JXB Advance Access originally published online on March 5, 2007
Journal of Experimental Botany 2007 58(6):1533-1543; doi:10.1093/jxb/erm027
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RESEARCH PAPER |
Analysis of leakage in IRGA's leaf chambers of open gas exchange systems: quantification and its effects in photosynthesis parameterization
1Laboratori de Fisiologia Vegetal, Grup de Recerca en Biologia de les Plantes en Condicions Mediterrànies, Universitat de les Illes Balears, Carretera de Valldemossa Km 7.5, 07122 Palma de Mallorca, Balears, Spain
2Instituto de Recursos Naturales y Agrobiología, CSIC, Apartado 1052, 41080 Sevilla, Spain
3Carnegie Institution of Washington, Department of Global Change Biology, 260 Panama Street, Stanford, CA 94305, USA
4Darmstadt University of Technology, Institute of Botany, Applied Plant Sciences. Schnittspahnstrasse 10, D-64287 Darmstadt, Germany
* To whom correspondence should be addressed. E-mail: jaume.flexas{at}uib.es
Received 8 December 2006; Revised 22 January 2007 Accepted 23 January 2007
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Abstract |
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The measurement of the response of net photosynthesis to leaf internal CO2 (i.e. A–Ci curves) is widely used for ecophysiological studies. Most studies did not consider CO2 exchange between the chamber and the surrounding air, especially at the two extremes of A–Ci curves, where large CO2 gradients are created, leading to erroneous estimations of A and Ci. A quantitative analysis of CO2 leakage in the chamber of a portable open gas exchange system (Li-6400, LI-COR Inc., NE, USA) was performed. In an empty chamber, the measured CO2 leakage was similar to that calculated using the manufacturer's equations. However, in the presence of a photosynthetically inactive leaf, the magnitude of leakage was substantially decreased, although still significant. These results, together with the analysis of the effects of chamber size, tightness, flow rate, and gasket material, suggest that the leakage is larger at the interface between the gaskets than through the gaskets. This differential leakage rate affects the parameterization by photosynthesis models. The magnitude of these errors was assessed in tobacco plants. The results showed that leakage results in a 10% overestimation of the leaf maximum capacity for carboxylation (Vc,max) and a 40% overestimation of day respiration (Rl). Using the manufacturer's equations resulted in larger, non-realistic corrections of the true values. The photosynthetic response to CO2 concentrations at the chloroplast (i.e. A–Cc curves) was significantly less affected by leakage than A–Ci curves. Therefore, photosynthetic parameterization can be improved by: (i) correcting A and Ci values for chamber leakage estimated using a photosynthetically inactive leaf; and (ii) using A–Cc instead of A–Ci curves.
Key words: Gas exchange, leakage, measurement errors, photosynthesis parameterization
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and DiscussionReferences |
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Since the introduction of the photosynthesis model by Farquhar et al. (1980), gas exchange measurements and, particularly, the response of net photosynthesis (A) to substomatal CO2 concentration (Ci), i.e. the A–Ci curve, have been widely used to parameterize leaf photosynthesis. The analysis of A–Ci curves allows the determination of rates of carboxylation, ribulose bisphosphate (RuBP) regeneration, or triose phosphate export out of the chloroplast, as well as quantification of the maximum carboxylation capacity (Vc,max_Ci), the maximum capacity for electron transport rate (Jmax_Ci), and the velocity for triose phosphate utilization (VTPU_Ci). These analyses have proven to be useful to develop prediction models of CO2 assimilation for crops (Díaz-Espejo et al., 2006) and natural vegetation (Xu and Baldocchi, 2003), to help predict the effects of climate change on photosynthesis (Sabaté et al., 2002), as a solid physiological basis for scaling up from leaf to whole plant and/or ecosystem carbon assimilation models (Woodward et al., 1995), and to assess the influence of several stresses, such as drought (Wilson et al., 2000), salinity (Centritto et al., 2003), extreme temperatures (Zhou et al., 2004), light constraints (Schultz, 2003), nutrient deficiency (Warren, 2004), or biotic stress (Sampol et al., 2003) on the photosynthetic capacity.
The importance and versatility of A–Ci curves for ecophysiological studies have raised debates on the technical aspects of their performance together with the technical limitations presented by the commercial instruments developed for their analysis (Long and Hällgren, 1993; Long and Bernacchi, 2003). Moreover, the introduction of combined gas exchange with isotope discrimination and/or with chlorophyll fluorescence analysis has allowed the estimation of leaf mesophyll conductance to CO2 (gm) and the chloroplastic CO2 concentration (Cc), which often differs from Ci as much as this differs from the atmospheric (Ca) CO2 concentration (Evans et al., 1986; Harley et al., 1992; Loreto et al., 1992). Because the model of Farquhar et al. (1980) is based on the assumption that the A–Ci curve can be mostly assimilated to Michaelis–Menten kinetics (i.e. as A being the velocity of an enzyme-mediated reaction and Ci being the substrate concentration at the site of the enzyme), the fact that Ci and Cc substantially differ has pointed to the convenience of taking into account gm in the parameterization of A–Ci curves (Bernacchi et al., 2002; Ethier and Livingston, 2004) or to convert them to A–Cc curves prior to assessing actual Vc_max and Jmax (Flexas et al., 2002, 2004; Terashima and Ono, 2002; Manter and Kerrigan, 2004; Warren et al., 2004; Galmés et al. 2006).
Several limitations of A–Ci curve analysis have been described (Long and Bernacchi, 2003; Galmés et al., 2006). For instance, under some conditions, water stress or feeding abscisic acid induces a heterogeneous closure of stomata (patchiness), which invalidates the estimations of Ci and, thus, the interpretation of A–Ci curves (Terashima, 1992; Buckley et al., 1997). Moreover, when stomatal conductance is very low, the influence of cuticular conductance may impair the estimation of Ci using the typical procedures (Boyer et al., 1997; Flexas et al., 2002). In addition to these limitations, inherent to the stomatal behaviour and the basic formulation of photosynthesis models, other limitations related to the design of the most commercially available gas exchange systems have also been described. For instance, because leaf chambers usually enclose a small leaf surface (2–6 cm2) surrounded by gaskets of a certain width, the illuminated photosynthesizing leaf area is always surrounded by an area darkened by gaskets, which respires and thus affects the measurement of the photosynthetic flux. The effects of this so-called ‘edge’ effect on respiration and photosynthesis rates have been quantified in detail (Jahnke and Krewitt, 2002; Pons and Welschen, 2002). Also, significant errors in the estimation of stomatal conductance, and subsequently in Ci, have been associated with errors in the measurement of leaf temperature (Tyree and Wilmot, 1990; Verhoef, 1997). Stomatal conductance can be overestimated by 20–60%, especially when gs values are low, as under stress conditions.
In addition, it has long been known but practically ignored that some CO2 could escape from or enter the chamber through the leaf gaskets (Long and Hällgren, 1993). This is especially important at the two ends of A–Ci curves when large CO2 gradients between the chamber and the surrounding air are created. Many recent studies are still using A–Ci curve analysis without taking the possible effects of leaks into account (Centritto et al., 2003; Bota et al., 2004; Warren et al., 2004; Zhou et al., 2004). Others, as recommended by some manufacturers, correct net photosynthesis values after measuring the apparent photosynthetic fluxes in the absence of a leaf, i.e. with an empty chamber (Bernacchi et al., 2001, 2002; Manter and Kerrigan, 2004; Li-COR, 2005). However, to the best of our knowledge, no detailed analyses of the occurrence and magnitude of gasket leakage under different conditions has been published. The effects of gasket leakage on photosynthesis parameterization using the Farquhar et al. (1980) model are largely unknown, as are its possible effects on A–Cc curves. On the other hand, it has been recognized that, when a leaf is placed in the chamber, additional leaks may occur that cannot be quantified by measuring fluxes on an empty chamber (Long and Bernacchi, 2003). Therefore, the objectives of the present study are: (i) to quantify the magnitude of leakage in the IRGA's chambers under different conditions; (ii) to compare the magnitude of these leaks with an empty chamber or with a chamber filled with leaves; and (iii) to quantify the effects of such leaks on the parameterization of both A–Ci and A–Cc curves performed in transgenic tobacco lines differing in photosynthesis.
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Materials and methods |
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Plant material
Tobacco (Nicotiana tabacum L.), grapevine rootstock (R-110, Vitis berlandierixrupestris), and cucumber (Cucumis sativus) plants were used for the experiments. Four different lines of transformed tobacco plants differing in the expression of aquaporin NtAQP1, as described by Siefritz et al. (2002) and Uehlein et al. (2003), were used. These plants were chosen because, having an identical leaf structure, they constitutively show large differences in their A–Ci response curve (Flexas et al., 2006). Because leaf structure and morphology may alter the magnitude of leaks (Long and Bernacchi, 2003), these plants are an ideal material allowing an analysis of the leakage effect using a single kind of leaf structure but at different photosynthetic capacities and without inducing any stress, which may result in patchy stomata closure and preclude a correct analysis of the A–Ci curves (Terashima, 1992; Buckley et al., 1997).
Antisense (AS) and overexpressing plants (O) were obtained from different lines: var. Samsun for AS and Hö 20.20 for O. Non-transformed plants of each line were used as controls (CAS and CO). AS lines were constitutive, whereas in O the NtAQP1 coding region was under the control of a tetracycline-inducible promoter (Uehlein et al., 2003). The plants were grown as described (Flexas et al., 2006).
Gas exchange and chlorophyll fluorescence measurements
Leaf gas exchange was determined simultaneously with measurements of chlorophyll fluorescence using the open gas exchange system Li-6400 (LI-COR Inc., Lincoln, NE, USA) with an integrated fluorescence chamber head (Li-6400-40; LI-COR Inc.). Unless stated, white gaskets were used in the leaf chamber. Measurements were made on the youngest fully expanded leaf. The actual photochemical efficiency of photosystem II (
PSII) was determined by measuring steady-state fluorescence (Fs) and maximum fluorescence during a light-saturating pulse of 
8000 µmol m–2 s–1 (F'm) following the procedures of Genty et al. (1989):
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The electron transport rate (J) was then calculated as:
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where PPFD is the photosynthetic photon flux density and 
is a term which includes the product of leaf absorptance and the partitioning of absorbed quanta between photosystems I and II. was previously determined as the slope of the relationship between PSII and CO2 obtained by varying light intensity under non-photorespiratory conditions in an atmosphere containing <1% O2 (Valentini et al., 1995).
In light-adapted leaves, photosynthesis was induced with saturating light (1000 µmol m–2 s–1) and 400 µmol mol–1 CO2 surrounding the leaf (Ca) with 15% blue light to maximize stomatal aperture. Leaf temperature was maintained at 25 °C, and leaf-to-air vapour pressure deficit was kept between 1 kPa and 2 kPa in all measurements. CO2 response curves were performed at steady state, at least 30 min after clamping the leaf. Six CO2 response curves were obtained for each plant line. Gas exchange and chlorophyll fluorescence were first measured at 400 µmol mol–1, then Ca was increased stepwise up to 1800 µmol mol–1 and returned to its original value, followed by a stepwise decrease down to 0 µmol mol–1. A was measured at 12 different Ca values for each curve.
The chloroplastic CO2 concentration (Cc) was estimated from combined gas exchange and chlorophyll fluorescence according to Epron et al. (1995) as:
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*) of 42.75 µmol mol–1, was taken after Bernacchi et al. (2001). The estimated Cc values were used to convert A–Ci curves into A–Cc curves (Terashima and Ono, 2002; Manter and Kerrigan, 2004). From A–Ci curves, the following photosynthetic parameters were calculated according to Long and Bernacchi (2003): mitochondrial respiration in the light (Rl), maximum carboxylation capacity (Vc,max_Ci), maximum capacity for electron transport rate (Jmax_Ci), and the velocity for triose phosphate utilization (VTPU_Ci). From A–Cc curves, only the maximum carboxylation capacity (Vc,max_Cc) and the maximum capacity for electron transport rate (Jmax_Cc) were calculable (Manter and Kerrigan, 2004). The temperature dependence of kinetic parameters of Rubisco for A–Cc curves was calculated following Bernacchi et al. (2002).
Leakage analysis
In order to establish the magnitude of gasket leakage, CO2 response curves using an empty chamber were run under different conditions. These curves were performed using two different chamber sizes, a 6 cm2 (6400-02B) and a 2 cm2 (6400-40) chamber. Additionally, two types of chamber gaskets, the white (spare part number 6400-30) and black (spare part number 6400-33) gaskets, were compared. Finally, different tightness of the chamber closure and different flow rates were compared.
Several methods attempting to minimize the magnitude of leakage were tested: (i) painting the entire external and internal surfaces of the gaskets with nail varnish; (ii) enclosing the entire chamber head in a plastic bag; and (iii) surrounding the chamber gaskets with a perforated tube ring connected to the exhaust of a second Li-6400 running an identical CO2 response curve in parallel.
Leakage of an empty chamber and of a chamber filled with an inert tobacco leaf was compared. To avoid large changes in leaf structure, leaves were thermally killed by immersion in boiling water until no variable chlorophyll fluorescence was detectable (typically a few minutes after boiling was started) measured using a PAM-2000 (Walz, Effeltrich, Germany), which was taken as evidence for total photosynthesis impairment and leaf death (Schreiber et al., 1998). While there was some unavoidable turgor loss in the heated leaf, it was obviously not as much as in the dried leaf used by Long and Bernacchi (2003). The general aspect and thickness of the boiled leaf resembled more those of a living leaf rather than of a dried leaf or a piece of paper.
The actual A values of living leaves were corrected by simple subtraction of the relationship between Ca and the ‘apparent’ photosynthesis of either an empty chamber or a chamber filled with a dead leaf. Ci and Cc values were then recalculated using the manufacturer's formulae.
Statistical analysis
One-way analysis of variance (ANOVA) was applied to assess the differences for each parameter between each separate control and its derivate line. Differences among means were established using a Duncan test (P <0.1). The data were analysed applying the SPSS 10.0 program for Windows.
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Results and Discussion |
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Magnitude of leakage through the gaskets of an empty chamber under different conditions
The magnitude of CO2 leakage in the IRGA chamber was determined from several A–Ci curves with an empty chamber. Linear relationships between ‘apparent’ net photosynthesis and Ca were obtained (Fig. 1A). Applying the manufacturer's equation to determine the diffusion coefficient k resulted in a k of about 0.44, which is very close to the value of 0.46 provided by the manufacturer (Li-COR, 2005). However, contrary to the manufacturer's suggestions (Li-COR, 2005), there were no differences between the round 2 cm2 chamber and the rectangular 6 cm2 chamber, as both yielded similar relationships for a given set-up (data not shown). Changes in flow rate had a relatively minor effect on the magnitude of leakage (Fig. 1B). Also, in contrast to the manufacturer's suggestions that leakage is detectable only at low flow rates (Li-COR, 2005), leakage was measurable at all flow rates, including a high flow rate of 700 µmol s–1.
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The effect of tightness of the chamber on the leakage was tested by performing A–Ci curves in an empty chamber at four different levels of tightness (initial, 1/4, 3/4, and a full turn of the nut). The tightness of closure did not significantly affect the magnitude of leakage (Fig. 1C). Therefore, reducing the gasket surface exposed to the interface between the inside and outside of the chamber did not significantly affect leakage. The magnitude of leakage through two different types of gaskets provided by LI-COR (spare part numbers 6400-30 and 6400-33) was also tested (Fig. 1D). Although black neoprene gaskets have a lower diffusivity to CO2 than the white gaskets (Li-COR, 2005), the magnitude of leakage was much larger when using the former (Fig. 1D). These results strongly suggest that most chamber leakage occurs between the two sealing gaskets and not through the gasket itself. Some researchers use grease to improve the seal between the leaf and the gasket which, in view of the results, may be a good way to reduce leakage.
Attempts to eliminate or minimize leakage
Several methods to avoid or reduce leakage were tested. These methods included (i) painting the entire external and internal surfaces of the gaskets with nail varnish; (ii) enclosing the entire chamber head in a plastic bag; and (iii) surrounding the chamber gaskets with a tube ring supplying external air to the vicinity of the gaskets with a CO2 concentration identical to the internal one. Painting the surfaces of gaskets with nail varnish—which is known to be largely impermeable to air—did not modify the magnitude of the leakage (data not shown), supporting the idea that most leakage occurs between the two gaskets. The effects of surrounding the gaskets with a CO2 flushing ring, or enclosing, in addition, the chamber in a plastic bag, are shown in Fig. 2. These manipulations contributed to reduce the magnitude of the leaks, so that the maximum reduction was achieved when combining chamber enclosure in a plastic bag with CO2 flushing surrounding the gaskets (Fig. 2). Even so, a large part of the total leakage (80%) remained even when taking these precautions, which suggests that these methods do not fully eliminate leaks. There could be several reasons for this. First, the typical time course of an A–Ci curve (i.e. a few minutes between each change in CO2 concentration), as used here, may not be sufficient to allow total equilibration between the CO2 concentrations outside and inside the chamber, particularly when a large bag (i.e. a large air volume) is used. Secondly, air pressure inside the chamber is always a bit higher than that outside to avoid CO2 entry, and therefore some leakage towards the outside may be unavoidable (Li-COR, 2005; Jahnke and Pieruschka, 2006). To achieve a perfect seal is quite difficult and would be even more difficult when measuring a living attached leaf. On the other hand, CO2 flushing around the gaskets also seems insufficient, not to mention the fact that the option of having the use of two IRGAs at the same time is not available to most researchers. Therefore, the proposed methods to minimize IRGA's chamber leakage do not seem a promising tool for the correct measurement of A–Ci curves; therefore, it must be concluded that it would be necessary to estimate them correctly and use such estimates to correct A–Ci data.
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Comparison of leakage from empty chambers and chambers filled with dead leaves
Because the magnitude of leakage was seemingly important, and the attempts to minimize it largely failed, it may seem that the most convenient way of performing A–Ci studies would be to quantify the magnitude of leakage in each condition, and then correct the resulting A–Ci curves accordingly, as previously suggested (Bernacchi et al., 2002; Long and Bernacchi, 2003).
However, when a leaf is placed in the chamber, the extent of leakage may differ from that in an empty chamber particularly if, as the above data suggest, most leakage occurs at the interface between the two sealing gaskets, where leaves are placed. Therefore, it must be necessary to obtain photosynthetically inert leaves and insert them in the chamber to quantify the leaks that may occur when measuring a living leaf. Long and Bernacchi (2003) recommend the use of a dead leaf obtained by rapidly drying a live specimen. However, drying may result in loss of some 80% mass and, thus, in important changes of leaf thickness. The overall structure of a leaf killed by this method may not be very comparable with that of an intact living leaf, therefore interfering with the interpretation of leakage. To avoid large changes in leaf structure, it was decided to kill a leaf thermally by submersing it in boiling water. Using this procedure, less water was lost from the leaf as compared with if it was dried, and the appearance of the dead leaf was much closer to that of a living one. The leaf was submersed in boiling water until no variable chlorophyll fluorescence was detectable (typically a few minutes after initiating boiling), which was taken as evidence for total photosynthesis impairment and leaf death.
Using this procedure, the magnitude of leakage in photosynthetically inactive leaves of V. berlandierixrupestris, C. sativus, and N. tabacum was analysed (Fig. 3). In contast to what was previously suggested (Long and Bernacchi, 2003), the magnitude of leakage was much lower when a leaf was inside the chamber than when using an empty chamber, regardless of the species analysed. These results largely reinforce the idea that leakage occurs between the two gaskets. If so, a leaf in the middle of the gaskets would increase the diffusion resistance of the system due to surface rugosity, while differences in leak rates between species (Fig. 3) could perhaps be interpreted as differences in leaf rugosity. Although the magnitude of leakage was reduced in the presence of a dead leaf, it was still significant. Moreover, the fact that the magnitude of leaks differs greatly among different leaves suggests that, in order to improve the accuracy and significance of A–Ci analysis, it would be important to estimate the extent of leakage for any particular species under any given condition, i.e. for any particular experimental conditions. The fact that leakage was larger in a heterobaric species such as Vitis than in homobaric tobacco suggests that leakage between the leaf and gasket surfaces is much larger than possible leakage through the leaf mesophyll (Jahnke and Krewitt, 2002; Pieruschka et al., 2005).
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Due to large differences in the magnitude of leakage in empty chambers as compared with chambers filled with a photosynthetically inactive leaf, using either basis for correction of true A and Ci values may yield different results. This is illustrated in Fig. 4 for a tobacco leaf. An A–Ci curve was performed, and the values were either not corrected, corrected using the manufacturer's diffusion coefficient k of 0.46, corrected using ‘apparent photosynthesis’ of the empty chamber, or corrected using ‘apparent photosynthesis’ of a boiled leaf. Clearly, all the corrections have some effect on A and Ci values. The corrections either using ‘apparent photosynthesis’ of the empty chamber or directly using the manufacturer's diffusion coefficient yielded similar results. The corrected photosynthesis differed greatly from uncorrected values, particularly at high CO2 concentrations (Fig. 4A). However, photosynthesis corrected using ‘apparent photosynthesis’ of the boiled leaf was not so different from uncorrected rates. Hence, using the manufacturer's corrections may suggest that leakage results in errors of ±10% in the estimations of A, depending on Ci, but using a photosynthetically inactive leaf reveals that the errors are only of approximately ±2% (Fig. 4B).
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Leakage effects on photosynthesis parameterization from A–Ci curves
Although the magnitude of leakage was lower when clamping a leaf than when using an empty closed chamber, it was still significant and differed among species. Therefore, an attempt was made to investigate the effects of such leakage on photosynthesis parameterization using the model of Farquhar et al. (1980). Transformed tobacco plants differing in the expression of aquaporin NtAQP1 were chosen for the analysis because, having an identical leaf structure, they constitutively show large differences in their net assimilation rates and A–Ci response curve (Flexas et al., 2006). The A–Ci curves for the four tobacco lines were either not corrected for leakage, corrected for the leakage estimated using the empty chamber, or corrected for the leakage estimated using a photosynthetically inactive tobacco leaf (Fig. 5). Although differences may be apparent but not large, significant differences appeared in some parameters (Table 1) when photosynthesis was parameterized following Long and Bernacchi (2003).
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Day respiration (Rl) was the most strongly affected (by up to 40%), suggesting that it may be difficult to estimate this parameter accurately even if a proper estimate of leaks is carried out. It has been already suggested that Rl estimates using parameterization of the Farquhar et al. (1980) model of photosynthesis are not reliable, and many authors prefer other methods to assess Rl (Brooks and Farquhar, 1985). Because other gas exchange methods, such as that described by Brooks and Farquhar (1985), are affected not only by leakage but also by many other technical problems (Galmes et al., 2006), only methods to measure Rl with labelled compounds (e.g. Haupt-Herting et al., 2001; Pinelli and Loreto, 2003) can be much more accurate and error-free in determining the true respiratory out-flux of CO2 in illuminated leaves. The differences in Vc,max_Ci were also significant (10%), which may be considered as an important bias. Leakage generally did not significantly affect estimations of Jmax_Ci or VTPU_Ci (Table 1). Corrections using leaks estimated on an empty chamber, as done in some studies (Bernacchi et al., 2001, 2002; Manter and Kerrigan, 2004), would lead to much larger, unrealistic differences in all these parameters (Table 1).
It is noticeable that the percentage error due to ignoring the effects of leakage did not depend on the photosynthetic rates. For instance, at ambient CO2 concentration, AN in AS plants was 17 µmol CO2 m–2 s–1, while in O plants it reached 22 µmol CO2 m–2 s–1. However, the percentage error for Vc,max_Ci and Jmax_Ci was identical. This may suggest that a relative comparison of species or treatments would be possible even without taking leakage into account. Nevertheless, the ranges of photosynthesis compared here were relatively small. It is suggested that, for larger differences in photosynthesis (e.g. when photosynthesis is reduced by any severe stress), the percentage error caused by ignoring leaks would differ between treatments, since the same absolute leak rate will represent a much larger percentage error in AN if the latter is largely reduced. On the other hand, when AN–Ci curves are performed to parameterize leaf or canopy models for prediction of photosynthesis (Walcroft et al., 1997; Cai and Dang, 2002; Medlyn et al., 2002a, b; Diaz-Espejo et al., 2006), the accuracy in the estimations of these parameters would be more important.
Leakage effects on photosynthesis parameterization from A–Cc curves
As already discussed (Flexas et al., 2006), A–Ci curves differed greatly from A–Cc curves (Fig. 6). Vc,max_Ci was 20–40% larger than Vc,max_Cc depending on the genotype (Tables 1 and 2). Jmax_Cc was less affected, yet it was 10% larger on a Cc basis for AS plants. There is now ample consensus that photosynthetic parameterization must be done on a Cc rather than on a Ci basis, since they both differ greatly in plants and the former more closely represents the CO2 concentration at the sites of carboxylation (Bernacchi et al., 2002; Ethier and Livingston, 2004; Manter and Kerrigan, 2004; Grassi and Magnani, 2005; Flexas et al., 2006). This may be particularly important under conditions where the ratio Ci/Cc is changing, such as under water or salt stress (Centritto et al., 2003; Flexas et al., 2004).
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Clearly, the effects of leakage on A–Cc curves (Fig. 6) and their parameterization (Table 2) were much lower than for A–Ci curves, and for the present data they were non-significant. This is because leakage affects A and Ci in opposite ways, while it affects A and Cc in the same direction. For instance, in a CAS plant at the lowest end of Ca (i.e. 100 µmol mol–1 air, where the percentage error in A is larger), A without correcting for leaks was 2 µmol CO2 m–2 s–1, while Ci was 80 µmol mol–1 air. Correcting for leaks resulted in a 26% increase in A with a 14% decrease in Ci. In this plant, the linear rate of electron transport was 97 µmol e– m–2 s–1. The estimated Cc without correcting for leaks was 64 µmol mol–1 air, but it increased by 7% when leaks were taken into account. Therefore, since both A and Cc are affected in the same way (although to a somewhat different extent), the effects of leakage on Vc,max_Cc have less impact.
Concluding remarks
CO2 leakage occurs when using small IRGA chambers, such as those of the Li-6400 (LI-COR Inc., Lincoln, NE, USA) and most other modern portable systems, especially when performing A–Ci curves. The present results suggest that most leakage takes place at the interface between the two sealing gaskets of the clamping chamber, and not through the chamber gaskets, as previously suggested. Attempting to equilibrate inside and outside CO2 concentrations has proven difficult. The presence of leaks induces errors in the measurement of both A and Ci, impairing the correct parameterization of photosynthesis following the Farquhar model. However, the magnitude of these errors is considerably reduced when A–Cc curves are used for parameterization. Therefore, if A–Cc curves are obtained, it may not be necessary to estimate the magnitude of leakage for most applications. If increasing accuracy by applying a correction is desired, the simplest method would be to use photosynthetically inert leaves after placing them in boiling water.
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Acknowledgements |
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This work was partly supported by grants AGL2005-06927-CO2-01/AGR, BFI2002-00772, and BFU2005-03102/BFI (Plan Nacional, Spain). MR-C and AD-E were beneficiaries of the Programa Ramón y Cajal (M.E.C.)
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References |
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