Relationships Between Carbonyl Sulfide (COS) and CO2 During Leaf

New Phytologist Research

Relationships between carbonyl sulﬁde (COS) and CO2 during leaf gas exchange

Keren Stimler1, Stephen A. Montzka2, Joseph A. Berry3, Yinon Rudich1 and Dan Yakir1 1Environmental Sciences and Energy Research, The Weizmann Institute of Science, Rehovot, Israel; 2NOAA-ESRL, Boulder, CO 80305, USA; 3Department of Global Ecology, Carnegie Institution of Washington, Stanford, CA 94305, USA

Summary

Author for correspondence: • Carbonyl sulﬁde (COS) exchange in C3 leaves is linked to that of CO2, providing Dan Yakir a basis for the use of COS as a powerful tracer of gross CO2 ﬂuxes between plants Tel: + 972 8 9342549 and the atmosphere, a critical element in understanding the response of the land Email: [email protected] biosphere to global change. Received: 2 December 2009 • Here, we carried out controlled leaf-scale gas-exchange measurements of COS Accepted: 25 January 2010 and CO2 in representative C3 plants under a range of light intensities, relative

humidities and temperatures, CO2 and COS concentrations, and following abscisic New Phytologist (2010) 186: 869–878 acid treatments. doi: 10.1111/j.1469-8137.2010.03218.x • No ‘respiration-like’ emission of COS or detectable compensation point, and no

cross-inhibition effects between COS and CO2 were observed. The mean ratio of ) COS to CO assimilation flux rates, As ⁄ Ac, was c. 1.4 pmol lmol 1 and the leaf Key words: biosphere–atmosphere 2 relative uptake (assimilation normalized to ambient concentrations, (As ⁄ Ac) exchange, carbonyl sulfide (COS), gross CO2 c s exchange, leaf gas exchange, stomatal (Ca ⁄ Ca )) was 1.6–1.7 across species and conditions, with significant deviations control. under certain conditions. Stomatal conductance was enhanced by increasing COS,

which was possibly mediated by hydrogen sulﬁde (H2S) produced from COS hydrolysis, and a correlation was observed between As and leaf discrimination against C18OO. • The results provide systematic and quantitative information necessary for the use of COS in photosynthesis and carbon-cycle research on the physiological to global scales.

Introduction considered the major sinks. Arguments for the lack of any significant secular trend in atmospheric COS concentra- Carbonyl sulfide (COS), is the most abundant sulfur-con- tion are consistent with a balance between sinks and taining gas in the troposphere with an average global mean sources (Chin & Davis, 1993; Watts, 2000; Kettle et al., ) mixing ratio of c.500 ± 100 pmol mol 1 (Montzka et al., 2002). However, recent improved measurements, such as 2007). Because of its long tropospheric lifetime, estimated in Antarctic firn air and air trapped in ice (Montzka et al., at 2–4 yr (Montzka et al., 2007; Suntharalingam et al., 2004), indicate contributions of anthropogenic sulfur 2008), it is transported into the stratosphere, and as a emissions over time. result of subsequent photolytic oxidation it is involved in Notably, the new measurements of COS and COS ⁄ CO2 the formation of stratospheric aerosol (Crutzen, 1983; ratios during the seasonal cycle in background atmospheric Chin & Davis, 1993; Kjellstrom, 1998). Most of the main measurements and modeling studies (Montzka et al., 2004, components of the global COS budget are known, but 2007; Blake et al., 2008; Campbell et al., 2008; Sunthara- uncertainties remain regarding the precise fluxes and sink ⁄ - lingam et al., 2008) show a large seasonal cycle, consistent source magnitudes involved. Emissions from the oceans, with that of the land biosphere photosynthetic activity anthropogenic emissions and biomass burning are believed (GPP). This clearly indicates the existing potential of COS, to be the major sources of atmospheric COS. Uptake by beyond the sulfur cycle and aerosol chemistry, as a new tra- terrestrial vegetation and soils, and reaction with OH are cer of photosynthesis, and the need for more research into

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processes associated with COS flux into plants, as per- although vegetation can produce dimethyl sulfide, which formed in the present study. can be rapidly oxidized to COS (Geng & Mu, 2004). Carbonyl sulfide uptake in plants and soil is likely domi- To date, quantitative aspects of the coupling of COS and nated by its reversible hydration, resulting in the formation CO2 exchange by plants are still uncertain and little is of H2SandCO2: known about the effects of various environmental parameters on these relationships. Such information, together with þ COS þ H2O $ HCOOS þ H $ H2S þ CO2 Eqn 1 new measurement technologies (Stimler et al., 2009), would help open the way for laboratory and field explora- tions of COS fluxes as a powerful new tracer for CO2 gross ) Under natural conditions, HCOOS and H2S are exchange in the land biosphere. The objective of this study greatly diluted in plant and soil water and the formation was to provide systematic, high-precision data on the rela- of H2S is exergonic (Schenk et al., 2004), making the tionships between COS and CO2 exchange in C3 leaves reverse reaction unfavorable. Hydration therefore repre- under controlled but variable conditions. sents an essentially one-way process for COS uptake. Note that any CO molecule taken up by leaves or wet soils 2 Materials and Methods undergoes similar, but fully reversible, hydration-dehydra- tion. In leaves, the reaction in Eqn 1 is dependent on Plant material catalysis by carbonic anhydrase (CA), because of the short residence time of COS or CO2 inside leaves (usually The C3 plant species, indigo spires sage (Salvia longispicata < 1 s). This dependence on CA activity has been demon- Martius Galeotti · Salvia farinacea Benth), hibiscus (Rosa 18 strated for CO2 in leaves based on O measurements sinensis Linn) and pepper (Capsicum annuum (L) Kuntze) (Gillon & Yakir, 2001). It has also been shown for COS were used in all experiments. The first two species were pur- using CA inhibitors in soils (Kesselemier et al., 1999), but chased in local nurseries and the last one was grown from the dependence on CA activity is not well quantified. seed in the glasshouse. All plants were kept in a glasshouse Enzymes other than CA, notably Rubisco (Lorimer & during the experimental period at c.25C under natural Pierce, 1989), can also consume COS, but their activities light. are very low compared with CA. Interestingly, it has been argued that CA’s affinity to COS is, in fact, greater than Leaf gas-exchange measurements its affinity to CO2 (Protoschill-Krebs et al., 1996). In plants, the correlations of COS fluxes with those of The experimental system consisted of a flow-through leaf CO2, as well as with water vapor, and photosynthetic active cuvette made of Teflon-coated stainless steel with a magnet- radiation (PAR), have been interpreted as evidence of sto- ically operated fan and a glass window at the top. A whole matal control over COS-uptake rates, which is supported leaf or branch was sealed in the cuvette (O-ring seal except by the daily pattern of this flux (Bartell et al., 1993; Hof- around the petiole, which was sealed with high-vacuum man, 1993; Kuhn et al., 1999; Xu et al., 2002). The depo- putty). If not otherwise indicated, measurements were sition velocity (flux normalized to concentration) for COS performed under a relative humidity (RH) of c. 70% and is greater than that for CO2 in most species. The mean ratio an air temperature of c. 25C. Light intensity was 55– ) ) of the deposition velocities at the leaf level is c. 3 (Sandoval- 1889 lmol photon m 2 s 1, regulated with layers of mira- Soto et al., 2005), but at the ecosystem level, the observed cloth and filtered through 5 cm water. Airflow was passed ratio ranges between 1 and 10, which is not well constrained through a magnesium perchlorate drying trap (Sigma- or understood (Xu et al., 2002; Geng & Mu, 2004; Sand- Aldrich) for drying before sampling in 180 ml flasks for oval-Soto et al., 2005; Montzka et al., 2007). Recently, the analysis of COS and isotopic composition of CO2. For Campbell et al. (2008) expressed these ratios as a leaf scale abscisic acid (ABA) treatments, intact leaves were immersed ) relative uptake (LRU), the ratio of the uptake of COS and in 10 6 M ABA solution (c. 98%, Sigma-Aldrich) and CO2 normalized to their respective ambient concentrations. 0.01% (w ⁄ v) Tween 20 (Fluka Chemie, Steinheim, Ger- Their findings point to a LRU of 2.2, compared with an many) as the surfactant for 20 min before measurements. ) ecosystem scale relative uptake of 5.7 (which includes eco- Synthetic air was purified by passing it (at 5 l min 1) system respiration). The overall flux from the atmosphere through a hydrocarbon filter (Super-Clean; Restek, Belle- to the leaves and soil is a diffusional flux that also depends fonte, PA, USA). COS and CO2 mixing ratios were adjusted on the ambient atmospheric COS concentrations. Some to the desired values by mixing the purified synthetic air measurements have also shown the existence of a compensa- with known gas mixtures produced from a permeation tion point for COS exchange, which seems to indicate a bal- device with a COS permeation tube (VICI Metronics, ance between uptake and emission (Kesselemier et al., 1999; Poulsbo, WA, USA), and a 1% CO2-pressurized cylinder, Xu et al., 2002). Little detail is available on these aspects, followed by a three-stage dilution system. All flow rates

New Phytologist (2010) 186: 869–878 The Authors (2010) www.newphytologist.com Journal compilation New Phytologist Trust (2010) New Phytologist Research 871 were regulated and measured by mass-flow controllers trapped using helium as the carrier gas. It was then passed (MKS, Andover, MA, USA). through a Carbosieve G packed column at 70C to separate N2O, and the eluted CO2 was analyzed on a Europa 20-20 continuous-flow isotope ratio MS (Europa Scientific Crewe, CO and COS analysis 2 UK). Batches of 15 flasks at a time were measured from an Carbon dioxide and H2O concentrations in the air entering automated manifold system, with five flasks of a standard and leaving the leaf cuvette were measured by infrared gas gas being measured for every 10 samples. Precision of the analyzer (Li-6262; Li-Cor, Lincoln, NE, USA), at a preci- measurements was ± 0.1 and ± 0.2‰ for 13C and 18O, )1 sion better than 1 lmol mol for CO2 and 0.1 mmol - respectively. Results are expressed in the small delta nota- )1 13 18 mol for water vapor. CO2 concentrations were based on tion (d‰) vs VPDB and VPDB-CO2 for C and O, periodical intercomparison of flask air samples with the respectively, where d%=(R ⁄ Rs –1)· 1000, and R and Rs NOAA Earth System Research Laboratory (Boulder, CO, are the isotope ratios of the sample and the appropriate USA). COS concentrations were determined by a GC-MS standard, respectively. Instantaneous leaf discriminations, (Varian model SATURN3) following an automated cryo- D, were calculated as described by Evans et al. (1986) for genic trap (normally c. 40 s trapping time, longer for low both 13C and 18O (Gillon & Yakir, 2000). The isotopic concentrations). The precision of the COS analysis was composition of CO2 in air supplied to the leaves during the ) 2.5% (< 14 pmol mol 1) at ambient atmospheric concen- experiments was c. d13C=)25% and d18O=)27%. trations. Leaf area was estimated by tracing leaves on paper and weighing the traces at the end of each experiment. Results and Discussion Photosynthetic parameters were calculated according to Von Caemmerer & Farquhar (1981). As for CO , COS- 2 COS uptake uptake rates were calculated based on the concentration difference between the inlet and outlet of the leaf cuvette, The potential use of COS as a tracer for gross CO2 uptake the flow rate and the leaf area. by leaves is associated with three basic assumptions: COS Conductance to CO2 was estimated from conventional co-diffuses with CO2 from the atmosphere through stomata gas-exchange measurements (Von Caemmerer & Farquhar, to the chloroplast along the same pathway; COS is not asso- 1981) and ‘online’ stable isotope measurements (Evans ciated with any respiration-like emission; and the two co- et al., 1986; Gillon & Yakir, 2000). Similarly, estimated diffusing species, CO2 and COS, do not interact (directly s leaf conductance to COS (gl ) was based on the leaf conduc- or indirectly). All three assumptions are well supported by tance to water vapor: the results presented here, but some exceptions were also noted. 1 g s ¼ Eqn 2 The importance of stomata in COS uptake could be l 1:94 þ 1:56 gsw gbl clearly seen in our experiments, as has been shown previously (Kluczewski et al., 1985; Goldan et al., 1988). First, in the )2 )1 where gsw and gbl (mol m s ) are the stomatal and dark when stomatal conductance, gs, is minimal, the rates of boundary layer conductance to water vapor, respectively, COS uptake were low (Table 1). Even under high ambient )1 1.94 is the COS ⁄ H2O binary gas phase diffusivity ratio COS concentrations of c. 3000 pmol mol , COS uptake in according to Fuller et al. (1966), yielding a COS ⁄ CO2 dif- the dark by sage, pepper and hibiscus was 1–17% as large as ) ) fusivity ratio of 0.827, and 1.56 is the ratio for the leaf the rates measured when light intensity was 500 lmol 2 s 1. boundary layer based on the two-thirds power effect in The residual uptake in the dark likely reflects incomplete sto- going from stagnant air to the laminar boundary layer, as matal closure. Conductance in the dark ranged between s )6 )5 )2 )1 done for CO2 (Farquhar & Lloyd, 1993). Ci could then be 6.4 · 10 and 1.8 · 10 mol m s . Second, a signifi- s s calculated from A and the estimated leaf conductance, cor- cant co-reduction in gs and COS-assimilation rate, A , was rected for the effects of the evaporation flux, also as conven- observed in the presence of light following leaf fumigation tionally done for CO2. with ABA. Reductions in gs were 91.4 ± 0.02 and 67.5 ± 0.03% in sage and hibiscus, respectively, leading to a propor- ) tional reduction in As from 80 ± 22 to 16 ± 0.7 pmol m 2 Isotopic analysis of CO ) ) ) 2 s 1 and from 93 ± 3 to 24 ± 0.7 pmol m 2 s 1 in these Carbon and oxygen isotopic analysis of CO2 was based on plants, respectively, consistent with previous results sampling CO2 in the air entering and exiting the leaf cuv- (Mawson et al., 1981; Sandoval-Soto et al., 2005). s ette by passing it through a 180 ml glass flask. The CO2 in For leaves not treated with ABA, a linear increase in A , s the flask was measured as described in Klein et al. (2005). as a function of ambient COS concentration, Ca , was Briefly, an aliquot of 1.5 ml was removed from each flask observed across species and across a wide range of concen- into a sampling loop and the CO2 was cryogenically trations (Fig. 1a). The apparent compensation point (ambi-

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s w Table 1 Mean rates of carbonyl sulﬁde (COS) assimilation (A ) and stomatal conductance (gs ) in three C3 species under dark and light ) ) (500 lmol photon m 2 s 1) conditions

Dark Light

s )2 )1 w )2 )1 s )2 )1 w )2 )1 s s Species A (pmol m s ) gs (mol m s ) A (pmol m s ) gs (mol m s ) A (dark) ⁄ A (light)

Rosa sinensis 0.5 ± 5.9 1.8E–05 71.3 ± 3.7 0.238 0.01 Capsicum annuum 15.9 ± 10.4 1.1E–05 93.7 ± 8.6 0.325 0.17 Salvia indigo spires 0.9 ± 4.1 6.4E–06 113.0 ± 13.4 0.395 0.01

Values for As are means ± SD. )1 )1 Experiments were conducted under CO2 and COS ambient concentrations of c. 380 lmol mol and c. 3000 pmol mol , respectively. ent COS concentration at zero uptake) estimated from the enhanced at high COS mixing ratios. As shown in Fig. 1(c), s best-fit line to data compiled from different experiments gs was enhanced by a factor of c. 3 by increasing Ca to four (r2 = 0.99, P < 0.0001) indicated an overall low mean of times typical ambient atmospheric concentrations. The ) 60.7 pmol mol 1 (statistically not different from zero), and response was consistent and linear for all three species stud- w 2 an average CO2 compensation point for the individual ies (gs = 0.0003[COS] + 0.07; r = 0.9, see Fig. 1c). ) experiments of 38.2 ± 1.6 lmol mol 1. Note that possible These observations are in fact consistent with an early report effects of soil COS transported via the xylem, as observed by Goldan et al. (1988), in which exposing leaves to low )1 for CO2 (Aubrey & Teskey, 2009) are likely negligible and high COS concentrations (100 and 700 pmol mol ) because of the essentially one-way hydration of COS to induced reduced leaf resistance by a factor of 2.8–5.5 at the H2S (see the Introduction section). higher COS concentration. While these values are associated with the regression sta- A possible mechanism for the stomatal response to COS tistics, the more direct approach of exposing the different is a H2S effect. H2S is produced from COS in the hydration plant species to COS-free air under illumination of reaction (Eqn 1) and while part of this product is converted )2 )1 1170 lmol photon m s and gs values ranging between to organic sulfur and used for protein synthesis (e.g. cyste- ) ) 0.42 and 0.64 mol m 2 s 1 showed no detectable COS ine; Rennenberg, 1984), the rest could accumulate in the emissions from any of the studied species. Previous reports leaves and eventually be emitted to the atmosphere (consti- )1 noted COS compensation points of 50–320 pmol mol tuting a significant global source of H2S; Watts, 2000). based on somewhat different methodology (Kesselemier & Some early studies reported a possible stomatal response to Merk, 1993,, but see also reanalysis in Seibt et al. (2009). H2S (Coyne & Bingham, 1978; Unsworth & Black, 1981). The results presented here therefore appear to confirm the According to those studies, at low concentrations of H2S, hypothesis that COS uptake in leaves is not associated with plant membranes may weaken, causing subsequent loss of any respiration-like emission. turgor in some epidermal cells and leading to stomatal As could be expected, the calculated average of intercellu- opening, similar to the effects of SO2 (Coyne & Bingham, s s lar-to-ambient concentration (Ci ⁄ Ca ) ratios for the C3 spe- 1978). cies used here under different conditions also showed a The observed gs response to COS clearly needs to be s linear correlation with Ca (Fig. 1b). In turn, these relation- further researched to establish the underlying mechanism ships indicated that under atmospheric concentrations of and its relevance under ambient conditions. We note, s COS, its intercellular concentrations (Ci ) in the leaf are however, that previous studies have indicated an c. 25% s s rather low and the mean Ci ⁄ Ca ratio in this case was increase in gs in response to exposure to H2S at a concen- )1 0.22 ± 0.12. Note that under COS concentrations three tration of 740 nmol mol (and reduced gs at concentra- )1 times higher than typical atmospheric values, Ci ⁄ Ca for tions of 3250 nmol mol ; Coyne & Bingham, 1978), COS reached 0.63 ± 0.13, indicating a significant retro- while atmospheric concentrations of H2S are only c. ) diffusion flux out of the leaves proportional to internal 7 pmol mol 1 (Watts, 2000). Considering that the COS concentrations and leaf conductance. The highly linear in the chloroplasts is converted to H2S and most of it dif- response shown in Fig. 1(b) could therefore be useful in fuses back to the substomatal air spaces, H2S concentra- s )1 s permitting the use of high-Ca conditions for calculating tions of c. 10–20 pmol mol (based on observed A leaf internal conductance (gm) to COS (see the section values and estimated internal conductance, as discussed ‘Co-diffusion of COS and CO2’). later) can be produced, potentially raising the concentration somewhat above ambient values. Nevertheless, if most of the continuous flux of COS taken up by the Stomatal response leaves is counteracted by a similar emission flux of H2S, While stomatal control of COS uptake seems unequivocal, its concentrations near the leaf surface could build up an unexpected effect was observed: gs was found to be under certain conditions (e.g. low turbulence, low OH)

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Co-diffusion of COS and CO2

Carbonyl sulfide and CO2 co-diffuse from the atmosphere into the chloroplasts of leaves, where both gas species undergo hydration catalyzed by CA. This is the terminal step for COS, which is converted to H2S with a strongly unfavorable reverse reaction, but it is a fully reversible step for CO2, allowing it to then be fixed by Rubisco. While the pathway for the two gases from the atmosphere to the site of CA is identical, there are chemophysical differences between the two gas species that influence their diffusion and reaction rates (see the Materials and Methods section). The diffusion steps from the atmosphere through a series of restricted conductance steps include conductance through the leaf boundary layer, gbl, the stomata, gs, and the mesophyll, gm (Fig. 2). The latter can be further divided into three additional steps associated with dissolution: gds, liquid-phase diffusion, gdf, and biochemical ‘conductance’, gbc. The drop in COS concentration from the intercellular spaces to the site of hydration is influenced by the internal s )1 ‘conductance’ gm (where gm =(1⁄ gds+1 ⁄ gdf+1 ⁄ gbc) ). The rate of CA’s reaction with COS at the very low concentrations used in this study is likely to be approximately proportional to the COS concentration at the site of the reaction (i.e. its partial pressure). We do not have sufficient information at this time to separate this from other limitations resulting from dissolution and diffusion of COS from the intercellular air spaces to its site of reaction. For the purpose of this study, we combine all of these steps into a single apparent conductance, gm, which can be evaluated as a residual from our gas-exchange analysis:

s 1 gm ¼ s Eqn 3 Ca 1 1 s : = w þ : = w A 1 94 gs 1 56 gbl

Using data from the four highest light intensities in the Carbonyl sulfide (COS) uptake rates (As) (a), ratio of COS Fig. 1 light-response experiments, we obtained gm values s s ) ) intercellular and ambient concentrations, expressed as Ci ⁄ Ca (b), (mol m 2 s 1) of 0.26 ± 0.21 (n = 27, range 0.11–0.70) and stomatal conductance (gs) (c), as a function of COS ambient s for sage and 0.30 ± 0.21 (n = 10, range 0.02–0.60) for concentration (Ca ). Data compiled from light response experiments in (b) and from different COS response experiments, under an ambi- hibiscus, and an overall average for all of our experiments of )1 ent concentration of c. 380 lmol mol CO2 and 1179 lmol pho- 0.27 ± 0.21. This estimate is similar to the more empirical ) ) ton m 2 s 1 light intensity, in (c). Inset: data from experiments one in which we estimated As (Eqn 3 rearranged) by simply conducted across a wider range of COS mixing ratios in C3 plants. ‘tuning’ for a mean gm value that provides the best fit to the C3 species used were Salvia indigo spires, Capsicum annuum and s observed A values (Fig. 3). In this case, the mean gm value Rosa sinesis. (panel a is S. indigo spires only). ) ) was 0.30 mol m 2 s 1. For CO2, the concentration at the site of the enzymes CA and potentially affect gs to a significant extent. Note that and Rubisco in the chloroplasts, Cc, was estimated from s while COS effect on gs can help explain increasing Ci ⁄ online stable isotope analysis of the CO2 during gas s s c Ca , as in Fig. 1(b), this should not affect LRU or A ⁄ A exchange (see the Materials and Methods section; Evans since, irrespective of cause, any changes in gs influence et al., 1986; Farquhar et al., 1989). The total internal con- both CO2 and COS (see the section ‘Leaf relative uptake ductance (gm) was then estimated from the measured flux, c ) of COS vs CO2’). A , and the concentration gradient Ci Cc.

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)1 )1 Fig. 2 Schematic representation of the resistance path to diffusion of both carbonyl sulﬁde (COS; pmol mol ) and CO2 (lmol mol )inC3 leaves. Values are estimated from mean values in light response measurements in Salvia indigo spires. Rates of COS and CO2 net assimilation ) ) ) ) ) ) were 20 pmol m 2 s 1 and 12 lmol m 2 s 1, respectively. Squares indicate concentrations; parentheses indicate conductance (mol m 2 s 1;

Eqn 2) and biochemical and dissolution limitations (Eqn 3) in the gas and liquid phase for COS. The partitioning of gm to wall conductance 18 13 (gw), and residual concentration within the chloroplast (gc) are based on stable isotope measurements of O and CofCO2.

estimate the internal conductance likely to be dominated by the cell wall and membranes, gw, and within the chloroplast, gch (see also Evans et al., 2009 for a recent review). Using the data from the light-response experiments for sage and hibiscus, we obtain gw = 0.15 ± 0.05 and 0.11 ± 0.04 and )2 )1 gch = 0.12 ± 0.003 and 0.09 ± 0.02 mol m s for the two species, respectively. The entire pathway, showing both the similarities and distinctions in the CO2 ⁄ COS diffusion steps as well as possible concentration gradients for a typical C3 leaf based on our measurements, is summarized in Fig. 2.

Leaf relative uptake of COS vs CO2 While testing the three basic assumptions noted earlier is a Fig. 3 Carbonyl sulfide (COS) uptake rates (As) derived from Eqn 3 prerequisite for the implementation of COS as a tracer for vs COS uptake rates observed during light response gas-exchange s gross CO2 uptake by leaves, our ability to predict the measurements with different C3 species. A values were estimated As ⁄ Ac ratio is required in practice. A priori, we could based on a single estimated value of COS conductance (gm) that combines the effects of dissolution and diffusion from the intercellu- expect the ratio to be relatively constant as long as gs is the lar air spaces to the reaction site. The best fit between model and primary control. But it would be equally expected that s )2 )1 observed A was obtained with a gm of 0.3 mol m s . Data were when factors other than gs are involved (e.g. humidity and taken from light-response experiments with leaf temperature at c. temperature effects on dissolution, diffusion and enzyme 24C, relative humidity of c. 70%, and CO2 and COS ambient )1 )1 reactions), the ratio will vary significantly. Fig. 4 shows concentrations of c. 380 lmol mol and c. 500 pmol mol , respectively. that, as expected, when compiling the gas-exchange data, an overall linear relationship between As and Ac was ) observed, with a mean As ⁄ Ac ratio of c. 1.4 pmol lmol 1, As proposed by Gillon & Yakir (2000), the CO2 concen- which seems quite robust across a wide range of assimila- trations at the boundary of CA activity (Ccs, e.g. at the chlo- tion rates. roplast surface) can be estimated by applying the same Note, however, that as the atmospheric concentrations of 13 18 isotopic approach used for C to analyze OinCO2. The both COS and CO2 are not constant, under either experi- additional gradients, Ci–Ccs and Ccs–Cc, are used to mental or natural conditions, the ratios of the normalized

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assimilation rates are often used. The LRU ratio (see the s c c s Introduction section) is estimated from (A ⁄ A )(Ca ⁄ Ca ). LRU as a function of light intensity, ambient COS and intercellular CO2 concentrations, RH and temperature is presented in Fig. 5. The average ratios for sage, hibiscus and pepper were 1.58 ± 0.12, 1.55 ± 0.20 and 1.74 ± 0.12, respectively, during the CO2, COS and light-response measurements, and were relatively constant across the experimental range (Fig. 5). This is probably because of the c linear response of A to [CO2] across this range. That is, c c s A ⁄ Ca is constant and A is insensitive to CO2, similar to s s the constant A ⁄ Ca across much of the COS range and c s c insensitivity of A to Ca .AtlowCa , the linear relationships break down, approaching the compensation point for CO2. In the light-response measurements (Fig. 5e), the stable Fig. 4 Relationship between assimilation rates of carbonyl sulfide s c ratios under high light probably reflect the dominance of (COS) (A ) and CO2 (A ) compiled from various gas-exchange measurements conducted with different C3 species measured under dif- the control of stomatal conductance on both CO2 and ferent environmental parameters (light and temperature, closed COS fluxes, but under low light, greater sensitivity of CO2 circles; relative humidity (RH), open circles). The linear best-fit line, assimilation (e.g. Rubisco, but not CA, sensitivity to light) excluding RH data, indicates a mean As ⁄ Ac ratio of 1.4 pmol l- ) results in a sharp increase in ratios. The temperature mol 1. Much of the scatter was contributed by data from the RH response measurements that used older plants and lower light inten- response (Fig. 5d) provides another example where the sities, with more limited photosynthetic response but no effect on LRU varies significantly, because of the differential temper- slope (linear-fit line including RH data: y = 1.41x + 8.17, r2 = 0.37, ature response of the two fluxes. While Ac peaks at c. 38C, P < 0.0001). and As at c. 25C the normalized ratio, LRU, peaks at

s c c s Fig. 5 Leaf relative uptake (LRU; (A ⁄ A )(Ca ⁄ Ca )) of carbonyl sulﬁde (COS) over CO2 under different experimental conditions in different C3 species in response to variations in ambient CO2 concentrations (a), ambient COS concentrations (b), relative humidity (RH) (c), air temperature (d) and light intensity (e). Plant species uses were: Capsicum annuum (closed circles), Salvia indigo spires (open circles) and Rosa sinesis (triangles). Two or three measurements were made for each point (SD indicated). Symbols without error bars have SD < 2%. Unless otherwise ) indicated, standard conditions maintained during experiments were as follows: temperature, c. 22C; RH, c. 70%; [COS], c. 550 pmol mol 1; )1 )2 )1 [CO2], c. 380 lmol mol ; light, 1180 (a, b, d) or 500 (c) lmol photon m s .

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c s Fig. 6 Changes in the rates of CO2 (A ) and carbonyl sulﬁde (COS, A ) assimilation, and of stomatal conductance (gs) in response to changes in )2 )1 CO2 internal concentrations (Ci), and ambient COS concentrations (under light intensity of 1180 lmol m s , air temperature of c. 24C and relative humidity (RH) of c. 70%). Plant species use were Salvia ofﬁcinalis (a, d), Capsicum annuum (b, e) and Rosa sinesis (c, f). Symbols

indicate CO2 (closed circles), COS (open circles), and gs (triangles). Symbols without error bars have SD < 2%. CO2 and COS ambient concen- ) ) trations were c. 380 lmol mol 1 and c. 500 pmol mol 1, respectively, where not otherwise indicated.

c 15C, for both sage and hibiscus. The results presented in species in Fig. 6. By contrast, at high [CO2], A continued Fig. 5 also represent a range of experiments with different to increase while As became saturated, in synchronization plants of different ages and somewhat different growing with stomatal conductance. No inhibitory effect of CO2 on conditions (even for the same species, reflecting standard As was apparent, and As was essentially constant at high s c glasshouse practices). This provides some perspective on the [CO2]. Similarly, in response to increasing Ca , A remained natural variability in LRU, irrespective of the response to essentially constant (light-saturated), while As increased ) experimental environmental parameters, for example, from nearly linearly in all species (to > 2000 pmol mol 1; a LRU of c. 1 in the plants used in the light-response mea- Fig. 6). surements, to > 3 in the plants used for the RH-response Integrating the results of this experiment across all three measurements (Fig. 5c; the latter were older plants mea- C3 species showed no apparent cross-interactions or toxicity sured in lower light intensities). Both the natural variability effects between CO2 and COS in the concentration ranges and the specific responses to environmental variables clearly used, probably since both were well below their Km values. show that LRU is far from constant. In most cases, however, This is significant because both CO2 and COS react with it may be predicted if the responses are well characterized. CA and could act as competitive inhibitors, or become toxic These LRU ratios are consistent with previous estimates in at very high concentrations (Miller et al., 1989; Goyal the range of 0.95–3.84 (Kesselemier et al., 1993; Sandoval- et al., 1991). Soto et al., 2005; cf. reanalysis of Seibt et al., 2009). 18 Link to O-CO2 Cross-interactions of COS and CO 2 Finally, we report on a preliminary exploration of the link c s s 18 At low [CO2], both A and A were well correlated with the between A and isotopic discrimination against Oin c 18 intercellular CO2 concentration (Ci ), as shown for all three CO2, D. Such a link is expected, as both processes criti-

New Phytologist (2010) 186: 869–878 The Authors (2010) www.newphytologist.com Journal compilation New Phytologist Trust (2010) New Phytologist Research 877

a strong stomatal response to COS, possibly mediated by H2S, which requires further research. More speciﬁc information is also needed on plant CA activities with respect to COS. The results provide quantitative basis for the applica- tion of COS ⁄ CO2 as a tracer in the terrestrial carbon cycle.

References

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