TY - JOUR
AU - Kawamitsu, Yoshinobu
AB - Abstract Intercellular CO2 concentration of leaves (Ci) is a critical parameter in photosynthesis. Nevertheless, uncertainties in calculating Ci arise as stomata close. Here, by modifying the assimilation chamber of a commercial gas-exchange equipment to directly measure Ci, we demonstrate overestimation of calculated Ci (i.e. Ci(c)) without stimulating stomatal closure. Gas exchange was measured on one side of the leaf while measured Ci (Ci(m)) was acquired simultaneously on the other side of the leaf in hypostomatous passion fruit (Passiflora edulis Sims) and amphistomatous sunflower (Helianthus annuus L.) and common bean (Phaseolus vulgaris L.). The adaxial surface showed comparable Ci(c) and Ci(m) in sunflower, whereas in common bean, where the adaxial surface has a low stomatal density, Ci(c) markedly differed from Ci(m) when the stomata remained open. However, the latter discrepancy disappeared when measuring the leaf flipped upside down so that the gas exchange was measured (i.e. Ci was calculated) on the abaxial side, which has a much higher stomatal density. The passion fruit showed the largest discrepancy on the astomatous side, indicating that the cuticle has a large impact on the calculation. Direct measurement of Ci is recommended as a more accurate estimate than the calculation when stomatal gas transport is restricted. Occurrence of overestimation and prospects for direct measurement are discussed. A–Ci curve, amphistomatous, cuticle, Heliunthus annuus, hypostomatous, patchy stomatal closure, Passiflora edulis, Phaseolus vulgaris, photosynthesis, stomata Introduction In land plants, leaves constitute the major interface with the atmosphere. Inside leaves, mesophyll cells consume CO2 during photosynthetic assimilation (A), and consequently the CO2 concentration in the intercellular airspace (Ci) is lower than in the bulk air outside the leaf (Ca). The CO2 enters leaves by diffusing through stomatal pores on the leaf surface, so Ci essentially indicates the CO2 substrate available for A (Farquhar et al., 1980). The cells inside leaves are wet, and the stomata allow water vapor to escape by transpiration. Although stomata can close if dehydration becomes excessive, CO2 entry is also restricted by stomatal closure, thereby diminishing A. Therefore, the response of A to various Ci (i.e. the A–Ci curve) is the foundation for relating photosynthetic biochemistry to prevailing environmental conditions experienced by leaves (Hanson et al., 2016). In general gas-exchange measurements, Ci is calculated from the relationship between water vapor exiting and CO2 entering through stomata (Moss and Rawlins, 1963). Instead, Sharkey et al. (1982) measured Ci directly. They put amphistomatous leaves between a chamber and a cup and measured the CO2 concentration inside the cup after it had equilibrated. Because no net CO2 exchange occurred through that side of the leaf, the CO2 concentration measured in the cup would be close to the concentration inside the leaf (measured Ci; Ci(m)). Indeed, the Ci(m) was identical to the value calculated (calculated Ci; Ci(c)) on the other side. Subsequently, close comparisons of Ci(m) and Ci(c) have highlighted the gradient of intercellular CO2 in the airspace owing to the finite conductance of the airspace (Mott and O’Leary,1984; Parkhurst et al., 1988). The gradient varies between species, depending on the anatomy of the mesophyll pores (Evans et al., 2009). For species having a fast assimilation rate, such as sunflower (Helianthus annuus L.), the gradient is generally too small to affect Ci(m) (Mott and O’Leary, 1984; Boyer and Kawamitsu, 2011). The small effect observed for sunflower leaves was confirmed recently when a direct-measurement system for Ci (Boyer and Kawamitsu, 2011) was incorporated into a commercial open gas-exchange apparatus (Tominaga and Kawamitsu, 2015a). Using a similar system, however, Tominaga and Kawamitsu (2015b) demonstrated that Ci(c) markedly differed from Ci(m) as stomata began to close after abscisic acid (ABA) was applied to this species. Tracing A–Ci curves, the Ci(c) showed an artefactual limitation of photosynthesis, as was previously indicated for ABA-fed leaves (Downton et al., 1988; Terashima et al., 1988; Meyer and Genty, 1998), whereas the Ci(m) showed a similar curve to leaves with open stomata. Therefore, these investigators attributed this difference to overestimation of the calculation. For the calculation, the diffusivity ratio of trace gases (water vapor, CO2) through the leaf surface is considered constant—assuming stomata are the dominant path for both gasses (Moss and Rawlins, 1963, von Caemmerer and Farquhar, 1981; Boyer and Kawamitsu, 2011). Using hypostomatous leaves of grape (Vitis vinifera L.), however, small gas fluxes on the astomatous adaxial side (i.e. cuticle/epidermis) were detected while the opposite stomatous surface was sealed (Boyer et al., 1997; Boyer, 2015a). At the same time, Ci(c) was much larger than expected (the photosynthetic CO2 compensation point). Evidently, this discrepancy was a consequence of the cuticle, which transmits water vapor 20- to 40-fold faster than it transmits CO2, a difference that is considerably larger than the constant 1.6 for stomata. Measuring Ci directly in sunflower, Boyer (2015b) calculated cuticular transpiration on the stomatous surface and suggested that it could significantly overestimate CO2 entry. Likewise, Tominaga and Kawamitsu (2015b) calculated the cuticular conductance of water vapor and reached the same conclusion. On the other hand, a number of studies have attributed the overestimation of Ci to a heterogeneous stomatal aperture or patchy stomatal closure. This likely occurs in response to acute stimuli (Gunasekera and Berkowitz, 1992), including application of ABA (Downton et al., 1988; Terashima et al., 1988), casting doubt upon the conclusion made about stomatous leaf surfaces. In fact, it is difficult to determine whether a suspicious value for Ci can be attributed to cuticular transport or to stomatal patchiness because both effects may appear simultaneously as stomata close. Another factor of concern is the intercellular CO2 gradient because any direct measurement inevitably limits CO2 entry from one side, thereby increasing the gradient. Finally, in evaluations of the cuticle effect on the calculation (Boyer, 2015b; Tominaga and Kawamitsu, 2015b), CO2 transport through the cuticle was assumed to be negligible; however, this might be an oversimplification. We therefore conducted experiments to investigate the effect of the cuticle on the calculation without stimulating stomatal closure. Gas exchange was measured separately on the adaxial and abaxial leaf surfaces of common bean (Phaseolus vulgaris L.), for which the stomatal density differs on each side. Simultaneously, Ci(m) was acquired for the other side. For comparison with common bean, we measured symmetric amphistomatous leaves of sunflower (Heliunthus annuus L.) and hypostomatous leaves of passion fruit (Passiflora edulis Sims). Materials and methods Plant material Common bean (P. vulgaris L. cv. Kentucky Blue) and sunflower (H. annuus L. cv. Hybrid sunflower) plants, each 5–6 weeks old, were sources of amphistomatous leaves, and 1-year-old purple passion fruit (P. edulis Sims) plants were the source of hypostomatous leaves. Passion fruit plants were grown in 12-liter plastic pots, and the bean and sunflower plants were grown in 4-liter plastic pots; each pot contained a soil mixture (soil:peat:sand=1:1:1) in a greenhouse located at the University of the Ryukyus, Okinawa, Japan (26°15′N, 127°45′E; elevation 127 m). The growth irradiance depended on solar radiation, and the daily maximum ranged from 300 to 1500 μmol m–2 s–1 photosynthetically active radiation (PAR) for sunflower and common bean. Passion fruit was grown under a shade net and received approximately 30% less irradiance than the other species. The daily maximum and minimum growth temperatures were, on average, 30.9 °C and 16.2 °C, respectively. Water was supplied whenever the soil surface was dry. All soil was saturated weekly with Hoagland’s nutrient solution composed of 4 mM KNO3, 6 mM Ca(NO3)2·4H2O, 2 mM MgSO4·7H2O, 2 mM KH2PO4, 0.5 µM CuSO4·5H2O, 10 µM MnSO4·H2O, 2 µM ZnSO4·7H2O, 25 µM H3BO3, 0.5 µM H2MoO4 and 0.5 µM Fe(III)-EDTA. All experiments with these plants were conducted with fully expanded leaves. Gas-exchange and direct-measurement systems The gas-exchange system has been reported in detail elsewhere (Tominaga and Kawamitsu, 2015a), and we used a modified system (the design drawings are available upon request). Briefly, the small cup of the Tominaga and Kawamitsu system was replaced with the bottom half of an assimilation chamber (LI6400-40; Li-Cor, Lincoln, NE, USA) of a commercial gas-exchange system (LI-6400XT; Li-Cor). Although use of the commercial assimilation chamber has certain advantages (e.g. combined fluorescence measurement), the small chamber is especially prone to diffusion leaks (Rodeghiero et al., 2007). To minimize the effect of leakage and detect small fluxes accurately, we used a larger chamber/cup that could also be attached to the sensor head (Fig. 1). This new laboratory-made chamber/cup with a window diameter of 6 cm encloses a 14-fold larger leaf area (28.3 cm2) than the LI-6400-40 chamber (2 cm2) with the window diameter of 0.8 cm. The chamber window is covered with Propafilm (polypropylene that is coated with Saran; 250-01885, Li-Cor). Fig. 1. Open in new tabDownload slide The lab-made chamber/cup for direct measurement of intercellular CO2 concentration (Ci). View from (A) front side and (B) back side, and (C) schematic diagram of the chamber/cup clamped on a leaf (represented by the green horizontal line). The brass chamber/cup was specially designed for the sensor head of the Li-Cor LI-6400XT open gas-exchange system. The leaf separates the cup from the chamber. The leaf-clamp seal is ensured by a coating of paraffin/lanolin. Mixing air passes through the upper chamber, allowing the leaf surface to exchange gases that are exhausted through the bypass (a). Water circulated inside the chamber wall (b) controls air temperature in the cup. A small blower circulates the air in the cup to the gas analyser (LI-840A; Li-Cor) through the inlet and outlet located at the bottom of the cup (c). The round chamber window (28.3 cm2) is covered with Propafilm (250-01885, Li-Cor). A leaf-temperature thermocouple (6400-04; Li-Cor) is inserted into the cup and attached to the lower surface of the leaf, as is the case in the standard LI-6400 chamber. Fig. 1. Open in new tabDownload slide The lab-made chamber/cup for direct measurement of intercellular CO2 concentration (Ci). View from (A) front side and (B) back side, and (C) schematic diagram of the chamber/cup clamped on a leaf (represented by the green horizontal line). The brass chamber/cup was specially designed for the sensor head of the Li-Cor LI-6400XT open gas-exchange system. The leaf separates the cup from the chamber. The leaf-clamp seal is ensured by a coating of paraffin/lanolin. Mixing air passes through the upper chamber, allowing the leaf surface to exchange gases that are exhausted through the bypass (a). Water circulated inside the chamber wall (b) controls air temperature in the cup. A small blower circulates the air in the cup to the gas analyser (LI-840A; Li-Cor) through the inlet and outlet located at the bottom of the cup (c). The round chamber window (28.3 cm2) is covered with Propafilm (250-01885, Li-Cor). A leaf-temperature thermocouple (6400-04; Li-Cor) is inserted into the cup and attached to the lower surface of the leaf, as is the case in the standard LI-6400 chamber. The new chamber/cup was replaced with the upper or lower half of the chamber (Fig. 1). A paraffin/lanolin mixture (2:8) was spread evenly (thickness ~1 mm) on the rims of both the chamber and the cup (i.e. the interface). This semi-solid coat sealed the chamber tightly when a leaf was clamped. All experimental leaves were large enough to cover the entire window area so that the upper chamber and lower cup were divided by the leaf. In the cup, CO2 equilibrated with that inside the stomatal pores (Ci(m)). The equilibrated air was smoothly circulated with a small fan to the infrared gas analyser (IRGA; LI-840A; Li-Cor) in a closed loop. To compensate for the volume of the larger cup, which potentially could delay the equilibration, the path length of the loop was kept as short as possible (~40 cm). The overall volume of the closed loop for the new system was 137 ml (1.2-fold larger than the previous system) determined by monitoring the concentration change with the LI-840A after injecting a small amount of 5% CO2 gas into the loop (see Supplementary Fig. S1 at JXB online). In the loop, Ci(m) was continuously measured while gas exchange through the opposite side of same leaf area was detected with the open gas-exchange system, which automatically calculated Ci (see ‘Calculations’). Accordingly, both Ci(m) and Ci(c) were obtained simultaneously for the same leaf area, allowing close comparison. Using a water-saturated filter paper (Advantec No. 1) as a leaf model (Ball, 1987), the boundary layer conductance for the lab-made chamber was estimated to be 1.38 mol m–2 s–1, which was less than one-third that of the LI-6400-40 and yet sufficiently greater than the stomatal conductance. Gas-exchange measurements After reaching steady state photosynthesis at the ambient CO2 concentration of 300–400 μmol per mole of air (hereafter μmol mol–1), the concentration was changed stepwise, allowing Ci to vary. CO2 concentration was regulated by mixing pure CO2 supplied to the LI-6400 console with CO2-free air humidified with a dew-point generator (LI-610; Li-Cor). Each leaf was illuminated with three metal-halide lamps (D-400, Toshiba) located approximately 1 m above the chamber. Fine net shades were inserted between the light source and the chamber so that the irradiance was adjusted to 300 μmol m–2 s–1 PAR, which was less than the 800 μmol m–2 s–1 PAR used in a previous study (Tominaga and Kawamitsu, 2015a,b). We reduced the irradiance to control leaf temperature as the light source containing a broad thermal ray otherwise warmed the leaf. As a result of the lower irradiance, the A for sunflower leaves was below saturation and yielded a lower A/Ci ratio of 0.064 (Ci=220 μmol mol–1) than the value of 0.09 (Ci=280 μmol mol–1) measured previously (Tominaga and Kawamitsu, 2015b). All gas-exchange measurements were carried out at a leaf temperature of 25 ± 0.1 °C, a leaf to air vapor pressure deficit of <1.5 kPa, and a constant gas flow rate of 500 μmol s–1. Common bean and sunflower leaves responded by slowly closing their stomata when the CO2 concentration was somewhat higher than the ambient level, which often required a longer measurement time. To help decrease the measurement time, the ambient CO2 concentration was increased to no more than 700 μmol mol–1. Diffusion leaks The upper chamber and lower cup were separated by clamping aluminum foil (instead of the leaf in Fig. 1C) using the paraffin/lanolin coat. For the open gas-exchange system, a test for diffusion leaks was carried out by estimating CO2 and the water vapor diffusion molar flow rate, KCO2 and KH2O, respectively, according to Rodeghiero et al. (2007). To maintain the concentration gradients of CO2 and water vapor between the outside and inside of the chamber, the sensor head was enclosed by a semi-closed cylinder in which air containing 400 μmol mol–1 CO2 and 20 mmol mol–1 water vapor (monitored with the LI-840A) flowed continuously at a rate of 15 l min–1 whereas dry air with 30 μmol mol–1 CO2 was supplied to the chamber from the console. KCO2 and KH2O were estimated as 0.21 ± 0.079 and 0.71 ± 0.13 μmol s–1, respectively, values that were comparable to those determined for the smaller LI-6400-40 chamber (Rodeghiero et al., 2007; Tominaga and Kawamitsu, 2015a), suggesting a similar rate of diffusion leak on a chamber basis. Accounting for the larger chamber interface (i.e. circumference of the chamber window), the new chamber had a better seal. Because the leakage can cause gas fluxes that can affect both assimilation and transpiration, which are measured on a leaf-area basis, the apparent fluxes (leak rate divided by leaf area) were significantly decreased in the larger chamber as compared with the previous system. The CO2 and water vapor concentrations outside the chamber were monitored by open-path IRGA (LI-7500; Li-Cor) set around the leaves, and were subsequently used to correct for leaks as described in Rodeghiero et al. (2007). The apparent fluxes in the open system were estimated to be <0.03 μmol m–2 s–1 for CO2 and <0.001 mmol m–2 s–1 for water vapor throughout the measurements. In most of our experiments, such small fluxes changed the calculation of Ci by only <1%. The CO2 leakage in the closed system increases or decreases the equilibrium of Ci(m) depending on the concentration gradient relative to the outside. To detect the leakage, we monitored the change of CO2 concentration in the empty cup (Ccup) after partially enclosing the expired air containing a high concentration of CO2 of ~1800 μmol mol–1 (see Supplementary Fig. S2). The leak was indicated by the Ccup decreasing slowly at a rate of 10.8 μmol mol–1 h–1 while the outside CO2 concentration (Croom) was constant at ~400 μmol mol–1. Based on this leak rate and the known volume of the closed system (137 ml), the rate of CO2 diffusion leak was estimated to be 0.0054 μmol m–2 s–1 on a leaf-area basis and should be even lower when Ccup is closer to Croom (c. 400–500 μmol mol–1) in our experiments. The CO2 leakage occurred too slowly to affect the equilibrium, and thus no correction was applied to the Ci(m). Stomatal density and size After gas-exchange measurements, nail polish was thinly spread at several points on both the upper and lower leaf surfaces within the measured leaf area. After waiting for the polish to dry, the polish was striped with clear double-sided tape and attached to a glass slide (i.e. a replica of the epidermis). Microphotographs of the replica were taken with a digital camera (HDR-SR12; Sony, Tokyo, Japan) attached to a microscope (Eclipse 80i; Nikon, Kawasaki, Japan). For each side of the leaves, 20 microphotographs were taken at random. We counted the number of stomata in each microphotograph (0.64–1.82 mm2) to calculate stomatal density. Mean stomatal size was calculated based on the length of the long axis of the stomata. Calculations Realizing that departing vapor behaved like entering CO2, Moss and Rawlins (1963) first calculated Ci (Ci(c)) from measured variables: Ci(c)=Ca−1.6AE(wi−wa)(1) where Ca is the CO2 concentration in the air (mol mol–1), A is the assimilation rate for CO2 moving into the leaf through stomata (mol m–2 s–1), E is the transpiration rate for water vapor moving out of the leaf through stomata (mol m–2 s–1), and wi and wa are the water vapor concentrations in the leaf and air outside the leaf, respectively (mol mol–1). The only other factor needed was the ratio of the diffusivity of water vapor to that of CO2, which is ~1.6 in air (Jarvis, 1971; Massman, 1998) assuming that stomata are the dominant path for both gases. Von Caemmerer and Farquhar (1981) rigorously extended the Moss–Rawlins relation to account for interactions between water vapor and CO2: Ci(c)=Ca−1.6A+C¯EE-w¯E(wi−wa)(2) where C¯Eand w¯E(⁠ C¯=(Ca + Ci(c))/2 and w¯=(wa + wi)/2) are interaction terms. This equation became the norm for determining Ci and is routinely used in commercial gas-exchange units including the LI-6400. Note that both Ca and wa were implicitly located in the boundary layer of the leaf surface in this study. The Ca and wa were, respectively, estimated with the boundary layer conductance of water vapor (1.38 mol m–2 s–1) and that of CO2 (1.01 mol m–2 s–1) estimated from the ratio of diffusivity in the ‘convective air’ (1.37) rather than ‘still air’ (1.6) for the stomatal pore (Ball, 1987). Von Caemmerer and Farquhar (1981) expressed the diffusion property of water vapor in Eq. (2) as a form of conductance (gw): gw=E−w¯Ewi−wa(3) In contrast to Eq. (2), the Ci(m) already reflected the interactions of CO2 and water vapor (Boyer and Kawamitsu, 2011): Ci(m)=Ca−1.6AE−w¯E(wi−wa)(4) where the only difference from Eq. (2) is the absence of the term C¯E ⁠. Boyer and Kawamitsu (2011) first expressed conductance of CO2 (gc) in terms of directly measured variables: gc=ACa−Ci(m)(5) In this study, the conductance of water vapor (gw) and of CO2 (gc) was derived from the directly measured variables using Eqs (3) and (5), respectively. Results Stomatal density and size In leaves of the common bean, the adaxial side had significantly fewer stomata (9 ± 5 mm–2) than the abaxial side (274 ± 31 mm–2), whereas in sunflower the stomata were distributed more evenly on both sides (Table 1). On the adaxial side, the stomatal density in sunflower (221 ± 24 mm–2) is much higher than in common bean. The stomatal ratio (adaxial/abaxial) was 0.03 for common bean leaves and 0.85 for sunflower. Stomatal size did not differ significantly between the two sides in sunflower, whereas in the common bean the adaxial surface had 33% larger stomata than the abaxial surface. Passion fruit leaves, which were observed to bear stomata only on the abaxial surface, had a stomatal density (209 ± 26 mm–2) comparable to that of sunflower. Table 1. Stomatal density and size in passion fruit, sunflower, and common bean . Adaxial . Abaxial . P . Ratio (adaxial/abaxial) . Stomatal density (mm–2)  Passion fruit 0 (n=80) 209 ± 26 (n=80)  Sunflower 188 ± 25 (n=80) 221 ± 24 (n=80) <0.001 0.85  Common bean 9 ± 5 (n=160) 274 ± 31 (n=160) <0.001 0.032 Long axis of stomata (μm)  Passion fruit 10.7 ± 1.3 (n=40)  Sunflower 12.4 ± 1.8 (n=40) 12.2 ± 2.7 (n=40) 0.583 1.02  Common bean 10.9 ± 1.7 (n=80) 8.2 ± 1.3 (n=80) <0.001 1.33 . Adaxial . Abaxial . P . Ratio (adaxial/abaxial) . Stomatal density (mm–2)  Passion fruit 0 (n=80) 209 ± 26 (n=80)  Sunflower 188 ± 25 (n=80) 221 ± 24 (n=80) <0.001 0.85  Common bean 9 ± 5 (n=160) 274 ± 31 (n=160) <0.001 0.032 Long axis of stomata (μm)  Passion fruit 10.7 ± 1.3 (n=40)  Sunflower 12.4 ± 1.8 (n=40) 12.2 ± 2.7 (n=40) 0.583 1.02  Common bean 10.9 ± 1.7 (n=80) 8.2 ± 1.3 (n=80) <0.001 1.33 The values are the mean ±SD. Open in new tab Table 1. Stomatal density and size in passion fruit, sunflower, and common bean . Adaxial . Abaxial . P . Ratio (adaxial/abaxial) . Stomatal density (mm–2)  Passion fruit 0 (n=80) 209 ± 26 (n=80)  Sunflower 188 ± 25 (n=80) 221 ± 24 (n=80) <0.001 0.85  Common bean 9 ± 5 (n=160) 274 ± 31 (n=160) <0.001 0.032 Long axis of stomata (μm)  Passion fruit 10.7 ± 1.3 (n=40)  Sunflower 12.4 ± 1.8 (n=40) 12.2 ± 2.7 (n=40) 0.583 1.02  Common bean 10.9 ± 1.7 (n=80) 8.2 ± 1.3 (n=80) <0.001 1.33 . Adaxial . Abaxial . P . Ratio (adaxial/abaxial) . Stomatal density (mm–2)  Passion fruit 0 (n=80) 209 ± 26 (n=80)  Sunflower 188 ± 25 (n=80) 221 ± 24 (n=80) <0.001 0.85  Common bean 9 ± 5 (n=160) 274 ± 31 (n=160) <0.001 0.032 Long axis of stomata (μm)  Passion fruit 10.7 ± 1.3 (n=40)  Sunflower 12.4 ± 1.8 (n=40) 12.2 ± 2.7 (n=40) 0.583 1.02  Common bean 10.9 ± 1.7 (n=80) 8.2 ± 1.3 (n=80) <0.001 1.33 The values are the mean ±SD. Open in new tab Hypostomatous leaves We placed the cup on the abaxial stomatous side while photosynthesis and transpiration were measured simultaneously in the chamber on the adaxial astomatous side of passion fruit leaves. Soon after clamping the leaf, both assimilation rate (A) and conductance of CO2 (gc) became too small to be detected on the astomatous side (Fig. 2A). In contrast, both transpiration rate (E) and conductance of water vapor (gw) were detectably large on the same side (Fig. 2B). These results demonstrated that the cuticle loses more water vapor than the amount of entering CO2. On the stomatous side, photosynthesis diminished the amount of CO2 inside the cup until equilibrium was reached (Fig. 2C). Because CO2 scarcely entered from the astomatous surface (Fig. 2A), the CO2 concentration at equilibrium (Ci(m)) ought to be equivalent to the photosynthetic CO2 compensation point where no net flux occurs in the leaf. In agreement with this supposition, Ci(m) equilibrated to ~43 μmol mol–1 at a leaf temperature (Tleaf) of 25 °C. In contrast, Ci calculated from the transpiration (Ci(c)) remained high, differing substantially from Ci(m). E on the astomatous surface denotes cuticular transpiration (Ecut), and on average Ecut was 4.25 μmol m–2 s–1 at a Tleaf of 25 °C. While keeping the ambient vapor concentration (wa) constant, the vapor concentration inside the leaf (wi) was raised stepwise by heating the leaf to 29 °C and subsequently to 33 °C (see Supplementary Fig. S3). The difference of the vapor concentration gradients across the cuticle (wi–wa) increased from 21 mmol mol–1 (at Tleaf of 25 °C) to 29 and 39 mmol mol–1, respectively. Ecut correlated positively and linearly with the vapor concentration gradients (Fig. 3). We calculated the cuticular conductance of water vapor (⁠ gwcut ⁠) on the astomatous surface according to gwcut=Ecut/(wi – wa) (Boyer et al. 1997). The gwcut was 0.21 mmol m–2 s–1 at each leaf temperature (Table 2) and also coincided well with the slope of the regression in Fig. 3. The constant gwcut indicated that the cuticle was stable at physiological temperatures (25–33 °C), supporting the previous study of isolated cuticles (Riederer and Schreiber, 2001). On the stomatous side, Ci(m) closely followed leaf temperature (Supplementary Fig. S3). The value and temperature dependence of Ci(m) (Table 2) were similar to those of the intercellular CO2 photocompensation point (Ci*) for C3 species (Walker and Ort, 2015). In this experiment, the smaller wa compared with wroom potentially caused leakage of water vapor into the chamber. The effect on Ecut was estimated to be greatest (7.6–13.3% of the apparent flux) when Ecut was small at a Tleaf of 25 °C and reached a minimum (4.6–6.0%) when Ecut was high at a Tleaf of 33 °C. Table 2. Temperature responses of gwcut ⁠, Ci(m), and Ci(c) Tleaf (°C) . gwcut (μmol m–2 s–1) . Ci(m) (μmol mol–1) . Ci(c) (μmol mol–1) . 25 213 ± 33 42.7 ± 0.4 469 ± 12 29 214 ± 23 51.4 ± 0.4 391 ± 10 33 211 ± 14 62.1 ± 1.0 330 ± 3 Tleaf (°C) . gwcut (μmol m–2 s–1) . Ci(m) (μmol mol–1) . Ci(c) (μmol mol–1) . 25 213 ± 33 42.7 ± 0.4 469 ± 12 29 214 ± 23 51.4 ± 0.4 391 ± 10 33 211 ± 14 62.1 ± 1.0 330 ± 3 The values are the mean ±SE (n=4). Open in new tab Table 2. Temperature responses of gwcut ⁠, Ci(m), and Ci(c) Tleaf (°C) . gwcut (μmol m–2 s–1) . Ci(m) (μmol mol–1) . Ci(c) (μmol mol–1) . 25 213 ± 33 42.7 ± 0.4 469 ± 12 29 214 ± 23 51.4 ± 0.4 391 ± 10 33 211 ± 14 62.1 ± 1.0 330 ± 3 Tleaf (°C) . gwcut (μmol m–2 s–1) . Ci(m) (μmol mol–1) . Ci(c) (μmol mol–1) . 25 213 ± 33 42.7 ± 0.4 469 ± 12 29 214 ± 23 51.4 ± 0.4 391 ± 10 33 211 ± 14 62.1 ± 1.0 330 ± 3 The values are the mean ±SE (n=4). Open in new tab Fig. 2. Open in new tabDownload slide Change in (A) assimilation rate (A) and conductance of CO2 (gc), (B) transpiration rate (E) and conductance of water vapor (gw), and (C) intercellular CO2 concentration (Ci) after clamping a passion fruit leaf. Numbers below species name indicate stomatal densities (Table 2) for the leaf side attached to the assimilation chamber (upper number) and the cup (lower number); gas exchange through the astomatous side was measured in the chamber while Ci was measured directly (Ci(m)) in the cup. Calculated Ci (Ci(c)) was simultaneously derived from the transpiration occurring within the same leaf area of the other adaxial surface. Note that A and gc overlap one another. In (A) and (C), erroneous data that were attributable to CO2 control by LI-6400 were removed. The experiments were conducted at a constant leaf temperature of 25 °C and ambient CO2 concentration of 400 μmol mol–1. Representative experiment from four replications. Fig. 2. Open in new tabDownload slide Change in (A) assimilation rate (A) and conductance of CO2 (gc), (B) transpiration rate (E) and conductance of water vapor (gw), and (C) intercellular CO2 concentration (Ci) after clamping a passion fruit leaf. Numbers below species name indicate stomatal densities (Table 2) for the leaf side attached to the assimilation chamber (upper number) and the cup (lower number); gas exchange through the astomatous side was measured in the chamber while Ci was measured directly (Ci(m)) in the cup. Calculated Ci (Ci(c)) was simultaneously derived from the transpiration occurring within the same leaf area of the other adaxial surface. Note that A and gc overlap one another. In (A) and (C), erroneous data that were attributable to CO2 control by LI-6400 were removed. The experiments were conducted at a constant leaf temperature of 25 °C and ambient CO2 concentration of 400 μmol mol–1. Representative experiment from four replications. Fig. 3. Open in new tabDownload slide Relationships between cuticular transpiration (Ecut) and gradient of water vapor concentration between the inside (wi) and outside (wa) of a passion fruit leaf. Ecut was measured on the adaxial astomatous surface. The water vapor gradient varied with leaf temperature (25, 29, 33 °C) when wa was kept constant. Means ±SE (n=4) and a regression line are shown. Fig. 3. Open in new tabDownload slide Relationships between cuticular transpiration (Ecut) and gradient of water vapor concentration between the inside (wi) and outside (wa) of a passion fruit leaf. Ecut was measured on the adaxial astomatous surface. The water vapor gradient varied with leaf temperature (25, 29, 33 °C) when wa was kept constant. Means ±SE (n=4) and a regression line are shown. Amphistomatous leaves The adaxial side of leaves was set up to allow gas exchange, and we measured A–Ci curves in sunflower—the species previously assessed with a smaller chamber and cup (Tominaga and Kawamitsu, 2015a,b). The adaxial stomata remained open during the experiments as indicated by the relatively constant gc (Fig. 4A). A was rapidly balanced with Ci(c) at each Ca. As expected for open stomata (gc=178 ± 24 mmol m–2 s–1), the calculation coincided well with the direct measurement for this species (Fig. 4B). It was clear that the larger cup facilitated a response of Ci(m) as fast as Ci(c). We then carried out the experiment using common bean leaves, for which the stomatal ratio contrasts with that of sunflower leaves (Table 1). With few stomata on the adaxial surface, gc was <20 mmol m–2 s–1 (Fig. 5A). A sensitive response of A to CO2 indicates open stomata. Comparison with gw suggested that gc became noisy when Ca was small (see Supplementary Fig. S4) due to the detection limit of A but not the Ci(m) (see Discussion). The gradient between Ca and Ci(m) appropriately directed the subtle CO2 flux, demonstrating the accuracy of the measurement, e.g. Ci(m) is always lower than the Ca when A is positive and vice versa. The Ci(c) differed largely from the Ci(m) from the outset (Fig. 5B). The effect of leak corrections on the Ci(c) was <4%. To further investigate the cause of this discrepancy, we flipped the leaf so that CO2 could readily diffuse into the abaxial side having much greater stomatal density (Fig. 6). The gc was 222 ± 15 mmol m–2 s–1, and the average CO2 conductance ratio (abaxial/adaxial) was 0.073. This value was more than double that of the stomatal ratio (0.032), suggesting that the stomata on the adaxial surface, although fewer in number, opened wider perhaps because of their larger size (Table 1). It took longer for Ci(m) to equilibrate for the flipped leaves because the CO2 in the cup diffused more slowly through the adaxial side with few stomata (cf. Figs 5 and 6). At equilibrium, the Ci(m) and Ci(c) were similar (Fig. 6B). Fig. 4. Open in new tabDownload slide Response of (A) assimilation rate (A) and conductance of CO2 (gc) to (B) various Ci for sunflower leaves. Gas exchange through the adaxial side was measured. The change in ambient CO2 concentration (Ca) is also shown with Ci. Values for Ci(m) and Ci(c) overlapped one another. For the gas-exchange measurement (A), the data acquired immediately after the change in Ca were removed owing to extreme values. Representative experiment from four replications. Fig. 4. Open in new tabDownload slide Response of (A) assimilation rate (A) and conductance of CO2 (gc) to (B) various Ci for sunflower leaves. Gas exchange through the adaxial side was measured. The change in ambient CO2 concentration (Ca) is also shown with Ci. Values for Ci(m) and Ci(c) overlapped one another. For the gas-exchange measurement (A), the data acquired immediately after the change in Ca were removed owing to extreme values. Representative experiment from four replications. Fig. 5. Open in new tabDownload slide As in Fig. 4, A–Ci curve measurement for common bean leaves. Gas exchange through the adaxial side was measured. Representative experiment from four replications. Fig. 5. Open in new tabDownload slide As in Fig. 4, A–Ci curve measurement for common bean leaves. Gas exchange through the adaxial side was measured. Representative experiment from four replications. Fig. 6. Open in new tabDownload slide A–Ci curve measurement for common bean leaves. Each leaf was flipped upside-down so that the gas exchange through the abaxial side could be measured. Representative experiment from four replications. Fig. 6. Open in new tabDownload slide A–Ci curve measurement for common bean leaves. Each leaf was flipped upside-down so that the gas exchange through the abaxial side could be measured. Representative experiment from four replications. Diffusivity ratio of water vapor and CO2 of the leaf surface After plotting A against Ci(m) and Ci(c) for the experiments described above, suppressed A–Ci curves were then derived from Ci(c) for the common bean leaves, for which gas exchange was measured on the adaxial side (Fig. 7A). The similar result for this species was also observed previously using the small chamber/cup system (see Supplementary Fig. S5). Evidently, the large discrepancy between the Ci(c) and Ci(m) was not induced by patchy stomatal closure because the adaxial stomata remained open (Fig. 5). Assuming that this discrepancy is created by the intercellular CO2 gradients (i.e. Ci(c)–Ci(m)), flipping the leaf should impose the gradient to a similar degree to achieve the same A while keeping the intercellular conductance unchanged. In fact, this was not true (abaxial in Fig. 7A), and even comparable Ci(c) and Ci(m) suggested minute gradients. Consequently, the calculation overestimated Ci by neglecting cuticular gas transport since other possibilities were likely ruled out. Fig. 7. Open in new tabDownload slide Data from Figs 2–4 using sunflower and common bean plotted as (A) A–Ci curves and (B) relationships derived from the measured variables in Eq. (6). In (A), closed circles denote Ci(m) and open circles denote Ci(c). Note that Ci(m) and Ci(c) are on top of each other for adaxial leaf side of sunflower and abaxial leaf side of common bean. The slope in (B) indicates the diffusivity ratio through the leaf surface (water/CO2). Fig. 7. Open in new tabDownload slide Data from Figs 2–4 using sunflower and common bean plotted as (A) A–Ci curves and (B) relationships derived from the measured variables in Eq. (6). In (A), closed circles denote Ci(m) and open circles denote Ci(c). Note that Ci(m) and Ci(c) are on top of each other for adaxial leaf side of sunflower and abaxial leaf side of common bean. The slope in (B) indicates the diffusivity ratio through the leaf surface (water/CO2). Upon measurement of leaf gas exchange, calculations essentially rely on the diffusivity ratio (water vapor/CO2) for stomata [1.6 in Eqs (1) and (2)], assuming that stomata are the only path for both water vapor and CO2. To measure the impact of the cuticle on the calculations, the diffusivity ratio through the leaf surface (Dw/c) was estimated from directly measured variables based on Eq. (4): Ca−Ci(m)=Dw/cAE−w¯E(wi−wa)(6) where Dw/c, instead of a theoretical constant 1.6 in Eq. (4), indicates the slope of the regression line for this relationship in Fig. 7B. For sunflower leaves, Dw/c was 1.58 ± 0.05. For common bean, values of Dw/c were 2.44 ± 0.37 and 1.79 ± 0.12 for the adaxial and abaxial side, respectively. Discussion Our results demonstrate that the cuticle is an essential contributor to the large discrepancy between Ci(c) and Ci(m), supporting the conclusion reached with different species using different approaches (Boyer, 2015b; Tominaga and Kawamitsu, 2015b). In those previous experiments, ABA was fed to sunflower leaves to close stomata, thereby simulating the astomatous leaf surface. Although the stomatal closure is imperfect, the calculation overestimates Ci whereas the direct measurement is unaffected by the open/closed status of stomata. Here, we employed common bean leaves with few stomata on one side, i.e. instead of manually inducing stomatal closure, and we also observed a large discrepancy (Figs 5 and 7A), as seen with stomatal closure in sunflower leaves (Boyer, 2015b; Tominaga and Kawamitsu, 2015b). These stomata remained open even wider than those on the opposite, more stomatous surface, rejecting the possibility of patchy stomatal closure. Moreover, the discrepancy was not a consequence of the intercellular CO2 gradient because it disappeared for the flipped leaf with the intercellular conductance unchanged. These results were consistent with the evidence for passion fruit that clearly shows that cuticular gas transport is not negligible (Fig. 2). Implications of the direct measurements Instead of diffusing through pores, at the outer cuticle layer both CO2 and water vapor diffuse through solid wax, which creates most of the resistance to cuticular gas transport (Šantrůček et al., 2004). Consequently, the cuticle has a much smaller conductance than stomata despite covering most of the leaf surface (Table 2), and therefore the impact on the calculation is not substantial unless the stomatal gas transport diminishes in stomatous surfaces. The cuticle effect appears because cuticle transports more water vapor than CO2 (Boyer et al., 1997; Boyer, 2015a), as can be measured in the astomatous gas exchange (Fig. 2). The effect was still detectable for the stomatous leaf surface having Dw/c larger than 1.6 (Fig. 7B). Those Dw/c values were much less than those of 20–40 for the astomatous side of grape leaves (Boyer et al., 1997; Boyer, 2015a) because the stomata still accounted for gas exchange to some extent. Similarly, Dw/c was 5.5 when the cuticular transpiration was estimated to be 5–16% of the total transpiration for sunflower leaves fed with ABA (Boyer, 2015b). To what extent does stomatal closure bring uncertainty to the calculation of Ci? According to Eq. (6), we estimated Dw/c for the A–Ci curve measurements in sunflower leaves fed with ABA (Fig. 4 in Tominaga and Kawamitsu, 2015b) and plotted it against various values of ‘stomatal conductance’ (gw) (Fig. 8A). The Dw/c generally appeared to rise sharply as the gw decreased from 100 mmol m–2 s–1. This trend is reasonable because cuticular conductance remains while stomatal conductance is reduced by stomatal closure. In addition, the Dw/c varied at a given gw in different leaves as stomata closed (shown as different symbols in Fig. 8A), indicating variable cuticular conductance among leaves of a single plant (Kerstiens, 1996). This is further supported by the gw–Dw/c relationship for the adaxial side of common bean having the largest Dw/c despite the highest gw (Fig. 8B) . The variation may be attributable to a lateral non-uniformity of cuticular conductance—the thinner cuticle of the guard cells likely has higher conductance than the cuticle on the surrounding epidermal cells (Šantrůček et al., 2004). The cuticular conductance varied on the stomatous side (Fig. 8) perhaps with the properties of guard-cells/stomata (e.g. number, size, density), whereas it was relatively constant without stomata (Table 2). This possibility can be tested by analysing the gw–Dw/c relationships for leaves having various stomatal properties. When using the regular assimilation chamber with gas flowing both adaxially and abaxially, Ci is calculated from the sum of fluxes on both sides. Therefore, hypostomatous or asymmetric amphistomatous leaves are not necessarily prone to the overestimation because the gas exchange occurring mostly on the stomatous side dilutes the gas exchange through the cuticle (as shown in the A–Ci measurements with a regular Li-Cor head set-up in Supplementary Fig. S5). However, the cuticular gas transport increases with the doubled cuticle. So, it is expected that the Dw/c illustrated in Fig. 8A departs from 1.6 more when gas-exchange measurements are configured normally. In A–Ci curve measurements, ABA caused either no depression (Raschke and Hedrich, 1985) or moderate depression in the broad bean, Vicia faba (Terashima et al., 1988). Likewise, Xanthium strumarium often showed significant depression (Fischer et al., 1986; Daley et al., 1989), but not always (Dubbe et al., 1978; Mott and Takemoto, 1989). Contrasting results were also observed during experiments (Raschke and Hedrich, 1985; Mott, 1995). These reported experimental inconsistencies were not necessarily attributable to stomatal patchiness but perhaps to the extent of stomatal closure and/or variable cuticular conductance. Stomatal patchiness may affect the calculation as lateral gas diffusion is obstructed in the intercellular airspace (Terashima et al., 1988; Terashima, 1992). This is a trait of heterobaric leaves in which bundle-sheath extensions span the gap between the upper and the lower epidermis. Homobaric leaves lacking this barrier, on the other hand, have the interconnected airspace. All our measurements so far have been limited to heterobaric leaves. However, because of a similar arrangement, the cuticle does affect the calculation regardless of the internal anatomy. Considerations of the direct measurement We technically altered the amphi- to hypostomatous leaves by attaching the cup on one side, thereby increasing the CO2 gradients (Parkhurst et al., 1988; Boyer and Kawamitsu, 2011). The Ci(m) reflects an equilibrium with the concentration just beneath the interior of epidermis on the cup-attached side. On the other hand, the site for the Ci(c) may be peristomata beneath the chamber-attached side (Sharkey et al., 1982; Mott and O’Leary, 1984; Parkhurst et al., 1988), or it may be deeper inside the leaf (Nonami and Schulze, 1989; Brodribb et al., 2007). In either case, Ci(m) represents the CO2 that has diffused through a somewhat longer path. This has been previously indicated by the Ci(m) being slightly but consistently lower than the Ci(c) in sunflower leaves with open stomata (Tominaga and Kawamitsu, 2015a,b; Boyer, 2015a,b). In contrast, the Ci(m) was almost identical to the Ci(c) in the present experiments (Figs 4 and 7A), suggesting little gradient. Because the gradients must be created by the mesophyll assimilation activity (i.e. A), this is likely due to slower assimilation under sub-saturating irradiance (300 μmol m–2 s–1). For the flipped leaves illuminated from the abaxial side, even slower mesophyll activity (indicated by the smaller A–Ci(m) slope) should have further contributed to the minor gradients (Fig. 7A). The larger chamber has advantages over the smaller chamber owing to the shorter length (circumference) of the gasket relative to the enclosed leaf area. The leak effect should be reduced because gaseous pores between the rubber gaskets and leaf are the major path for leakage in commercial chambers (Flexas et al., 2007). Diffusion leak could be further suppressed by using semi-solid materials for the gaskets. Consequently, our lab-made chamber enabled commercial open gas-exchange equipment to measure in vivo cuticle properties (Fig. 3). Nevertheless, the gc derived from the Ci(m) became erratic as Ca decreased (Figs 5A and 6A). As Ca decreases, gc becomes increasingly sensitive to the small A, for which the noise/signal ratio increases accordingly whereas the CO2 gradient (Ca–Ci) decreases [Eq. (5)]. On the other hand, the water vapor gradient (wi–wa) is relatively stable, and thus the calculation of gw [Eq. (3)] is insensitive to change in Ca (see Supplementary Fig. S4). The signal noise in A also affects the estimation of Dw/c [Eq. (6)]. Thus, Dw/c is recommended to be estimated from multiple measurements when gw is stable (Fig. 7B), or otherwise discarded in the region where A is small (as implemented in Fig. 8). Fig. 8. Open in new tabDownload slide Relationships between conductance of water vapor (gw) and diffusivity ratio through the leaf surface (Dw/c) estimated in A–Ci curve measurements for adaxial side of (A) sunflower leaves fed with 10 μM ABA and (B) common bean leaves. Different symbols represent replications with different leaves. Data shown in (A) are representative analysis of the previous experiments (Fig. 4 in Tominaga and Kawamitsu, 2015b). Data are removed when A <±1.5 μmol m−2 s−1 in (A) and A <±0.4 μmol m−2 s−1 in (B) due to incorrect Dw/c (see Discussion). In (B), vertical and horizontal bars indicate ±SD (n=3). Fig. 8. Open in new tabDownload slide Relationships between conductance of water vapor (gw) and diffusivity ratio through the leaf surface (Dw/c) estimated in A–Ci curve measurements for adaxial side of (A) sunflower leaves fed with 10 μM ABA and (B) common bean leaves. Different symbols represent replications with different leaves. Data shown in (A) are representative analysis of the previous experiments (Fig. 4 in Tominaga and Kawamitsu, 2015b). Data are removed when A <±1.5 μmol m−2 s−1 in (A) and A <±0.4 μmol m−2 s−1 in (B) due to incorrect Dw/c (see Discussion). In (B), vertical and horizontal bars indicate ±SD (n=3). In clamp-on chambers, gas-exchange measurements may be affected by the edge—respiratory CO2, originating from the leaf areas below the gaskets, laterally diffuses through the intercellular airspace to the leaf areas inside the chamber (Jahnke and Krewitt, 2002; Pieruschka et al., 2005). Because this internal CO2 supply tends to increase Ci around the edge while reducing the apparent A there (Pieruschka et al., 2006), both measured flux and Ci(m) can be affected. The effect is detectable in homobaric leaves but not heterobaric leaves with negligible lateral diffusion (Pieruschka et al., 2005, 2006). As with leakage through the gasket, the larger chamber/cup helps to avoid the intercellular leakage by decreasing the edge-to-area ratio of the enclosed leaf part. Implications for the photosynthesis model parameters The impact of the cuticle carries over to the critical parameters in photosynthetic models (Hanson et al., 2016). Because mesophyll conductance (gm) describes assimilatory CO2 drawdown from intercellular spaces to chloroplast (Cc; i.e. gm=A/(Ci–Cc)), overestimation of Ci immediately leads to underestimation of gm. Ci* and day respiration (Rd) can also be affected by the overestimation of Ci as they are commonly estimated from the initial A–Ci slopes under multiple sub-saturating irradiances (Walker and Ort, 2015). Our Fig. 7A clearly shows the slope tilted by the overestimation . Recently, a new method for rapid A–Ci response (RACiR) greatly reduced the measurement time of an A–Ci curve to ~5 min, and succeeded in estimating the same model parameters, namely the maximum rate of Rubisco carboxylation (Vc,max) and electron transport (Jmax), as a standard A–Ci methodology (Stinziano et al., 2017). The technique relies on technological advancements of a new instrument (LI-6800, Li-Cor) that allow high temporal resolution measurements under rapidly changing CO2 concentrations (Stinziano et al., 2017). Whereas this rapid technique opens up the opportunity of high-throughput, large-scale, and on-site A–Ci measurements, the uncertainty of Ci still remains because the method similarly relies on the calculation. The direct measurement may be applicable to the rapid technique because the time required for the Ci(m) to reach equilibrium was as fast as the Ci(c) when CO2 was changed quickly (Figs 4B and 5B). However, the Ci(m) lagged behind the Ci(c) when the stomatal conductance for the cup-attached side would be low (Fig. 6B). For the RACiR featuring the direct measurement, the volume of the closed system ought to be reduced until the time lag is negligible. Conclusions and prospects Overestimation of Ci is a general problem in leaf gas-exchange measurements. Obviously, there should be caution when stomatal closure is stimulated, e.g. under drought and salinity (Flexas et al., 2004). Current techniques cannot distinguish cuticular gas transport from measured gas exchange on stomatous leaf surfaces. This makes the overestimation unpredictable. Assuming cuticle is a perfect barrier to CO2 (i.e. cuticular conductance to CO2 is zero), gwcut can be estimated on the stomatous surface and used for correction (Boyer, 2015b; Tominaga and Kawamitsu, 2015b). However, the correction would be obstructed by the variability among leaves (Fig. 8). We conclude that direct measurements are a better option—especially when stomatal gas transport is restricted (Boyer, 2015a,b; Tominaga and Kawamitsu, 2015a,b; Hanson et al., 2016). Given climate change will likely increase water scarcity worldwide, it is urgent to predict plant response to water deficit that primarily affects photosynthesis (Hanson et al., 2016). To achieve this goal, it is essential to accurately estimate the photosynthetic model parameters in response to stressful environments. However, uncertainties in the calculation of Ci have affected interpretation of gas-exchange measurements (Flexas et al., 2004), thereby preventing scientists from exploring a broad range of species and environmental conditions. The direct measurement may now be a solution. To play safe, Ci calculated from values of gw <100 mmol m−2 s−1 should not be used (Fig. 8A). This range of gw occurs in C3 leaves experiencing ‘severe drought’ that potentially imposes biochemical limitations (Medrano et al., 2002; Flexas et al., 2004). Recently, cuticular gas transport was found to be diminished by turgor loss due to a shrinkage effect (Boyer, 2015a). This may favor a minor cuticle effect on the calculation for dehydrated leaves. In this regard, the drought response of the cuticle needs to be investigated especially in intact leaves. Now that improvement of gas exchange analysis largely depends on the capability of commercial instruments (Stinziano et al., 2017), the built-in system is advantageous (Tominaga and Kawamitsu, 2015a). In addition, it is available to many scientists. The challenge is to match the system with emerging techniques. Supplementary data Supplementary data are available at JXB online. Fig. S1. Measurement of the volume of the closed system. Fig. S2. Leak test in the closed system. Fig. S3. Traces of temperature responses of cuticular transpiration and Ci. Fig. S4. Comparisons of gc with gw. Fig. S5. Plotting of A–Ci curves with our previous system. Acknowledgements Thanks are due Yasuhiko Teruya (Faculty of Engineering, University of the Ryukyus) for kindly fabricating the chamber and cup. Our deepest appreciation goes to Professor John S. Boyer (Division of Plant Sciences, University of Missouri) for his insightful comments and warm encouragement. We would also like to thank three anonymous reviewers for thoughtful comments that greatly broadened the scope of our analysis and discussion. References Ball JT . 1987 . Calculations related to gas exchange . In: Zeiger E, Farquhar GD, Cowan IR, eds. Stomatal function . Palo Alto : Stanford University Press , 445 – 476 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Boyer JS . 2015a. Turgor and the transport of CO2 and water across the cuticle (epidermis) of leaves . Journal of Experimental Botany 66 , 2625 – 2633 . Google Scholar Crossref Search ADS PubMed WorldCat Boyer JS . 2015b. Impact of cuticle on calculations of the CO2 concentration inside leaves . Planta 242 , 1405 – 1412 . Google Scholar Crossref Search ADS PubMed WorldCat Boyer JS , Kawamitsu Y. 2011 . Photosynthesis gas exchange system with internal CO2 directly measured . Environment Control in Biology 49 , 193 – 207 . Google Scholar Crossref Search ADS WorldCat Boyer JS , Wong SC, Farquhar GD. 1997 . CO2 and water vapor exchange across leaf cuticle (epidermis) at various water potentials . Plant Physiology 114 , 185 – 191 . Google Scholar Crossref Search ADS PubMed WorldCat Brodribb TJ , Feild TS, Jordan GJ. 2007 . Leaf maximum photosynthetic rate and venation are linked by hydraulics . Plant Physiology 144 , 1890 – 1898 . Google Scholar Crossref Search ADS PubMed WorldCat Daley PF , Raschke K, Ball JT, Berry JA. 1989 . Topography of photosynthetic activity of leaves obtained from video images of chlorophyll fluorescence . Plant Physiology 90 , 1233 – 1238 . Google Scholar Crossref Search ADS PubMed WorldCat Downton WJS , Loveys BR, Grant WJR. 1988 . Stomatal closure fully accounts for the inhibition of photosynthesis by abscisic acid . New Phytologist 108 , 263 – 266 . Google Scholar Crossref Search ADS WorldCat Dubbe DR , Farquhar GD, Raschke K. 1978 . Effect of abscisic acid on the gain of the feedback loop involving carbon dioxide and stomata . Plant Physiology 62 , 413 – 417 . Google Scholar Crossref Search ADS PubMed WorldCat Evans JR , Kaldenhoff R, Genty B, Terashima I. 2009 . Resistances along the CO2 diffusion pathway inside leaves . Journal of Experimental Botany 60 , 2235 – 2248 . Google Scholar Crossref Search ADS PubMed WorldCat Farquhar GD , von Caemmerer S, Berry JA. 1980 . A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species . Planta 149 , 78 – 90 . Google Scholar Crossref Search ADS PubMed WorldCat Fischer E , Raschke K, Stitt M. 1986 . Effects of abscisic acid on photosynthesis in whole leaves: changes in CO2 assimilation, levels of carbon-reduction-cycle intermediates, and activity of ribulose-1,5-bisphosphate carboxylase . Planta 169 , 536 – 545 . Google Scholar Crossref Search ADS PubMed WorldCat Flexas J , Bota J, Loreto F, Cornic G, Sharkey TD. 2004 . Diffusive and metabolic limitations to photosynthesis under drought and salinity in C3 plants . Plant Biology 6 , 269 – 279 . Google Scholar Crossref Search ADS PubMed WorldCat Flexas J , Díaz-Espejo A, Berry JA, Cifre J, Galmés J, Kaldenhoff R, Medrano H, Ribas-Carbó M. 2007 . Analysis of leakage in IRGA’s leaf chambers of open gas exchange systems: quantification and its effects in photosynthesis parameterization . Journal of Experimental Botany 58 , 1533 – 1543 . Google Scholar Crossref Search ADS PubMed WorldCat Gunasekera D , Berkowitz GA. 1992 . Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon . Plant Physiology 98 , 660 – 665 . Google Scholar Crossref Search ADS PubMed WorldCat Hanson DT , Stutz SS, Boyer JS. 2016 . Why small fluxes matter: the case and approaches for improving measurements of photosynthesis and (photo)respiration . Journal of Experimental Botany 67 , 3027 – 3039 . Google Scholar Crossref Search ADS PubMed WorldCat Jahnke S , Krewitt M. 2002 . Atmospheric CO2 concentration may directly affect leaf respiration measurement in tobacco, but not respiration itself . Plant, Cell & Environment 25 , 641 – 651 . Google Scholar Crossref Search ADS WorldCat Jarvis PG . 1971 . The estimation of resistances to carbon dioxide transfer . In: Sestak Z, Catsky J, Jarvis PG, eds. Plant photosynthetic production: Manual of methods . The Hague : Junk , 566 – 631 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Kerstiens G . 1996 . Cuticular water permeability and its physiological significance . Journal of Experimental Botany 47 , 1813 – 1832 . Google Scholar Crossref Search ADS WorldCat Massman WJ . 1998 . A review of the molecular diffusivities of H2O, CO2, CH4, CO, O3, SO2, NH3, N2O, NO, and NO2 in air, O2 and N2 near STP . Atmospheric Environment 32 , 1111 – 1127 . Google Scholar Crossref Search ADS WorldCat Medrano H , Escalona JM, Bota J, Gulías J, Flexas J. 2002 . Regulation of photosynthesis of C3 plants in response to progressive drought: stomatal conductance as a reference parameter . Annals of Botany 89 Spec No , 895 – 905 . Google Scholar Crossref Search ADS PubMed WorldCat Meyer S , 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 Physiology 116 , 947 – 957 . Google Scholar Crossref Search ADS PubMed WorldCat Moss DN , Rawlins SL. 1963 . Concentration of carbon dioxide inside leaves . Nature 197 , 1320 – 1321 . Google Scholar Crossref Search ADS WorldCat Mott KA . 1995 . Effects of patchy stomatal closure on gas exchange measurements following abscisic acid treatment . Plant, Cell & Environment 18 , 1291 – 1300 . Google Scholar Crossref Search ADS WorldCat Mott KA , O’leary JW. 1984 . Stomatal behavior and CO2 exchange characteristics in amphistomatous leaves . Plant Physiology 74 , 47 – 51 . Google Scholar Crossref Search ADS PubMed WorldCat Mott KA , Takemoto JY. 1989 . Syringomycin, a bacterial phytotoxin, closes stomata . Plant Physiology 90 , 1435 – 1439 . Google Scholar Crossref Search ADS PubMed WorldCat Nonami H , Schulze ED. 1989 . Cell water potential, osmotic potential, and turgor in the epidermis and mesophyll of transpiring leaves . Planta 177 , 35 – 46 . Google Scholar Crossref Search ADS PubMed WorldCat Parkhurst DF , Wong SC, Farquhar GD, Cowan IR. 1988 . Gradients of intercellular CO2 levels across the leaf mesophyll . Plant Physiology 86 , 1032 – 1037 . Google Scholar Crossref Search ADS PubMed WorldCat Pieruschka R , Schurr U, Jahnke S. 2005 . Lateral gas diffusion inside leaves . Journal of Experimental Botany 56 , 857 – 864 . Google Scholar Crossref Search ADS PubMed WorldCat Pieruschka R , Schurr U, Jensen M, Wolff WF, Jahnke S. 2006 . Lateral diffusion of CO2 from shaded to illuminated leaf parts affects photosynthesis inside homobaric leaves . New Phytologist 56 , 857 – 864 . Google Scholar OpenURL Placeholder Text WorldCat Raschke K , Hedrich R. 1985 . Simultaneous and independent effects of abscisic acid on stomata and the photosynthetic apparatus in whole leaves . Planta 163 , 105 – 118 . Google Scholar Crossref Search ADS PubMed WorldCat Riederer M , Schreiber L. 2001 . Protecting against water loss: analysis of the barrier properties of plant cuticles . Journal of Experimental Botany 52 , 2023 – 2032 . Google Scholar Crossref Search ADS PubMed WorldCat Rodeghiero M , Niinemets U, 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 & Environment 30 , 1006 – 1022 . Google Scholar Crossref Search ADS PubMed WorldCat Šantrůček J , Šimáňová E, Karbulková J, Šimková M, Schreiber L. 2004 . A new technique for measurement of water permeability of stomatous cuticular membranes isolated from Hedera helix leaves . Journal of Experimental Botany 55 , 1411 – 1422 . Google Scholar Crossref Search ADS PubMed WorldCat Sharkey TD , Imai K, Farquhar GD, Cowan IR. 1982 . A direct confirmation of the standard method of estimating intercellular partial pressure of CO2 . Plant Physiology 69 , 657 – 659 . Google Scholar Crossref Search ADS PubMed WorldCat Stinziano JR , Morgan PB, Lynch DJ, Saathoff AJ, McDermitt DK, Hanson DT. 2017 . The rapid A–Ci response: photosynthesis in the phenomic era . Plant, Cell & Environment 40 , 1256 – 1262 . Google Scholar Crossref Search ADS PubMed WorldCat Terashima I . 1992 . Anatomy of non-uniform leaf photosynthesis . Photosynthesis Research 31 , 195 – 212 . Google Scholar Crossref Search ADS PubMed WorldCat Terashima I , Wong SC, Osmond CB, Farquhar GD. 1988 . Characterization of non-uniform photosynthesis induced by abscisic acid in leaves having different mesophyll anatomies . Plant and Cell Physiology 29 , 385 – 394 . Google Scholar OpenURL Placeholder Text WorldCat Tominaga J , Kawamitsu Y. 2015a. Tracing photosynthetic response curves with internal CO2 measured directly . Environmental Control in Biology 53 , 27 – 34 . Google Scholar Crossref Search ADS WorldCat Tominaga J , Kawamitsu Y. 2015b. Cuticle affects calculations of internal CO2 in leaves closing their stomata . Plant & Cell Physiology 56 , 1900 – 1908 . Google Scholar Crossref Search ADS PubMed WorldCat von Caemmerer S , Farquhar GD. 1981 . Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves . Planta 153 , 376 – 387 . Google Scholar Crossref Search ADS PubMed WorldCat Walker BJ , Ort DR. 2015 . Improved method for measuring the apparent CO2 photocompensation point resolves the impact of multiple internal conductances to CO2 to net gas exchange . Plant, Cell & Environment 38 , 2462 – 2474 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology.
TI - Direct measurement of intercellular CO2 concentration in a gas-exchange system resolves overestimation using the standard method
JF - Journal of Experimental Botany
DO - 10.1093/jxb/ery044
DA - 2018-04-09
UR - https://www.deepdyve.com/lp/oxford-university-press/direct-measurement-of-intercellular-co2-concentration-in-a-gas-GyUvXyNHwU
SP - 1981
EP - 1991
VL - 69
IS - 8
DP - DeepDyve
ER -