Cuticle Affects Calculations of Internal CO2 in Leaves Closing
Their Stomata
Jun Tominaga1,2 and Yoshinobu Kawamitsu1,*
1
Regular Paper
Faculty of Agriculture, University of the Ryukyus, Okinawa, 903-0213 Japan
The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima, 890-8580 Japan
2
*Corresponding author: E-mail, [email protected]; Fax, +81-98-895-8734.
(Received April 19, 2015; Accepted July 18, 2015)
Analyzing the assimilation rate (A) relative to the CO2 concentration inside leaves (Ci) has been a useful approach for
investigating plant responses to various environments.
Nevertheless, there are uncertainties in calculating Ci when
stomata close, restricting the application. Here, A–Ci curves
were traced in sunflower (Helianthus annuus L.) leaves using
a method for directly measuring Ci. The method was incorporated into an LI-6400 open gas exchange system, and stomata were closed by feeding 10 mM ABA through petioles.
The conductance to CO2 was derived from the directly measured Ci and compared with the conductance from the
water vapor flux (i.e. the standard calculation). When
stomata were open, measured and calculated Ci gave similar
A–Ci curves. When stomata were closed, the curves differed
because measured Ci departed from the calculated value.
This difference caused the calculation to trace an artifactual
limitation of photosynthesis. The direct measurement
avoided this problem and followed the curve for leaves
with open stomata. Largely because of the cuticle, the calculation overestimated CO2 entry into the leaf because the
cuticle transmitted more water vapor than CO2, and the
calculation relied on water vapor. Consequently, the standard calculation gave conductances larger than those from
directly measured Ci. Although the cuticle conductance to
water vapor remained constant as stomata closed, it increasingly contributed to the overestimation of Ci. The system
provided here is not affected by these cuticle properties and
thus is expected to open up the opportunity for A–Ci analysis in plant physiology.
Keywords: ABA Gas exchange Helianthus annuus L LI6400 Photosynthesis Stomata.
Abbreviations: A, assimilation rate; Ca, ambient CO2 concentration; Ci, CO2 concentration inside leaves; Ci(c), calculated
Ci; Ci(m), directly measured Ci; E, transpiration rate; gbw,
boundary layer conductance to water vapor; gCO2, conductance to CO2; gCO2(c), gCO2 estimated with property of conductance to water vapor; gCO2(m), gCO2 calculated from Ci(m);
gcw, cuticle conductance to water vapor; gsw, stomatal conductance to water vapor; gsw0 , strict sense of stomatal conductance to water vapor; gw, conductance to water vapor;
IRGA, infrared gas analyzer; PPFD, photosynthetic photon
flux density.
Introduction
In leaves of terrestrial plants, most gas exchange with the atmosphere depends on diffusion through stomatal pores.
Diffusion is a passive physical process and can be regulated
when stomata open or close. Closure prevents excessive
amounts of water diffusing outward, but at the same time
hinders CO2 diffusing inward because the stomata are the
common gates for both gases. This leads to a decrease in the
CO2 inside leaves (Ci) and a diminished rate of photosynthesis
due to a shortage of substrate.
The photosynthetic CO2 response curve in which assimilation
(A) is determined as a function of Ci, namely the A–Ci curve, has
been widely used to assess photosynthetic performance in various environments. Because the Ci is a result of diffusion already
passed through stomata, changes in the curve must indicate
non-stomatal limitation of photosynthesis (Boyer 1971,
Farquhar and Sharkey 1982, Graan and Boyer 1990). The
method allows the limitations on photosynthesis to be analyzed
separately from stomatal effects; however, to trace the curve
correctly, the Ci needs to be determined accurately.
Basically, Ci is routinely calculated from the outward diffusion of water vapor (Moss and Rawlins 1963, Jarman 1974, von
Caemmerer and Farquhar 1981, Boyer and Kawamitsu 2011).
The calculations assume a common gas phase path for CO2 and
water vapor (i.e. stomata). However, there is evidence that both
gases can move across the cuticle although at a lower rate than
through stomata (Boyer et al. 1997, Boyer 2015). This can potentially bias the calculation (Boyer et al. 1997, Meyer and
Genty 1998, Boyer 2015). Patchy stomatal closure (Terashima
et al. 1988, Mott 1995) also might be a problem because the
calculations assume a uniform distribution of stomatal apertures (Buckley et al. 1997). The calculations become more affected as the stomata close because these influences increase
accordingly.
While stomata are open, the calculation appears reasonably
accurate. Sharkey et al. (1982) and Boyer and Kawamitsu (2011)
measured Ci directly and compared it with that measured with
the standard gas exchange parameters. The two measurements
were similar for open stomata and the similarity was recently
supported when a direct measurement system for Ci was incorporated into a LI-6400 open gas exchange apparatus (Tominaga
and Kawamitsu 2015). In the present work, we applied this
Plant Cell Physiol. 56(10): 1900–1908 (2015) doi:10.1093/pcp/pcv109, Advance Access publication on 23 July 2015,
available online at www.pcp.oxfordjournals.org
! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
µ
µ
Plant Cell Physiol. 56(10): 1900–1908 (2015) doi:10.1093/pcp/pcv109
Fig. 1 Ambient CO2 (Ca), measured internal CO2 [Ci(m)] and calculated internal CO2 [Ci(c)] on a leaf with stomata open. A cup was
attached to this leaf, but the measurements were simultaneous on
the same leaf area. In Ci(c), the data for about 1 min immediately after
the change in Ca were removed due to extreme values. Data are typical
for six replications.
method to sunflower leaves whose stomata were being closed
by feeding 10 mM ABA. The results showed that calculated Ci
[Ci(c)] departed from measured Ci [Ci(m)] in these conditions.
Results
Leaves with open stomata
To measure Ci directly, the bottom half of the leaf chamber
fluorometer (LI-6400-40; Li-Cor) was replaced with a handmade chamber (cup; see the Materials and Methods). The
cup enclosed the same cross-sectional area inside the gaskets
(2 cm2) as the standard bottom half. While the bottom cup
measured CO2 concentration equilibrated with that in the
intercellular spaces of the leaf [Ci(m)], the upper half simultaneously measured standard gas exchange parameters and
calculated internal CO2 [Ci(c)]. In some experiments, measurements were carried out with the standard bottom half for the
chamber, allowing gas exchange through both surfaces of the
leaf (free leaf), which contrasted with the cup-attached leaf
allowing gas exchange only through the adaxial surface. In the
free leaf, only the Ci(c) could be determined.
When stomata were open, measured and calculated Ci responded similarly to the change in external CO2 concentration
(Fig. 1). The Ci(m) slightly lagged behind the Ci(c). At an ambient
CO2 concentration (Ca) of 400 mmol mol1, A was about
25 mmol m2 s1 and the Ci(c) was 280 ± 10 mmol mol1 and
slightly higher than the Ci(m) of 268 ± 13 mmol mol1. This indicated that CO2 gradients were present internally due to finite
CO2 conductance in the intercellular space (Sharkey et al.
1982). Although the cup-attached leaf would enhance these
gradients as it limits the CO2 entry from one side (Parkhurst
et al. 1988, Parkhurst and Mott 1990), comparable A–Ci curves
were obtained regardless of whether the cup was attached or
not (Fig. 2A). A saturated at about 800 mmol mol1 for both
measurements, although the free leaf had slightly higher Ci(c)
than Ci(m). When the cup was attached, the conductance to
water vapor (indicated as gsw in Fig. 2B) was maintained at
>70% of that for the free leaf, perhaps because stomata
µ
Fig. 2 (A) Data of Fig. 1 showing assimilation (A) for the leaf and
plotted as an A–Ci curve. Measured internal CO2 [filled circles, Ci(m)]
and calculated internal CO2 [open circles, Ci(c)] are indicated for the
cup-attached leaf, and for the same leaf area after the measurement
with the cup (triangles, Free). (B) Conductance to water vapor (gsw)
calculated for the leaf in Fig. 1 using data for Ci(c) (open circles) with
the cup attached or the free leaf without a cup (triangles). A representative experiment from three replications is shown.
opened wider when the cup suppressed gas exchange from
one side (Boyer and Kawamitsu 2011). Similarly to the free
leaf, the gsw was diminished as Ca increased above the ambient
concentration (Fig. 2B). So, the stomata closed somewhat
when CO2 increased. These results confirmed that the equilibrated CO2 in the cup [i.e. Ci(m)] was at the end of the gaseous
diffusion path for CO2 and was dominantly controlled by stomata. Also, the cup scarcely altered diffusion into the leaf.
Leaves with closed stomata
In contrast to leaves with open stomata, Ci(m) differed from Ci(c)
when stomata closed (Fig. 3). After the petiole was cut under
water, gsw and A dropped as stomata closed temporarily
(Fig. 3A). The gsw and A gradually recovered to 90–110% of
the original within <2 h. After recovery, ABA was added to the
water, and gsw and A dropped similarly but did not recover.
Ci(m) also decreased (Fig. 3B), suggesting that CO2 was depleted
rapidly by photosynthesis while the photosynthetic demand for
CO2 was retained (Lauer and Boyer 1992). The Ci(m) did not
1901
µ
µ
µ
J. Tominaga and Y. Kawamitsu | Internal CO2 measured directly in leaves
Fig. 3 (A) Assimilation rate (A) and stomatal conductance for water vapor (gsw) at various times after excising the petiole under water followed
by 10 mM ABA to close the stomata. (B) Measured internal CO2 [Ci(m)] and calculated internal CO2 [Ci(c)] when ambient CO2 (Ca) is varied for the
leaf in (A). A cup was attached to this leaf, but the measurements were simultaneous on the same area in (A) and (B). Note the large difference in
Ci(m) and Ci(c) after ABA was fed. The data for about 1 min immediately after the change in Ca were removed due to extreme values. Data are
typical for six replications.
respond to the change in Ca as much as in leaves with open
stomata (compare Figs. 1 and 3). At the higher Ca range above
900 mmol mol1, Ci(m) hardly followed the further increase in Ca
because stomata closed more tightly at the higher Ca (gsw in
Fig. 3A). When stomata began to close more tightly, Ci(m) actually became slightly depleted, causing the traces to be skewed
(in the inset of Fig. 3B). On the other hand, the Ci(c) continued
to increase with increasing Ca (Fig. 3B) and became markedly
different from Ci(m). It was also seen that the Ci(c) became erratic
as stomata closed. While the leakage of the gas exchange system
potentially alters the calculation (Rodeghiero et al. 2007), it
could not explain the departure of Ci(c) here (Supplementary
Fig. S1).
As a consequence of this difference, two distinguishable A–
Ci curves appeared when stomata closed (Fig. 4A). The Ci(c)
departed from Ci(m) as Ca increased, and the A–Ci(c) curve was
far below the original before feeding ABA (Fig. 4A). In contrast,
1902
the Ci(m) gave a curve like that before feeding (Fig. 4A). Similar
results were obtained in all replications (n = 10), and the maximum Ci(m) was no more than 200 mmol mol1 when Ca was at
the highest (2,000 mmol mol1). Conductance to CO2 [gCO2(m)]
could be estimated from the measured Ci and compared
with that [gCO2(c)] derived by the water vapor with the
standard calculation (see the Materials and Methods). As
shown in Fig. 4B, the gCO2(c) was larger than the gCO2(m), but
declined with increasing external CO2, suggesting that the stomata still responded to CO2 as for the leaves without ABA
treatment. ABA closed stomata to varying degrees (i.e. gCO2)
between replications (Supplementary Fig. S2). The depression
of the Ci(c) slope became greater when stomata closed more
tightly (compare Fig. 4A and Supplementary Fig. S2).
In order to confirm that the difference between Ci(m) and
Ci(c) was not an artifact of our measurement system, we conducted an experiment with a single leaf in which A–Ci curves
µ
µ
Plant Cell Physiol. 56(10): 1900–1908 (2015) doi:10.1093/pcp/pcv109
µ
µ
Fig. 4 (A) Data of Fig. 3 plotted as an A–Ci curve. (B) CO2 conductance (gCO2) plotted at various concentrations of ambient CO2 (Ca).
Different curves are obtained from directly measured [filled circles,
Ci(m)] or calculated [open circles, Ci(c)] concentrations of CO2. Arrows
indicate the data before feeding ABA to close the stomata. A representative experiment from 10 replications is shown.
were taken (i) on the free leaf with open stomata; (ii) on the
same leaf but with cup attached and ABA fed (i.e. cup-attached
leaf with closed stomata); and subsequently (iii) on the same
but free leaf (i.e. free leaf with closed stomata). Step (ii) was the
same as in the previous experiment shown in Fig. 3. When
stomata were closed with ABA, A saturated with CO2 at a
lower rate (Fig. 5A, +ABA) than when stomata were open
(Fig. 5A, –ABA). However, for Ci(c) the saturated rate extended
to higher Ci than indicated by Ci(m). The Ci(c) curve led to the
misconception that there was non-stomatal limitation of
photosynthesis (Fig. 5A, +ABA for free leaf). The Ci(m) curve
was superimposed on that for the free leaf with stomata open
(Fig. 5A), indicating no non-stomatal limitation of photosynthesis. These results were reflected in the conductances. When
stomata closed, both the gCO2(m) and gCO2(c) were lower than
the gCO2(c) for open stomata (Fig. 5B). However with stomata
closed, gCO2(m) was always lower than gCO2(c) (Fig. 5B). As Ca
rose, stomata closed more tightly (Fig. 5B and inset) and eventually became so low that A did not increase despite
2,000 mmol mol1 outside the leaf (Fig. 5A). Note that the
µ
µ
Fig. 5 (A) Comparison of A–Ci curves when stomata are open (–ABA)
or closed (+ABA) and the internal CO2 was calculated. Included in the
comparison are internal CO2 concentrations directly measured with
an attached cup [filled circles, Ci(m) + ABA]. Note that several filled
data points lie on top of each other at A of 14 mmol m2 s1. (B)
Conductance to CO2 (gCO2) at various concentrations of ambient
CO2 (Ca) for the leaf in (A). Data were obtained in a series of measurements for a single leaf (see text). Arrows indicate the data before
feeding ABA. A representative experiment from four replications is
shown.
slope of the Ci(c) curve in Fig. 5A was not depressed after the
ABA application (+ABA for free leaf) as much as that shown in
Fig. 4A whose stomata closed more tightly. Also, the free leaf
illustrated a Ci(c) curve similar to the corresponding cupattached leaf (data not shown). These results confirm the previous experiment showing that ABA closed stomata without
changing the A–Ci curve. It further indicates that a substantial
problem exists in calculated Ci with closed stomata (Fig. 5A,
+ABA).
Estimation of cuticle conductance to water vapor
According to Boyer et al. (1997) and Boyer (2015), the amount
of CO2 moving through the cuticle is much smaller than for
water vapor. Consequently, the CO2 can be considered to diffuse essentially through only stomata, while water vapor moves
1903
µ
J. Tominaga and Y. Kawamitsu | Internal CO2 measured directly in leaves
µ
µ
Fig. 6 (A) Conductance to CO2 (gCO2) from measured internal CO2
[Ci(m)] or calculated from water vapor flux [Ci(c)] at various concentrations of ambient CO2 (Ca) when stomata were closed with ABA. (B)
Cuticle conductance to water vapor (gcw) in (A) calculated from
Equation (10). Data are means ± SE (n = 10).
through the cuticle plus stomata. Expressing the difference between the calculated and measured conductances to CO2 thus
approximates the cuticle conductance to water vapor as shown
in Equation (10). In agreement, the gCO2(c) was consistently
larger than the gCO2(m) by, on average, 10–40% over the range
of Ca (Fig. 6A). Assuming that the difference of conductance to
CO2 resulted solely from the cuticle component, the cuticle
conductance to water vapor (gcw) estimated from Equation
(10) ranged from 5 to 9 mmol m2 s1 in most of the Ca
range except 30 mmol m2 s1 at the lowest Ca of
30 mmol mol1 (Fig. 6B). At the lowest Ca, the gCO2(m)
decreased to some extent while the gCO2(c) did not. It was probable that small errors in the measurement of Ci(m) (e.g. leakage)
contributed to the larger gcw at the lowest Ca.
In turn, we incorporated the gcw values to correct Ci(c) and
test if the correction would allow Ci(c) to approach Ci(m).
Generally, corrected Ci(c) approached Ci(m) (Fig. 7). Upon a Ca
of >600 mmol m2 s1, however, it gradually became lower
than it should be [i.e. lower than Ci(m)]. This was attributed
to the gcw which assumed no CO2 transfer across the cuticle
[Equation (9)]. Though it was much slower for CO2 to move
1904
Fig. 7 Typical examples of correction for cuticle conductance to
water vapor (gcw) in A–Ci(c) curves. The calculation was corrected
with the estimated gcw at each measurement by estimating the
strict sense of stomatal conductance to water vapor (gsw0 ) according
to Equation (6). (A) Data for Fig. 4A and (B) another experiment are
shown. The corrected Ci(c) gradually became lower than the Ci(m) as
ambient CO2 (Ca) increased.
across the cuticle than for water vapor (Boyer 2015), CO2 must
be forced into the leaf through the cuticle to some extent as the
external concentration increased. This created overcorrection
of conductance for CO2. In fact, decreasing gcw values down to
approximately 9 % gave a closer approximation of Ci(m).
Discussion
Diffusion of CO2 and water vapor in leaves
In this work, the CO2 concentration inside leaves was directly
measured as well as calculated by the standard method from
the water vapor flux. The measurements were simultaneous on
the same leaf area and made in a commercially available open
system for determining the gas exchange of leaves. The system
was slightly modified to include the direct measurement and,
by making determinations on the same area of leaf, a close
comparison was possible. The data showed that the internal
CO2 became progressively different in the two measurements
as open stomata began to close. Plotting the assimilation
Plant Cell Physiol. 56(10): 1900–1908 (2015) doi:10.1093/pcp/pcv109
against the internal CO2 concentration (A–Ci curve) gave different conclusions. Leaves with closed stomata either had no
change in photosynthetic capacity [A–Ci(m)] or had a large
change [A–Ci(c)]. This created a problem for interpreting gas
exchange measurements. It suggests that great caution should
be used when measuring A–Ci curves on leaves with closing
stomata. Importantly, both methods gave essentially the same
A–Ci curve when stomata were open.
Generally, the standard calculation considers stomata as the
dominant path for CO2 and water vapor (Moss and Rawlins
1963, von Caemmerer and Farquhar 1981). While this can be
essentially true when stomata are open, water vapor and CO2
also move through the cuticle. The CO2 moves slowly, probably
because it has to move through the water in the epidermal cells
in addition to cuticle wax before entering the intercellular
spaces of the leaf. In contrast, water moves only through the
waxes and is more rapid (Boyer 2015). As a result, basing the
standard calculation on water movement overestimates CO2
entry. It is proposed that the differences in Ci(m) and Ci(c) as well
as the differences in gCO2(m) and gCO2(c) are attributable to this
difference in cuticle transport.
The values for the cuticle conductance to water vapor were
calculated from these differences in cuticle transport and were
5–30 mmol m2 s1, i.e. comparable with those reviewed by
Kerstiens (1996) for 200 plant species. Because the method
involved cup attachment, it could be used only with amphistomatous species, but may be an alternative to conventional
sealing methods (Tominaga and Kawamitsu 2015).
In dehydrating leaves, water transport across the cuticle
decreased when leaf water potentials decreased (Boyer et al.
1997, Burghardt and Riederer 2003). Boyer (2015) recently
found that the cuticle tightened when turgor was lost and
the leaf shrank at these low potentials. The permeability diminished for both CO2 and water, suggesting that anything affecting stomatal closure and in turn leaf turgor will affect cuticle
properties. This was not the case for the present study as stomata were closed by ABA alone (i.e. without associated reductions in leaf water potential or turgor), which is consistent with
the constant gcw at various Ci (Fig. 6B).
Any other factor affecting the calculated or directly measured conductances might cause them to differ in Equation
(10). Because the conductance to CO2 in the intercellular airspace is finite, vertical gradients of CO2 must be present in a
leaf. As a result, the Ci(m) may be lower than the actual one. We
tested this possibility using sunflower leaves with stomata
open, and estimated the conductance of the mesophyll to be
1,200 mmol m2 s1 (Tominaga and Kawamitsu 2015). This is
large compared with the other conductances controlling CO2
diffusion into the leaf and suggests that Ci(m) was only slightly
lower than the actual value in the free leaf (Fig. 2). Because the
gradients must be created by the mesophyll assimilation activity that in turn was affected by the substrate concentration, it
gradually disappeared as Ci decreased.
The standard calculation also assumes lateral uniformity
over the leaf surface (i.e. uniform stomatal aperture, leaf temperature and metabolic capacity) and in the leaf (i.e. uniform
CO2 distribution). Patchy stomatal closure would diminish this
uniformity (Terashima et al. 1988, Buckley et al. 1997, Meyer
and Genty 1998). Internal water vapor is assumed to be nearly
saturated (i.e. uniform) regardless of stomatal apertures
(Farquhar 1978). In contrast, CO2 diffuses so little through
the cuticle that it may be considered to move into leaves
almost entirely through stomata. Given patchy stomatal closure, internal CO2 may not be distributed uniformly inside the
leaf [CO2 diffusion would be restricted in the lateral direction
(Terashima et al. 1988, Terashima 1992, Morison et al. 2005)].
Consequently, A would vary from patch to patch depending on
the local Ci. Also, leaf temperature [i.e. the factor determining wi
in Equation (1)] might not be distributed uniformly over the
leaf surface with patchy stomatal closure (West et al. 2005),
which potentially would alter the calculation as well.
In contrast, the Ci(m) reasonably represented actual A–Ci
curves even with severely closed stomata. If patchy stomatal
closure occurred, this result could not have been obtained because the Ci(m) would presumably average across patches (i.e.
provide a uniform value) while the calculated Ci(c) would not.
The lack of change in A–Ci curves with closed stomata most
probably suggests that the overestimation was caused by the
water transfer across the cuticle (Meyer and Genty 1998) rather
than patchiness.
It was indeed surprising that a small cuticle conductance can
make such a large overestimation in the calculations; however,
because the cuticle contributes increasingly to leaf conductances as stomata close, the overestimation was more critical
when stomata were closed than when they were open.
Barrier against CO2 diffusion
Because the cuticle barrier to CO2 diffusion is substantial when
stomata close, photosynthesis could not recover to the original
rates with elevated CO2 outside of the leaf because the stomata
closed more tightly. In order to overcome this problem, Graan
and Boyer (1990) used a gas exchange system to increase external CO2 around the leaf to the %-level, allowing CO2 to
penetrate the leaf despite closed stomata, whereupon the
photosynthetic activity increased. In their experiment, Ca was
increased to 3,000 mmol mol1 to recover the photosynthetic
rate for ABA-fed leaves. Similarly, we estimated from Equation
(8) with minimum gCO2(m) (5–12 mmol m2 s1) that Ca has to
increase to 2,300–5,200 mmol mol1 to recover A
(25 mmol m2 s1). Saturating A requires still further increases
in external CO2 concentration. So far, these high CO2 concentrations cannot be achieved with commercially available gas
exchange systems (ADC Bioscientific 2004, Walz 2005, Li-Cor
Biosciences 2008).
Applications of the system
Calculated Ci traced an artifactual non-stomatal limitation of
photosynthesis when stomatal conductance to water vapor
was low. The problem appears to be caused by including cuticle
water loss while scarcely any CO2 is transported through the
cuticle. Moreover, the water loss varies when leaf turgor (i.e.
water status) varies. Furthermore, the correction for the cuticle
water loss seems insufficient unless the property for CO2 transfer is known (Fig. 7). The direct measurement of Ci can avoid
1905
J. Tominaga and Y. Kawamitsu | Internal CO2 measured directly in leaves
those complexities. Although the system provided here has the
potential to open up the opportunity for A–Ci analysis for plant
physiology, it critically relies on the LI-6400 open gas exchange
system. A great advantage is its simplicity and availability to
many scientists. Its limitations are the leakage, the moderate
range of external CO2 concentrations and restriction to hypostomatous leaves. As for the leakage, using a larger chamber (e.g.
2 3 cm chamber) instead of the 2 cm2 fluorometer chamber
might moderate the potential effect (Rodeghiero et al. 2007).
Materials and Methods
Plant material
Plant materials were the same as those used for a detailed description of the
method (Tominaga and Kawamitsu 2015). Sunflower (Helianthus annuus L. cv.
Hybrid sunflower from Kaneko Seeds Co., Ltd.) plants were grown in a glasshouse located in the Faculty of Agriculture, University of the Ryukyus, Okinawa,
Japan (26 150 N, 127 45E; altitude 127 m). In December 2013, seeds were germinated in a fertilized seeding soil with 380, 290 and 340 mg l1 of N:P:K (Takii
& Co., Ltd.). After 10 d, seedlings were transplanted and grown in 4 liter plastic
pots containing a soil mixture consisting of 1 : 1 : 1 soil : peat : sand. The plants
were automatically watered three times each day and were fertilized weekly
with 500 ml of Hoagland’s nutrient solution composed of 4 mM KNO3, 6 mM
Ca(NO3)24H2O, 2 mM MgSO47H2O, 2 mM KH2PO4, 0.5 mM CuSO45H2O,
10 mM MnSO4H2O, 2 mM ZnSO47H2O, 25 mM H3BO3, 0.5 mM H2MoO4 and
0.5 mM Fe(III)-EDTA. Fluorescent light was supplemented when the photosynthetic photon flux density (PPFD) above the plants fell below
800 mmol m2 s1. Daylength in the glasshouse was extended to 15 h to prevent
flowering. The day and night temperatures ranged between 17 and 24 C and 13
and 22 C, respectively. Only upper fully expanded leaves (130–180 cm2) from 7to 8-week-old plants were used.
Gas exchange systems
The gas exchange system was described earlier (Tominaga and Kawamitsu
2015). The entire system resembled that developed by Sharkey et al. (1982)
in which leaf gas exchange was measured in the open flow (LI-6400XT; Li-Cor),
but included a direct measurement of Ci from Boyer and Kawamitsu (2011) in
which Ci was determined in a closed system (Fig. 8A). The latter involved a
small cup incorporated into an integrated fluorescence chamber head (LI-640040; Li-Cor) (Fig. 8B). Because the apparatus was also used for the other experiments, we used this small chamber which can be particularly prone to leaks (see
below) among the chambers provided by the manufacturer (Rodeghiero et al.
2007). When leaf gas exchange was measured, the cup was attached to the
abaxial surface of the amphistomatous leaves, and CO2 in the cup was equilibrated with that in the stomatal pores adjacent to the airspace (i.e. directly
inside the abaxial surface). The equilibrated air was gently circulated with a
small fan to an infrared gas analyzer (IRGA; LI-840A; Li-Cor) in a closed loop
without pulses. The smooth and continuous air movement in the loop led to
stable and fast responses of the equilibrated CO2. This helped to retain the fast
response and environmental control of the LI-6400 system. While the directly
measured internal CO2 [Ci(m)] was continuously traced, normal CO2 and water
vapor exchange through the same section of adaxial surface were simultaneously detected in the open gas exchange system, which allowed the internal
CO2 to be calculated by the standard method [Ci(c)]. Consequently, both Ci(m)
and Ci(c) were obtained in a single measurement with a cup-attached leaf.
The CO2 concentration was regulated with pure CO2 in a tank connected
to the LI-6400 console and with CO2-free air primarily passed through soda
lime. Humidity was controlled by a dew point generator (LI-610; Li-Cor) in the
CO2-free air. We modified the system to attain low CO2 concentration
(<50 mmol mol 1) according to Li-Cor Biosciences (2010). Both IRGAs for LI6400 and LI-840A were calibrated using the same standard gases. For LI-6400,
calibration was performed with 0 and 400 mmol CO2 mol1 air, whereas
for LI-840A additional 2,000 mmol CO2 mol1 air was used for the higher CO2
range.
1906
A–Ci curves
A–Ci measurements were made using either the cup or the standard bottom
half as shown in Fig. 1. After clamping on the leaf, at an ambient CO2 concentration (Ca) of around 400 mmol mol1, photosynthesis and Ci(m) became steady
within 40–60 min depending on the leaf. Thereafter, the photosynthesis response to varying Ci was measured. The Ca was lowered stepwise down to
30 mmol mol1 and then returned to 400 mmol mol1 to re-establish the initial
steady-state value of photosynthesis. The Ca was then increased stepwise up to
1,400–2,000 mmol mol1. For each curve, 8–10 measurements were made. After
steady-state photosynthesis and Ci(m) were achieved at each Ca, which usually
occurred within 10–20 min (Fig. 1), standard gas exchange parameters were
measured. After these measurements were completed, the standard bottom
half was used for measurements every 10 min at each Ca step (free leaf).
Steady-state photosynthesis tended to be achieved more rapidly with the
standard bottom half (usually within 10 min). After stomatal closure was
induced (see below), it required more time for photosynthesis and Ci(m) to
reach steady state, especially at higher Ca.
Photosynthesis was measured at a PPFD of 800 mmol m2 s1, which was
80–90% saturating for A and prevented photoinhibition by often prolonged
measurements. All measurements were carried out at a leaf temperature of
25 C and a leaf to air vapor pressure difference (VPD) of 1.0–2.0 kPa, using a
constant flow rate of 250 mmol s1. In the early morning, plants were taken
from the glasshouse to the laboratory (room temperature of 25 C). There,
plants were illuminated with fluorescent lamps that delivered a PPFD of
150–400 mmol m2 s1 at leaf height. The plants were acclimated under the
light at least 1 h before the measurement started.
Stomatal closure
Stomata were closed by feeding ABA through a petiole. The petiole was excised
under degassed water in a 100 ml glass cup, and photosynthesis was measured
in the excised leaf. After photosynthesis was recovered and became steady, the
stomata were closed by mixing 100 mM ABA with the water in the cup to give a
final concentration of 10 mM. The (±)-cis, trans-ABA was purchased from
Sigma. The stock solution was made by bringing the pH to 10–11 with KOH
to dissolve the ABA, then neutralizing to pH 7 with HCl (Lauer and Boyer 1992).
Calculation of photosynthesis parameters and
cuticle conductance to water vapor
Water vapor conductance (gw) was calculated according to von Caemmerer
and Farquhar (1981):
gw ¼
Eð1wÞ
wi wa
ð1Þ
where E is the transpiration flux (mol m2 s1), wi and wa are the respective
water vapor concentrations of the intercellular spaces inside the leaf and the
bulk air outside of the leaf boundary layer, and w is the average water vapor
concentration in these two locations (mol mol1). The wi was assumed to be
saturating at leaf temperature.
The gw would be decomposed as:
1
1
1
¼
+
gw gsw gbw
ð2Þ
where, in series, gsw and gbw are the stomatal and the boundary layer conductance to water vapor (mol m2 s1), respectively. The boundary layer conductance for each side of the leaf chamber was 4.64 mol m2 s1 (calibrated for the
LI-6400-40 chamber by Li-Cor Biosciences 2008). The conductance to CO2
[gCO2(c)] was estimated with the property of conductance to water vapor as:
1
1:6 1:37
¼
+
gCO2ðcÞ gsw gbw
ð3Þ
where 1.6 and 1.37 are the ratio of diffusivities of CO2 and water vapor in air and
in the boundary layer, respectively (von Caemmerer and Farquhar 1981).
Analogous to Equation (1), gCO2(c) can be expressed as:
gCO2ðcÞ ¼
ðA+cEÞ
Ca CiðcÞ
ð4Þ
Plant Cell Physiol. 56(10): 1900–1908 (2015) doi:10.1093/pcp/pcv109
A
B
Fig. 8 (A) Schematic diagram of the gas exchange system with internal CO2 (Ci) directly measured. In the cup attached to the abaxial surface,
the CO2 equilibrated with that in the stomatal pores adjacent to the airspace, whereas the gas exchange occurs in the open gas exchange system
(LI-6400XT; Li-Cor) attached to the adaxial surface. The equilibrated CO2 concentration [i.e. Ci(m)] was measured in the closed loop with the
IRGA (LI-840A; Li-Cor) while the micro blower (109P0412H309; Sanyo Denki Co., Ltd.) allowed the air to circulate gently around the loop
(300 ml min1). The condenser ensured atmospheric pressure and that the loop path and the cup would be free of condensation. (B) The cup
specially designed for the bottom half of an integrated fluorescence chamber head (LI-6400-40; Li-Cor), having a round airspace with 2 mm depth
surrounded by the black neoprene gaskets (LI-6400-41; Li-Cor) which shares the same leaf area (2 cm2) with the upper half. Leaf temperature was
measured with a fine 0.13 mm chromel–constantan thermocouple (CHCO-005; Omega Engineering) appressed to the underside of the leaf by
the flexed stainless wire in the cup. The bypass to the exhaust for the open path allowed matching the two IRGAs during measurements. The
approximate total volume of the closed system was 100 ml (including LI-840A) with a total path length of 1.8 m.
where A is the CO2 flux (mol m2 s1) and Ca is the CO2 concentration outside
of the boundary layer (mol mol1). The cE value represents the interaction of
CO2 diffusing into the leaf with water vapor diffusing out to the air (von
Caemmerer and Farquhar 1981).
The Ci(c) was then given as:
gCO2ðcÞ E=2 Ca A
ð5Þ
CiðcÞ ¼
gCO2ðcÞ +E=2
According to Boyer et al. (1997) and Boyer (2015), water vapor is also
transpired through the cuticle, and the influence of cuticle conductance to
water vapor (gcw) cannot be neglected as stomata close. The gcw value is
included in gsw as:
gsw ¼ gsw 0 +gcw
ð6Þ
0
where gsw is the strict sense of stomatal conductance to water vapor, and in
parallel with cuticle conductance (Jarvis 1971).
Substituting Equation (6) into Equation (3) gives:
1
gCO2ðcÞ
¼
1:6
1:37
+
gsw 0 +gcw gbw
ð7Þ
It should be noted that in Equation (7) the gas diffusivity ratio of
1.6 is applied to the cuticle conductance despite the fact the cuticle is
solid. This gives the cuticle effect as the equivalent gas phase conductance in order to examine how the cuticle alters the gas phase calculation
of gCO2(c).
In contrast to gCO2(c) in Equation (4), the conductance to CO2 [gCO2(m)]
was also calculated from the directly measured Ci [Ci(m)] regardless of
water vapor flux because the equilibrated CO2 in the cup already
experienced the interaction with water vapor (Boyer and Kawamitsu 2011,
Boyer 2015):
gCO2ðmÞ ¼
A
Ca CiðmÞ
ð8Þ
Although the gCO2(m) contains the cuticle conductance to CO2, the cuticle
transmits 1/20th to 1/40th as much CO2 as water vapor (Boyer et al. 1997, Boyer
2015). Assuming that the cuticle conductance to CO2 was negligibly small, the
gCO2(m) would be expressed as:
1
1:6 1:37
¼
+
gCO2ðmÞ gsw 0 gbw
ð9Þ
Considering the large boundary layer conductance in the total conductance
to CO2, the last term on the right-hand side of Equations (7) and (9) may be
neglected. The boundary layer conductance accounts for 7% of the total conductance to CO2 when gCO2 is 250 mmol m2 s1 (i.e. stomata opened relatively), and <1% when gCO2 is <50 mmol m2 s1 (i.e. stomata closed
relatively).
Then the difference between the Equations (7) and (9) gives the cuticle
conductance to water vapor as:
ð10Þ
gcw ¼ 1:6 gCO2ðcÞ gCO2ðmÞ
Correction for leakage
Tests and corrections for the leakage of CO2 and water vapor into and out of
the gas exchange apparatus have been applied as described elsewhere
(Tominaga and Kawamitsu 2015). Briefly, the chamber was tested for leaks
with various sealants in addition to the rubber gasket supplied by Li-Cor, according to Flexas et al. (2007). The effects of neither the sealants nor the cup
attachment were indicated, so the rubber gasket was used in further leak tests
and in this work. The leakage in the direct Ci measurement system (i.e. closed
loop) was separately evaluated by monitoring the CO2 concentration in the
closed loop after a CO2 injection. No leak was detected, and the system was
considered leak free in this experiment (i.e. no correction for leakage).
A leak in the Li-Cor open gas exchange system was corrected by estimating
the CO2 and water vapor diffusion molar flow rate, KCO2 and KH2O, respectively,
following Rodeghiero et al. (2007). Average values of 0.21 mmol s1 for KCO2 and
2.0 mmol s1 for KH2O were obtained and used in this study. In principle, the leak
affects the measurements of fluxes for CO2 and water vapor, depending on the
concentration gradients of CO2 and water vapor between inside and outside
the chamber. To know these gradients, the concentrations outside the chamber
1907
J. Tominaga and Y. Kawamitsu | Internal CO2 measured directly in leaves
were monitored during all the measurements by an open path IRGA (LI-7500;
Li-Cor) set around the leaves. The corrections of the fluxes can alter the calculation as in Equation (5), and thus the A–Ci curve (Supplementary Fig. S1).
Statistics
The number of replications is presented in the figure legends for each experiment. The results are given as means with SDs unless otherwise indicated. All
the presented results were already corrected for leakage as described above.
Supplementary data
Supplementary data are available at PCP online.
Acknowledgements
We are deeply grateful to Dr. J.S. Boyer who has given us constructive comments and warm encouragement. Without his
guidance and persistent help, this paper would not have been
possible.
Disclosures
The authors have no conflicts of interest to declare.
References
ADC Bioscientific (2011) LCpro-SD Portable Photosynthesis System:
Instruction Manual. ADC BioScientific Ltd., Hoddesdon, UK.
Boyer, J.S. (1971) Nonstomatal inhibition of photosynthesis in sunflower at
low leaf water potentials and high light intensities. Plant Physiol. 48:
532–536.
Boyer, J.S. (2015) Turgor and the transport of CO2 and water across the
cuticle (epidermis) of leaves. J. Exp. Bot. 66: 2625–2633.
Boyer, J.S. and Kawamitsu, Y. (2011) Photosynthesis gas exchange system
with internal CO2 directly measured. Environ. Control Biol. 49: 193–207.
Boyer, J.S., Wong, S.C. and Farquhar, G.D. (1997) CO2 and water vapour
exchange across leaf cuticle (epidermis) at various water potentials.
Plant Physiol. 114: 185–191.
Buckley, T.N., Farquhar, G.D. and Mott, K.A. (1997) Qualitative effects of
patchy stomatal conductance distribution features on gas exchange
calculations. Plant Cell Environ. 20: 867–880.
Burghardt, M. and Riederer, M. (2003) Ecophysiological relevance of
cuticular transpiration of deciduous and evergreen plants in relation to stomatal closure and leaf water potential. J. Exp. Bot. 54:
1941–1949.
Farquhar, G.D. and Raschke, K. (1978) On the resistance to transpiration of
the sites of evaporation within the leaf. Plant Physiol. 61: 1000–1005.
Farquhar, G.D. and Sharkey, T.D. (1982) Stomatal conductance and photosynthesis. Annu. Rev. Plant Physiol. 33: 317–345.
Flexas, J., Dı́az-Espejo, A., Berry, J.A., Cifre, J., Galmes, J., Kaldenhoff, R., et al.
(2007) Analysis of leakage in IRGA’s leaf chambers of open gas exchange
systems: quantification and its effects in photosynthesis parameterization. J. Exp. Bot. 58: 1533–1543.
1908
Graan, T. and Boyer, J.S. (1990) Very high CO2 partially restores photosynthesis in sunflower at low water potentials. Planta 181: 378–384.
Jarman, P.D. (1974) The diffusion of carbon dioxide and water vapour
through stomata. J. Exp. Bot. 25: 927–936.
Jarvis, P.G. (1971) The estimation of resistances to carbon dioxide transfer.
In Plant Photosynthetic Production: Manual of Methods. Edited by
Sestak, Z., Catsky, J. and Jarvis, P.G. pp. 566–631. Junk, The Hague.
Kerstiens, G. (1996) Cuticular water permeability and its physiological
significance. J. Exp. Bot. 47: 1813–1832.
Lauer, M.J. and Boyer, J.S. (1992) Internal CO2 measured directly in leaves.
Abscisic acid and leaf water potential cause opposing effects. Plant
Physiol. 98: 1310–1316.
Li-Cor Biosciences (2008) Using the LI-6400/ LI-6400XT Portable
Photosynthesis System. LI-COR Biosciences, Inc., Lincoln, NE.
Li-Cor Biosciences (2010) Application Note 7 Modification of LI-6400/LI6400XT to Control at Low [CO2]. LI-COR Biosciences, Inc., Lincoln, NE.
Meyer, S. and Genty, B. (1998) Mapping intercellular CO2 mole fraction
(Ci) in Rosa rubiginosa leaves fed with abscisic acid by using chlorophyll
fluorescence imaging. Significance of Ci estimated from leaf gas exchange. Plant Physiol. 116: 947–957.
Morison, J.I., Gallouët, E., Lawson, T., Cornic, G., Herbin, R. and Baker, N.R.
(2005) Lateral diffusion of CO2 in leaves is not sufficient to support
photosynthesis. Plant Physiol. 139: 254–266.
Moss, D.N. and Rawlins, S.L. (1963) Concentration of carbon dioxide inside
leaves. Nature 197: 1320–1321.
Mott, K.A. (1995) Effects of patchy stomatal closure on gas exchange
measurements following abscisic acid treatment. Plant Cell Environ.
18: 1291–1300.
Parkhurst, D.F. and Mott, K.A. (1990) Intercellular diffusion limits to CO2
uptake in leaves. Studies in air and helox. Plant Physiol. 94: 1024–1032.
Parkhurst, D.F., Wong, S.C., Farquhar, G.D. and Cowan, I.R. (1988)
Gradients of intercellular CO2 levels across the leaf mesophyll. Plant
Physiol. 86: 1032–1037.
Rodeghiero, M., Niinemets, U. and Cescatti, A. (2007) Major diffusion leaks
of clamp-on leaf cuvettes still unaccounted: how erroneous are the
estimates of Farquhar et al. model parameters? Plant Cell Environ. 30:
1006–1022.
Sharkey, T.D., Imai, K., Farquhar, G.D. and Cowan, I.R. (1982) A direct
confirmation of the standard method of estimating intercellular partial
pressure of CO2. Plant Physiol. 69: 657–659.
Terashima, I. (1992) Anatomy of non-uniform leaf photosynthesis.
Photosynth. Res. 31: 195–212.
Terashima, I., Wong, S.C., Osmond, C.B. and Farquhar, G.D. (1988)
Characterization of non-uniform photosynthesis induced by abscisic
acid in leaves having different mesophyll anatomies. Plant Cell Physiol.
29: 385–394.
Tominaga, J. and Kawamitsu, Y. (2015) Tracing photosynthetic response curves with internal CO2 measured directly. Environ. Control
Biol. 53: 27–34.
von Caemmerer, S. and Farquhar, G.D. (1981) Some relationships between
the biochemistry of photosynthesis and the gas exchange of leaves.
Planta 153: 376–387.
Walz (2005) Portable Gas Exchange Fluorescence System GFS-3000:
Handbook of Operation. Heinz Walz GmbH, Effeltrich, Germany.
West, J.D., Peak, D., Peterson, J. and Mott, K.A. (2005) Dynamics of stomatal patches for a single surface of Xanthium strumarium L. leaves
observed with fluorescence and thermal images. Plant Cell Environ 28:
633–641.