Plant, Cell and Environment (2015) 38, 2 417 –2 4 32

doi: 10.1111/pce.12564

Original Article 13

CO2/12CO2 exchange fluxes in a clamp-on leaf cuvette: disentangling artefacts and flux components Xiao Ying Gong, Rudi Schäufele, Wolfgang Feneis & Hans Schnyder

Lehrstuhl für Grünlandlehre, Technische Universität München, 85354, Freising, Germany

ABSTRACT Leaks and isotopic disequilibria represent potential errors and artefacts during combined measurements of gas exchange and carbon isotope discrimination (Δ). This paper presents new protocols to quantify, minimize, and correct such phenomena. We performed experiments with gradients of CO2 concentration (up to ±250 μmol mol−1) and δ13CCO2 (34‰), between a clamp-on leaf cuvette (LI-6400) and surrounding air, to assess (1) leak coefficients for CO2, 12CO2, and 13CO2 with the empty cuvette and with intact leaves of Holcus lanatus (C3) or Sorghum bicolor (C4) in the cuvette; and (2) isotopic disequilibria between net photosynthesis and dark respiration in light. Leak coefficients were virtually identical for 12CO2 and 13CO2, but ∼8 times higher with leaves in the cuvette. Leaks generated errors on Δ up to 6‰ for H. lanatus and 2‰ for S. bicolor in full light; isotopic disequilibria produced similar variation of Δ. Leak errors in Δ in darkness were much larger due to small biological : leak flux ratios. Leak artefacts were fully corrected with leak coefficients determined on the same leaves as Δ measurements. Analysis of isotopic disequilibria enabled partitioning of net photosynthesis and dark respiration, and indicated inhibitions of dark respiration in full light (H. lanatus: 14%, S. bicolor: 58%). Key-words: bundle sheath leakiness; CO2 transfer conductance; diffusive leak; isotopic disequilibrium; isotope ratio mass spectrometry; labelling; Li-Cor LI-6400; mesophyll conductance; photosynthesis; respiration.

INTRODUCTION Measurements of ‘online’ carbon isotope discrimination (Δ) in combination with CO2 and H2O gas exchange provides insight to limitations of discrete steps in the transfer of CO2 from the air to ribulose 1·5-bisphosphate carboxylase/ oxygenase (Rubisco) in both C3 and C4 plants (Evans et al. 1986; Farquhar et al. 1989). For instance, the method allows studies of mesophyll conductance in C3 plants (Evans et al. 1986; Pons et al. 2009; Tholen et al. 2012) and bundle sheath leakiness in C4 plants (Henderson et al. 1992; Cousins et al. 2007; Kromdijk et al. 2010). Moreover, carbon isotope discrimination during dark respiration provides clues on resCorrespondence: R. Schäufele. e-mail: [email protected]; H. Schnyder. e-mail: [email protected] © 2015 John Wiley & Sons Ltd

piratory metabolism and the chemical identity of the internal substrates used in dark respiration (Tcherkez et al. 2003; Gessler et al. 2009). However, the measurement of online Δ is a formidable task. Errors can occur and can lead to false physiological interpretations. Such artefacts may arise from leaks in the measurement system (Long & Bernacchi 2003; Flexas et al. 2007) or unaccounted isotopic disequilibria between photosynthetic and respiratory 13CO2/12CO2 fluxes (Schnyder et al. 2003). Leaks are potentially a major issue in online Δ measurements with clamp-on leaf cuvettes, as is suggested by net CO2 exchange measurements (Long & Bernacchi 2003; Li-Cor 2008). Two (complementary) procedures have been tested by the manufacturer, Li-Cor Inc., and other scientists to minimize the error on net CO2 exchange measurements. One is using a leak coefficient for CO2 derived from tests with an empty cuvette or with a cuvette with dead leaves in it (Flexas et al. 2007; Rodeghiero et al. 2007; Li-Cor 2008; Wang et al. 2012). However, the usefulness of the correction has been questioned, as it yielded corrections with high uncertainty or non-realistic results (Flexas et al. 2007; Rodeghiero et al. 2007). As of now, leak coefficients for 12CO2 and 13CO2, for corrections of leak effects on Δ measurements, have not been determined. In addition, there are presently no protocols to estimate leak coefficients for total CO2, 12CO2, and 13 CO2 with intact leaves present in the cuvette during the determination. This is unfortunate, as leaf geometry (or other factors) may influence leak rates (Jahnke & Krewitt 2002; Rodeghiero et al. 2007). Probably the most useful and effective recommendation is to minimize the leak by reducing the gradient of CO2 concentration and isotope composition between the cuvette and surrounding air (e.g. by placing the instrument in a plastic bag flushed with the exhaust air of the instrument; Flexas et al. 2007; Rodeghiero et al. 2007). Isotopic disequilibrium artefacts – that result from unaccounted artificial isotopic disequilibria between CO2 fixation and dark respiration in illuminated leaves – occur when the carbon isotope composition of CO2 used in the online Δ measurements differs from that in the growth environment. Measured ΔA, the Δ during net CO2 exchange in light, is a flux-weighted function of the isotopic composition of the net photosynthetic flux (δP) and the isotopic composition of CO2 released during concurrent dark respiration (δRL). In isotopic equilibrium conditions, with the same CO2 used for plant growth and Δ measurements, δP and δRL differ only by the intrinsic carbon isotope discrimination of dark respiration, e 2417

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X. Y. Gong et al.

(Farquhar et al. 1982). When the carbon isotope composition of the CO2 used for measurements (δcuvette) differs from that in the growth environment (δenviron) of the plant, the difference between δP and δRL is enhanced by the offset between δcuvette and δenviron (Gillon & Griffiths 1997; Tcherkez et al. 2005). These isotopic disequilibria cause artefacts if a simplified Δ model that does not include the fractionation associated with dark respiration is used. To disentangle the contribution of respiration, the rate of respiration in light needs to be known. Interestingly, this enhanced offset between δP and δRL – induced by the momentary change in the isotopic composition of CO2 in the system – can be used to partition net photosynthesis and dark respiration in light, as has been shown in mesocosms (Schnyder et al. 2003; Tcherkez et al. 2010). More than 40 years ago, that principle was pioneered to disentangle gross photosynthesis and (photo)respiration by means of 14CO2/12CO2 mixtures with constant composition (Ludwig & Canvin 1971a,b). The present study aimed at (1) a systematic quantitative evaluation of leakage and isotopic disequilibrium artefacts on Δ estimation using a widely-used clamp-on leaf cuvette gas exchange system; (2) establishing methods to minimize or to correct for leak artefacts on Δ during gas exchange in light (ΔA) and darkness (ΔRD); and (3) developing a procedure to eliminate the effect of isotopic disequilibria (associated with dark respiration in light) on estimates of photosynthetic Δ (ΔP). The work was performed with the LI-6400 (Li-Cor Inc., Lincoln, NE, USA) gas exchange system. Experiments were performed with a system that enabled manipulation and monitoring of both CO2 concentration and δ13CCO2 in the leaf cuvette (LI-6400) and surrounding atmosphere. The paper describes leak tests with the entire system (including the console), elimination of leaks in the console, leak tests with the empty cuvette and with fake leaves, and intact leaves of a C3 (Holcus lanatus L.) and a C4 grass (Sorghum bicolor L.). We estimated leak coefficients for CO2 (KCO2), 12CO2 (K12CO2) and 13CO2 (K12CO2) with the empty cuvette and with a cuvette with intact leaves in it. The leak coefficients allowed for corrections of 12CO2 and 13CO2 leaks (into or out of the leaf chamber) during Δ-measurements with leaves.After leak correction, photosynthetic Δ was estimated from short-term isotopic disequilibria between photosynthetic and respiratory CO2 fluxes.

MATERIALS AND METHODS

an inward leak flux and a negative value to an outward leak. Thus,

NL = A − L s .

(1)

For measurements in the dark, the net CO2 exchange rate (calculated as the negative of Eqns 1–17 in Li-Cor 2008), ND (μmol m−2 s−1), is the sum of dark respiration rate (RD, μmol m−2 s−1) and the leak flux (L, μmol s−1) per unit leaf area (s, m2). Thus,

N D = RD + L s .

(2)

In Eqns 1 and 2, A and RD have positive signs. So, neglect of an inward leak causes an underestimation of A and an overestimation of RD. According to Fick’s first law, CO2 diffuses through the gaskets according to the concentration gradient across the gaskets and the diffusion coefficient (or leak coefficient, KCO2) of the gaskets. A leak coefficient of CO2 can be quantified with an empty leaf cuvette as suggested by the manufacturer (See Fig. 4–12 in Li-Cor 2008):

K ′ CO2 = u (Cout − C in ) (CM − Cout ) ,

(3)

with Cout, Cin and CM the actual (i.e. not humidity corrected) CO2 concentration (μmol mol−1) in the sample cell and reference cell of the analyser, and in the surrounding air, and u the incoming air flow rate (mol s−1). Flexas et al. (2007) and Rodeghiero et al. (2007) assessed leak coefficients with inactive leaves and noted that the leak coefficient was altered when a leaf was present in the cuvette. Here, we propose a new method to quantify the leak coefficient with active, attached leaves placed between the gaskets (KCO2) during measurements of CO2 exchange in the dark. If we represent the leak by KCO2 (CM − Cout), Eqn 2 becomes:

N D = RD + KCO2 (CM − Cout ) s .

(4)

The slope of the linear relationship between ND and (CM − Cout)/s provides the estimate of KCO2 and can be obtained by manipulation of (CM − Cout) and concurrent measurements of ND. The (leak-corrected) estimate of RD is given by the intercept of the relationship between ND and (CM − Cout)/s, or can be obtained from Eqn 4. Similarly, Eqn 1 can be expressed as:

Theory

N L = A − KCO2 (CM − Cout ) s .

Separation of leak and leaf net CO2 exchange fluxes

Again, the (leak-corrected) estimate of A can be obtained from Eqn 5 using KCO2 measured in the dark.

In the case of a non-leak-free leaf cuvette and measurements of CO2 exchange in the light, the net CO2 exchange rate per unit leaf area observed by LI-6400 (Eqns 1–17 in LI-6400 manual; Li-Cor 2008), NL (μmol m−2 s−1), is the sum of net assimilation rate per unit leaf area (A, μmol m−2 s−1) and the leak flux (L, μmol s−1) per unit leaf area (s, m2). For clarification of the direction of the leak, we assign a positive value to

Correction of leak effects on Δ measurements in light and darkness

(5)

The above rationale can be expanded to 13CO2 and 12CO2 flux measurements, to correct leak effects on Δ measurements in light and darkness.Thus, if we apply Eqn 4 to 12CO2 and 13CO2 fluxes in the dark, we obtain:

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2417–2432

13

CO2/12CO2 leak and isotopic disequilibrium artefacts in clamp-on leaf cuvettes

12

N D = 12 RD + K12 CO2 ( 12 CM − 12Cout ) s and

(6)

13

N D = 13 RD + K13CO2 ( 13 CM − 13Cout ) s .

(7)

In all cases, the superscripts 12 and 13 refer to the CO2 concentration or flux of the respective carbon isotope; while no superscript is given, where concentration or flux refers to total CO2. Concentrations of 12CO2 (12C) and 13CO2 (13C) in a given sample (with sample referring to the CO2 measurements at the inlet or outlet of the leaf cuvette, or the mesocosm, as appropriate) is calculated as 12

C sample = C (1 +

13

C sample = C

sample

sample

(1 +

) and sample

(8)

),

(9)

with Ʀsample, the ratio of 13C to 12C (Ʀ) in that CO2 sample. Ʀsample is obtained as sample

= (δ sample + 1)

VPDB

,

(10)

with δsample, the δ13C of the sample and ƦVPDB the ratio of 13C to 12C in the international VPDB standard. ND, used to solve the left sides of Eqns 6 and 7, is measured by LI-6400, and δND is obtained as:

δ ND = (δ inC in dry − δ outCout dry ) (C in dry − Cout dry )

(11)

with subscript ‘dry’ indicating that Cin and Cout measured by LI-6400 were corrected to zero humidity; δin and δout are the δ13C of the air at the inlet and outlet of the leaf cuvette. δNL is calculated in the same way as δND (Eqn 11). Knowing ND and δND, 12ND and 13ND can be calculated; and the ratio of 13 C/12C in RD, ƦRD, is obtained by combining Eqns 6 and 7: RD

=

N D − K13CO 2 (13C M − 13Cout ) s . 12 N D − K12 CO 2 (12C M − 12C out ) s 13

(12)

δRD is then obtained using Eqn 10. ΔRD, 13C discrimination during respiration in the dark, is obtained as:

Δ RD = (δ S − δ RD ) (1 + δ RD ) ,

(13)

with δS the δ13C of the substrate that is used in respiration. For gas exchange measurements in light, Eqn 5 can be applied for 12CO2 and 13CO2 fluxes, thus, 12

N L = 12 A − K12 CO2 ( 12 CM − 12Cout ) s and

(14)

13

N L = 13 A − K13CO2 ( 13 CM − 13Cout ) s .

(15)

Again, 12NL and 13NL are calculated from observed NL and δNL. Thus, 13A (and 12A) can be calculated from knowledge of K13CO2 (K12CO2) and measurements of 13NL (12NL) 13Cin (12Cin), 13 Cout (12Cout), 13CM (12CM) and s. The ratio of 13C/12C in A, ƦA, is obtained as: A

=

13 12

N L + K13CO 2 (13C M − 13Cout ) s N L + K12 CO 2 (12C M − 12Cout ) s

(16)

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δA, the δ13C of net assimilation, is calculated from ƦA using Eqn 10. Discrimination during net assimilation in light (ΔA) is obtained as:

Δ A = (δ out − δ A ) (1 + δ A ) .

(17)

So, ΔA and ΔRD are discriminations corrected for leak effects (but not for isotopic disequilibrium effects, which may affect ΔA, see below).

Partitioning of net photosynthesis and dark respiration in light, and estimation of carbon isotope discrimination during net photosynthesis In this section, we show that the use of CO2 sources with different δ13C can help us to separate the two flux components of the net assimilation rate, leaf net photosynthesis (P, gross photosynthesis minus photorespiration, Gifford 2003) and dark respiration in light, RL; A = P − RL. This flux separation is based on the condition that dark respiration is fed entirely by substrate formed in the growth environment, prior to the gas exchange in the leaf cuvette. Thus, the flux separation effectively exploits the isotopic disequilibrium between the net photosynthetic and respiratory CO2 fluxes, which arises when plants are transferred between environments with different δ13CCO2. The δ13C of the net photosynthetic CO2 flux, δP, corresponds to the flux-weighted balance of δA and δRL, the δ13C of dark respiration in light (Yakir & Wang 1996; Schnyder et al. 2003):

δP =

δ A A + δ RL RL . A + RL

(18)

δP depends on the δ13C of CO2 in the cuvette (δout) and 13C discrimination in net photosynthesis (ΔP), which is independent of the δ13C of the CO2 source (Farquhar et al. 1989):

δ P = (δ out − Δ P ) (1 + Δ P ) .

(19)

During gas exchange measurements with a leaf chamber, δP is directly influenced by the δ13C of CO2 inside the chamber (δout), while δRL reflects the isotopic composition of the respiratory substrate, which has been formed previously in the growth environment. For the flux separation, we used CO2 from a mineral (δ13CCO2 = −5‰) and an organic source (−39‰), and the approximation that δP ≈ δout − ΔP. As ΔP is independent of δout, the difference between δPm and δPo (‘m’ denoting mineral and ‘o’ organic), δPm − δPo, must equal the δ-difference between the two CO2 sources, δout m − δout o. Replacement of δPm and δPo with the right hand expression of Eqn 18, leads to:

δ Am Am + δ RLm RLm δ Ao Ao + δ RLo RLo = δ out m − δ out o. − Am + RLm Ao + RLo

(20)

The isotopic composition of CO2 does not affect the gas exchange rates; therefore, Am = Ao and RLm = RLo. Further,

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2417–2432

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X. Y. Gong et al.

assuming δRLm = δRLo in leaves, which are only briefly exposed to CO2 with altered δ13C, RL can be determined as:

RL = A ((δ Am − δ Ao ) (δ out m − δ out o ) − 1) .

(21)

Knowing RL, and assuming δRL = δRD, Eqns 18 and 19 can be solved to yield estimates of δP and ΔP, which account and correct for the effect of isotopic non-steady-state (i.e. differential ‘labelling’ of P and RL). For effects of uncertainties of δRL (relative to δRD), see Discussion. The above method is developed from a mesocosm-scale 13 C differential labelling approach (Schnyder et al. 2003; Tcherkez et al. 2010), and the basic assumption is similar. If some of the substrate feeding RL is supplied by current carbon fixation in the leaf chamber, then RL (defined by Eqn 21) is the respiration using substrates manufactured in the growth environment. The photorespiratory substrate pool is rapidly turned over by current photosynthesis [half-life of a few minutes (Ogren 1984; Gillon & Griffiths 1997)]; hence the photorespiratory CO2 flux attains a new isotopic steady-state soon after the beginning of gas exchange measurements in light. A mass balance can also be established for A = G − RL − F (G, gross photosynthesis; F, photorespiration), and developed in the same way as Eqn 21 to solve for RL (Supporting Information Appendix S4). Hence, a few minutes after the transfer to the cuvette, the isotopic disequilibrium separates two groups of fluxes: G and F (reflecting the δ13C of CO2 in the cuvette), and RL (reflecting the δ13C of respiratory substrate formed in the prior growth environment).

Gas exchange measurement systems The study employed a mesocosm 13CO2/12CO2 gas exchange facility (Schnyder et al. 2003) and a portable CO2 exchange system with a clamp-on leaf cuvette (LI-6400). The air supplied to the mesocosm and leaf cuvette was mixed from CO2-free, dry air and CO2 of known carbon isotope composition (Schnyder et al. 2003). CO2 concentration [CO2] inside the mesocosm (CM) was monitored with an infrared gas analyser (LI-6262, Li-Cor Inc.). The mesocosm and cuvette systems were coupled to a continuous-flow isotope ratio mass spectrometer, IRMS (Deltaplus Advantage equipped with GasBench II, ThermoFinnigan, Bremen, Germany) via a stainless steel capillary. Sample air was drawn through the capillary with a peristaltic pump and passed through a 0.25 mL sample loop attached to the 8-port Valco valve of the GasBench II. Sample air in the loop was introduced into the IRMS via an open split after passage of a dryer (Nafion) and a GC column (25 m × 0.32 mm Poraplot Q; Chrompack, Middleburg, the Netherlands). After every second sample, a VPDB-gauged CO2 reference gas was injected into the IRMS via the open split. The whole-system precision (SD) of repeated measurements was 0.10‰ (n = 50).

Quantifying the leak with an empty leaf cuvette Experiments were preceded by leak tests with the whole portable leaf gas exchange system, including the console and

clamp-on cuvette. Leaks were localized and then eliminated by exchanging (spare) parts (Supporting Information Appendix S1). After that, the console and the tubing outside of the cuvette of the LI-6400 were leak-free. Leakage in the leaf cuvette of the LI-6400 was assessed by 13CO2/12CO2 gas exchange measurements with the empty cuvette or with a strip of filter paper (0.5 × 4 × 80 mm; Schleicher & Schuell, Munich, Germany) placed between the gaskets to mimic a metabolically inactive leaf. In all the tests, the manufacturer’s black neoprene gaskets were used. These have a lower permeability to CO2 than the white gaskets (Boesgaard et al. 2013). The sensor head of the LI-6400 system was placed in a mesocosm with controlled [CO2] and carbon isotope composition of the CO2. The [CO2] in the mesocosm (CM) and in the leaf cuvette (measured at the outlet of the cuvette, Cout), were controlled independently. Five target-[CO2]-gradients (CM − Cout: −250, −100, 0, +100, +250 μmol mol−1) between leaf cuvette and mesocosm air were created by supplying the cuvette with a constant [CO2] of 250 μmol mol−1 and varying CM between 0 and 500 μmol mol−1. In addition, the cuvette was supplied with CO2 with a δ13C of −5‰, whereas the mesocosm received CO2 with a δ13C of −39‰. Thus, any (net) leakage of CO2 – into or out of the empty cuvette or cuvette with the strip of filter paper – was detected as a difference between the [CO2] in the sample cell (Cout) and that in the reference cell (Cin, CO2 fed into the cuvette) of the LI-6400 leaf cuvette. Leaks of 13CO2 and 12CO2 were detected by the concurrent measurements of δ13C of CO2 in the sample cell (δout) and reference cell (δin). Empty cuvette tests were performed with three air flow rates (u) through the cuvette (60, 200 or 500 μmol s−1), and the same tests were repeated with the strip of filter paper (results are shown in the Supporting Information). All tests were done with the cuvette tightly closed; that is the leaf cuvette was closed and the adjustment knob turned to be tight, then the cuvette was opened and the knob turned a further one half turn, and then closed again, as recommended by the LI-6400 manual. IRGA readings of sample cell and reference cell were carefully checked in the ‘match mode’ of the LI-6400 after measurement conditions were changed. During empty cuvette tests dry air was supplied to the mesocosm and cuvette, thus, H2O diffusive leaks had no effect on the measurements.

Calculation of leak coefficient with empty leaf cuvette Calculation of the CO2 leak (LCO2, pmol s−1) in an empty cuvette was done according to the equations given by LI-COR (LI-COR 2008):

LCO 2 = u (Cout − C in ) = K ′ CO 2 (CM − Cout )

(22)

This equation is similar to Eqn 4, but excludes the biological flux component (RD). LCO2 was plotted against the [CO2] gradient (CM − Cout), and the slope of the linear correlation yielded the leak coefficient of the empty cuvette (K’CO2). Furthermore, L12CO2 was plotted against 12CM − 12Cout, the slope yielding K′12CO2, and L13CO2 was plotted against 13 CM − 13Cout to estimate K′13CO2.

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2417–2432

13

CO2/12CO2 leak and isotopic disequilibrium artefacts in clamp-on leaf cuvettes

Quantifying the leak artefact with intact leaves in the cuvette Effects of different leak scenarios on Δ were tested with a C3 (Holcus lanatus) and a C4 grass (Sorghum bicolor). Plants were grown individually in plastic pots filled with soil, kept in a growth chamber (Conviron E15, Conviron, Winnipeg, Canada) and supplied with water and nutrients as in Lehmeier et al. (2008). Ambient air was supplied to the growth chamber, thus plants were grown in atmospheric CO2 with a δ13C of approx. −10‰. During measurements, the LI-6400 sensor head and plant were held in the same mesocosm as used for the empty cuvette tests, and the control of the air supply to the leaf cuvette and the mesocosm was performed in a similar manner. Gas exchange and the concurrent carbon isotope discrimination in light (ΔNL) and in the dark (ΔND) were measured on the youngest fully expanded leaves using the clamp-on leaf cuvette of the same LI-6400. Under each measurement condition, we waited until a stable gas exchange rate was observed (c. 15–30 min), then the δ13C measurement was triggered and gas exchange data were stored.

same species (in one case only three leaves were measured, C3 first run, Scen-I). Each leaf was measured only once for online Δ in the light (and for no more than 50 min). For the measurements of a leaf, four consecutive records of [CO2] and δ13C in both inlet and outlet air of the leaf cuvette of LI-6400 were performed as repeated measurements on the same leaf. Measurements in light were performed at a photosynthetically active photon flux density of 1600 μmol m−2 s−1. Block temperature of the cuvette was kept at 25 °C. The relative humidity of the mesocom was held near 70%, and the upstream of the inlet of LI-6400 was humidified to a relative humidity of about 30%. The relative humidity of air inside the leaf cuvette was dependent on transpiration rate and air flow rate and varied between 75% ∼ 90% at an air flow rate of 60 μmol s−1 (Supporting Information Appendix S2, Table S2). For each leaf, [CO2] and the δ13C in the incoming (Cin and δin) and outgoing cuvette air (Cout and δout) were measured continuously ([CO2]) or with an offset of 6 min (δ13C). Measured δ13C were corrected considering the non-linearity effect of the IRMS (Elsig & Leuenberger 2010). ΔNL, reporting Δ during net gas exchange in light including the leak effect, was calculated according to equation 10 of Evans et al. (1986):

Experimental scenarios Measurements were done with three scenarios in which the [CO2] gradient and the δ13CO2 gradient between the leaf cuvette and the mesocosm were manipulated (Table 1). In scenario I, the [CO2] gradient and the δ13CO2 gradient between the inside and outside of leaf cuvette was maintained close to nil; in scenario II, the air outside the cuvette was kept close to CO2-free; and in scenario III, a small [CO2] gradient and a strong δ13C gradient was maintained between the inside and outside of the cuvette. The main aims of these different scenarios were to minimize opportunities for leak effects on gas exchange and Δ measurements (Scen-I); to provoke losses of CO2 from the cuvette (Scen-II); and to evaluate bidirectional fluxes of CO2 with different δ13CCO2 through leaks in the cuvette (Scen-III).

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Δ NL =

ξ (δ out − δ in ) . 1 + δ out − ξ (δ out − δ in )

(23)

ξ was calculated as:

ξ=

C in dry , C in dry − Cout dry

(24)

where Cin dry and Cout dry are [CO2] that have been corrected to zero humidity (Evans et al. 1986). δ13C of net CO2 exchange in light (δNL) and dark (δND) were obtained using Eqn 11. ΔND was obtained as:

Δ ND =

δ s − δ ND , 1 + δ ND

(25)

All scenarios were performed with three experimental runs (except for Scen-II, which had only two experimental runs), again with runs using different combinations of CO2 sources with different δ13CCO2 (−5‰ and −39‰). Scen-I employed CO2 with a δ13C −5‰ in both the mesocosm and cuvette in the first run, and −39‰ in the second and third runs. Scen-II – which maintained a CO2-free mesocosm – used either a δ13C of −5‰ (first run) or −39‰ (second run) in the cuvette. Finally, Scen-III combined a δ13C of −39‰ in the mesocosm and −5‰ in the leaf cuvette (first run), or −39‰ in the leaf cuvette and −5‰ in the mesocosm (second and third runs).

with δs the δ13C of the substrate used in respiration. δs was assumed to be equal to the δ13C of plant biomass, that is −28‰ for the C3, and −12‰ for the C4 grass. High ξ are associated with a low precision of ΔNL measurements. In our measurements ξ were lower than 10, with the exceptions of C3 leaves measured at an air flow rate of 200 μmol s−1 where ξ ranged between 15 and 30. Similarly, large values of Cin dry/(Cout dry − Cin dry) in the dark are associated with a low precision of ΔND measurements.Therefore, we used a cut-off criterion of Cin dry/(Cout dry − Cin dry) > 70 to eliminate the least precise measurements. This eliminated all ΔND measurements made at a flow rate of 200 μmol s−1 and in Scen-II.

Determination of 13C discrimination during leaf gas exchange (ΔN)

Quantifying the leak – accounting for the leak artefact

Δ measurements in each scenario in each experimental run were performed on four leaves from four individuals of the

The three measurement scenarios provided a range of [CO2] gradients across the gaskets and therefore produced different

Experimental runs

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2417–2432

Third

Second

First

Third

60 60 60 60 60 60 200 – 200 60 60 60 60 60 60 200 – 200

Flow rate μmol s−1

−5 −39 – −5

−39 −39

−5 −39 – −5 −5

−39 −39

−5

δM ‰ −5 −5 −5 −39 −39 −39 −39 – −39 −5 −5 −5 −39 −39 −39 −39 – −39

δin ‰ Cin μmol mol−1 390 390 390 390 390 390 390 – 390 390 390 390 390 390 390 390 – 390

CM μmol mol−1 ≈Cout 0 390 ≈Cout 0 390 ≈Cout – 390 ≈Cout 0 390 ≈Cout 0 390 ≈Cout – 390 314 320 314 318 306 307 368 – 363 182 171 185 178 182 206 332 – 331

Cout μmol mol−1 15.2 (1.1) a a 17.5 (0.4) a a 14.9 (1.1) a a 13.7 (0.3) a a A 16.3 (1.1) a a 15.2 (0.6) a a A 11.5 (1.2) a A – 15.2 (1.2) a A 24.3 (1.6) a a 27.1 (1.0) a a 23.0 (0.7) a a 24.6 (1.1) a a A 24.1 (0.8) a a 21.2 (1.5) a a A 21.5 (1.8) a A – 22.0 (1.4) a A

NL μmol m−2 s−1 16.3 (1.0) a b 15.5 (0.5) a b 12.5 (1.8) a b 20.9 (0.8) b a A 19.1 (0.6) b a 26.2 (0.9) a a A 22.1 (1.7) a A – 26.2 (1.2) a A 3.3 (0.1) a b 3.2 (0.2) a b 1.8 (0.1) b b 4.1 (0.1) b a A 4.4 (0.1) b a 6.5 (0.2) a a A 4.8 (0.4) b A – 7.8 (0.7) a A

ΔNL ‰

15.1 (1.1) a a 15.4 (0.7) a a 15.4 (1.1) a a 13.7 (0.3) a a A 14.4 (1.2) a a 15.7 (0.7) a a A 11.4 (1.2) a A – 15.3 (1.3) a A 24.4 (1.6) a a 26.4 (1.0) a a 23.8 (0.8) a a 24.6 (1.1) a a A 23.6 (0.8) a a 21.8 (1.5) a a A 21.4 (1.8) a A – 22.2 (1.4) a A

A μmol m−2 s−1

16.7 (1.0) a b 17.8 (0.5) a b 18.2 (1.3) a a 21.5 (0.8) a a A 21.6 (0.3) a a 20.9 (0.8) a a A 22.5 (1.7) a A – 20.4 (1.2) a A 3.4 (0.2) a b 3.3 (0.2) a b 3.8 (0.2) a b 4.2 (0.1) a a A 4.4 (0.1) a a 4.6 (0.1) a a A 4.8 (0.5) a A – 5.4 (0.5) a A

ΔA ‰

δM and δin represent the carbon isotope composition of CO2 in the mesocosm and reference cell (inlet) of the LI-6400 clamp-on leaf cuvette; CM, Cin and Cout give the CO2 concentration in the mesocosm, reference cell (inlet) and sample cell (outlet) of the LI-6400. Values in brackets are standard errors of means calculated from four replicates (on the youngest fully expanded leaves of four individuals). Different lowercase letters (test effect of scenarios) indicate a significant difference between scenarios within an experimental run (P < 0.05); different underlined lowercase letters (test effect of CO2 source) indicate significant difference between first and second runs within the same scenario; and different capital letters (test effect of flow rate) indicate significant difference between second and third runs within the same scenario.

C4

I II III I II III I II III I II III I II III I II III

First

C3

Second

Scen.

Exp. runs

C3/C4

Table 1. Experimental runs and scenarios, and the results of gas exchange (net exchange rate, NL; and net assimilation rate, A) and carbon isotope discrimination in net exchange (ΔNL) and in net assimilation (ΔA) measurements with leaves of H. lanatus (C3) and S. bicolor (C4) at a photosynthetically active photon flux density of 1600 μmol m−2 s−1

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13

CO2/12CO2 leak and isotopic disequilibrium artefacts in clamp-on leaf cuvettes

magnitudes of leaks between scenarios. KCO2, K12CO2, K13CO2 with intact (attached) leaves in the cuvette were obtained by the gas exchange measurements in the dark using Eqns 4, 6 and 7. The leak effect was eliminated to obtain RD and ΔRD using Eqns 4, and 6–13. Similarly, A and ΔA were obtained using Eqns 5, and 14–17.

Accounting for the isotopic disequilibrium − determination of RL Δ was measured using CO2 with different δ13CCO2 in the different runs (−5‰ in the first run and −39‰ in the second and third runs). This meant different contrasts of δ13CCO2 between the cuvette and the growth environment in the first run relative to the second and third run. This led to differential labelling of the net photosynthetic flux and was used to partition P and RL (Eqn 21). Prior to that, all data were corrected for leak artefacts (see above). Eqns 18 and 19 were solved to yield estimates of δP and ΔP. Leaf temperature differed by about 2 °C between measurements of gas exchange in light and darkness in this study (Tlight > Tdark, Supporting Information Appendix S2, Table S2). In order to compare dark respiration rate in light and darkness, RL was temperature-normalized with respect to RD by assuming a Q10 of 2 (Atkin et al. 2005). The normalization yielded RL corr and was expressed relative to RD as:

RL corr RD =

RL . ( T light − T dark) 10 Q10 × RD

(26)

Estimates of ΔP were plotted against Ci/Ca (the ratio of internal to atmospheric CO2 concentration, obtained from gas exchange measurements), and compared with the ‘simple’ Farquhar models of C3 and C4 carbon isotope discrimination (Farquhar et al. 1982; Farquhar 1983). Any leak leads to an incorrect estimation of A, and therefore a small artefact on Ci estimation. Therefore, a correction was applied to observed Ci (Ci obs) as: Ci = Ca − A (Ca − Ci obs)/NL.

Estimation of mesophyll conductance (C3) and bundle sheath leakiness (C4) Mesophyll conductance (gm, cf. Evans et al. 1986) is defined as gm = A/(Ci − Cc), with Cc the CO2 concentration in the chloroplast. Estimates of Cc were obtained by comparing observed ΔP and estimates of ΔPi for infinite gm. A modified model of C3 discrimination including photorespiration was used:

C Ca − C i Γ* +b i − f Ca Ca Ca

(27)

C C − Cc Ca − C i Γ* + am i +b c − f Ca Ca Ca Ca

(28)

Δ Pi = a

ΔP = a and

Δ Pi − Δ p = (b − am )

C i − Cc , Ca

(29)

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where a = 4.4‰, b = 28.9‰, am = 1.8‰ (Evans et al. 1986; Pons et al. 2009) and f = 11‰ (Ghashghaie et al. 2003). Γ*, the CO2 compensation point in the absence of dark respiration, was calculated from leaf temperature (T) as Γ* = 42.7 + 1.68(T − 25) + 0.012(T − 25)2 in Brooks & Farquhar (1985). Bundle sheath leakiness (ϕ) in S. bicolor was estimated using the simple model of C4 carbon isotope discrimination (Farquhar 1983):

Δ p = a + (b4 + φ (b3 − S ) − a)C i Ca

(30)

with a = 4.4‰, b4 = −5.7‰, b3 = 28.9‰, and S = 1.8‰. In the analysis of ΔP of C3 and C4 photosynthesis, leaf boundary layer conductance was assumed to be infinite and the ternary effect (Farquhar & Cernusak 2012) was ignored. The reasons for these simplifications are (1) we did not measure boundary layer conductance and all measurements were done on similar leaves under similar conditions; and (2) the contributions of the ternary effect were small in this study (t = 0.005 for C3 and 0.002 for C4; maximum error is 0.02 mol m−2 s−1 for gm and 0.001 for ϕ), and were similar (with a similar error in Δ estimations) for all measurements. Accordingly, the simplifications did not influence the interpretation of scenarios and artefacts.

Statistical analysis Statistical analysis was performed using SPSS version 16 (SPSS Inc., Chicago, IL, USA). One-way anova was performed for the analysis of variance, and multiple comparisons of means were done with the Tukey test. For all parameters in tests with leaves, the means of repeated measurements (four measurements) on the same leaf were used for statistical analysis. Thus, each data point in the results had four real replicates (four different leaves from different individuals, with one exception in ΔA of H. lanatus in Scen-I, first experimental run, in which three leaves were measured).

RESULTS Leak tests with empty chamber Empty cuvette tests with gradients of [CO2] between the mesocosm (CM; varying between 0 and 500 μmol mol−1) and inlet of the cuvette (Cin; held constant at 250 μmol mol−1) had no statistically detectable effect on the difference in [CO2] between the inlet (Cin) and outlet (Cout) of the cuvette, Cin − Cout, when the measurements were performed at air flow rates of 500 μmol s−1 or 200 μmol s−1. Similarly, the contrast of δ13CCO2 between the mesocosm (−39‰) and cuvette (−5‰) had no significant effect on the δ13C-difference between the inlet (δin) and outlet (δout), δin − δout, of the cuvette, if measurements were performed at air flow rates of 500 μmol s−1 or 200 μmol s−1 (Fig. 1). However, statistically significant CO2 leakage into or out of the cuvette was evident at a flow rate of 60 μmol s−1: when [CO2] in the mesocosm (CM) was higher than in the empty cuvette (CM > Cout), Cout

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Figure 1. Effect of CO2 concentration gradient between leaf cuvette (Cout) and mesocosm (CM), CM − Cout, on differences between CO2 concentration in the reference cell (Cin) and sample cell (Cout), Cout − Cin (upper panels), and differences in carbon isotope composition of CO2 in the reference cell (δin) and sample cell (δout), δout − δin (lower panels), of an empty leaf cuvette (LI-6400). Tests were performed with air flow rates of 500 μmol s−1, 200 μmol s−1 and 60 μmol s−1 as indicated on top of each panel. The cuvette was supplied with CO2 with a δ13C of −5‰, while the mesocosm received CO2 with a δ13C of −39‰. [CO2] gradients were created by supplying a constant [CO2] of 250 μmol mol−1 to the leaf cuvette and changing the [CO2] in the mesocosm (CM). Error bars represent standard errors (n = 4). Stars mark significant differences (P < 0.05) between Cin and Cout, or δin and δout.

was higher than Cin, showing that CO2 was leaking into the cuvette. Conversely, when CM was lower than Cout, Cout was lower than Cin, demonstrating that CO2 was leaking out of the cuvette. Similarly, the contrast of δ13CCO2 between the mesocosm (−39‰) and the cuvette (−5‰) caused an increasing divergence between δin and δout as CM was increased from 0.9 in all cases. Error bars represent standard errors (n = 4). Experimental conditions as in Fig. 1.

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2417–2432

Second

First

60 60 60 60 60 60 60 60 60 60 60 60 −5

−39 −39

−5 −5

−39 −39

−5

‰

μmol s−1 −5 −5 −5 −39 −39 −39 −5 −5 −5 −39 −39 −39

‰

δin μmol mol−1 390 390 390 390 390 390 390 390 390 390 390 390

≈Cout 0 390 ≈Cout 0 390 ≈Cout 0 390 ≈Cout 0 390

Cin

μmol mol−1

CM

399 386 399 395 384 393 403 389 402 398 387 396

μmol mol−1

Cout

1.5 (0.1) b a −1.0 (0.2) a a 1.4 (0.1) b a 1.3 (0.1) b a −0.8 (0.3) a a 1.2 (0.1) b a 1.4 (0.1) b a −0.2 (0.1) a a 1.1 (0.1) b a 1.3 (0.1) b a 0.01 (0.1) a a 1.1 (0.1) b a

μmol m−2 s−1

ND

−36.3 (8.1) b b

48.8 (4.4) a a −3.1 (1.4) a a

−70.1 (6.3) b b −2.7 (2.0) b a

55.6 (7.7) a a −8.4 (5.5) a a

−2.8 (3.4) b a

‰

ΔND

1.5 (0.1) a a 1.5 (0.2) a a 1.4 (0.1) a a 1.3 (0.1) a a 1.3 (0.1) a a 1.2 (0.1) a a 1.5 (0.1) a a 1.3 (0.1) a a 1.2 (0.1) a a 1.3 (0.1) a a 1.2 (0.1) a a 1.1 (0.1) a a

μmol m−2 s−1

RD

−0.6 (7.0) a a

2.1 (2.1) a a −3.6 (1.4) a a

−9.1 (11.0) a a −2.6 (2.0) a a

−5.7 (0.9) a a −8.5 (5.4) a a

−2.4 (3.4) a a

‰

ΔRD

CO2/12CO2 leak and isotopic disequilibrium artefacts in clamp-on leaf cuvettes

See Table 1 for explanation of experimental procedures and statistical information.

C4

I II III I II III I II III I II III

First

C3

Second

Scen.

Exp. runs

C3/C4

δM

Flow rate

Table 2. Experimental runs and scenarios, and the results of gas exchange (net exchange rate, ND; and respiration rate, RD) and carbon isotope discrimination in net exchange (ΔND) and in respiration (ΔRD) measurements with H. lanatus (C3) and S. bicolor (C4) in the dark

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C3/C4

Exp. runs

KCO2 μmol s−1

R2

K12CO2 μmol s−1

R2

K13CO2 μmol s−1

R2

C3

First Second First Second

1.964 (0.140) 1.877 (0.076) 1.913 (0.156) 1.583 (0.167)

0.95 0.98 0.94 0.90

1.964 (0.140) 1.877 (0.076) 1.913 (0.156) 1.583 (0.167)

0.95 0.98 0.94 0.90

1.963 (0.141) 1.869 (0.076) 1.921 (0.159) 1.576 (0.167)

0.95 0.98 0.94 0.90

C4

Table 3. Leak coefficients for CO2 (KCO2), 12 CO2 (K12CO2) and 13CO2 (K13CO2) in gas exchange measurements with leaves of H. lanatus (C3) and S. bicolor (C4) held in the dark

Values in brackets are standard errors. R2 of the linear regressions used to determine normalized leak rate are shown. For details of experimental runs, see Table 1.

Scen-II, and sometimes even negative (Table 2).These results clearly pointed to outward leakage of CO2 from the cuvette in Scen-II. Increasing the flow rate through the cuvette in the third run did not change NL or ND. Leak coefficients for CO2 (KCO2) with intact leaves were assessed using Eqn 4. KCO2 were not different between species and between experimental runs (Table 3): KCO2 of H. lanatus leaves ranged between 1.88 and 1.96 μmol s−1; KCO2 of S. bicolor leaves ranged from 1.58 to 1.91 μmol s−1. After applying corrections for leak artefacts using the normalized leaks determined during the measurement of dark respiration (Table 3), estimated A and RD were not different between the scenarios (Tables 1 and 2).

Isotopic disequilibrium during Δ measurements in light ΔA, obtained after elimination of the leak artefact, differed between the two runs that used different δ13CCO2 (δin = −5‰ in the first run and δin = −39‰ in the second run). The effect of δ13CCO2 on ΔA occurred in all scenarios with both H. lanatus and S. bicolor (Table 1). This effect was interpreted in terms of a differential labelling of the photosynthetic and dark respiration CO2 fluxes, and was used to disentangle the two fluxes. This provided estimates of RL of 1.33 μmol m−2 s−1 in H. lanatus and 0.59 μmol m−2 s−1 in S. bicolor (Fig. 3a). These values were then used to estimate net photosynthesis. Dark respiration in light was compared

Carbon isotope discrimination during CO2 exchange in light and dark Observations of ΔN (ΔNL or ΔND) differed systematically (although not always significantly) between Scen-I (which maintained the same δ13C of CO2 in the mesocosm and cuvette) and III (which maintained a different δ13C of CO2 in the mesocosm and cuvette, Tables 1 and 2). All other measurement conditions were the same, and important physiological parameters, that is, Ci/Ca and leaf temperature did not differ between the scenarios (Supporting Information Appendix S2, Table S2). The different estimates of ΔN in Scen-I and -III were clearly related to artefacts caused by the contrasting δ13C of CO2 in mesocosm and leaf cuvette air: if CO2 in mesocosm air was 13C-depleted relative to cuvette air (Scen-III, first run of H. lanatus and S. bicolor), then ΔNL was smaller than in Scen-I and ΔND was greater (and generally un-physiologically large). These effects were significant in all comparisons, except for one case (ΔNL of H. lanatus). Conversely, if CO2 in the mesocosm was 13C-enriched relative to cuvette air (Scen-III of second and third runs) then ΔNL was greater than in Scen-I and ΔND was smaller. Again, the effect was systematic, statistically significant in five out of six comparisons, and pointed to an effect of differential bidirectional fluxes of 13CO2 and 12CO2 through gaskets on Δ estimates. Notably, estimates of ΔNL in Scen-II (which maintained CO2free air around the cuvette) did not differ significantly from Scen-I. Leak coefficients for 12CO2 (K12CO2) and 13CO2 (K13CO2) obtained with intact leaves in the dark were assessed using Eqns 6 and 7 (Table 3). K12CO2 and K13CO2 ranged between 1.58 and 1.96 μmol s−1, but were virtually identical for each species within an experimental run. After application of corrections for leak artefacts, estimated ΔA and ΔRD did not differ significantly between the scenarios (Tables 1 and 2).

Figure 3. Dark respiration rate in light (RL, a) and the ratio of dark respiration in light to that in darkness (RL corr/RD, b) in leaves of H. lanatus (C3) and S. bicolor (C4). Measured RL was corrected to RL corr for the same temperature as in the dark (RL corr) by applying a Q10 of 2. Error bars represent standard errors; n = 23 for H. lanatus and n = 24 for S. bicolor.

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CO2/12CO2 leak and isotopic disequilibrium artefacts in clamp-on leaf cuvettes

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with respiration in the dark after correcting RL for the effect of light on leaf temperature, RL corr/RD (cf. Eqn 26). The ratio of RL corr/RD was 0.86 in H. lanatus and 0.42 in S. bicolor (Fig. 3b), indicating a smaller inhibition of dark respiration by light in the C3 (14%) than in the C4 grass (58%). When plotting ΔNL against observed Ci/Ca, the relationships were poor because of the large variation in ΔNL for both H. lanatus (Fig. 4a) and S. bicolor (Fig. 5a). After correction of the leak effect on Δ, ΔA of both species were again plotted against corrected Ci/Ca. Still, the data of the first and second run did not follow the same relationship because of the isotopic disequilibrium effect on ΔA. Finally, when the effect of respiration-related isotopic imbalance was accounted for by estimation of ΔP (using Eqns 18 and 19), the data of both experimental runs with H. lanatus converged on the same relation of ΔP versus Ci/Ca (Fig. 4c). On average, ΔP was 4.6 ± 0.2 (SE) ‰ more negative than was expected for infinite gm and gave an estimate of gm of 0.30 ± 0.02 (SE) mol CO2 m−2 s−1. In a similar way, the values of ΔP for S. bicolor collapsed on the same relationship of ΔP versus Ci/Ca and indicated a bundle sheath leakiness (ϕ) of 0.324 ± 0.004 (SE) (Fig. 5c).

DISCUSSION CO2 leaks can be quantified with intact leaves inside clamp-on cuvettes This work presents a new method for the assessment of the CO2 leak coefficient (KCO2) during measurement of CO2 exchange with intact leaves in a clamp-on leaf cuvette in the dark. These measurements, performed at a flow rate of 60 μmol s−1 with leaves of H. lanatus (C3) and S. bicolor (C4), demonstrated leak coefficients for CO2 in the range of 1.6 to 2.0 μmol s−1, 7–9 times greater than those obtained with the empty cuvette or with a fake inactive leaf. So far, there are no reports of leak coefficients determined with intact leaves, limiting opportunities for discussion. However, our observed K’CO2 with the empty cuvette (0.22 μmol s−1) was similar to that reported by others (0.1 to 0.4 μmol s−1; Rodeghiero et al. 2007), although lower than that communicated by the manufacturer (0.46 μmol s−1). Studies of leak coefficients with empty cuvette and with dead leaves obtained different results: a greater leak coefficient with a dead leaf (relative to the empty cuvette) was found in one study (Rodeghiero et al. 2007), and a smaller leak coefficient was found in another study (Flexas et al. 2007). Our study found no difference in KCO2 between live leaves of H. lanatus and S. bicolor, although the two species differed strongly in leaf width. However, leaf (surface) geometry or (internal) anatomy may affect leak coefficients, possibly via gas movement through the leaf (Jahnke & Pieruschka 2006). Given the possible variability of K-values between different objects/leaves, we suggest that leak coefficients should be studied on the same intact leaves that are used in gas exchange studies in light or darkness. In empty cuvette tests at flow rates of ≥200 μmol s−1, leaks of CO2 were not detectable as Cin − Cout was not significantly different from zero. However, our inability to detect a leak in

Figure 4. Correlation between carbon isotope discrimination during net CO2 exchange in light (ΔNL, a), net CO2 assimilation (ΔA, b) and net photosynthesis (ΔP, c) and Ci/Ca in leaves of H. lanatus (C3). Ci/Ca were corrected for leak effect in panels b and c. Different symbols indicate carbon isotope discrimination measured in the first experimental run with a δ13C of CO2 of −5‰ in air feeding the cuvette (filled symbols) and carbon isotope discrimination measured in the second experimental run with a δ13C of CO2 of −39‰ (open symbols). Dashed lines in panel (b) are linear regressions for each experimental run; the grey line in panel (c) is the linear regression through data from both experimental runs. The black solid lines in all panels represent the theoretical prediction of C3 carbon isotope discrimination from the equation Δ = a + (b − a) Ci/Ca with a = 4.4‰, and b = 28.9‰.

these observations may have been due to limited sensitivity of the IRGA. If that is true, then a high air flow rate would only mask the CO2 leak rather than abolish it. As KCO2 with intact leaves was up to nine times higher than that with an

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Figure 5. Correlation between carbon isotope discrimination during net CO2 exchange in light (ΔNL, a), net CO2 assimilation (ΔA, b) and net photosynthesis (ΔP, c) and Ci/Ca in leaves of S. bicolor (C4). Ci/Ca were corrected for leak effects in panels b and c. The solid line represents the theoretical prediction of C4 carbon isotope discrimination from the equation Δ = a + (b4 + ϕ (b3 − S) − a) Ci/Ca with a = 4.4‰, b4 = −5.7‰, b3 = 28.9‰, S = 1.8‰ and ϕ = 0. For meanings of symbols and dashed lines, see Fig. 4.

empty cuvette, significant leak artefacts should also be suspected for flow rates higher than 60 μmol s−1 (see also below).

Quantifying and correcting 12CO2 and effects on Δ measurements

13

CO2 leak

Here, we report the first quantification of leak coefficients of 12 CO2 and 13CO2 with an empty cuvette and with a cuvette with intact leaves included in it. KCO2, K12CO2 and K13CO2,

determined at a flow rate of 60 μmol s−1 were all highly significant and practically indistinguishable. Again, the leak coefficients (K12CO2 and K13CO2) were 7–9 times greater with intact leaves in the cuvette than that with an empty cuvette. The observed K12CO2 and K13CO2 were statistically equivalent. If the leak through the gaskets was completely diffusive, it should be associated with a fractionation of 4.4‰ (implying a K12CO2/K13CO2 ratio of 1/0.9956). This effect is much smaller than the measurement error of the IRGA and IRMS measurements. In the empty cuvette test with an air flow rate of 60 μmol s−1 and a [CO2] of 250 μmol mol−1 at the inlet of the cuvette, the leak led to a maximum Cout − Cin of 1 μmol mol−1, thus contributing 0.9 in all cases. Error bars represents standard errors (n = 4). Figure S5. δ13C of net CO2 assimilation in light (δA) and respiration in the dark (δRD) in leaves of H. lanatus (C3) and S. bicolor (C4). Measurements from two experimental runs in Scen-I are compared (see Table 1). In the first run, the cuvette was supplied with CO2 with a δ13C of −5‰; in the second run, the cuvette received CO2 with a δ13C of −39‰. In both cases, the cuvette and the mesocosm received air with the same δ13CCO2. Error bars are standard errors for four replicate measurements.

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Table S1. Parts exchanged to eliminate leaks in the console of LI-6400. Table S2. Experimental runs and scenarios, and parameters (Ci/Ca, the ratio of internal to atmospheric CO2 concentration; E, transpiration rate; rHout, relative humidity in the leaf cuvette; Tleaf, leaf temperature) in gas exchange measurements with H. lanatus (C3) and S. bicolor (C4) in light. δM and δin represent the carbon isotope composition of CO2 in the mesocosm and reference cell (inlet) of the LI-6400 clamp-on leaf cuvette; CM, Cin and Cout give the CO2 concentration [CO2] in the mesocosm, reference cell (inlet) and sample cell (outlet) of the LI-6400.Values in brackets are standard deviations of means.

Appendix S1. Leak tests with CO2-free air and pure Argon. Appendix S2. Additional gas exchange parameters in measurements with H. lanatus and S. bicolor. Appendix S3. Using Eqns 4, 6 and 7 to determine leak coefficients (K) with an intact leaf held in the leaf cuvette in the dark. Appendix S4. Using two CO2 sources with distinct δ13C to estimate respiration in light (RL). Appendix S5. Leak tests with a strip of filter paper placed between the gaskets of the clamp-on leaf cuvette (LI-6400). Appendix S6. δ13C of net CO2 assimilation in light (δA) and respiration in the dark (δRD) during measurements with contrasting δ13CCO2 in the air supply of the leaf cuvette.

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2417–2432