Measurement of Gross Photosynthesis, Respiration in the · Breakthrough Technologies Measurement of Gross Photosynthesis, Respiration in the Light,andMesophyllConductanceUsingH 2

Breakthrough Technologies

Measurement of Gross Photosynthesis, Respiration in theLight, andMesophyll Conductance UsingH2

18O Labeling1[OPEN]

Paul P. G. Gauthier,a,2 Mark O. Battle,b Kevin L. Griffin,c,d and Michael L. Bendera,e

aDepartment of Geosciences, Princeton University, Princeton, New Jersey 08544bDepartment of Physics and Astronomy, Bowdoin College, Brunswick, Maine 04011cDepartment of Earth and Environmental Sciences, Columbia University, New York, New York 10027dDepartment of Ecology, Evolution, and Environmental Biology, Columbia University, New York, New York 10027eInstitute of Oceanology, Shanghai Jiao Tong University, Shanghai, China

ORCID ID: 0000-0002-2893-7861 (P.P.G.G.).

A fundamental challenge in plant physiology is independently determining the rates of gross O2 production by photosynthesisand O2 consumption by respiration, photorespiration, and other processes. Previous studies on isolated chloroplasts or leaveshave separately constrained net and gross O2 production (NOP and GOP, respectively) by labeling ambient O2 with 18O whileleaf water was unlabeled. Here, we describe a method to accurately measure GOP and NOP of whole detached leaves in acuvette as a routine gas-exchange measurement. The petiole is immersed in water enriched to a d18O of ;9,000‰, and leaf wateris labeled through the transpiration stream. Photosynthesis transfers 18O from H2O to O2. GOP is calculated from the increase ind18O of O2 as air passes through the cuvette. NOP is determined from the increase in O2/N2. Both terms are measured by isotoperatio mass spectrometry. CO2 assimilation and other standard gas-exchange parameters also were measured. Reproduciblemeasurements are made on a single leaf for more than 15 h. We used this method to measure the light response curve ofNOP and GOP in French bean (Phaseolus vulgaris) at 21% and 2% O2. We then used these data to examine the O2/CO2 ratio of netphotosynthesis, the light response curve of mesophyll conductance, and the apparent inhibition of respiration in the light (Kokeffect) at both oxygen levels. The results are discussed in the context of evaluating the technique as a tool to study andunderstand leaf physiological traits.

Gross O2 production (GOP; the rate of water splittingat PSII), mitochondrial respiration in the light (RL), theoxygenation of Rubisco, and the photosynthetic carbonoxidation cycle (i.e. photorespiration; Hagemann et al.,2013) are fundamental rate properties of plants. Whenassessed as net O2 fluxes, the rates of these processes areconfounded. Since each of the component processesrepresents a distinct biochemical pathway with im-portant cellular functions, quantifying the contributionof these processes to the net assimilatory flux is ofparticular interest. The utilization of O2 as an electronacceptor around PSI via the Mehler reaction also cancontribute to O2 uptake (Badger et al., 2000). Precisemeasurements of O2 fluxes from leaves would provide

a better understanding of these important processesand shed light on diverse metabolic responses to envi-ronmental variation (Furbank et al., 1982; Badger et al.,2000; Hillier, 2008; André, 2011).

A variety of methods have been developed in leavesand phytoplankton to measure net O2 production(NOP; Kok, 1948; Cornic et al., 1989; Laisk et al., 1992;Laisk and Oja, 1998). GOP has been measured directlyusing an 18O tracer and mass spectrometry (Mehler andBrown 1952; Hoch et al., 1963; Ozbun et al., 1964;Radmer and Ollinger, 1980; Ruuska et al., 2000). Twogeneral approaches have been used to measure GOP:(1) measuring the increase of 16O2 from unlabeled waterin a chamber containing dioxygen only as 18O2; and (2)measuring 18O16O of photosynthetic O2, produced fromsplitting 18O-labeled water. All published GOP data forleaves use the first approach to determine GOP and,givenNOPmeasurements, O2 uptake (Berry et al., 1978;Gerbaud and André, 1979; Canvin et al., 1980; Peltierand Thibault, 1985; Biehler and Fock, 1995; Haupt-Herting and Fock, 2002; André, 2011). Typically, thismethod relies on leaf fractions or small leaves that areplaced in a gas-tight gas-exchange chamber connectedto a membrane inlet mass spectrometer (Berry et al.,1978; Biehler and Fock, 1995). The chamber air is thenreplaced with O2 present as

18O2, and the evolution of16O2 and

18O2 is monitored. The decrease of 18O2 con-centration in the system constrains O2 uptake, and the

1 This work was funded by the Princeton Environmental InstituteGrand Challenge Grant Program.

2 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Paul P.G. Gauthier ([email protected]).

P.P.G.G. designed the experiments, analyzed the data, and pre-pared the article; P.P.G.G., M.O.B., K.L.G., and M.L.B. designed thegas-exchange cuvette; M.O.B. built the cuvette; K.L.G. and M.L.Bsupervised the experiments; all authors discussed the results andworked on the article.

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increase of 16O2 gives GOP. GOP, determined in thisway, is a reliable measure of the electron transport rate(J; André, 2013), which then can be used to estimatemesophyll conductance (gm; Flexas et al., 2012) andchloroplastic [CO2] (Cc; Renou et al., 1990; André, 2011).Nevertheless, among current methods used to measuregm (for review, see Flexas et al., 2012), the 18O methodwas limited by a restriction associated with obtaining18O2 and the need for specific equipment (e.g. mem-brane inlet mass spectrometry). As a consequence, todate, measurements of NOP and GOP have receivedlittle attention.The second approach, relying on 18O-labeled water,

has been used to measure NOP and GOP in phyto-plankton (Grande et al., 1989; Goldman et al., 2015).With this method, water in the cells is labeled withH2

18O. If implemented with a whole leaf in a flow-through gas-exchange system, the leaf would be la-beled with H

2

18O via the transpiration stream. In thecuvette, NOP raises the O2 concentration, and GOP alsoraises the d18O of O2. The rates of NOP and GOP aremeasured from the difference in O2/N2 (Grande et al.,1989) and d18O of O2 in air entering and leaving thecuvette. O2 uptake is the difference between these tworates (GOP – NOP). The measurement of the change inO2 concentration is a standard practice for high-precision measurements: changes in O2 concentrationare routinely measured from changes in the ratio of O2/N2 rather than from the O2 concentration or partialpressure, which also depend on temperature and hu-midity.The advantage of this method is that gas-exchange

properties are determined with a steady-state gas-exchange protocol rather than in a closed gas-exchange system. The challenge of this technique isthat there are only small differences in the O2/N2 ratioand d18O of O2 between outgoing and incoming air.These small changes can bemeasured by high-precisionisotope ratio mass spectrometry. For example, a rate ofNOP resulting in a CO2 drawdown or O2 rise of 100 mLL21 would cause O2/N2 in air exiting the chamber to beincreased by 0.47‰. If water molecules were labeled to+10,000‰, GOP of 100 mmolmol21 would cause d18O ofO2 to increase by 5‰. For comparison, dO2/N2 can bemeasured by isotope ratio mass spectrometry to aprecision of 60.005‰ and d18O of O2 can be measuredto a precision of 60.03‰ or better (Bender et al., 1994).Thus, one can measure [O2] and d18O changes associ-ated with NOP and GOP to a precision of about 61 mLL21 out of 210,000 mL L21 (Bender et al., 1998). OurH2

18O labeling approach, as described below, allows usto simultaneously measure a full set of fundamentalgas-exchange properties, including NOP and GOP. Wealso can measure light and CO2 response curves, thuscharacterizing many important physiological proper-ties of plants simultaneously. As will be shown, we canregulate flow rate, temperature, humidity, and otherproperties of air in the chamber.As a demonstration of the utility of this technique, we

measured GOP and NOP of French bean (Phaseolus

vulgaris) as a function of irradiance at 21% and 2% O2.We also measured CO2 assimilation. We then usedthese results to examine ΔO2/ΔCO2 during exposure tolight, the light response of gm, and the Kok effect (ap-parent inhibition of respiration in the light; Kok, 1948,1956; Hoch et al., 1963) under normal and non-photorespiratory conditions.

RESULTS

Measurement of Net and Gross Photosynthesis

Gas-exchange equations predict that net assimilation(Anet) increases linearly at low light intensity, wherelight is limiting, and plateaus at high light levels, whereCO2 assimilation is limited by Rubisco activity. In Fig-ure 1, the linear portion of the curve is restricted to lightintensities lower than 160 mmolm22 s21. The asymptoteof the photosynthesis light response curve representsthe maximum rate of Anet, NOP, or GOP. The asymp-tote is determined using a nonrectangular quadraticfunction. At 21% O2, GOPmax (the subscript max refersto the asymptotic value) is 34.2 mmol m22 s21 andNOPmax is 13.5 mmol m22 s21. At 2% O2, GOPmax is19mmolm22 s21 andNOPmax is 15.8mmolm22 s21. At anygiven [O2], NOPmax and Amax are indistinguishable, asalso suggested by Figure 2. The difference betweenGOP and NOP at 21% O2 is greatest at maximum lightintensity. This point corresponds to a reduction in Civalues (Supplemental Fig. S1). In this discussion, weacknowledge that ambient CO2 (Ca) changed in the leafchamber (Supplemental Fig. S1). During the experi-ments at 21%O2,Ci increases from 2456 5 mmolmol21 atmaximum light intensity to 458 6 8 mmol mol21 indarkness. The variation in Ci with irradiance is a con-sequence of changes in net photosynthesis and respi-ration as well as variable stomatal closure and Ca in thecuvette (from 379 6 4 to 426 6 3 mmol mol21). As aconsequence, Ca is slightly higher in the dark, whererespiration adds CO2 to ambient air, than in the light,where photosynthetic assimilation removes CO2. Theambient CO2 in the cuvette depends on the differencebetween net CO2 assimilation by the leaf and the flux ofCO2 into the cuvette. At 2% O2, Ci increases with de-creasing light intensity. The range of increase is en-hanced by the higher gross assimilation rate at highirradiances (from 1296 19 to 4376 13mmolmol21). Forirradiances above 200 mmol photosynthetically activeradiation (PAR) m22 s21 at 2% O2, Ci was lower than200 mmol mol21 and considered limiting for photo-synthesis. The discussion of the results focuses on thelower irradiance range (0–200 mmol PAR m22 s21).

All measured responses of GOP to increasing lightintensities follow a similar nonrectangular quadraticfunction (Fig. 1, C andD). As suggested by the higher SD

of fluxes in the replicate experiments (Fig. 1), GOP ismost variable from leaf to leaf at high light intensities.However, this variability is less pronounced for mea-surements at 2% O2 (Fig. 1). Nevertheless, the precisionof the measurements is better at 2% than at 21% O2 (for

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description about d18O accuracy and precision, seeSupplemental Material).

Figure 2 shows a positive correlation of nearly 1:1between NOP andAnet. At 21%O2, the linear regressionbetween NOP and Anet (photosynthetic quotient) is1.066 6 0.013 (r2 = 99.6%, P , 0.01) O2 per CO2. At 2%O2, it is 0.9786 0.013 (r2 = 99.5%, P, 0.01) O2 per CO2.The ratio NOP/Anet for values obtained in the linearportion of the curve (light intensities lower than160 mmolm22 s21) is 0.9826 0.097 (r2 = 90.2%, P, 0.01)at 21% O2 and 0.9566 0.013 (r2 = 99.5%, P, 0.01) at 2%O2. The correlation between NOP and Anet in theRubisco-limited part of the curve (light intensity above160 mmolm22 s21) is 1.0266 0.027 (r2 = 99.2%, P, 0.01)O2 per CO2 at 21%O2 and 0.9996 0.067 (r2 = 97.3%, P,0.01) O2 per CO2 at 2% O2. The correlation betweenNOP and Anet for all the data presented in Figure 2 is1.006 6 0.012 (r2 = 99.1%, P , 0.01). This correlationvalidates the measurement of net photosynthesis usingeither O2 or CO2 fluxes.

Stomatal Conductance and gm

Stomatal conductance (gs) increases with light in-tensity to reach a maximum around 0.1 mol m22 s21

(Fig. 3A), but gs is higher at 2% O2 than at 21%O2. Lightsaturation of gs occurs at about 160 mmol PAR m22 s21

at 2% O2 and 500 mmol PAR m22 s21 at 21% O2.At 21% O2, gm increases with irradiance to an

asymptotic value. As irradiance rises above about

110mmolm22 s21,Cc decreases slightly from 1616 16 to1096 5mmolmol21 (Fig. 3, B andC).Nevertheless,Cc tendsto be conserved, and variation in Ci seems to be com-pensated for by an increase of gm with increasing lightintensities. In fact, gm reaches an asymptote by400 to 450 mmol PAR m22 s21. At 21% O2, this as-ymptote is around 0.1 mol CO2 m

22 s21. At 2% O2, be-cause O2 uptake is similar to RL, gm and Cc cannot becalculated (see Eq. 18 in “Materials and Methods”).

The Kok Effect under Photorespiratory andNonphotorespiratory Conditions

Considering the light-limited part of the experiment,Figure 4 summarizes the data for the linear portionof the light response curve. Linear regression ofGOP and NOP in the low-light-intensity part of thephotosynthesis-irradiance curve (0–160 mmol PAR m22

s21) is used for leaves exposed to either 21% or 2% O2.At 21% O2, the linear portion of the curve occurs be-tween 0 and 90mmol PARm22 s21. At 21%O2, NOP andGOP diverge as light intensities rise, reflecting in-creasing photorespiration at higher rates ofAnet or GOP.The slope of GOP versus irradiance for values between0 and 110 mmol m22 s21 is 0.07 6 0.004 mmol O2 pro-duced per quanta (r2 = 98.2, P , 0.01) at 21% O2. Theslope of NOP is 0.054 6 0.002 O2 molecules producedper quanta (r2 = 99.7%, P , 0.01). The linear portion ofGOP and NOP versus irradiance curves at 2% O2 liesbelow 110 mmol m22 s21. At 2% O2, the slopes of GOP

Figure 1. Light response of net CO2 assimilation and NOP for French bean leaves exposed to 21% O2 (black symbols) or 2% O2

(white symbols). A and B, Light responses of net CO2 assimilation. C and D, Light responses of NOP. Nonrectangular quadraticcurves are represented by solid lines for net fluxes and by dashed lines for GOP. Errors bars represent SE for n = 3.

64 Plant Physiol. Vol. 177, 2018

Gauthier et al.




and NOP with increasing light intensities were similar,0.066 6 0.002 O2 molecules produced per quanta (r2 =99.7, P , 0.01).Similarly, using linear regressions of the NOP and

Anet values obtained for light intensities in the linearportion of the NOP and Anet light response curvesabove 35 mmol PAR m22 s21 (as defined above) for in-dividual leaves, a rate of respiration in the light can beobtained following the Kok method. On average, Ro-apparent is 0.54 6 0.16 mmol m22 s21. In comparison,the averaged value of dark respiration (Rdark) is 1.06 60.07 mmol m22 s21. As a consequence, Ro-apparent for21% O2 is inhibited by 46% at light intensities above thebreak point in the light response curve. At 2%O2, linearregressions of GOP and NOP against irradiance areparallel (Fig. 4B), and the linear regression of NOP in-tercepts the y axis at the average value of dark respi-ration (Rdark = 0.79 6 0.12 mmol m22 s21). Therefore, at2% O2, there is no discernible Kok effect and no ap-parent light inhibition of mitochondrial respiration.Dark respiration values (1.06 6 0.07 mmol m22 s21 at

21% O2 and 0.79 6 0.12 mmol m22 s21 at 2% O2) arecompared with Ro-corrected or Rc-corrected valuescalculated from either O2 or CO2, respectively (Fig. 5).At 21% O2, the Ci correction increases Ro slightly from0.59 6 0.05 to 0.68 6 0.04 mmol m22 s21. Thus, aftercorrection, respiration is 36% lower in the light than inthe dark. For Rc, the apparent value is 0.746 0.14 mmolm22 s21 and the corrected value is 0.646 0.04 mmolm22

s21. Although the impact of the Ci correction on the truevalues of Ro and Rc are different, at 21% O2, these dif-ferences are not statistically significant using a Stu-dent’s t test comparison with P . 0.05. At 2% O2, thecorrection for Ci variations decreases Rc from 1.15 60.39 to 0.646 0.04 mmol m22 s21 and decreases Ro from

0.866 0.28 to 0.726 0.04 mmolm22 s21. The respiratoryquotient for apparent respiration in the light is then1.06 6 0.14 at 21% O2 and 1.12 6 0.05 at 2% O2.

DISCUSSION

Assessment of the Gas-Exchange Method

The technique developed here is a robust gas-exchange method combining cavity ring-down spec-trometers (CRDS) with a high-precision isotope ratiomass spectrometer (IRMS) to more fully characterizeleaf carbon, oxygen, and water vapor exchanges undernear-ambient conditions. This technique takes advan-tage of the ability of the IRMS to achieve a precision of60.03‰ in the measurement of d18O and 60.005‰ indO2/N2. This technique does not enable the measure-ment of previously unmeasured properties. For exam-ple, GOP has been measured before by 18O2 labeling(Berry et al., 1978; Canvin et al., 1980; Peltier and Thi-bault, 1985; Biehler et al., 1997; Badger et al., 2000;Haupt-Herting and Fock, 2000; Ruuska et al., 2000).However, our technique has twomain virtues. First, theuse of a steady-state gas-exchange system allows themeasurement of leaf properties such as RL and gm thatcould not be easily measured in the closed-systemchambers used previously to measure GOP. Second,this instrument allows us to constrain and vary majorenvironmental properties such as O2 concentrations,irradiance, and temperature in a systematic way and,thus, to study a variety of leaf physiological processes.

Compared with previously used closed gas-exchanges systems (Laisk et al., 1992; Badger et al.,2000; Haupt-Herting and Fock, 2002), the technique

Figure 2. Correlation betweenNOPand net CO2 assimilation for leavesexposed to 21% O2 (black symbols) or 2% O2 (white symbols). Thedashed line represents a 1:1 relation.

Figure 3. Light response of gs, Cc, and gm for French bean leaves ex-posed to 21% O2. A, gs. B, Cc. C, gm. Dotted lines represent asymptoticvalues for each parameter. Errors bars represent SE for n = 3.





developed here presents significant improvements thatovercome several previous limitations. However, spe-cial care needs to be taken when preparing detachedleaves. Detaching leaves from their main branch candisrupt the continuity of the water stream and lead toxylem embolism and leaf wilting (Savvides et al., 2012;Tombesi et al., 2014). As in Gauthier et al. (2010), wheredetached leaves were maintained alive for up to 16 h,the protocol described here (see above) routinelyallowed measurements for more than 15 h. In past ex-periments using membrane inlet mass spectrometry,the cost of 18O2 limited the length of experiments to onlya few hours (Atkins and Canvin, 1971; Harris et al.,1983), also limiting the range of measurements and

their application. The technique presented overcomesthis limitation by using ambient air and a limitedamount ofH2

18O. Second, the utilization of entire leaveshas two advantages: it limits leaf desiccation (Flexaset al., 2006) and reduces stress reactions associated withleaf cutting. Furthermore, it increases the differencebetween entering and exiting air and increases repeat-ability and precision (Supplemental Figs. S4 and S5).Finally, comparedwith the techniques used on attachedleaves, enclosing a leaf, petiole, and water supply re-duces potential system leaks (Yakir, 1992; Haupt-Herting and Fock, 2000).

The limitations associated with working on detachedleaves also need to be recognized. There is evidence that

Figure 4. Low-light response of NOP (squares) andGOP (triangles) for individual leaves of French bean exposed to 21%O2 (blacksymbols) or 2% O2 (white symbols). A through F show individual low-light responses, and G and H show averaged values. NOPvalues are corrected forCi variation using Kirschbaum and Farquhar (1987). Dashed-dotted lines represent the linear regression ofthe values obtained for light intensities between 40 and 100 mmol m22 s21. Error bars represent SE for n = 3.


Gauthier et al.




gs is regulated bywater potential and plant water status(Buckley, 2017), yet in our experiments here, leaf waterpotential was not monitored. Thus, the low gs andresulting Ci observed in our experiments may be asso-ciated with detaching the leaves. Alternatively, the lowgs also could have been generated by stomatal patchi-ness (Buckley et al., 1997; Mott and Buckley, 2000). Fi-nally, we note the limitation associated with variable Cainside the chamber, which led to limiting Ci values (lessthan 200 mL L21) under nonphotorespiratory condi-tions. This limiting Ci could lead to a limitation of car-bamylation and the apparent inactivation of Rubisco(Sage et al., 2002). Future experiments should continueto quantify and minimize the overall stomatal responsewhile attempting to study leaf physiological parame-ters under near-ambient conditions and constant Ca.This system enables the measurement of GOP and

NOP concurrently with many other commonly mea-sured properties, such as Anet, gs, and carbon isotopediscrimination. There are a number of ways in whichthis instrument could be applied to important topics inplant physiology. For example, the measurement ofGOP and electron flow through the photosyntheticelectron transport chain also can be used to quantify thequantum yield of photosynthesis if light absorption ismeasured (Singsaas et al., 2001). Similarly, precisemeasurements of GOP coupledwithNOP can constrainO2 uptake and provide some insight into the role of theMehler reaction (Badger et al., 2000; Haupt-Herting andFock, 2002). Finally, GOP can be compared directly

with fluorescence, which is its proxy for electron flow(Genty et al., 1989; von Caemmerer, 2000; André, 2011).Calibrating fluorescence against measured values ofGOPwould allow accurate estimates of this property ina wide range of studies.

NOP data, together withmeasurements ofAnet, allowus to determine the stoichiometry of photosynthesisand respiration, expressed as ΔO2/ΔCO2. Doing soadds the capacity to assess processes such as nitrateassimilation (Bloom et al., 1989), anaplerotic carbonfixation (Oja et al., 2007; Angert et al., 2012), or theproduction of lipids (Aubert et al., 1996; Devaux et al.,2003; Tcherkez et al., 2003), in addition to potentialchanges in the respiratory substrates and cellular en-ergetics (Tcherkez et al., 2003; Nogués et al., 2004). Inour experiments, the photosynthetic quotient wasnearly constant and very close to 1, as seen by the strong1:1 relationship betweenNOP andAnet (Fig. 2). This is tobe expected if both the photosynthates created and therespiratory substrates used during the measurementwere Glc, starch, and other basic carbohydrates. How-ever, considering the low rate of RL, the influence of therespiratory substrates on the net assimilation quotientis relatively small. In addition, the ratio NOP/Anet at21%O2was slightly higher than 1 by a few percent. Thissmall imbalance in O2 production compared with CO2assimilation could emerge from O2 produced duringnitrogen assimilation in leaves (Bloom et al., 1989). Inaddition, we are mindful that, in low light, calculationsof ΔO2/ΔCO2 due to respiration in the light from CO2and O2 exchange are inherently uncertain. This is be-cause they rely on extrapolating NOP and Anet to zeroirradiance. Although photosynthesis and respiratoryquotients of leaves are rarelymeasured in gas-exchangeexperiments, they have the potential to add valuableinformation. As discussed here, with our system, it ispossible to measure these properties under a variety ofambient conditions.

Response of gm to Light Intensity

Measuring GOP allows for the calculation of both Ccand gm (Renou et al., 1990; André 2011; Flexas et al.,2012). We found, in French bean, that gm doubled whenlight increased from 200 to 1,100 mmol PAR m22 s21.Similar responses have been reported previously fromC isotope discrimination (D13C; Hassiotou et al., 2009;Douthe et al., 2011, 2012) or fluorescencemeasurements(Flexas et al., 2007; Xiong et al., 2015). Douthe et al.(2011) found that, when light increased from 200 to1,100 mmol PAR m22 s21, gm increased by 60% for threeEucalyptus spp. In a similar light intensity range, Flexaset al. (2007) found a 40% increase of gm with irradiancefor Olea and Vitis spp. Finally, Hassiotou et al. (2009)reported an increase with irradiance of 22% of gm in sixspecies of Banksia. The response of gm to irradiance islikely to be species dependent andmay be a strategy forplants to maintain a positive carbon balance in leaves(Flexas et al., 2006). However, no consensus exists

Figure 5. Comparison of respiration rates measured in the dark (blackbars) and apparent respiration rate in the light using the Kok method onAnet (gray bars) or using the Kok method on NOP (white bars) for Frenchbean leaves exposed to 21% O2 or 2% O2. Values of RLight were cor-rected for variations in Ci. Results of Student’s t test are represented bydouble asterisks when the difference from the other group is very sig-nificant (P # 0.01) and by single asterisks at P , 0.05. Each group isrepresented by a different letter, a or b. Error bars represent SE for n = 3.





regarding the mechanism for the rapidity of the re-sponse (Douthe et al., 2011). Our results demonstratethe capacity of our system to determine gm, addingto the short list of studies using alternative approachesto the most commonly used techniques (fluorescenceor 13C) to study environmental responses of gm.

Major differences exist between previously reportedgm methods and the current O2 method. The O2 methodhas some distinct advantages for studying gm responsesto environmental factors. Carbon isotope approachesuse a combination of isotope effects associated withcarbon metabolism (Tazoe et al., 2011) to estimate gm.The relevant fractionation processes that need to beconsidered include the diffusion of CO2 through thestomata (a = 4.4‰), the fractionation for dissolution anddiffusion through water (a1 = 1.8‰), fractionationcaused by Rubisco (b = 30‰), and fractionation asso-ciated with respiration and photorespiration (e = 5.1‰and f = 11.6‰, respectively; Gong et al., 2015). gm is thencalculated according to Tazoe et al. (2011):

gm ¼�b2 a1 2 eRL

AnetþRL

�Anetpa

aþ ðb2 aÞpipa

2Dobs 2eRLðpi 2G∗ÞðAnetþRLÞpa 2

fG∗

pa

ð21Þ

where Anet is net CO2 assimilation, pi and pa are CO2partial pressure of the ambient and intercellular air-spaces, G* is the compensation point in the absence ofRL, and Dobs is the observed CO2 discrimination calcu-lated from the difference in C isotopes entering andleaving an open gas-exchange system (Evans et al.,1986). Photorespiratory and respiratory fluxes are de-termined using G* and RL. By making these measure-ments at 2% O2, the impact of RL and G* on the estimateof gm remains small and negligible (Tazoe et al., 2009).Comparing Equations 18 and 21, we can see that bothequations constraining gm invoke four common terms:RL, Anet, G*, and Ci. The O2 method also requires deter-mining U 2 RL, while the 13C method requires estimat-ing five isotope effects and the apparent discrimination(D). Furthermore, Gong et al. (2015) have shown thatdifferences in the d13C between the tank gas used in gas-exchange experiments and the ambient air the plantswere grown in can lead to errors in calculated valuesof photosynthetic discrimination. This problem is

avoided when using the O2 method, where the onlyunique term needed to calculate gm is U (Eq. 16), deter-mined frommeasurements of GOP and NOP. The mainassumption of the O2 method is that oxygen consump-tion is negligible for processes other than photorespir-ation and respiration (such as photoreduction bychlororespiration or theMehler reaction). Finally, Equa-tion 21 has been shown to overestimate gm (Farquharand Cernusak, 2012), and the ternary effect of transpi-ration rate on Anet needs to be considered for the esti-mates of gm using the 13C method.

Mitochondrial Respiration in the Light

We found that, at 21% O2, respiration was lightinhibited (based on the nonlinearity in the light re-sponse curve of Anet), as has been commonly found inother experiments (Atkin et al., 2005; Tcherkez et al.,2008; Heskel et al., 2013). However, at 2% O2, light didnot inhibit mitochondrial respiration (i.e. there was noKok effect). The lack of a Kok effect under ambient[CO2] has been found previously in studies of maize(Zea mays; Cornic and Jarvis 1972) and rice (Oryza sativa;Ishii and Murata, 1978). In the case of maize, a C4 plantwhere photorespiration is insignificant, Cornic andJarvis (1972) measured the light response curve at lowirradiances using 0%, 21%, and 100% O2 and did notobserve a Kok effect. They concluded that the lack of aKok effect could be explained by the lack of photores-piration under natural conditions. For rice, Ishii andMurata (1978) did not observe a Kok effect at 2% O2 butobserved a larger Kok effect with 50%O2. This led themto conclude that photorespiration was the proximatecause of the Kok effect.

However, contrary to the two studies on maize andrice, Sharp et al. (1984) found the occurrence of a Kokeffect in sunflower (Helianthus annuus) leaves underelevated [CO2]. Since photorespiration is inhibited un-der high CO2, the explanation of a direct influence ofphotorespiration on the Kok effect may be too simple.Sharp et al. (1984) also reported a reduction of darkrespiration under low O2. However, they suggestedthat it originated from the limitation of O2 uptake by themitochondrial electron transport chain when O2 wasless than 2% (their low-O2 partial pressures were 1%).

Figure 6. Schematic of the gas-exchange sys-tem used for the experiments. MFC, Mass flowcontroller; WT, water trap; CRDS, cavity ring-down spectrometer; IRMS, isotope ratio massspectrometer; MFM,mass flowmeter. Subscriptv is water vapor.


Gauthier et al.



Our data also suggested a lower dark respiration rate at2% O2, but the difference was not statistically significant(Student’s t test, P = 0.056). Furthermore, Griffin andTurnbull (2013) and Yin et al. (2011) reported significantlight inhibition of respiration in both C3 and C4 plants.Tcherkez et al. (2008) found that the inhibition of respira-tion in the light is independent of the CO2/O2 ratio butthat, under low-O2 conditions, the degree of inhibitionmay diminish. More recently, Farquhar and Busch (2017)suggested that most of the Kok effect could be explainedby a variation in Cc under limiting irradiances. However,in Vicia faba, this explanation was not confirmed. Instead,Buckley et al. (2017) found that (1) the break point (Kokeffect) was unaffected by [O2], confirming the findings ofTcherkez et al. (2008), and (2) the activity of the oxygencomponents of mitochondrial respiration decline pro-gressivelywith irradiance (Buckley et al., 2017). In fact, thedegree of inhibition of respiration in the light may reflect astrong plasticity of the respiratory system in C3 plants.Comparison of this flexibility among C3 plants is needed,and the gas-exchange system presented here can contrib-ute physiological information for future investigations.

CONCLUSION

We have presented a method that allows the stan-dard gas-exchange techniques to be extended to themeasurement of NOP and GOP. With this method, abroad suite of gas-exchange properties can be mea-sured while regulating light, CO2, O2, temperature, andhumidity. The light response curves presented herewere used to demonstrate the method and to determineseveral key parameters that could help improve ourunderstanding of fundamental leaf processes. Twosignificant insights are highlighted. First, mitochondrialrespiration was not inhibited in the light when leaves ofFrench bean were exposed to nonphotorespiratoryconditions. This result reinforces some earlier conclu-sions that photorespiration could indirectly play amajor role in regulating the intensity of the Kok effect insome plants, but it remains to be further investigated.Second, gm increases with an increase in light intensity.gm inferred fromO2 fluxes represents an alternative andcomplementary approach thatmay clarify uncertaintiesin its measurement. For example, Farquhar and Busch(2017) demonstrated that the uncertainty associatedwith the measurement of respiration in the light usingthe Kok effect is of the same order of magnitude as theuncertainty in Cc using conventional CO2 gas ex-changes. Comparable calculations using the O2 methodare currently missing, and a better understanding of thelimitations of O2 diffusion toward the chloroplast isneeded. Major uncertainties remain around the natureof the Kok effect and gm.Measuring O2 fluxes could adduseful information to compute more accurate Cc valuesand thus examine the Kok effect with sufficient preci-sion (Tcherkez et al., 2017a, 2017b). Finally, this tech-nique creates opportunities to study the long-termresponse of gross photosynthesis to environmental

changes and to investigate the balance between pho-tochemical energy production by the electron transportchain and the utilization of this energy for carbon as-similation and other cell processes.

MATERIALS AND METHODS

Plant Material

Seeds of French bean (Phaseolus vulgaris) were germinated onwet filter paperin petri dishes for 5 to 6 d in the laboratory. Once true leaves appeared, plantletswere transferred to a greenhouse and placed in trays containing potting mix for1 week before being transferred to 10-L pots. Photosynthetic photon flux den-sity during a 16-h photoperiod was maintained above 500 mmol m22 s21 usingsupplemental high-pressure sodium lighting. Ambient temperature wasmaintained at approximately 23°C/14°C day/night. All pots were watered for5 to 10 min, two to three times per day depending on plant size, by drip irri-gation. Nutrient solution (Miracle-Gro enriched with iron-EDTA; Scotts) wasapplied twice per week. Mature green leaves (approximately 3 to 4 weeks old)were detached from the plants under water, and the apical leaflet was used forthe experiment.

Gas-Exchange System

Gas-exchangemeasurementsweremadeon fully expanded leavesplaced inaglass and aluminum chamber similar to that described by von Caemmerer andHubick (1989). The chamber’s internal dimensions were 1503 1903 15 mm fora volume of 427.5 cm3. The air within the chamberwaswellmixedwith a 15-cm-long cross-flow fan driven by an external DC-powered, magnetically coupleddrive (MagneDrive; Autoclave Engineers). The upper surface of the cuvette wassealed with a 10-mm-thick tempered glass plate measuring 170 3 260 mm. Anair-tight seal was achieved by pressing the glass lid against a Viton O-ring witheight clamps to apply homogenous pressure. Air temperature inside thechamber was controlled by a thermoelectric (Peltier) temperature controller(TC-36-25; TE Technology) in thermal contact with the bottom of the cuvette.Two type K thermocouples coupled to amplifier breakout boards (MAX31850K;Adafruit Industries), interfaced to an Arduino Uno R3 (http://arduino.cc),were used to record air and leaf temperatures. The cuvette was uniformly il-luminated by three controllable customized SPYDRx LED systems (FluenceBioengineering) placed above the chamber. A small quantum sensor (VersatileMini; Heinz Walz) inside the cuvette was used to measure PAR reaching theadaxial side of the leaf.

During each experiment, a constant flow of compressed air entered thechamber after passing through twoDrierite columns (W.A. HammondDrierite)and a glass cryogenic trap (at250°C) to remove moisture. Some air exiting thechamber also was dried. A schematic of the gas-exchange system is presented inFigure 6. Dried compressed air was allowed to flow through a glass capillaryinto the reference side of the changeover valve of the mass spectrometer (Fin-nigan Delta plus XP; Fig. 6). A mass-flowmeter (Aalborg) was used to measurethe flow rate of air exiting the chamber. A second small fraction of exiting airwas sent to an isotopic H2O CRDS (L2130-I; Picarro) for analysis of the H2Omixing ratio in air (Supplemental Figs. S6 and S7C) and d18O of water vapor (d18

Ov; Supplemental Figs. S6 and S7D). Transpiration rate (E) and gs were calcu-lated according to Buck (1981) and von Caemmerer and Farquhar (1981).Exiting air that was not sent to thewater analyzerwas dried by passage througha Drierite column and a glass water trap. A fraction of this air was sent to aCRDS CO2 analyzer, which measured CO2 (Supplemental Figs. S6 and S7A)and d13C in CO2 (Supplemental Figs. S6 and S7A). Note that the d13C data werenot used in any of the calculations of this article. A separate fraction of driedexiting air flowed through a glass capillary tube to the sample side of the massspectrometer to measure dO2/N2 and d18O of O2 (Supplemental Figs. S6 and S7,B and E, respectively).

Petiole Box

For each experiment, the leaf’s petiole was placed in a 40- 3 30- 3 20-mmsealed aluminum box containing 22 cm3 of 18O-labeled water. The entire leafand petiole box was placed inside the leaf chamber. In this way, any potentialleak caused by the petiole or leaf blade crossing the side wall of the chamberwas avoided (as described previously by von Caemmerer and Hubick [1989]




http://arduino.cc








and Yakir et al. [1994]). The aluminum lid of the petiole box had a 10-3 10-mmaperture that allowed for the passage of the petiole. Underneath the lid, a 38-328- 3 1-mm Viton sheet with smaller aperture was custom fit to the variablediameter of each leaf’s petiole. The aluminum plate and Viton sheet suppressedevaporation from the petiole box to the point where it did not have a discernibleinfluence on the concentration or d18O of water vapor exiting the cuvette. Thus,transpiration was the only significant source of water vapor (v) leaving thecuvette.

Leaf Water Labeling

An entire fully expanded leaf was detached from a plant, and the petiole wasimmediately immersed in tap water. The petiole was then recut under water toremove any potential embolism. After 30 min in darkness, the side leaflets wereremoved and the apical leaflet was used in the experiment. The leaf wastransferred to the petiole box filled with labeled water (as described above) at8,806‰ and 8,735‰ d18O for 21% O2 and 2% O2 experiments, respectively. Thepetiole was again recut under water inside the box, and the entire system (leaf +petiole box) was sealed inside the chamber. The course of leaf water labelingwas followed using the CRDS (Fig. 6) to measure d18Ov of transpired watervapor exiting the chamber (Supplemental Figs. S6 and S7D).

Calculating GOP, NOP, and O2 Uptake

GOP is defined as the rate ofO2 production bywater splitting andNOPas thesum of all O2 fluxes between air and the leaf. The d18O of leaf water (d18OL) iscalculated first. Leaf water is the substrate for photosynthesis, and the d18O ofphotosynthetic O2 approximates d18OL (Guy et al., 1987). Mass balance equa-tions constrain GOP and NOP, given the observed production rate of the twomajor isotopologues of O2,

16O2 and18O16O. O2 uptake is then calculated by

difference. Other isotopologues of O2 have very low abundances, make a trivialcontribution to O2 fluxes, and are neglected. Errors introduced in GOPby this neglect are 0.04% for 17O16O and less for the less abundant 17O2,

18O17O,and 18O2.

Air is run through the cuvette until d18O of water vapor exiting the chamberreaches a constant value, at which point the water balance is at steady state. Then,the isotopic composition of water entering the leaf through the petiole should besimilar to that of water exiting the leaf through the transpiration stream. Thus, theflux of H2

18O derived from transpiration (i.e. F18O-v), is calculated as:

F18O2 v ¼ F16O2 v

�H2

180H2

160

�v

ð1Þ

However, the H218O/H

2

16O ratio of water vapor (v) is less than its liquid sourcedue to kinetic and equilibrium isotope fractionation during evaporation(Harwood et al., 1998). Applying the standard Craig-Gordon model of evap-oration (Farquhar et al., 1989):

Re ¼ aþðakRsð12 hÞ þ RvhÞ ð2Þwhere Re is the isotopic ratio at the site of evaporation, Rs is the isotopic ratio ofthe source of water (here, xylem),Rv is the isotopic ratio of the water vapor, a+ isthe equilibrium isotope fractionation at the leaf temperature (1.009; Majoube1971), ak is the kinetic isotope effect for the evaporation of water from the leafinto dry air (1.032; Cernusak et al., 2016), and h is relative humidity.

We initiated themeasurement of the light response after 5 to 6 h atmaximumillumination, when the d18O of transpired water reached an asymptotic value.We assumed that the leaf was at steady state, and so the water entering by thepetiole is equivalent to the water exiting the leaf, in this case, Rs = Rv.

We further assumed that the water at the site of evaporation is a goodrepresentation of bulk leaf water, such that Re = Rleaf, where Rleaf is the isotopicratio of leaf water. Here, the 18O/16O ratio of H2O will be represented as�

H218O

H216O

�, and so Rv =

�H2

18OH2

16O

�v

and Rleaf =�

H218O

H216O

�L

Equation 2 can nowbe rearranged and solved for the ratio ofH218O/H

2

16O inthe evaporating liquid (leaf water) as a function of the ratio in the vapor, thekinetic and equilibrium O isotope fractionations, and the humidity:

�H2

18OH2

16O

�L¼�H2

18OH2

16O

�vaþðak þ ð12akÞhÞ ð3Þ

(H218O/H

2

16O)v is the ratio of water vapor measured by the CRDS in the airexiting the chamber. This equation shows that the H2

18O/H216O of the liquid

will be 1% to 2% higher than the ratio in the vapor, regardless of whether waterentering the petiole is labeled. When calculating the gross rate of photosyn-thesis, this leaf water enrichment is taken into account.

The basicmass balance equation for O2 and itsmajor isotopologues (16O2 and18O16O) flowing through the cuvette is:

FO2 out 2 FO2 in ¼ GOP2U16O2ð4Þ

where GOP is gross O2 production and U is O2 uptake by respiration, photo-respiration, and all other processes. To distinguish this flux from individualisotope fluxes, the production of each isotopologue will be represented by P.

An analogous equation can be written for 16O2:

F16O2 out2 F16O2 in

¼ P16O2 in2U16O2

ð5Þ

In calculating O2 fluxes, we consider only two isotopologues, 16O2 and18O16O.

The ratio of 18O16O/16O2 is nominally 1:250. We do not consider 18O2 because itsabundance is so low (18O2/

16O2 ; 1:250,000). Equation 5 can be expanded to:

F18O16Oout2 F18O16Oin

¼ 2�H2

18OH2

16O

�LP16O2

2

�18O16O16O2

�cuv

UO2aU ð6Þ

The factor of 2 in the first term on the right-hand side reflects the fact that an O2

molecule has two chances to acquire an 18O atom from water splitting. Thesubscript cuv indicates ambient air inside the cuvette. The term aU representsthe isotope effect associated with respiration and all other uptake processes(photorespiration, alternative pathway, Mehler reaction.). Calculated fluxesare essentially insensitive to aU (0.982), because the d18O of photosynthetic O2

differs from that of ambient O2 more than the d18O of O2 consumed by respi-ration.

Solving Equation 5 for U16O2, substituting into Equation 6, and solving forP16O2 gives:

P16O2¼

DF18O16O 2�

18O16O16O2

�aUDF16O2

2�H2

18OH2

16O

�L2�

18O16O16O2

�cuv

aU

ð7Þ

In Equation 7, H218O/H

2

16OL = (H218O/H2

16OVSMOW)(1 + d18OL-VSMOW/10

3) and18O16O/16O2cuv = 2(H2

18O/H216OVSMOW)(1 + d18Oair-VSMOW/10

3)(1 + d18Ocuv-air/103). The subscripts air and cuv refer to O2 in ambient air and air within thecuvette, respectively, as above. VSMOW indicates that the isotopic compositionis expressedwith Vienna StandardMeanOceanWater as the reference.DF is theflux out of the chamber minus the flux into the chamber. This flux is the flux of 16

O2 or18O16O out of the chamber minus the flux in. These fluxes are calculated

from the change in 16O2/14N2 and

18O16O/14N2 as air passes through the cuvette,measured by the IRMS. In calculating these fluxes, we assume that the N2

concentration of entering and exiting air is the same, the O2 mixing ratio of air is0.0201784, and the 18O16O/16O2 ratio of ambient air is 0.0040104.

Considering that (1 + 18O/16O)2 is very close to 1, the production of 18O2 (andother isotopologues) is negligible and the GOP is:

GOP ¼ P16O2

"1þ 2

H2

18OH2

16O

!L

#ð8Þ

NOP is simply the sumof the outgoingminus incomingfluxes of 16O2 (Eq. 5) and18O16O (Eq. 6):

NOP ¼ DF16O2þ DF18O16O ð9Þ

The difference between outgoing and incoming fluxes of 16O2 is determinedusing dO2/N2 (see below). Net O2 uptake is the difference between GOP andNOP (GOP 2 NOP).

Mass Spectrometric Measurements of GOP and NOP

TodetermineGOPandNOP,weusedaThermoDeltaplusXP IRMS (ThermoFinnigan) configured to simultaneouslymeasuremasses 28, 29, 32, 33, 34, 38, 40,and44. The changeover valveof themass spectrometerwas connected to thegas-exchange system using two 25-mm-i.d. glass capillaries (SGE Analytical Sci-ence). The connections of the capillaries with the chamber and the changeovervalve were secured with vespal ferrules (Agilent Technologies). To equalizesample and reference side ion currents, the capillary length was set to 149 cm onthe sample side and to 145 cm on the reference side. Each measurement con-sisted of a block of 16 to 25 cycles (reference/sample/reference changeovers).


Gauthier et al.




For each cycle, dO2/N2 (32/28) and d18O of O2 (34/32) were calculated from thedifference in isotope ratios of air leaving and entering the cuvette. Both ele-mental and isotopic ratios were expressed in the standard d notation as:

d�‰� ¼ 1000

�Ratiosample

Ratioreference2 1�

ð10Þ

Ratiosample and Ratioreference are the measured ion current ratios of 16O2/14N2 and

18O16O/16O2.

Light Response Curves

Light response curves of net CO2 assimilation, NOP, andGOPwere obtained bydecreasing PARmeasured at the top of the leaf from high light to darkness. Typicaldata collected from an experiment at 21% O2 are presented in Figure 1, based onresults from 10 decreasing light levels (1,100, 800, 500, 200, 100, 80, 70, 60, 40, and0 mmol m22 s21). Similar experiments were done using air containing 2% O2 tominimize photorespiration. For the 2%O2 experiments, maximal light intensity wasreduced to 700mmolm22 s21 andfluxesweremeasuredat agreater number of lowerlight intensities (900, 700, 420, 300, 150, 100, 90, 80, 70, 60, 40, 35, and 0mmolm22 s21).Typical data collected from an experiment at 2%O2 are presented in Figure 1. Lightresponse curvedatawerefittedwith thenonrectangular quadratic functionofÖgrenand Evans (1993) using the curve-fit routine of SciPy (Oliphant, 2007; Millman andAivazis, 2011; van derWalt et al., 2011). This routine uses nonlinear least squares forthe specified functional form.

Mesophyll Conductance

gm was evaluated using the common approach of adopting a single resis-tance (gm) to characterize the transport of CO2 between the intercellular airspaceand the site of carboxylation (Tholen et al., 2012). gmwas calculated using the O2equations developed by Renou et al. (1990). O2 uptake (U) is given by the sum ofthe rate of Rubisco oxygenation (Vo), the rate of oxygen consumption by theoxidation of glycolate during the C2 cycle (0.5Vo), andmitochondrial respirationand other processes (Mehler reaction, plastoquinone terminal oxidase.; des-ignated as Ro):

U ¼ Vo þ 0:5 Vo þ Ro ð11ÞA similar equation was developed from Farquhar et al. (1980) for CO2 assimi-lation:

Anet ¼ Vc 2 0:5Vo 2 Rc ð12Þwhere Vc is the velocity of carboxylation and Rc is the rate of decarboxylation inthe light associated with mitochondrial respiration. Solving Equation 11 for Vo,combining with Equation 12, and solving for Vc gives:

Vc ¼ Anet þ ðU2RoÞ3

þ Rc ð13Þ

In addition, Farquhar and von Caemmerer (1982) defined Cc as:

Cc ¼ 2G∗�Vc

Vo

�ð14Þ

G* is the CO2 compensation point in the absence of respiration in the light. Bycombining Equations 13 and 14, we obtain:

Cc ¼ G�ð3Anet þU2Ro þ 3RcÞðU2RoÞ ð15Þ

The limitation of photosynthesis through gm can be written, following Fick’slaw, as:

Cc ¼ Ci 2Anet

gmð16Þ

And so:

gm ¼ AnetðU2RoÞCiðU2RoÞ2G∗ð3Anet þU2Ro þ 3RcÞ ð17Þ

Here, G* was not measured but assumed to be 42.75 mmol mol21 at 25°C, asestimated by Bernacchi et al. (2001). A standardized Arrhenius function and

assumed activation energy of 37.8 kJmol21 were used to account for the effect oftemperature on G*, as in Medlyn et al. (2002).

In the case where Rc = Ro, as assumed by Renou et al. (1990), Equation 17 canbe simplified as in Flexas et al. (2012):

gm ¼ AnetðU2RÞCiðU2RÞ2G∗ð3Anet þU þ 2RÞ ð18Þ

where R is respiration rate in the light.

Determining Terms Needed to Calculate gm

The rate of respiration in the light (R) was determined by extrapolating the linearportion of the Anet versus irradiance curve to zero irradiance (Kok, 1948). It is im-portant to note that this extrapolation assumes that it is possible to extrapolate fromconditions where Cc , Ca, above the break point in the light response curve, toconditions where Cc . Ca, below the light compensation point. This approach toassessing the respiration rate in the light was challenged recently (Farquhar andBusch 2017; but see Buckley et al., 2017) but is provisionally retained.

The zero-irradiance intercepts of the light response curves of NOP werecalculated fromnetC assimilation ratesmeasured between 40 and 110mmolm22

s21. These values were then equated to both Ro and Rc. To account for the de-pendence of Anet or NOP on changes in internal CO2 concentration, Ro and Rcwere recalculated according to Kirschbaum and Farquhar (1987). This correc-tion uses an estimate of the rate of ribulose 1,5-bisphosphate regeneration, Vj,calculated from Anet or NOP. It constrains Rc and Ro when the intercept of thelinear regression of Vj versus light is zero. Uncorrected values are reported asRo-apparent and Rc-apparent. Corrected values are reported as Ro-correctedand Rc-corrected. Ro-corrected and Rc-corrected values were used in Equations11 and 12 and in all further calculations. The parametersVj and Ro were used tocorrect NOP for variable Ci; Vj and Rc were used to correct Anet.

Typical Experimental Protocol

Before and during each experiment, the reference gas (incoming air) was an-alyzed against itself to assess the magnitude and drift in the zero enrichment(shaded areas in Supplemental Figs. S6 and S7). The zero enrichment is the arti-factual difference in the composition of incoming and outgoing air when there isno source or sink for O2 within the chamber. Leaves were initially placed in thedark for 30min. The light intensitywas then raised tomaximize transpiration andaccelerate leaf labeling. Leavesweremaintained in this condition until d18O of thewater vapor reached isotopic steady state. At this point, the d18O of transpiredwater leaving the chamberwas constant (Supplemental Figs. S6 and S7D), and themeasure of O2 and CO2 fluxes began, alongwith their light response, as describedabove. For the duration of an experiment, leaves were exposed to either 21% O2(Supplemental Fig. S6) or 2% O2 (Supplemental Fig. S7) in air. Air temperaturewas maintained at 20°C 6 1°C. Averaged leaf area was 46.23 6 1.3 and 81.06 62.44 cm2 at 21% O2 and 2% O2, respectively. Average flow rate was measured at2.47 6 0.1 and 2.64 6 0.1 L min21 at 21% O2 and 2% O2, respectively, under ourtypical conditions (1 atm pressure and 20°C 6 1°C).

Comparative Estimates of Photosynthetic Properties

Alternative formulations from the literature were used to calculate Jg and gmin order to evaluate the accuracy of our calculations from GOP measurements.The theoretical electron transport rate, Jg, was calculated as by Flexas et al.(2012; Eq. 12.18):

Jg ¼ ðAþ RdÞ4ðCc þ 2G∗ÞCc 2 G∗ ð19Þ

Cc was arbitrarily calculated from ambient CO2 exiting the chamber, Ca, as Cc =0.4 Ca following Roupsard et al. (1996).

To determine the accuracy of gm, theoretical gm was calculated usingEquation 7 fromHarley et al. (1992), which considers a variable J (for review, seeFlexas et al., 2012, and refs. therein):

gm ¼ Anet

Ci 2G∗ðJaþ8ðAnetþRcÞÞJa 2 4ðAnetþRcÞ

ð20Þ

Usually, Ja is obtained indirectly using fluorescencemeasurements. In the absenceof such measurements, we assumed Ja = 4*GOP, as in von Caemmerer (2000),









since four electrons are produced for each molecule of O2 released by PSII. Thisestimate of gm is referred to as gm Harley (Supplemental Figs. S2 and S3).

Statistical Analysis

Linear regressionswereused todetermine the linearity in the light responseofNOP, GOP, and Anet. These regressions also were used to calculate the Kokeffect. Uncertainties correspond to a confidence interval of 95%. Linear re-gressions also were used to determine the correlation of O2 versus CO2. Inde-pendent Student’s t tests were used to assess the statistical difference betweendark respiration and RL values and between RL values from Anet and NOP.Differences between means were considered significant at p-value , 0.05 andhighly significant at p-value , 0.01.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. The variation of Ca and Ci with light intensity forFrench bean leaves exposed to 21% O2 and 2% O2.

Supplemental Figure S2. Correlation between gm calculated using the O2method and theoretical gm calculated using the variable J method ofHarley et al. (1992).

Supplemental Figure S3. gm versus irradiance for French bean leavesexposed to 21% O2.

Supplemental Figure S4. dO2/N2, NOP, d18O, and GOP for each of threereplicate incubations versus irradiance for French bean leaves exposed to21% O2.

Supplemental Figure S5. dO2/N2, NOP, d18O, and GOP for each of threereplicate incubations versus irradiance for French bean leaves exposed to2% O2.

Supplemental Figure S6. Measurements for a French bean leaf exposed to21% O2.

Supplemental Figure S7. Measurements for a French bean leaf exposed to2% O2.

ACKNOWLEDGMENTS

We thank Guillaume Tcherkez, Joseph Berry, and Graham Farquhar forcommentaries on an early version of this article. We also thank the anonymousreviewers for their valuable comments on the article, Robert Stevens formachining the aluminum chamber, and Karina Graeter for assisting with itsdesign. P.P.G.G. thanks Jordan Lubkeman for technical assistance.

Received May 9, 2016; accepted March 5, 2018; published March 27, 2018.

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