll
fluorescence were allowed to stabilize before the next pulse
was given. For a particular leaf, the CO2 response was
recorded at high irradiance (1500 lmol quanta m2 s1) and
then at low irradiance (500 lmol quanta m2 s1) without
removing the leaf from the gas exchange chamber and after
allowing the stomata to reopen. Tests showed that the order
of the high and low irradiances had no effect on the CO2
response. Possible leakage into and out of the cuvette was
checked and the correction approach suggested by Flexas
et al. (2007a) was deemed unnecessary. To improve the
gasket seal around the midrib for some leaves, an additional
flexible modelling compound (Terastat) was used. Lateral
diffusion through the leaf is unlikely to have happened due
to the heterobaric nature of these leaves.
The C3 photosynthesis model (Farquhar et al., 1980) was
fitted to the A–Ci measurements, whereby A is the minimum
of the RuBP-saturated rate of photosynthesis (Ac) and the
RuBP-limited rate (Aj):
A ¼ min Ac ; Aj Rd
ð2Þ
where Ac and Aj are computed as:
Ac ¼
Vc ðCi C Þ
Rd
Ci þKc 1þ KOo
ð3Þ
JðCi C Þ
Rd
4Ci þ8C
ð4Þ
Aj ¼
Vc is the maximum rate of RuBP carboxylation, C* is
the photo-compensation point in the absence of
2306 | Hassiotou et al.
mitochondrial respiration, Rd is respiration in the light,
Kc and Ko are the Michaelis–Menten constants for CO2
and O2, respectively, O is the O2 concentration, and J is
the rate of electron transport.
Calibration of the relationship between Jf and JCO 2
The rate of electron transport estimated from chlorophyll
fluorescence prior to any calibration (Jf,uncal) is given by the
equation:
Jf;uncal ¼ UPSII PPFDab
ð5Þ
where PPFD is the photosynthetic photon flux density, a
is leaf absorptance, and b represents the proportion of
quanta absorbed by Photosystem II (this has been
assumed to be 0.5 for C3 plants; Ögren and Evans,
1993). A Taylor integrating sphere (Taylor, 1935) and
a spectroradiometer (LI1800, Li-Cor, Lincoln, NE, USA)
were used to obtain estimates of a for the LED light
source. Two leaves per species (originating from two
different plants, with the exception of B. paludosa and B.
integrifolia, where two leaves of the same plant were
used) were measured and their average absorptance was
used as the a value of that species.
The relationship between Jf,uncal and the rate of electron
transport obtained from gas exchange (JCO2 ) bears some
fundamental uncertainties. Part of these uncertainties can be
eliminated by measuring a and b (although the latter is rarely
measured; Warren, 2006). However, even when these measurements are made, there is still much uncertainty in the
relationship between Jf and JCO2 which may originate from
alternative electron sinks and/or the fact that the fluorescence
signal primarily emanates from the upper mesophyll
layers and thus, most probably, is not representative of the
whole leaf, in contrast to the gas exchange signal (Warren,
2006). To partially deal with the first uncertainty, fluorescence was calibrated by relating UPSII to A measured under
non-photorespiratory
conditions. UCO2 was calculated as
UCO2 ¼ 4 ðA þ Rd =PPFD. This approach assumes that under low O2 conditions, photorespiration is suppressed; hence
the true relationship between Jf and JCO2 can be established
(Epron et al., 1995). Thus, A–Ci curves (for Ca of 100, 200,
400, 800, and for some species 1000 lmol mol1), combined
with chlorophyll fluorescence measurements, were generated
at 2% O2 at both low (500 lmol quanta m2 s1) and high
(1500 lmol quanta m2 s1) irradiances. The CO2 responses
under non-photorespiratory conditions were collected after
the CO2 responses under photorespiratory conditions for
a particular leaf, without removing it from the gas exchange
chamber. Initial tests showed that this order of data
collection did not influence the results.
The relationship between UCO2 and UPSII at 2% O2 at each
irradiance was used to calibrate Jf, which was calculated as:
Jf ¼ ½ðUPSII sÞþc PPFD
ð6Þ
where s is the slope and c is the intercept of the
relationship between UCO2 and UPSII at 2% O2.
Estimation of gm using the ‘variable J method’
The ‘variable J method’ (Harley et al., 1992), based on
simultaneous measurements of gas exchange and chlorophyll
fluorescence, was used to obtain estimates of gm for each
point of the A–Ci curves at low (500 lmol quanta m2 s1)
and high (1500 lmol quanta m2 s1) irradiances. Initially,
the A–Ci curves were converted to A–Cc curves using the
equation:
C Jf þ8ðAþRd Þ
Cc ¼
ð7Þ
Jf 4ðAþRd Þ
gm was then calculated as:
gm ¼
A
Ci Cc
ð8Þ
A Rd of 0.8 lmol m2 s1 and a C* of 36.9 lmol mol1
(Brooks and Farquhar, 1985), adjusted using the temperature dependence stated by the authors, were used. For the
temperature corrections, the average temperature of the leaf
during the construction of the A–Ci curve was used. A
sensitivity analysis was also done to examine the effects of
different Rd and C* on gm.
Statistical analyses
To test whether gm at 500 lmol quanta m2 s1 (gm500) was
significantly lower than gm at 1500 lmol quanta m2 s1
(gm1500), a paired t test was carried out comparing gm for
the two irradiances at ambient CO2 concentration. This test
was done separately for each species and by considering all
the replicates of all species. A regression analysis, based on
the exponential model gm¼emCi+k, was done to determine
whether gm decreased significantly with increasing Ci. The
data from each leaf were fitted to this model to examine
whether the slope parameter m was significantly different
from zero in each case. Moreover, the relationship between
LMA and the rate at which gm decreased with increasing Ci
was examined using a mixed-effects model to predict
log(gm), with Ci and LMA as the explanatory variables and
individual leaves as random effects, and then considering
the significance of the Ci–LMA interaction. The above
regression analyses were carried out using the R programming language (R Development Core Team, 2007), the first
using the generalized linear modelling function with Gaussian errors and an exponential link function, and the second
using the linear mixed-effects modelling function (Pinheiro
et al., 2007).
Results
gm in seven Banksia species was estimated using gas
exchange and chlorophyll fluorescence at different CO2
concentrations and irradiances. Results from two of the
species studied, B. solandri and B. integrifolia, are presented
in greater detail. The relationships between CO2 assimilation rate per unit leaf area (A) or electron transport rate (Jf)
Mesophyll conductance in sclerophylls | 2307
and intercellular CO2 concentration (Ci) are shown for three
replicate leaves (Fig. 1). Greater Ci values were reached in
B. integrifolia compared with B. solandri because stomatal
conductance declined less at high ambient CO2 concentration (Ca). For both species, CO2 assimilation rates were
reduced across the range of Ci values used at a PPFD of 500
compared with 1500 lmol quanta m2 s1. The transition
from Rubisco to RuBP regeneration limitation of A at a Ci
of about 300 lmol mol1 is apparent from the calculated
rate of electron transport (Fig. 1B, D). In the RuBP
regeneration-limited region, Jf was essentially constant for
B. integrifolia, but gradually increased for B. solandri. In
some species (e.g. B. repens) the RuBP regeneration-limited
region was not reached at a Ca of 800 lmol mol1. A did
not decrease at high CO2 concentrations in any of the leaves
measured. Rubisco-limited and RuBP regeneration-limited
curves were fitted to A–Ci curves for one replicate leaf of
each of the two species (Fig. 2). The RuBP regenerationlimited region was probably not reached by the B. solandri
leaf at a Ca of 800 lmol mol1, but a curve was fitted
through the two highest Ci points by way of illustration.
The apparent transition between the two limitations occurred
at a Ci of 350–400 lmol mol1, which was higher than that
indicated from Jf. While the Rubisco-limited region is well
described by the model, the model curves fit poorly around
the transition region. Comparison of the relationship
between the gas exchange-based quantum yield (UCO2 ) and
the photochemical efficiency of Photosystem II (UPSII) at
Fig. 1. Response of net photosynthesis (A) and electron transport rate estimated from fluorescence (Jf) to intercellular CO2 concentration
(Ci) in Banksia solandri (A, C) and B. integrifolia (B, D), at a PPFD of 1500 lmol quanta m2 s1 (open symbols) and 500 lmol quanta
m2 s1 (filled symbols). Measurements from three replicate leaves (circles, squares, triangles) of each species at each PPDF are shown.
Fig. 2. Responses of net photosynthesis (A) to leaf intercellular CO2 concentration (Ci) fitted to the C3 model of photosynthesis (Farquhar
et al., 1980) in a leaf of Banksia solandri (A) and a leaf of B. integrifolia (B) at a PPFD of 1500 lmol quanta m2 s1 (open circles) and 500
lmol quanta m2 s1 (filled circles). Tleaf (oC) is the leaf temperature, Vc (lmol m2 s1) is the RuBP-carboxylation rate, J (lmol m2 s1)
is the electron transport rate, and Rd (lmol m2 s1) is the respiration rate in the light. For each of these parameters, the first number
corresponds to the value of the parameter at a PPFD of 1500 lmol quanta m2 s1, whereas the second number is the value at a PPFD
of 500 lmol quanta m2 s1.
2308 | Hassiotou et al.
2% O2 and different irradiances (Fig. 3) revealed that
separate calibrations were required for each leaf at each
irradiance to accurately estimate Jf, and thus gm (see
Supplementary Table S1 at JXB online).
A 2-fold range in CO2 assimilation rate per unit leaf area
was found between the Banksia species examined, with
a trend to lower rates as LMA increased (Fig. 4A).
Mesophyll conductance declined from 0.17 mol m2 s1 in
B. attenuata to 0.09 mol m2 s1 in B. elderiana as LMA
increased 4-fold (Fig. 4B). Increasing LMA did not alter the
draw-down in the CO2 concentration from the atmosphere
to the sites of carboxylation (Ca–Cc), and no correlation
was found between LMA and the draw-down in the CO2
concentration from the atmosphere to the substomatal
cavities (Ca–Ci) or from the substomatal cavities to the sites
of carboxylation (Ci–Cc) (Fig. 4C, D, E). Strong correlations were found between A and both gs and gm (Fig. 5).
Scatter in these relationships was also evident, as both B.
integrifolia and B. attenuata had low gm relative to A.
At 1500 lmol quanta m2 s1, a significant decrease in gm
with increasing Ci was found in all the replicates of the
seven species examined (see Supplementary Table S2 at JXB
online). gm declined by 35–60% in response to increasing
CO2 concentration (Ca values of 50–800 lmol mol1) at
1500 lmol quanta m2 s1, with a more variable response
(0–60%) at 500 lmol quanta m2 s1. Despite the variability
of gm amongst some replicates at very low and/or very high
CO2 concentrations, a decline in gm with increasing CO2
concentration was evident at both irradiances in B. integrifolia and at high irradiance in B. solandri (Fig. 6). At 500
lmol quanta m2 s1, a significant decrease in gm with
increasing ambient CO2 concentration was seen in all the
species examined, with the exception of B. solandri (all
replicates) (Fig. 6C) and two out of three replicates of B.
paludosa, for which trends were absent (see Supplementary
Table S2 at JXB online). Mixed-effects modelling showed
that the interaction between LMA and Ci was not
significant (P¼0.07) at high irradiance (1500 lmol quanta
m2 s1) but highly significant (P <0.0001) at low irradiance (500 lmol quanta m2 s1), indicating that the rate at
which gm decreased with increasing Ci was more pronounced in species with high LMA.
To illustrate the sensitivity of the gm calculation to the
fluorescence calibration, two calculations are shown for a B.
solandri leaf at two irradiances (Fig. 7): (i) fluorescence was
calibrated using the slope and intercept of the UCO2 UPSII
relationship under non-photorespiratory conditions; (ii) the
rate of electron transport was calculated from equation 5.
Surprisingly, under high irradiance, gm values calculated
from equation 5 were more stable over the Ci values tested
than when the calibrated fluorescence was used. The latter
resulted in a high gm value at a Ci of around 150 lmol
mol1. Under low irradiance, gm values calculated from
equation 5 increased dramatically once Ci exceeded 500
lmol mol1, whereas gm values calculated from calibrated
fluorescence were consistent over the Ci range tested. The
uncertainty of gm estimates increases at low and high CO2
concentrations.
A sensitivity analysis of the effects of Rd and C* on the
estimation of gm showed that the lower the Ci the greater
the impact of these parameters on gm (see Supplementary
Table S3 at JXB online). Although the degree to which Rd
and C* influenced the estimation of gm changed with
irradiance, this was of minor importance (see Supplementary Table S3 at JXB online). Regardless of Ci, gm was
always more sensitive to changes in Rd at lower than at
higher irradiance. The effect of C* on gm, however, was
somewhat greater at higher than at lower irradiance at Ci
values lower than 200 lmol mol1, but the opposite was
found at Ci values higher than 200 lmol mol1. Reduction
of C* from 36.9 lmol to 28 lmol mol1 reduced gm by
22.5% and 25.2%, on average, at 1500 and 500 lmol
quanta m2 s1, respectively, whereas increasing C* from
36.9 lmol to 40 lmol mol1 increased gm by 11.7% and
14.3%, on average, at 1500 and 500 lmol quanta m2 s1,
respectively.
Fig. 3. Calibration curves for Banksia solandri (A) and B. integrifolia (B) showing the relationship between electron transport rate
estimated from gas exchange and electron transport rate estimated from fluorescence under non-photorespiratory conditions (2% O2).
Data are expressed as gas exchange-based quantum yield (UCO2 ¼4(A+Rd)/PPFD) versus photochemical efficiency of Photosystem II
(UPSII). Each calibration was done at four Ca values (100, 200, 400, and 800 lmol mol1) and at PPFDs of 1500 lmol quanta m2 s1
(open symbols) and 500 lmol quanta m2 s1 (filled symbols). Measurements from three replicate leaves (circles, squares, triangles) of
each species at each PPFD are shown.
Mesophyll conductance in sclerophylls | 2309
Fig. 5. Relationship of mesophyll conductance (gm) (filled symbols)
and stomatal conductance (gs) (open symbols) with rates of net
photosynthesis (A) at a PPFD of 1500 lmol quanta m2 s1. A
was normalized to a common intercellular CO2 concentration (Ci of
262 lmol mol1), which was the average Ci of the seven Banksia
species examined at ambient CO2 concentration (Ca), to take into
account any variation in gs (B. serrata, squares; B. integrifolia,
crosses; B. attenuata, circles; B. solandri, triangles; B. paludosa,
stars; B. repens, inverted triangles; B. elderiana, diamonds). A
significant positive correlation was found between gs–A (P <0.01)
and gm–A (P <0.05).
For each leaf, A–Ci curves were measured at PPFD levels
of 1500 and 500 lmol quanta m2 s1 which allowed us to
examine whether gm varied with irradiance (Fig. 8). On
average, gm at a PPFD of 500 lmol quanta m2 s1 (gm500)
was 22% less than that at 1500 lmol quanta m2 s1
(gm1500). This compares with a decrease in A of 27%. The
decrease in gm with decreased irradiance was significant
(P <0.01 or P <0.05) for all the species studied, with the
exception of B. solandri, in which one out of the three leaves
measured had higher gm500 than gm1500.
Discussion
Fig. 4. Relationship between (A) rate of net photosynthesis (A), (B)
mesophyll conductance (gm), (C) the draw-down in the CO2 concentration from the atmosphere to the substomatal cavities (Ca–Ci),
(D) the draw-down in the CO2 concentration from the substomatal
cavities to the sites of carboxylation (Ci–Cc), and (E) the draw-down
in the CO2 concentration from the atmosphere to the sites of
carboxylation (Ca–Cc), and leaf dry mass per area (LMA) at a PPFD of
1500 lmol quanta m2 s1 and ambient CO2 concentration in the
seven Banksia species examined (B. serrata, squares; B. integrifolia,
crosses; B. attenuata, circles; B. solandri, triangles; B. paludosa,
stars; B. repens, inverted triangles; B. elderiana, diamonds).
The combined effects of leaf structure (LMA), ambient CO2
concentration, and irradiance on gm in sclerophylls of the
genus Banksia with a wide range of LMA were investigated.
Whilst gm decreased with increasing LMA, it scaled with A
such that gm imposed a similar limitation on A in all species.
After discussing the species differences in leaf attributes, the
dynamic changes of gm in response to CO2 and irradiance
and uncertainties associated with the method are then
addressed.
Effects of LMA on gm
In a recent review, Flexas et al. (2008) drew an upper bound
for gm that decreased linearly with increasing LMA, reaching zero at a LMA value of about 240 g m2. Our gm values
for Banksia species with LMA values ranging from 130–480
g m2 lie well above this upper bound. Therefore, the
2310 | Hassiotou et al.
Fig. 6. Response of mesophyll conductance (gm) to intercellular CO2 concentration (Ci) at a PPFD of 1500 lmol quanta m2 s1 (A, B)
and 500 lmol quanta m2 s1 (C, D) in Banksia solandri (A, C) and B. integrifolia (B, D). Measurements from three replicate leaves
(circles, squares, triangles) of each species at each PPFD are shown.
Fig. 7. Effect of chlorophyll fluorescence calibration on mesophyll
conductance (gm) in a leaf of Banksia solandri. The response of gm
to leaf intercellular CO2 concentration (Ci) is presented at a PPFD
of 1500 lmol quanta m2 s1 (A) and 500 lmol quanta m2 s1
(B). Open symbols show gm calculated from uncalibrated fluorescence, whereas filled symbols show gm calculated from calibrated
fluorescence.
notional upper bound is redrawn as a concave curve with an
unknown asymptotic value at LMA greater than 500 g m2
(Fig. 9).
Banksia species with high LMA values showed significantly (P < 0.05) lower gm than low-LMA species at 1500
lmol quanta m2 s1. Leaf structure may constrain gm (Fig.
4) either through gias (conductance to diffusion in the
mesophyll intercellular air spaces), gliq (conductance to
diffusion through the cell walls, the cytosol, and the
chloroplast envelope) or both. Lower mesophyll porosity
and greater leaf thickness of the species with higher LMA
may reduce gias. Chloroplast surface area exposed to the
intercellular air spaces (Sc) has been shown to positively
correlate with gm (Evans et al., 1994; Terashima et al., 2006)
and one would expect Sc to increase with increasing LMA.
However, high-LMA species may have photosynthetic cells
with thicker cell walls which would reduce gliq (Kogami
et al., 2001; Terashima et al., 2006; JR Evans et al.,
unpublished data). The highest-LMA species for which gm
has been examined, B. elderiana, has a mesophyll cell wall
thickness of about 0.35 lm (F Hassiotou, unpublished
data), which is towards the upper end of the 0.15–0.4 lm
range that has been previously reported (Hanba et al., 1999,
2001, 2002). However, it is very unlikely that mesophyll cell
wall thickness is proportional with LMA, as the LMA of B.
elderiana is eight times greater than the tree leaf LMA
values from the above studies for which cell wall thickness
was measured.
The range of gm values recorded in this study at ambient
CO2 concentration and high irradiance (0.084–0.169 mol
m2 s1) for Banksia species falls within the range reported
previously for woody evergreen species (Flexas et al., 2008).
Interestingly, Ci–Cc did not differ noticeably among the
Mesophyll conductance in sclerophylls | 2311
Fig. 8. Effect of irradiance on mesophyll conductance (gm).
Mesophyll conductance at ambient CO2 concentration and
a PPFD of 500 lmol quanta m2 s1 (gm500) and 1500 lmol
quanta m2 s1 (gm1500) for all replicates of six Banksia species is
shown (B. serrata, squares; B. integrifolia, crosses; B. attenuata,
circles; B. solandri, triangles; B. paludosa, stars; B. repens,
inverted triangles). The dotted line is the 1:1 line.
tissue and/or mesophyll cell wall thickening. Increased
investment in structural tissue will have no consequence on
a mesophyll trait like gm. However, if higher LMA is a result
of mesophyll cell wall thickening, this would reduce gm per
unit of exposed mesophyll surface. This reduction could be
offset by increases in exposed mesophyll surface associated
with thicker leaves, which could maintain a constant rate of
photosynthesis per unit leaf area. To maintain the constant
draw-down in CO2 concentration between the intercellular
airspaces and the chloroplasts (Ci–Cc) that was observed, it
was concluded that leaves with thicker mesophyll cell walls
and greater mesophyll cell surface area must have a lower
amount of Rubisco per unit of chloroplast surface area
exposed to intercellular airspace.
The stability in Cc found across a considerable range of
LMA is consistent with co-variation of gm with photosynthetic capacity. The relationship between gm and A was
linear, which is consistent with what has often been
reported in the literature for both mesophytic and sclerophyllous species (von Caemmerer and Evans, 1991; Loreto
et al., 1992; Evans and von Caemmerer, 1996).
Effects of CO2 concentration and irradiance on gm
Fig. 9. Relationship between mesophyll conductance (gm) and
leaf dry mass per area (LMA) in the absence of stress in different
species. Open symbols represent leaves of high-altitude plants,
old leaves, shade leaves, and other leaves, data that have been
compiled from 17 different studies (Flexas et al., 2008). The
Banksia species examined in this study are presented as filled
symbols. Taking into account only the data presented in the open
symbols, Flexas et al. (2008) drew an upper bound for gm that
decreased linearly with increasing LMA, extrapolating to zero at
a LMA value of about 240 g m2. The current study has extended
the LMA range where gm has been examined to a LMA value of
480 g m2. Thus, this notional upper bound is redrawn as
a concave curve with an unknown asymptotic value at LMA
greater than 500 g m2.
species despite a 4-fold range in LMA and a 2-fold range in
A. It is evident from anatomical observations (data not
shown) that variation in LMA is due to changes in leaf
thickness, or structural sclerified tissue relative to mesophyll
The variation in gm with CO2 that was observed in seven
Banksia species was consistent with responses reported by
Flexas et al. (2007b) for six species (Olea europaea, a hybrid
of Vitis berlandieri3Vitis rupestris, Cucumis sativus, Arabidopsis thaliana, Nicotiana tabacum, Limonium gibertii). The
relative response of gm to CO2 (as a percentage of maximum
values) in the above species that was presented by Flexas
et al. (2008) was very similar to that of the Banksia species
studied here. The physiological explanation for the reduced
gm at high CO2 concentration is not known. Consistent with
optimization of resource use, it is to be expected that gs
would increase at low CO2 concentrations, to allow for
more efficient photosynthesis, and would decrease at CO2
concentrations that are not limiting photosynthesis in order
to reduce water loss from the leaf through transpiration;
however, it is not clear why gm would decrease at high CO2
concentrations. Is there a disadvantage to high CO2
diffusion or a cost related to maintenance of high gm?
This is still unknown. Recent evidence involving aquaporins
in gm regulation was given by Miyazawa et al. (2008)
who showed that the decrease in gm in tobacco leaves
acclimated to long-term water stress was not associated with
a decrease in aquaporin content. Rather, there was a loss of
mercury sensitivity which was suggested to reflect deactivated aquaporins. The cost-related hypothesis behind the
decrease of gm at high CO2 concentrations remains to be
elucidated.
Irradiance was also found to impact on gm. At ambient
CO2 concentration, gm at low irradiance (500 lmol quanta
m2 s1) was, on average, 22% lower than that at 1500
lmol quanta m2 s1. The effect of irradiance on gm in our
high-LMA species was much less than that reported for N.
tabacum (Flexas et al., 2007b). The speed and reversibility
of the changes in gm suggest a change in membrane
2312 | Hassiotou et al.
permeability. Aquaporins may provide a mechanism for this
through rapid changes in their gating, insertion or removal
from membranes (Katsuhara et al., 2008). However, it is
also possible that the irradiance dependence of gm is
a consequence of the experimental technique used, since
fluorescence samples the leaf differently to gas exchange.
This is discussed in the next section.
Methodological issues
The ‘fluorescence method’ that is often used to derive gm
bears some assumptions that may explain the dependence of
gm on irradiance observed in this study. This analysis treats
the leaf as a ‘big chloroplast’ which assumes that the ratio
of photosynthetic capacity to light absorbed is the same for
each chloroplast. In the few cases where profiles of
photosynthetic properties have been measured with respect
to depth from the leaf surface, the ratio of photosynthetic
capacity to light absorbed is only constant with depth
through a leaf in green light (Evans and Vogelmann, 2003,
2006). Since chlorophyll fluorescence and photosynthesis
were measured under red light, CO2 fixation occurs predominantly near the adaxial surface under low irradiance,
with fixation occurring deeper within the mesophyll under
high irradiance (Evans and Vogelmann, 2003, 2006).
Assuming that gias>> gliq and that the profile of gliq
through the leaf is proportional to the profile of Rubisco,
under dim red light there is a large draw-down (Ci–Cc) near
the adaxial surface decreasing towards the abaxial surface.
As irradiance increases, additional CO2 fixation occurs
deeper within the mesophyll, which increases the drawdown deep within the mesophyll. However, the profile of
chloroplasts sampled by chlorophyll fluorescence stays the
same. Consequently, while CO2 assimilation rate increases
by 33% under 1500 compared to 500 lmol quanta m2 s1,
the apparent draw-down increases by only 15% (JR Evans,
unpublished data). This translates into a gm500 value that is
15% less than the gm1500 value, consistent with the trend
shown in Fig. 8. Inevitably, there are many assumptions in
this modelling approach in the absence of detailed anatomical information and measurements of gm at such a fine
scale. A more comprehensive modelling effort will be
presented elsewhere as it is beyond the scope of this paper.
Another consequence of the ‘big chloroplast’ model is
that the estimation of gm involves the application of Fick’s
Law to an average point in the mesophyll. In reality, there
are distributed sinks, and a network of resistances in series
and parallel would be a better analogy. While this would
confer more complex behaviour, it is also less tractable
given the limited spatially resolved information available. In
contrast to our finding that gm estimated using the
fluorescence method varies with irradiance, gm estimated by
carbon isotope discrimination was found to be independent
of irradiance in wheat leaves (Tazoe et al., 2009). Given that
both the fluorescence and isotope methods for calculating
gm require a number of assumptions, that the short-term
response of gm to irradiance has only been observed for
tobacco (Flexas et al., 2007b) and Banksia (Fig. 8), but not
wheat (Tazoe et al., 2009) and that both methods have not
been applied to the same leaves, more experimentation is
needed to elucidate the response of gm to irradiance.
The fluorescence signal for each leaf was calibrated to
avoid the assumptions needed when calculating Jf. The
sensitivity of gm to this calibration is illustrated in Fig. 7.
The non-zero intercept of the relationship between UCO2 and
UPSII at a low O2 concentration may be indicative of
alternative electron sinks. These could include pathways in
the chloroplasts that use reduced ferredoxin or NADPH
(Genty and Harbinson, 1996), the oxaloacetate–malate
shuttle (Scheibe, 1987), nitrate assimilation and biosynthetic
activities, or cyclic electron flow around Photosystem I
(Heber et al., 1978; Furbank and Horton, 1987).
A sensitivity analysis of the effects of Rd and C* on the
estimation of gm has been presented (see Supplementary
Table S3 at JXB online). Although the absolute values of
gm are affected by Rd and C*, these effects are significantly
(P <0.01) lower at Ci values between 200 lmol and 400
lmol mol1. Most importantly, this exercise showed that
error in neither Rd nor C* altered the dependence of gm on
CO2, irradiance or LMA.
Conclusions
This study extended the range of LMA values where gm had
been measured from 220 to 480 g m2, presenting the effects
of both leaf structure (through LMA) and key environmental factors (CO2 and irradiance) on gm in sclerophyllous
Banksia species. Increased CO2 concentration and decreased
irradiance caused gm to significantly decline. gm showed
a strong correlation with LMA, with high-LMA species
having a significantly lower gm than low-LMA species.
Interestingly, across the LMA range that was examined and
across a wide variation in photosynthetic capacity, the
leaves had a similar draw-down in the CO2 concentration
from the atmosphere to the chloroplasts, i.e. they had
similar Ci and Cc values. This supports the notion that
variation in gs and gm across different species and functional groups results in a highly conserved Cc. Future
studies must focus on how leaf structural parameters such
as cell wall thickness and exposed chloroplast surface area
influence gm.
Supplementary data
Three supplementary tables are available at JXB online:
Supplementary Table S1. Slopes and intercepts of the
calibration curves.
Supplementary Table S2. Exponential regression analysis
of the response of mesophyll conductance (gm) to leaf
intercellular CO2 concentration (Ci).
Supplementary Table S3. Sensitivity analysis of the
effect of the respiration in the light (Rd) and the photocompensation point in the absence of mitochondrial
Mesophyll conductance in sclerophylls | 2313
respiration (C*) on the estimation of gm in the Banksia
species examined.
Acknowledgements
Many thanks are extended to Professor Marilyn Ball for
providing us with the Li-Cor fluorescence chamber head
and to Professor Hans Lambers and Professor Paul
Kriedemann for critically reading the manuscript. We are
grateful to Mrs Stephanie McCaffery for her assistance with
part of the long-day gas exchange measurements in
Canberra. The authors acknowledge the Australian Research Council–New Zealand (ARC–NZ) Research Network for Vegetation Function for funding part of the study.
FH was the recipient of an Australian Postgraduate Award.
This manuscript was improved after insightful discussions
during the workshop ‘Mesophyll conductance to CO2:
mechanisms, modelling and ecological implications’ that
FH and JRE attended in Palma de Mallorca in September
2008. Thanks to Dr J Flexas who was the main organizer of
the workshop and the European Science Foundation (ESF)
for funding support.
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