Journal of Experimental Botany, Vol. 60, No. 8, pp. 2235–2248, 2009
doi:10.1093/jxb/erp117 Advance Access publication 23 April, 2009
REVIEW PAPER
Resistances along the CO2 diffusion pathway inside leaves
John R. Evans1,*, Ralf Kaldenhoff2, Bernard Genty3 and Ichiro Terashima4
1
Environmental Biology Group, Research School of Biological Sciences, The Australian National University, GPO Box 475, Canberra
ACT 2601, Australia
2
Department of Applied Plant Sciences, Institute of Botany, Darmstadt University of Technology, D-64287 Darmstadt, Germany
3
CEA, CNRS, Université Aix-Marseille, UMR 6191 Biologie Végétale et Microbiologie Environnementale, Laboratoire d’Ecophysiologie
Moléculaire des Plantes, CEA Cadarache, 13108 Saint Paul lez Durance, France
4
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033,
Japan
Received 10 December 2008; Revised 12 March 2009; Accepted 17 March 2009
Abstract
CO2 faces a series of resistances while diffusing between the substomatal cavities and the sites of carboxylation
within chloroplasts. The absence of techniques to measure the resistance of individual steps makes it difficult to
define their relative importance. Resistance to diffusion through intercellular airspace differs between leaves, but is
usually of minor importance. Leaves with high photosynthetic capacity per unit leaf area reduce mesophyll resistance
by increasing the surface area of chloroplasts exposed to intercellular airspace per unit leaf area, Sc. Cell walls impose
a significant resistance. Assuming an effective porosity of the cell wall of 0.1 or 0.05, then cell walls could account for
25% or 50% of the total mesophyll resistance, respectively. Since the fraction of apoplastic water that is unbound and
available for unhindered CO2 diffusion is unknown, it is possible that the effective porosity is <0.05. Effective porosity
could also vary in response to changes in pH or cation concentration. Consequently, cell walls could account for >50%
of the total resistance and a variable proportion. Most of the remaining resistance is imposed by one or more of the
three membranes as mesophyll resistance can be altered by varying the expression of cooporins. The CO2
permeability of vesicles prepared from chloroplast envelopes has been reduced by RNA interference (RNAi) expression
of NtAQP1, but not those prepared from the plasma membrane. Carbonic anhydrase activity also influences mesophyll
resistance. Mesophyll resistance is relatively insensitive to the manipulation of any step in the pathway because it
represents only part of the total and may also be countered by pleiotropic compensatory changes. The parameters in
greatest need of additional measurements are Sc, mesophyll cell wall thickness, and the permeabilities of the plasma
membrane and chloroplast envelope.
Key words: Aquaporin, cell wall, cooporin, internal conductance, mesophyll conductance.
Introduction
The consumption of CO2 by Rubisco reduces the partial
pressure of CO2, p(CO2), in the stroma of the chloroplasts,
Cc, below that in the ambient air, Ca. The magnitude of the
draw-down in p(CO2) between Ca and Cc depends on
the balance between the net rate of CO2 assimilation and
the conductance to CO2 transfer. The first steps along the
diffusion pathway, namely the boundary layer and stomatal
conductance, can be quantified by gas exchange analysis
and used to calculate the p(CO2) in the substomatal cavities,
Ci (von Caemmerer and Farquhar, 1981). The p(CO2) at the
sites of carboxylation, Cc, can also be calculated by a variety
of techniques (Evans and Loreto, 2000; Warren, 2006). Using
Fick’s law, mesophyll conductance, gm, has been defined as
the CO2 assimilation rate, A, divided by Ci–Cc. The term
mesophyll conductance (Harley et al., 1992) is synonymous
with internal conductance (Lloyd et al., 1992), wall and liquid
phase conductance (Evans, 1983), and CO2 transfer conductance (von Caemmerer and Evans, 1991). In this review, the
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2236 | Evans et al.
components of mesophyll conductance are examined, with
particular emphasis on their relative importance (see also
Evans and Loreto, 2000; Ethier and Livingston, 2004;
Terashima et al., 2006; Flexas et al., 2008; Warren, 2008).
It is important to note that mesophyll conductance is
defined as A/(Ci–Cc). This definition assumes that the leaf
can be simplified to one sink at the average position in the
mesophyll and necessarily ignores the complexity of a leaf
(see Parkhurst, 1994 for extensive discussion). In reality, the
mesophyll is a complex structure that varies greatly between
species and growth irradiance. The diffusion pathway
through intercellular airspaces can be influenced by leaf
thickness, cell shape, and packing relative to the position of
stomata (Fig. 1). For example, a tobacco leaf has a single
layer of cells in the palisade tissue whereas a Camellia
japonica leaf has three layers. While the C. japonica leaf is
twice as thick as the tobacco leaf, in both leaves the palisade
tissue occupies the upper 40% of the mesophyll. The
impression that palisade cells are tightly packed with narrow
vertical channels between cylindrical cells conceals the fact
that numerous lateral channels may also be present (Fig. 1B).
To enable analysis, mesophyll conductance can be separated into its two phases, gaseous and liquid. It is conceptually
convenient to consider the reciprocal of conductance, that is,
resistance, since the total resistance of a pathway is given by
the sum of the resistances along a series.
rm ¼ rias þrliq
ð1Þ
where, rm, rias, and rliq represent the mesophyll, intercellular
airspace, and liquid resistances, respectively.
Gaseous phase
The complex problem of modelling CO2 diffusion through
intercellular airspaces within a 3D leaf with distributed
sinks has seldom been solved (Aalto and Juurola, 2002).
However, the dramatic consequence of diffusion limitations
can be seen with spatially resolved fluorescence imaging
(Morison et al., 2005). By placing spots of silicone grease
onto the leaf surface to prevent CO2 entry across the
epidermis, the only pathway available for CO2 to diffuse to
mesophyll cells underneath the spot was laterally through
the intercellular airspaces. The inferred lateral CO2 gradient
could be modelled using a 2D approach (Morison et al.,
2005).
Lateral diffusion in leaves can be restricted to patches
bounded by bundle sheath extensions to veins that reach
both epiderms. If stomata in a given patch are closed, then
CO2 assimilation can be restricted (Terashima et al., 1988).
There are two extremes in leaf type associated with bundle
sheath extensions: one forming a complete vertical barrier,
termed a heterobaric leaf; and the other where no barrier is
present, termed a homobaric leaf (Terashima, 1992). Leaves
can also have incomplete barriers, such that a continuum
exists between the two extreme types. It is also possible to
form a paradermal barrier to diffusion through the centre of
a leaf which isolates the upper and lower intercellular
airspaces, e.g. maize (Long et al., 1989).
Lateral diffusion conductance has recently been explored
in several studies which demonstrate that it is of the same
order of magnitude as vertical conductance through intercellular airspaces (Morison et al., 2005, 2007; Pieruschka
et al., 2005, 2006; Lawson and Morison, 2006). Resistance
to diffusion through intercellular airspaces has been investigated by comparing gas exchange under normal air
with that in helox (air where helium replaces nitrogen to
increase diffusivity). Parkhurst and Mott (1990) found
a significant increase in CO2 assimilation rate in helox at
the same substomatal Ci (0–27%) and the enhancement was
greater in hypostomatous leaves. In contrast, Genty et al.
Fig. 1. Leaf anatomy varies between species and with growth irradiance. The paths for CO2 diffusion through intercellular airspaces from
substomatal cavities to cell walls adjacent to chloroplasts depend on mesophyll thickness, porosity, and location of stomata. Nicotiana
tabacum transverse (A) and paradermal (B) sections (Evans et al., 1994), Camellia japonica transverse section (C) (Hanba et al., 1999).
Resistances along the CO2 diffusion pathway inside leaves | 2237
(1998) observed no detectable change in leaves of Rosa
rubiginosa and Populus koreana3trichocarpa. A theoretical
diffusion analysis of leaves suggested that resistance
through intercellular airspaces represented between 8% and
42% of mesophyll resistance for Phaseolus vulgaris and
Pinus sylvestris, respectively (Niinemets and Reichstein,
2003). It is likely that the importance of gaseous diffusion
limitation varies between species and during leaf expansion,
but, as few studies have been reported, it is generally
assumed that resistance through the gaseous phase is small
relative to that through the liquid phase.
The liquid pathway through which CO2 diffuses to reach
Rubisco comprises many elements (Fig. 2). CO2 dissolves in
the water-filled pores of the cell wall and diffuses to the
plasma membrane where it enters the cytosol. It then diffuses
to the chloroplast, through the envelope and stroma to reach
Rubisco. CO2 is also released inside mitochondria during
respiration and photorespiration and diffuses out into the
cytosol. The total resistance through the liquid phase can be
expressed as the sum of the resistance of each element in
series (Evans et al., 1994; Niinemets and Reichstein, 2003;
Terashima et al., 2006):
r#liquid ¼ r#wall þr#plasma membrane þr#cytosol
þr#chloroplast envelope þr#stroma
Liquid phase
Gas exchange parameters are usually expressed per unit
projected leaf area but, as CO2 enters the liquid phase, it is
useful to scale the fluxes to the surface area of chloroplasts
exposed to intercellular airspaces. The importance of
chloroplast location was well stated by Haberlandt (1914):
‘chloroplasts adhere.to those walls which abut upon airspaces; by this means they evidently obtain the most
favourable conditions for the absorption of carbon-dioxide’.
Laisk et al. (1970) hypothesized that mesophyll conductance
should depend on the surface area of chloroplasts exposed to
intercellular airspaces, and this was observed for tobacco
(Evans et al., 1994).
In this review, resistances (or conductances) expressed on
the basis of the exposed chloroplast surface area are
denoted by r#x (or g#x), where the x stands for the element
of the pathway. They can be converted to a projected leaf
area basis using the surface area of chloroplasts exposed to
intercellular airspace per unit leaf area, Sc,
rx¼ r#x =Sc or gx¼ g#x Sc
ð2Þ
ð3Þ
At present, none of these elements can be independently
measured in the intact leaf. However, by considering all of
the components together, it is possible to speculate on their
relative importance. The resistance to CO2 diffusion imposed by each element, r#x, can be expressed following
a rationale introduced in previous studies (Hall, 1971;
Niinemets and Reichstein, 2003; Terashima et al., 2006) as:
hx s x
¼
ð4Þ
r#diffusion
x
ux Dx jx
where hx is the thickness of element x, sx is the tortuosity of
the pathway through the element, ux is the porosity of the
element when relevant, Dx is the diffusivity of CO2 in the
element’s solvent (water or lipid), and jx is the solvent to
water partitioning coefficient for CO2 in the element (for
water and lipids, j equals 1 and 1.5–1.7, respectively,
Gutknecht et al., 1977; Missner et al. 2008).
An additional term is required to account for the
potential effect of facilitation of CO2 transport such that r#x
is expressed as:
Fig. 2. Transmission electron micrograph illustrating the liquid pathway for CO2 diffusion within a leaf of Nicotiana tabacum (Frederick
and Newcomb, 1969). Inset at top left are micrographs of onion cell wall with the same magnification (McCann et al., 1990), top, fresh
unfixed; below, pectins extracted with CDTA, Na2CO3, and 1 M KOH. Both white scales are 0.2 lm.
2238 | Evans et al.
hx s x
1
1
¼ r#diffusion
r#x ¼
x
ð1 þ ex Þ
ux Dx jx ð1 þ ex Þ
ð5Þ
where ex is defined as the enhancement due to facilitation
processes in that element.
In membranes, facilitation of CO2 transport can result from
specific protein channelling (i.e. porin), and e can be defined as
the enhancement associated with porins. Note that when
facilitation by porin is absent (e¼0), Equation 5 reduces to the
traditional view that membrane resistance is simply related to
the solubility of the molecule in the lipid phase (Equation 4).
This view is invalid when porins are involved (Al-Awqati, 1999),
hence the introduction of an additional term in Equation 5.
For modelling purposes, CO2 transport through a membrane is treated as two resistances in parallel, one associated
with the bulk membrane, r#x_B.Memb, and the other associated with cooporins, r#x_Coop (see section on Plasma
membrane, Terashima et al., 2006), where the x represents
the plasma membrane or the chloroplast inner or outer
envelope. It is assumed membranes have sufficiently low
permeability to bicarbonate that it can be ignored. Thus,
the resistance and the corresponding conductance through
a specific membrane element are:
1
r#x membrane ¼ ðr#1
x B:Memb þ r#x Coop Þ
1
and
g#x membrane ¼ g#x B:Memb þ g#x Coop
ð6Þ
Using Equation 6, g#x_membrane can be rearranged as:
!
g#x Coop
ð7Þ
g#x membrane ¼ g#x B:Memb 1 þ
g#x B:Memb
and, r#x
membrane
as:
r#x membrane ¼ r#x B:Memb r#x membrane ¼ r#x B:Memb 1
1þ
g#x Coop
g#x B:Memb
ð8Þ
1
1þ
or
r#x B:Memb
r#x Coop
Since by definition only non-facilitated passive transport
occurs at the bulk membrane, r#B.Memb equals r#diffusion
membrane and
Equation 8 become:
1
or
r#x membrane ¼ r#diffusion
x membrane g#x Coop
1 þ g#x B:Memb
ð9Þ
1
diffusion
r#x membrane ¼ r#x membrane r#
1 þ r#xx B:Memb
Coop
The facilitation term, e for a specific membrane element
in Equation 5, can now be described from Equation 9 as:
g#x Coop
r#x B:Memb
ex membrane ¼
¼ ð10Þ
g#x B:Memb
r#x Coop
In the aqueous phase, facilitation is due to the contribution of HCO–3 to CO2 transport, which is related to the
efficiency of the interconversion between HCO–3 and CO2
catalysed by carbonic anhydrase (CA). Under steady state,
facilitated CO2 diffusion requires (i) rapid interconversion
between CO2 and HCO–3 and H+, which depends on CA
activity; and (ii) equal diffusion traffic of H+ and HCO–3
which implies facilitated H+ diffusion, i.e. diffusion of
mobile buffers carrying H+ (Gros and Moll, 1974;
Gutknecht et al., 1977). Since HCO–3 concentration
increases with increasing pH, the potential efficiency of the
facilitation process increases along the CO2 diffusion
pathway from the weakly acidic apoplast to the weakly
alkaline chloroplast stroma. Considering that CO2 and
HCO–3 are the major inorganic carbon species at the pH of
the mesophyll, CO2 transport is treated as two resistances in
parallel, one associated with the CO2 diffusion, r#x_aqueous_C
and the other associated with HCO–3 diffusion, r#x_aqueous_B.
Using the same rationale as in Equations 6–10, e can be
described for a given aqueous element as:
ex aqueous ¼
g#x aqueous B r#x aqueous C
¼
g#x aqueous C r#x aqueous B
ð11Þ
Assuming that the [HCO–3]/[CO2] is close to the equilibrium for the pH and that the diffusion pathway is similar
for CO2 and HCO–3, then e can be calculated for a given
aqueous element as:
HCO
DB
3
ex aqueous ¼
ð12Þ
½CO2 x D
where DB/D is the ratio of diffusivities of bicarbonate to
CO2 in water (Farquhar, 1983). At 25 C, DB/D¼0.56
(Kigoshi and Hashitani, 1963).
In C3 plants, there is both theoretical and experimental
evidence for significant CA-dependent facilitation of CO2
transport across chloroplast stroma even though the CO2
assimilation rate appears insensitive to changes in CA
activity (Cowan, 1986; Price et al., 1994; Gillon and Yakir,
2000). In these studies, leaf CA activity was attributed to
the main chloroplastic bCA isoform (bCA1), which is
localized in the stroma (Badger and Price, 1994; Moroney
et al., 2001). However, several other CA isoforms showing
specific subcellular targeting relevant to CO2 transport and
recycling in the mesophyll have been identified in Arabidopsis
(Fabre et al., 2007): bCA2 and bCA3 are targeted to the
cytosol, bCA4 to the plasma membrane, bCA5 to the
chloroplast, and bCA6 to the mitochondria. The cytosolic
isoform bCA2 can be relatively abundant, as its activity
represents 13% of total CA activity estimated in potato leaf
(Rumeau et al., 1996). While there is strong experimental
evidence for the occurrence of CAs in all of the compartments involved along the CO2 diffusion pathway with the
exception of the cell wall, the potential facilitation role of
these recently identified CA isoforms has yet to be reported.
Cell wall
There are a growing number of species for which both
mesophyll conductance and quantitative anatomical data
Resistances along the CO2 diffusion pathway inside leaves | 2239
exist. For Nicotiana tabacum, mesophyll conductance varied
almost in direct proportion with the surface area of
chloroplasts exposed to intercellular airspace per unit leaf
area (Evans et al., 1994). However, when many species were
examined together, it became apparent that mesophyll
conductance per unit of exposed chloroplast area varied
considerably (Evans et al., 2004; Terashima et al., 2006).
The resistance to CO2 diffusion imposed by cell walls,
r#wall, can be estimated from Equation 5. Values for the cell
wall porosity and tortuosity of the pores through cell walls
are unknown. However, some values can be approximated
by assuming a water content of 50%, some simple geometry,
and the images of onion cell walls (McCann et al., 1990). The
tangled web of microfibrils, which have a diameter of 8 nm,
create pores 10–20 nm in diameter (Fig. 3A, B). By assuming
the microfibrils are straight cylinders aligned parallel to each
other, separated by their radius and that the alignment of
each successive layer is normal to its neighbours, this
simplified matrix contains 50% water (Fig. 3C). The pores
occupy 11% of the plan area at their narrowest plane, which
is illustrated by changing the black threshold in Fig. 3B.
When viewed in cross-section, the pathway curves around the
cylinder, which results in a tortuosity up to p/2. In reality, the
Fig. 3. Cell wall structure. (A) Fresh, unfixed onion cell wall
(McCann et al., 1990). (B) 11% of image A set as an apparent
pore. (C) Diagram of a cell wall in plan view showing a microfibril
array spaced one radius apart. This results in a structure with 50%
water content and direct pores occupying 11% of the plan area.
(D) Cross-sectional view of the wall showing the changing size of
each pore and the tortuosity with depth through the matrix.
microfibrils are not arranged in such regular order, but this is
likely to alter mainly the pore size and not the proportion of
plan area that the pores represent. Irregular orientation
between layers may also affect the tortuosity. This simple
model suggests that for a wall with a 50% water content, the
effective porosity, u/s, could be 0.1132/p¼0.07.
If cell walls represent a significant proportion of the liquid
phase resistance, then one would expect that leaves with
thicker cell walls would have lower mesophyll conductance
per unit of exposed chloroplast surface area. This is apparent
when leaves are divided into two classes of cell wall thickness
(Fig. 4). Leaves with thin mesophyll cell walls have much
greater mesophyll conductance for a given exposed chloroplast surface area than leaves with thick mesophyll cell walls.
The data were therefore reanalysed by calculating mesophyll resistance per unit of exposed chloroplast surface area,
r#m. When all the data available are compared, r#m
increased approximately linearly with increasing mesophyll
cell wall thickness (Fig. 5A). Solid lines from Equation 4 are
drawn for various values of effective porosity (u/s). This
has previously been assumed to be between 0.1 (Terashima
et al., 2006) and 0.3 (Evans et al., 1994; Nobel, 1999). If one
assumes an effective porosity of 0.1 or 0.05, then cell walls
would account for 25% or 50% of the total mesophyll
resistance, respectively. The remaining proportion of mesophyll resistance was virtually independent of cell wall
thickness. This suggests, surprisingly, that other elements in
the resistance pathway co-vary with cell wall thickness. For
example, the rate of CO2 assimilation under high irradiance
per unit of exposed chloroplast surface area declines with
increasing cell wall thickness (Fig. 5B). This implies that
leaves with thicker mesophyll cell walls have less Rubisco
per unit of exposed chloroplast surface area and thinner
chloroplasts because if the content of Rubisco per unit of
exposed chloroplast surface area did not decrease with
Fig. 4. Relationship between mesophyll conductance, gm, and the
surface area of chloroplasts exposed to intercellular airspace per
unit leaf area, Sc, for leaves with thin or thick mesophyll cell walls
(Evans et al., 1994; Evans and Vellen, 1996; Hanba et al., 1999,
2001, 2002; Kogami et al., 2001; Vyas et al., 2007; Tholen et al.,
2008).
2240 | Evans et al.
could potentially play a slight role in facilitating CO2
diffusion through the wall to the plasma membrane.
Apoplastic pH can be influenced by transpiration rate and
whether nitrate or ammonium was added to the transpiration stream (Jia and Davies, 2007), so it is possible that e
(Equation 12) could vary. The hydration of cell wall
materials creates a lot of bound water in the pectic gels
which may restrict the free diffusion of molecules. Extraction of onion cell walls with CDTA and Na2CO3 to remove
pectic polymers clearly increased the size of the pores
(McCann et al., 1990) (Fig. 2, top left). The dimensions of
aqueous channels through the wall may be able to change
when either the concentration of cations or pH changes in
the apoplast. In turn, this could alter the diffusion resistance through the cell wall.
Plasma membrane
Fig. 5. (A) Relationship between mesophyll resistance per unit of
exposed chloroplast surface area and mesophyll cell wall thickness. Solid lines are shown for various effective porosities.
Regression: y¼471x+16, r2¼0.54, n¼56. Data sources are given
in the legend to Fig. 4. (B) CO2 assimilation rate per unit of
exposed chloroplast surface area in relation to mesophyll cell wall
thickness. (C) Draw-down between Ci and Cc in relation to cell wall
thickness. Regression: y¼119x+68, r2¼0.14, n¼56.
increases in cell wall thickness, then the draw-down Ci–Cc
would have been much greater than that observed. If a 0.1
lm thick cell wall accounted for 50% of the total mesophyll
resistance in leaves, then a cell wall 0.3 lm thick would
increase Ci–Cc by 100%, whereas the limited data available
suggest that it increases by only 30% (Fig. 5C). At this
stage, there are too few measurements from which to
generalize. The net result of the decline in CO2 assimilation
rate per chloroplast area is to offset the increase in
mesophyll resistance such that the draw-down Ci–Cc is only
weakly related to cell wall thickness (Fig. 5C).
The slightly acidic pH of the apoplast (5.5–6; Jia and
Davies, 2007) means that bicarbonate and apoplastic CA
The concept of CO2 diffusion through the plasma membrane has recently been changed by the possibility that there
are two parallel pathways: through the bulk membrane and
through cooporins (aquaporins that are permeable to CO2;
Terashima et al., 2006). Traditionally it was thought that
because CO2 is lipophilic, it could readily pass through lipid
bilayers. The permeability required to satisfy the CO2
assimilation rate of a chloroplast is so large that it is
technically difficult to measure. With the discovery of
aquaporins, evidence has been growing in support of their
role not only as water channels, but in increasing membrane
permeability to CO2. Evidence first appeared using Xenopus
oocytes where expression of the human red blood cell
aquaporin gene AQP1 increased their permeability to CO2
(Nakhoul et al., 1998). The effect could be inhibited by
p-chloromercuriphenylsulphonic acid (Cooper and Boron,
1998). There are multiple aquaporin genes in plants and,
whereas the water permeability of the plasma membrane
intrinsic protein (PIP) PIP1 types are insensitive to mercury,
the PIP2 types can be inhibited by mercury (Biela et al.,
1999; Suga and Maeshima, 2004). Terashima and Ono
(2002) exploited this to provide the first evidence that
suggested aquaporins might enhance CO2 permeability in
Vicia faba and P. vulgaris leaves. They found that mercury
altered the response of the CO2 assimilation rate relative to
intercellular CO2, and calculated that mesophyll conductance had been reduced. Then, as had been found with
human AQP1, when N. tabacum NtAQP1 was expressed in
Xenopus oocytes, the CO2 permeability was increased
(Uehlein et al., 2003). More direct evidence was forthcoming with antisense expression of a PIP gene from Hordeum
vulgare, HvPIP2;1 in Oryza sativa plants (Hanba et al.,
2004), and NtAQP1 in N. tabacum (Flexas et al., 2006). In
both cases, antisense suppression was associated with
a reduction in mesophyll conductance. Given that these
proteins could alter CO2 permeability, the term aquaporin
seems to be a misnomer, and Terashima et al. (2006)
proposed the more appropriate name of cooporin. When
the CO2 permeability of membrane vesicles was compared,
Resistances along the CO2 diffusion pathway inside leaves | 2241
antisense suppression of NtAQP1 did not affect the CO2
permeability of plasma membrane vesicles (Uehlein et al.,
2008). Rather, there was a significant reduction in the CO2
permeability of membrane vesicles prepared from the chloroplast envelope (discussed further below). The permeability
associated with PIP aquaporins appears to depend on the
formation of multimeric complexes, and this also changes
whether the complex can be regulated by phosphorylation
(Harvengt et al., 2000; Fetter et al., 2004; Temmei et al.,
2005). By expressing artificially linked tetramers with different ratios of NtAQP1 and NtPIP2;1 in Saccharomyces
cerevisiae it was found that increased CO2 permeability
required the presence of three or four out of four copies of
multiple NtAQP1, whereas increased water permeability
required only a single copy of NtPIP2;1 and water permeability increased in proportion with additional copies
(Fischer, 2007; Pede, 2008). PIP1 does not affect water
permeability in leaves and heterologous systems; however, in
roots, PIP1 appears to alter water permeability because it
affects PIP2 integration (R Kaldenhoff, unpublished).
Cytosol
Chloroplasts are appressed against mesophyll cell walls in C3
leaves with little cytosol in between. However, the thickness
of the intervening layer of cytosol is somewhat uncertain
because of the risk of fixation artefacts. It is not uncommon
to see a considerable gap between the chloroplast and cell
wall in electron micrographs. The question is whether this
reflects the actual chloroplast position in vivo. Chloroplasts
are attached to actin filaments that are involved in chloroplast movement in response to light and other stimuli
(Takagi, 2003; Wada et al., 2003). An indication that the
thickness of the cytosolic layer could influence mesophyll
conductance is found in the comparison of tobacco expressing excess phytochrome (Sharkey et al., 1991). Relative to the
wild type, leaves with additional phytochrome were thicker,
contained more Rubisco and chlorophyll per unit leaf area,
but had lower CO2 assimilation rates and mesophyll
conductance. A feature noted in the transgenic leaves was
the cupping of chloroplasts, with 40% of chloroplasts having
a layer of cytosol that was >0.7 lm. While a thicker layer of
cytosol would be expected to reduce mesophyll conductance,
quantitative analysis is not possible without knowing the
surface area of chloroplasts exposed to intercellular airspace
per unit leaf area for these two leaf types.
The pH of the cytosol is ;7.5 (Kurkdjian and Guern,
1989), so interconversion of CO2 and bicarbonate by CA
can facilitate CO2 diffusion. To date, there is no evidence
that variation in cytosolic CA significantly influences
mesophyll conductance.
Chloroplast envelope
Chloroplasts are bounded by an envelope which consists of
two membranes. As mentioned for the plasma membrane,
CO2 movement across these membranes can occur via the
bulk membrane and/or via cooporins. Confocal microscopy
of cells expressing cooporin–green fluorescent protein
(GFP) fusion proteins, in situ immunology studies, and
protein identification in isolated membranes have demonstrated that NtAQP1 is present in the chloroplast inner
membrane as well as in the plasma membrane (Uehlein
et al., 2008). When membrane vesicles were assayed, RNA
interference (RNAi)-mediated reduction in NtAQP1 reduced the CO2 permeability of chloroplast envelopes by
90% compared with only a 10% reduction for plasma
membranes. The CO2 permeability of the chloroplast
envelope was ;5 times less than that of the plasma
membrane, suggesting that this was a major component of
the resistance pathway (Uehlein et al., 2008). However,
when mesophyll conductances for the control and RNAi
leaves were compared, there was only a 20% reduction. On
the other hand, Niinemets and Reichstein (2003) concluded
that the permeability of the plasma membrane was less than
that of the chloroplast envelope based on phytohormone
uptake rates measured on protoplasts and chloroplasts by
Gimmler et al. (1981). Thus, the quantitative values for
permeability are still uncertain, and further work is required
to obtain values that are consistent with the constraint
placed by measurements of mesophyll conductance with
intact leaves. One of the difficulties with manipulating
aquaporin genes is that altering the expression of one gene
alters the expression level of other aquaporins (Li et al.,
2008), but whether this translates into altered protein
abundance in the membrane is unclear. The ultimate
location of PIPs expressed in cells also seems to depend on
which other PIPs are present. In Zea mays, when both
ZmPIP1 and ZmPIP2 were expressed in mesophyll protoplasts, then ZmPIP1 relocated from the endoplasmic reticulum to the plasma membrane in association with
ZmPIP2 (Zelazny et al., 2007). The resultant permeability
of the PIPs also depends on the composition of multimeric
complexes (Fischer, 2007; Pede, 2008).
Chloroplast stroma
Having entered the chloroplast, CO2 then faces the longest
diffusion distance in the liquid phase. The diffusion is
facilitated by CA (Cowan, 1986) which interconverts CO2
and bicarbonate, because at the pH of the stroma (pH up to
8), the ratio of bicarbonate to CO2 is ;50. The majority of
CA activity in leaves is thought to be localized within
chloroplasts. Antisense reduction of CA in N. tabacum
reduced mesophyll conductance by ;30% (Price et al., 1994;
Williams et al., 1996). By comparing 13C and 18O discrimination in CO2, Gillon and Yakir (2000) gained two
estimates of CO2 concentration in the liquid phase. They
calculated that chloroplast resistance accounted for 76% of
mesophyll resistance in Glycine max, 55% in N. tabacum,
and 24% in Quercus robur leaves.
It is unclear whether CO2 diffusion is restricted in any
way by thylakoid membranes or starch granules that are
2242 | Evans et al.
frequently prominent inside chloroplasts. The volume of
chloroplasts is likely to be closely related to the Rubisco
content of a leaf. Variation in Rubisco content per unit leaf
area is likely to result in an increase in chloroplast surface
area per unit leaf area and/or an increase in chloroplast
thickness. Limited data exist, but for Triticum aestivum, N.
tabacum, and Chenopodium album, an increase in Rubisco
per unit of exposed chloroplast surface area is associated
with increased chloroplast thickness (Fig. 6A). The diagonal
lines suggest that Rubisco active site concentration is
between 0.5 and 2 mM, but the difference between species
may reflect the fact that Rubisco was quantified by different
methods in each study. The increase in Rubisco per
chloroplast surface area should result in greater fluxes of
CO2 per chloroplast surface area for leaves with thicker
chloroplasts. However, as mesophyll resistance per chloroplast surface area does not appear to vary with chloroplast
thickness in tobacco (Fig. 6), this results in greater drawdowns in CO2 between intercellular airspaces and the sites
of carboxylation in leaves with thicker chloroplasts. Somewhat paradoxically, although the results from analysing the
role of CA suggested that the chloroplast stroma must
impose a significant resistance, this resistance appears to be
independent of pathlength because it does not vary with
chloroplast thickness. It contrasts with the significant
increase in mesophyll resistance per chloroplast surface area
observed when cell wall thickness increases (Fig. 5A).
Integration
The range in CO2 permeability values published for various
membranes spans four orders of magnitude (Table 1). It is
likely that permeability values depend to some extent on
how and in which system they are measured. For example,
values obtained from planar lipid bilayers tend to be much
greater than those from vesicles. For any of the assays,
diffusion through the unstirred boundary layer can reduce
the apparent permeability. Boundary layer diffusion is in
fact the rate-limiting step if theoretical maximum permeability values >3 cm s1 are considered. Since biophysical
studies on bilayers provide direct evidence for transport
rates in this range, the data attained on cellular systems
were called into question (Missner et al., 2008). It remains
to be elucidated if the experimental approach using a twin
Teflon chamber system reflects the situation of cellular CO2
transport. The discrepancy in measured membrane permeability could also reflect general differences between bilayer
and cellular membranes containing integral and associated
proteins. It is possible that these proteins cover a large
proportion of the membrane (Engelman, 2005) and reduce
the CO2 transport rate by physical occlusion. In this
environment, cooporins could facilitate CO2 transport and
enable higher rates of CO2 exchange. It is of significance but
also not known whether other membrane proteins could
change the properties of cooporins.
The range in resistance associated with each element
along the pathway can be calculated using Equation 5
(Table 2). The greatest uncertainty at present is associated
with the membranes. To be consistent with measured
mesophyll conductance, a membrane permeability of 0.35
cm s1 was required, based on the assumption that the
permeability of the plasma membrane and the inner and
outer chloroplast envelope were similar (Evans et al., 1994).
Cell walls could account for up to 50% of the total
resistance.
Although a resistance analogue without spatial representation could be misleading (Parkhurst, 1994), in the
Table 1. CO2 permeabilities reported for various membranes
Permeability
Fig. 6. Relationships between chloroplast thickness and (A)
Rubisco site content per unit of exposed chloroplast surface area,
(B) mesophyll resistance per unit of exposed chloroplast surface
area. In (A), the diagonal lines show varying Rubisco site
concentrations. Data sources: Nicotiana tabacum (n, wild-type;
, antisense SSu; h, antisense SSu, double copy) (Evans et al.,
1994), Chenopodium album ; (Oguchi et al., 2003), Triticum
aestivum d (JR Evans, unpublished).
Membrane
Reference
Alga plasma
membrane
Colon epithelia
Gimmler et al. (1990)
(cm s1)
(mol m2
s1 bar1)
0.0001
0.00004
0.0015
0.0006
0.002
0.008
0.0008
0.0032
0.012
0.08
0.0048
0.032
Chloroplast envelope
Tobacco plasma
membrane
Erythrocyte
Yeast
0.35
0.14
Lipid bilayer
1.6
0.64
Lipid bilayer
Endeward and
Gros (2005)
Uehlein et al. (2008)
Uehlein et al. (2008)
Yang et al. (2000)
R Kaldenhoff
(unpublished)
Gutknecht et al.
(1977)
Missner et al. (2008)
Resistances along the CO2 diffusion pathway inside leaves | 2243
Table 2. Characteristics of elements involved in the liquid phase transfer of CO2
h is the thickness of the element, s is the tortuosity of the CO2 pathway, u is the porosity of the element when relevant, D is the diffusivity of
CO2 in the element solvent (water or lipids), j is the solvent to water partitioning coefficient for CO2, and e is the enhancement due to
facilitation. The resistance for each element is calculated with Equation 5.
Element/compartment
u (lm)
t
u
D* (m2 s1)
k
ey
r#x (m2chlor s bar mol1)z
Cell wall
Plasmalemma
Cytosol
Envelope
Stroma
Total, r#liquid
0.05–0.4
0.005–0.01
0.05–0.7§
0.01–0.02**
0.5–3.0
1–1.4
>1c(1)
>1{(1)
>1{(1)
>1{ (1,2,3)
0.05–0.2
<1c(1)
<1{ (1)
<1{(1)
<1{ (1,2,3)
1.73109
1014–1011
1.73109
1014–1011
1.73109
1
1.5–1.7
1
1.5–1.7
1
0.1–0.2
0–6
5–7.8
0–6
9.5–24.1
3–156
2.1–12500
0–1
4.2–25000
0.3–6.5
9.6–37664yy
* For aqueous compartments, the diffusivity of carbon dioxide in water at 25 C is used. For membrane elements, an apparent diffusivity of D
is calculated from the permeability range given in Table 1 as D¼Phs/[uj(1+e)] where P is the permeability. A value approaching 1011 is required to
bring the total resistance within the observed range, which equates to a P of 0.35 cm s1.
y
pH ranges for cell wall 5.5–6.0, cytosol 7.3–7.5, and stroma 7.6–8.0, have been used to calculate eaqueous according to Equation (12); emembrane
is uncertain.
z
1 s m1 converts to 0.025 m2 s bar mol1.
§
Value for an abnormal chloroplast from a plant expressing excess phytochrome.
{
Due to non- or less permeable macromolecules and complexes in the pathway, e.g. proteins1, starch2, thylakoids3.
**
Approximate thickness is estimated as twice that of a single chloroplast envelope membrane, neglecting the compartment in between the
membranes.
yy
Observed range 25–300 (Fig. 5A).
absence of the necessary detailed information on which to
formulate a more complex analysis, it is still useful to
examine the consequence on rm of changing the relative
magnitude of the resistances. To make the problem
tractable, rather than use Equations 1 or 3, it is necessary
to group the terms into two resistances, namely rmembrane
and rother:
rm¼ rmembrane þrother
ð13Þ
For the sake of argument, all three membranes have
been assumed to be equivalent even though this is unlikely
to be the case. Even this simple resistance model has more
parameters than we have information with which to
constrain it. However, we can use the reduction in gm
observed when cooporins have been manipulated by
antisense (Hanba et al., 2004; Flexas et al., 2006; Uehlein
et al., 2008) and mercury inhibition (Miyazawa et al.,
2008) as an indication of the relative contribution from
cooporins. If complete functional suppression of all
cooporin isoforms in leaves of a knockout plant is
assumed [keeping in mind that (i) full knockout is unlikely
in antisense plants; (ii) suppression of a specific cooporin
isoform could alter the function of other cooporin isoforms present in the element; and (iii) the three membranes
may not behave identically], then membrane resistance
(Equation 6) simplifies to
KO
r#membrane¼ r#B:Memb
ð14Þ
This can be expressed using Equation 2 and substituting
into Equation 13 as,
KO
r#m¼ r#B:Memb þr#other
ð15Þ
The inhibition of gm can be defined by knocking out
cooporin function, I, as
WT
I¼ 1 KO
KO
rm
gm
¼ 1 WT
rm
gm
ð16Þ
By combining and rearranging Equations 6, 10, and
13–16, we obtain
I
rmembrane 1
e membrane¼
ð1 IÞ
rm
ð17Þ
Experiments where gm has been manipulated either by
mercury inhibition or by antisense/overexpression of cooporins are shown in Table 3. Since values for I obtained
with strong antisense suppression ranged from 0.2 to 0.32,
to illustrate Equation 17, two examples are given where
knocking out cooporins inhibited gm by 25% or 50% (Fig.
7). For a leaf where gm was inhibited by 25%, the CO2 flux
through cooporins must exceed that through the bulk
membrane whenever membrane resistance accounts for
<33% of rm (Fig. 7A). Interestingly, regardless of whether
a smaller or larger r#Cooporin is added relative to r#Bulk
Membrane, the addition of cooporins always reduces membrane resistance. If cooporin inhibition reduces gm by 50%,
then the flux through cooporins in the wild type always
exceeds that through the bulk membrane (Fig. 7B). The
ratio of conductance through cooporins to that of the bulk
membrane, emembrane, is inversely related to the ratio of
membrane to total resistance (Equation 17, Fig. 7C).
Therefore, cooporins have to enhance membrane permeability to a greater extent as the membrane accounts for less
of the total mesophyll resistance. Since the cell wall could
account for 25–50% of the total resistance, the most likely
physiological region is when membrane resistance accounts
for <70% of the total resistance. It is evident from Fig. 7C
that this is the region where membrane resistance should be
most responsive to the addition of cooporins.
2244 | Evans et al.
Table 3. Values for gm reported for plants where cooporins have been inhibited by mercury, reduced by antisense, or increased by
overexpression
The mean value for the inhibition of gm, I (see Equation 16), or the relative increase in conductance associated with overexpression (to compare
with Fig. 8) are shown.
Treatment
Genus
WT
Hg
Vicia
Phaseolus
Nicotiana
Oryza
Oryza
Nicotiana
Nicotiana
Nicotiana
Nicotiana
Oryza
Nicotiana
Nicotiana
Nicotiana
Antisense
Overexpression
gm (mol m2 s1)
gm (mol m2 s1)
KO
I
0.457
0.3
0.29
0.191
0.191
0.247
0.339
0.21
0.3
0.2
0.12
0.2
0.167
0.146
0.179
0.231
0.15
0.24
0.56
0.60
0.31
0.13
0.24
0.28
0.32
0.29
0.20
0.191
0.328
0.323
0.20
0.27
0.409
0.451
0.29
This simple model can also be used to examine the extent to
which enhancing the cooporin pathway will increase mesophyll
conductance. For the two conditions presented above, the
impact of doubling the expression of cooporins was also
calculated by assuming that OEr#Cooporin¼WTr#Cooporin/2
(Fig. 8). In the physiological range of rmembrane/rm (i.e.
<0.7), doubling the amount of cooporin increases mesophyll
conductance by up to 35%. The range observed in experiments
where cooporins have been overexpressed is 25–45% [Table 3,
HvPIP2 in rice (Hanba et al., 2004) or NtAQP1 in tobacco
(Flexas et al., 2006)], suggesting either that the flux through
cooporins has more than doubled or another pleiotropic
effect. Alternatively, it indicates that membrane resistance
accounts for the majority of mesophyll resistance, which is at
odds with the likely proportion associated with the cell wall
and intercellular airspace. Quantitative interpretations of the
change in mesophyll conductance are hampered by the fact
that pleiotropic changes are generally observed in addition to
the specific gene that has been targeted. For example, in rice,
overexpression of cooporin was associated with altered
mesophyll cell shape, decreased Sc, and thicker cell walls
(Hanba et al., 2004). For Nicotiana, overexpression of
NtAQP1 was not associated with changes in LMA (leaf mass
per area) or Sc, but cell wall thickness was not measured
(Flexas et al., 2006). These additional changes would need to
be included to analyse the impact of overexpression of
cooporins on membrane conductance properly. It is necessary to quantify Sc, mesophyll cell wall thickness, and
Rubisco in order to provide cross-checks.
A final factor that needs to be remembered is that
mesophyll resistance per chloroplast area can change as leaves
age. When leaves of T. aestivum were followed through time,
both photosynthetic capacity and mesophyll conductance
decreased in parallel (Loreto et al., 1994; Evans and Vellen,
1996), but there was no change in the surface area of
chloroplasts exposed to intercellular airspace. This suggests
OE
gm/WTgm
Reference
Terashima and Ono (2002)
Terashima and Ono (2002)
Miyazawa et al. (2008)
Hanba et al. (2004)
Hanba et al. (2004)
Flexas et al. (2006)
Flexas et al. (2006)
Flexas et al. (2006)
Uehlein et al. (2008)
1.41
1.25
1.40
1.45
Hanba et al. (2004)
Flexas et al. (2006)
Flexas et al. (2006)
Flexas et al. (2006)
that the resistance of the mesophyll cell wall and/or
membranes increased over time. It is also possible that the
anchoring by actin is not as strong in senescent leaves,
because floating chloroplasts were observed in senescing
wheat leaf mesophyll cells (K Ono, unpublished results). This
could lead to an increase in the thickness of cytosol through
which CO2 has to diffuse.
Conclusion
At this stage there are too few data and species with which
to constrain the relative importance of different elements
along the CO2 diffusion pathway. However, it is clear that
the measurements that have been made on the surface area
of chloroplasts exposed to intercellular airspace per unit leaf
area, mesophyll cell wall thickness, and by manipulating
CA, Rubisco, and cooporins allow us to begin to subdivide
the liquid phase into distinct functional elements. Most of
the components can be shown to contribute to the overall
resistance. It is likely that their relative importance differs
between species and possibly with growth conditions. In
order to improve the quantitative analysis of the various
elements along the pathway, greater effort should be given
to: (i) measuring and reporting the surface area of
chloroplasts exposed to intercellular airspace; (ii) measuring
mesophyll cell wall thickness; (iii) measuring membrane
permeability to CO2; and (iv) quantifying CA activity and
Rubisco.
The first three parameters are crucial for integrating the
total mesophyll resistance and where we have the least
amount of information or the greatest uncertainty. Quantifying CA activity and Rubisco for these leaves would
provide a check for facilitation efficiency mediated by CA
and for internal consistency, respectively.
Resistances along the CO2 diffusion pathway inside leaves | 2245
Fig. 8. The effect of overexpressing cooporins on mesophyll
conductance. The calculation is made by halving rCooporin relative
to the wild type for the two values of I used in Fig. 7. The increase
in gm relative to that of the wild type, OEgm/WTgm, increases to I
when membranes account for the total mesophyll resistance, i.e.
WT
rmembrane/WTrm¼1.
D
DB
e
gx
g#x
Fig. 7. Relative magnitudes of a three-component resistance
model for the liquid phase of mesophyll conductance (Equations 8
and 13). Mesophyll resistance, rm, is the sum of two resistances in
series: membrane and non-membrane, rmembrane and rother, respectively. The membrane resistance comprises two resistances in
parallel: bulk membrane and cooporins, rBulk Membrane (rB. Memb in
equations) and rCooporin (rCoop in equations), respectively. (A) The
relative change in the membrane components as a function of the
proportion of total mesophyll resistance associated with membranes. The inhibition of mesophyll conductance by knocking out
cooporins, I, was assumed to be 0.25 (Equation 16, Table 3). (B)
The value of I assumed to be 0.5. (C) The ratio of conductance
through cooporins to that through the bulk membrane, e (Equations 10 and 17), calculated for the two values of I shown in A and
B. A horizontal line is shown at 1 to illustrate where cooporin
conductance equals bulk membrane conductance.
Acknowledgements
We would like to thank Jaume Flexas for organizing and
the ESF for supporting the workshop which led to this
work, Professors Newcomb and McCann and Dr Hanba for
allowing us to use their micrographs, and Professor von
Caemmerer for insightful comments. B.G. thanks the ANR
agency (contract BLAN07-1-192876) for funding.
List of symbols
A
Cx
rate of CO2 assimilation
partial pressure of CO2 where x represents a, the
surrounding air, i, intercellular airspaces, c, the
sites of carboxylation within the chloroplast
j
u
rx
r#x
Sc
h
s
diffusivity of CO2 in water
diffusivity of bicarbonate in water
enhancement associated with facilitation through
membranes or aqueous elements
conductance to CO2 through m (mesophyll), cooporins or bulk membrane expressed on a projected
leaf area basis
conductance to CO2 per unit surface area of
chloroplast exposed to intercellular airspace, where
x represents bulk membrane, chloroplast envelope,
cooporin, cytosol, liquid, membrane, m (mesophyll), other, plasma membrane, stroma or wall
solvent to water partitioning coefficient for CO2
porosity of the element
resistance to CO2 on a projected leaf area basis,
where x represents bulk membrane, chloroplast
envelope, cooporin, cytosol, ias (intercellular airspace), liq (liquid), membrane, m (mesophyll),
other, plasma membrane, stroma or wall
resistance to CO2 per unit surface area of chloroplast exposed to intercellular airspace for element x
surface area of chloroplasts exposed to intercellular
airspace per unit projected leaf area
thickness of the element
tortuosity of the element
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