the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 10 Feb 2025
Isotope discrimination of carbonyl sulfide (34S) and carbon dioxide (13C, 18O) during plant uptake in flow-through chamber experiments
Abstract. Carbonyl sulfide (COS) has been proposed as a proxy for gross primary production (GPP), as it is taken up by plants through a comparable pathway as CO2. COS diffuses into the leaf and undergoes an essentially one-way reaction in the mesophyll cells, catalyzed by the enzyme carbonic anhydrase (CA), and does not exit the leaf again. In order to use COS as a proxy for GPP, however, the mechanisms of COS uptake and its coupling to CO2 uptake need to be well understood. Characterizing the isotopic discrimination of COS during plant uptake can provide useful information on the COS uptake process and can help to constrain the COS budget.
This study presents joint measurements of isotope discrimination during plant uptake for COS (CO34S) and CO2 (13CO2 and C18O16O). A C3 plant, sunflower (Helianthus annuus), and a C4 plant, papyrus (Cyperus papyrus), were enclosed in a flow-through plant chamber and exposed to varying light levels. The incoming and outgoing gas compositions were measured online, and discrete air samples were taken for isotope analysis.
The COS uptake flux was around 75 pmol mol–1 for sunflower and between 99 and 110 pmol mol–1 for papyrus. The corresponding 34Δ for COS was 3.4 ± 0.8 ‰ for sunflower and 2.6 ± 0.3 ‰ for papyrus. For CO2, a negative relationship was observed between the uptake flux and the isotopic discriminations 13Δ and 18Δ. The CO2 uptake and Δ values indicate that our sunflower behaved as expected for a C3 plant, while the papyrus was not displaying typical C4 behavior, perhaps due to the relative low light conditions during our experiments.
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The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
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Preprint
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The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
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Interactive discussion
Status: closed
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RC1: 'Comment on egusphere-2025-215', Anonymous Referee #1, 10 Mar 2025
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AC1: 'Reply on RC1', Sophie Baartman, 14 Apr 2025
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2025/egusphere-2025-215/egusphere-2025-215-AC1-supplement.pdf
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AC1: 'Reply on RC1', Sophie Baartman, 14 Apr 2025
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RC2: 'Comment on egusphere-2025-215', Anonymous Referee #2, 14 Mar 2025
General
The paper presents the first isotopic measurements of COS and CO2 made in flow-through chamber for one papyrus, a C4 plant and sunflower, a C3 plant. The setting allows them to derive the COS and CO2 plant uptake, the internal and mesophyll concentrations of CO2 and COS respectively, the photosynthetic discrimination against 13CO2, C18O2 and to explore how they respond to increasing light intensity. They conclude about a distinct behavior was observed between the C3 and the C4 plant.
The strength of the paper stands on the perspectives that such isotopic measurements could provide insights into the underlying processes of the COS and CO2 plant uptake. Especially, the isotopic discrimination of COS gives insight into the CA activity. However, the paper needs to be written in a rush, and the authors should add more contexts and perspectives. In short, I would recommend publishing this paper with major revisions, which consist in improving the storyline. The comments below go around those lines.
The storyline (objectives, method, conclusion) needs to be clarified: . Which complementary piece of information each isotope discrimination can bring? The benefits of using isotopes discrimination of carbon and COS are not clearly explained. Also, in the method section, the photosynthetic discriminations against 13CO2 and C18O2 are not presented. The author should also some perspectives in their conclusion.
Clarity of the Figures: The green and the dark green colors are hard to distinguish. I would recommend putting the same colors for each PAR for papyrus and sunflower. The number of plant replicate is confusing. What is a replicate? I would suggest making a tabular with the number of measurement/replicates for each measured or computed quantity. Likewise, the observations made at PAR equal to 0 are not shown on Figure 4.Representativeness of the experiments: The papyrus, a C4 plant, in the experiment grows in tropical swamps and in arid light saturated environment. How this C4 plant is representative of all the C4 plants? The experiment is far from reality as, because of time constrain, the author did not repeat the experiments with higher light intensity than PAR=400. The effects of soil water level, VPD and nutrients availabilities are also not discussed. Likewise, the chamber was well aerated, which results in infinite boundary layer conductance. Is it realistic, especially at night when the boundary layer becomes stable?
More in-depth analysis of the results is needed: Stomatal and total conductances are needed to explain the results for the C3 and C4 plants (as done in Stimler et al. 2011). The paper only presents experimental results and lacks interpretation in terms of processes.
Specific comments
Abstract
Line 24 – “does not exit the leaf again”. Confusing, sounds like it is the same CO2 molecule that enters and leaves the leaf.
Line 25 – Precise why performing such isotopic measurements and how can isotopic discrimination of COS and CO2. Only the isotopic discrimination of COS is mentioned.
Line 30 -35: Mention what are the implications of these results
Line 35: “The papyrus was not … experiments” Explain a bit better. How is the C4 plant supposed to behave? And how does the papyrus behave in the experiment?
Introduction
- You only describe the processes and the equations underlying the isotope discrimination of COS. As the title mentions it, you should also describe , here or in the method, the processes underlying the isotope discrimination of CO2.
- You should add a tabular or a section showing which piece of information each isotope discrimination or molecule can give to guide the reader.
- Make a tabular with the Davidson et al. (2022) to compare their measurements with yours, add a column for the experimental conditions (CO2, COS mole fractions ec)
Line 40 Add a citation
Line 43 The SIF, satellite retrievals of GPP, net co2 fluxes estimated by atmospheric inversion are not mentioned. Which advantage has COS plant uptake compared to these mentioned methods?
Line 45 Add Wehr et al. 2015 (https://www.sciencedirect.com/science/article/abs/pii/S0168192315007145) who quantified respiration from GPP thanks to isotopic measurements.
Line 49 Lack of fundamental papers about COS
Line 52 Cite Montzka et al. 2007 https://agupubs.onlinelibrary.wiley.com/doi/full/10.1029/2006JD007665
Line 56 «Assuming that there is no COS emissions” Discuss the validity of this assumption: it has been shown that Beliso et al. (2018) (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0278584) showed that rapeseed emit COS. Some plants in swamps also emit COS.
Line 62: How negligible is the daytime respiration? (https://www.sciencedirect.com/science/article/abs/pii/S0168192315007145)
Figure 70 Please make the Figure more understandable. What is the blue and red lines in the middle? What are the zigzag lines in the middle? Add the name of the conductance’s. Some accolades near the name of the space could help to separate the cell/space.
Line 75 The first two sentences, “COS discrimination…factors” and “The discrimination …” are not logical. You should explain which beneficial information COS isotope discrimination can give based on literature and go to the next line, for the definition.
Line 88 Add As the reaction with CA is supposed to be irreversible,
Line 93 Explain why this may be a too crude simplification of the diffusion processes taking place based on literature studies (https://bg.copernicus.org/articles/20/2573/2023/bg-20-2573-2023.pdf)
Line 95 In this case = for C4 species?
Line 101 “ambient COS and CO2 concentrations”. You just said above the experiments were caried at high CO2 and COS mole fractions. And how high?
Line 165 Why do you use to instruments to measure both air in and out of the chamber? What is the specifities of each instrument?
Line 182 Why choosing these temperatures? Are they representative of the place where these two plants live or is it associated with maximal CA activity?
Line 185 remove the space
Line 189 add coma
Line 194 There is a mistake in the formula, please verify. Wa is not wo?
Line 261 – 264 These two sentences should be in the method. This is not clear either. Why some sample must be treated as duplicate?
Line 264 Can you compare your COS uptake fluxes with those from the literature by making a distinction between C3/C4 plant? Can you find some COS uptake flux measured at the ecosystem level (fluxnet type)?
Line 266 Some measurements are also made at the ecosystem level and not in controlled chamber. Please mention that.
Figure 3. Why the As is lower in C4 plant than in C3 plant at PAR=0?
Line 289 Would be nice to have a plot for LRU as well on Figure 3 (3 panels)
Line 294 Please make a distinction between C3 (LRU=1.68) and C4 plant (1.21). only 4 values for C4….
Line 297 Your results cannot be directly compared with Davidson 2022 as they used both high CO2 and high COS concentrations. For instance, Wu Sun 2021 showed that LRU increases with CO2 and that can also affect Davidson 2022 results.
Line 302 Cite Wu Sun for the dependance of LRU to various environmental conditions and be more specific.
Line 304 Explain first why the quantity ci/ca is interesting to study.
Line 370 What is the typical PAR that other experiments use?
Line 371-377, not clear, explain better…As papyrus grow in swamps, this setting is closer to reality?
Line 435 Remind that CO2 assimilations in C4 plants are expected to be more efficient
Line 421 These two sentences are contradictory. First, you sat that the CA reaction is light independent and then you say that the COS uptake was lower during the dark experiment.
Conclusion
Lower discrimination against C18O2 is observed, which is consistent with previous measurements (Stimler et al., 2011). Why not for 14COS?
Can you bring some conclusions about the CA activity?
It is necessary to add a perspective section to pave the way for future experiments.
Citation: https://doi.org/10.5194/egusphere-2025-215-RC2 -
AC2: 'Reply on RC2', Sophie Baartman, 14 Apr 2025
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2025/egusphere-2025-215/egusphere-2025-215-AC2-supplement.pdf
Interactive discussion
Status: closed
-
RC1: 'Comment on egusphere-2025-215', Anonymous Referee #1, 10 Mar 2025
Review of Baartman et al., Isotopic discrimination of carbonyl sulfide…
The paper deals with an important and exciting topic. The use of COS as a unique tracer of photosynthesis and the rare measurements of the isotopic discrimination, D34S, associated with COS uptake. The paper presents a unique measurement system for gas exchange, COS, and isotopic analysis, and it is well-written.
However, the paper has some rather significant issues that need attention. This is partly so as there seems to be a gap between the impressive analytical measurements and the experimental, plant gas exchange, part . Below are some of the concerns noted as I was reading the paper (i.e., in no special order) that I hope will help to improve the paper.
In general, the motivation is to introduce D34S to “provide useful information on the COS uptake process and help to constrain the COS budget” (upfront in the abstract). However, at the end, the paper does not tell us what we learned in either aspect. At least some discussion of these aspects is needed, or these should be strongly toned down.
In fact, the paper goes on to declare another much more modest and specific goal: To verify the published D34S data obtained in a ‘closed system’ (Davisson et al.) in their new ‘open steady-state system’. The paper generally confirms the earlier data but in a way that does not provide additinal confidence due to the experimental difficulties. In its present form, therefore, it is uncertain whether the paper will advance the field in that respect. Better focusing on what exactly is the bottom line/take-home message for D34S is needed.
On the methodological side, it is not clear how many plants were used as replicates. In the Method, three papyrus cuttings and “a sunflower plant” were noted. In Fig. 3, n=2 is indicated (with SE…). In Fig. 4, no replication is indicated; in Fig. 5, 6, some individual replications are plotted, each with its own SE. While the information is incomplete and confusing, the impression is that only a few actual replications were made, and there is no clear distinction between the precision (repeating the measurements) and replications. There is also missing information on Blank Testing of the chambers, which seems to be critical in COS experiments. The inlet COS concentration (2-3 ppb) is 4-5 times the atmospheric level) is indicated but not the chamber ambient concentrations (outlet). [BTW, in the Abstract, fluxes are reported in pmo mol-1, which are not flux units.] More information seems to be required.
The aspects noted above are significant as many of the observations are somewhat unexpected or uncharacteristic, and a range of particular explanations are required, such as non-uniform light level (“low light” in parts), “not optimal behavior”; “stomata not fully open”; increasing Ci with increasing light and increasing A, no response of COS assimilation to light, mostly constant D34S, constant Cm, etc. In fact, the feeling is that more measurements would help to get more conventional results.
Fig. 3 presents a nearly complete insensitivity of COS uptake to light level (in sharp contrast to CO2 uptake), and it is explained by the light in-sensitivity of carbonic anhydrase. However, COS should still respond to light for the same CA activity because of its effect on conductance (g). No information on conductance is given in this paper.
Details of leaf gas exchange equations are presented, including conductance, internal concentrations, etc. However, all those were developed strictly for the leaf scale, which may not apply here. The photo in the Appendix shows that this was a rather ‘dense canopy scale’ experiment. The authors note this can explain some of the non-typical observations, but there is no discussion on how to scale from leaf to canopy (or vice versa). The photo clearly indicates no uniformity in conditions and, in turn, in activities. This scaling gap should be addressed, and if it can be overcome, it should be explained in more detail. By the way, it seems there are some publications on branch scale measurements, which can be helpful to compare (likely also Yang et al. 2017 or 2018 who tried to scale between leaf to canopy).
Along these lines, internal concentrations (Ci) are estimated using the leaf-scale equations, and Cm is calculated based on the D34S estimates. The difference between the Ci and Cm is interesting but not defined or discussed, except that very different values are reported for Ci/Ca and Cm/Ca.
Note also that the physiological calculations of conductance, g, based on E and ci, depend on leaf temperature and water vapor saturation assumption. This is tricky in the present study, which uses a dense canopy in a different light and temperature in the chamber. It seems that COS flux, as long as it is based on the assumption of near-zero internal concentrations (i.e., no compensation point, an issue that is ignored in this paper), may offer a simpler alternative to total conductance, which could at least be compared (i.e., As=gCa…).
The LRU estimates are important. However, it is clearly sensitive to the high ambient COS used as COS uptake can generally be linearly related to Ca-cos, and it also seems that some information on this response may be available in the literature. In this case, the effect could be estimated to some extent, and an attempt to correct the LRU for comparison with literature values at ambient COS could be made, and discussed. In fact, a good agreement on the uncorrected LRU does not add confidence, as noted above.
Citation: https://doi.org/10.5194/egusphere-2025-215-RC1 -
AC1: 'Reply on RC1', Sophie Baartman, 14 Apr 2025
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2025/egusphere-2025-215/egusphere-2025-215-AC1-supplement.pdf
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AC1: 'Reply on RC1', Sophie Baartman, 14 Apr 2025
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RC2: 'Comment on egusphere-2025-215', Anonymous Referee #2, 14 Mar 2025
General
The paper presents the first isotopic measurements of COS and CO2 made in flow-through chamber for one papyrus, a C4 plant and sunflower, a C3 plant. The setting allows them to derive the COS and CO2 plant uptake, the internal and mesophyll concentrations of CO2 and COS respectively, the photosynthetic discrimination against 13CO2, C18O2 and to explore how they respond to increasing light intensity. They conclude about a distinct behavior was observed between the C3 and the C4 plant.
The strength of the paper stands on the perspectives that such isotopic measurements could provide insights into the underlying processes of the COS and CO2 plant uptake. Especially, the isotopic discrimination of COS gives insight into the CA activity. However, the paper needs to be written in a rush, and the authors should add more contexts and perspectives. In short, I would recommend publishing this paper with major revisions, which consist in improving the storyline. The comments below go around those lines.
The storyline (objectives, method, conclusion) needs to be clarified: . Which complementary piece of information each isotope discrimination can bring? The benefits of using isotopes discrimination of carbon and COS are not clearly explained. Also, in the method section, the photosynthetic discriminations against 13CO2 and C18O2 are not presented. The author should also some perspectives in their conclusion.
Clarity of the Figures: The green and the dark green colors are hard to distinguish. I would recommend putting the same colors for each PAR for papyrus and sunflower. The number of plant replicate is confusing. What is a replicate? I would suggest making a tabular with the number of measurement/replicates for each measured or computed quantity. Likewise, the observations made at PAR equal to 0 are not shown on Figure 4.Representativeness of the experiments: The papyrus, a C4 plant, in the experiment grows in tropical swamps and in arid light saturated environment. How this C4 plant is representative of all the C4 plants? The experiment is far from reality as, because of time constrain, the author did not repeat the experiments with higher light intensity than PAR=400. The effects of soil water level, VPD and nutrients availabilities are also not discussed. Likewise, the chamber was well aerated, which results in infinite boundary layer conductance. Is it realistic, especially at night when the boundary layer becomes stable?
More in-depth analysis of the results is needed: Stomatal and total conductances are needed to explain the results for the C3 and C4 plants (as done in Stimler et al. 2011). The paper only presents experimental results and lacks interpretation in terms of processes.
Specific comments
Abstract
Line 24 – “does not exit the leaf again”. Confusing, sounds like it is the same CO2 molecule that enters and leaves the leaf.
Line 25 – Precise why performing such isotopic measurements and how can isotopic discrimination of COS and CO2. Only the isotopic discrimination of COS is mentioned.
Line 30 -35: Mention what are the implications of these results
Line 35: “The papyrus was not … experiments” Explain a bit better. How is the C4 plant supposed to behave? And how does the papyrus behave in the experiment?
Introduction
- You only describe the processes and the equations underlying the isotope discrimination of COS. As the title mentions it, you should also describe , here or in the method, the processes underlying the isotope discrimination of CO2.
- You should add a tabular or a section showing which piece of information each isotope discrimination or molecule can give to guide the reader.
- Make a tabular with the Davidson et al. (2022) to compare their measurements with yours, add a column for the experimental conditions (CO2, COS mole fractions ec)
Line 40 Add a citation
Line 43 The SIF, satellite retrievals of GPP, net co2 fluxes estimated by atmospheric inversion are not mentioned. Which advantage has COS plant uptake compared to these mentioned methods?
Line 45 Add Wehr et al. 2015 (https://www.sciencedirect.com/science/article/abs/pii/S0168192315007145) who quantified respiration from GPP thanks to isotopic measurements.
Line 49 Lack of fundamental papers about COS
Line 52 Cite Montzka et al. 2007 https://agupubs.onlinelibrary.wiley.com/doi/full/10.1029/2006JD007665
Line 56 «Assuming that there is no COS emissions” Discuss the validity of this assumption: it has been shown that Beliso et al. (2018) (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0278584) showed that rapeseed emit COS. Some plants in swamps also emit COS.
Line 62: How negligible is the daytime respiration? (https://www.sciencedirect.com/science/article/abs/pii/S0168192315007145)
Figure 70 Please make the Figure more understandable. What is the blue and red lines in the middle? What are the zigzag lines in the middle? Add the name of the conductance’s. Some accolades near the name of the space could help to separate the cell/space.
Line 75 The first two sentences, “COS discrimination…factors” and “The discrimination …” are not logical. You should explain which beneficial information COS isotope discrimination can give based on literature and go to the next line, for the definition.
Line 88 Add As the reaction with CA is supposed to be irreversible,
Line 93 Explain why this may be a too crude simplification of the diffusion processes taking place based on literature studies (https://bg.copernicus.org/articles/20/2573/2023/bg-20-2573-2023.pdf)
Line 95 In this case = for C4 species?
Line 101 “ambient COS and CO2 concentrations”. You just said above the experiments were caried at high CO2 and COS mole fractions. And how high?
Line 165 Why do you use to instruments to measure both air in and out of the chamber? What is the specifities of each instrument?
Line 182 Why choosing these temperatures? Are they representative of the place where these two plants live or is it associated with maximal CA activity?
Line 185 remove the space
Line 189 add coma
Line 194 There is a mistake in the formula, please verify. Wa is not wo?
Line 261 – 264 These two sentences should be in the method. This is not clear either. Why some sample must be treated as duplicate?
Line 264 Can you compare your COS uptake fluxes with those from the literature by making a distinction between C3/C4 plant? Can you find some COS uptake flux measured at the ecosystem level (fluxnet type)?
Line 266 Some measurements are also made at the ecosystem level and not in controlled chamber. Please mention that.
Figure 3. Why the As is lower in C4 plant than in C3 plant at PAR=0?
Line 289 Would be nice to have a plot for LRU as well on Figure 3 (3 panels)
Line 294 Please make a distinction between C3 (LRU=1.68) and C4 plant (1.21). only 4 values for C4….
Line 297 Your results cannot be directly compared with Davidson 2022 as they used both high CO2 and high COS concentrations. For instance, Wu Sun 2021 showed that LRU increases with CO2 and that can also affect Davidson 2022 results.
Line 302 Cite Wu Sun for the dependance of LRU to various environmental conditions and be more specific.
Line 304 Explain first why the quantity ci/ca is interesting to study.
Line 370 What is the typical PAR that other experiments use?
Line 371-377, not clear, explain better…As papyrus grow in swamps, this setting is closer to reality?
Line 435 Remind that CO2 assimilations in C4 plants are expected to be more efficient
Line 421 These two sentences are contradictory. First, you sat that the CA reaction is light independent and then you say that the COS uptake was lower during the dark experiment.
Conclusion
Lower discrimination against C18O2 is observed, which is consistent with previous measurements (Stimler et al., 2011). Why not for 14COS?
Can you bring some conclusions about the CA activity?
It is necessary to add a perspective section to pave the way for future experiments.
Citation: https://doi.org/10.5194/egusphere-2025-215-RC2 -
AC2: 'Reply on RC2', Sophie Baartman, 14 Apr 2025
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2025/egusphere-2025-215/egusphere-2025-215-AC2-supplement.pdf
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Sophie L. Baartman
Steven M. Driever
Maarten Wassenaar
Linda M. J. Kooijmans
Nerea Ubierna Lopez
Leon Mossink
Maria E. Popa
Ara Cho
Lisa Wingate
Thomas Röckmann
Steven M. A. C. van Heuven
Maarten C. Krol
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
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Review of Baartman et al., Isotopic discrimination of carbonyl sulfide…
The paper deals with an important and exciting topic. The use of COS as a unique tracer of photosynthesis and the rare measurements of the isotopic discrimination, D34S, associated with COS uptake. The paper presents a unique measurement system for gas exchange, COS, and isotopic analysis, and it is well-written.
However, the paper has some rather significant issues that need attention. This is partly so as there seems to be a gap between the impressive analytical measurements and the experimental, plant gas exchange, part . Below are some of the concerns noted as I was reading the paper (i.e., in no special order) that I hope will help to improve the paper.
In general, the motivation is to introduce D34S to “provide useful information on the COS uptake process and help to constrain the COS budget” (upfront in the abstract). However, at the end, the paper does not tell us what we learned in either aspect. At least some discussion of these aspects is needed, or these should be strongly toned down.
In fact, the paper goes on to declare another much more modest and specific goal: To verify the published D34S data obtained in a ‘closed system’ (Davisson et al.) in their new ‘open steady-state system’. The paper generally confirms the earlier data but in a way that does not provide additinal confidence due to the experimental difficulties. In its present form, therefore, it is uncertain whether the paper will advance the field in that respect. Better focusing on what exactly is the bottom line/take-home message for D34S is needed.
On the methodological side, it is not clear how many plants were used as replicates. In the Method, three papyrus cuttings and “a sunflower plant” were noted. In Fig. 3, n=2 is indicated (with SE…). In Fig. 4, no replication is indicated; in Fig. 5, 6, some individual replications are plotted, each with its own SE. While the information is incomplete and confusing, the impression is that only a few actual replications were made, and there is no clear distinction between the precision (repeating the measurements) and replications. There is also missing information on Blank Testing of the chambers, which seems to be critical in COS experiments. The inlet COS concentration (2-3 ppb) is 4-5 times the atmospheric level) is indicated but not the chamber ambient concentrations (outlet). [BTW, in the Abstract, fluxes are reported in pmo mol-1, which are not flux units.] More information seems to be required.
The aspects noted above are significant as many of the observations are somewhat unexpected or uncharacteristic, and a range of particular explanations are required, such as non-uniform light level (“low light” in parts), “not optimal behavior”; “stomata not fully open”; increasing Ci with increasing light and increasing A, no response of COS assimilation to light, mostly constant D34S, constant Cm, etc. In fact, the feeling is that more measurements would help to get more conventional results.
Fig. 3 presents a nearly complete insensitivity of COS uptake to light level (in sharp contrast to CO2 uptake), and it is explained by the light in-sensitivity of carbonic anhydrase. However, COS should still respond to light for the same CA activity because of its effect on conductance (g). No information on conductance is given in this paper.
Details of leaf gas exchange equations are presented, including conductance, internal concentrations, etc. However, all those were developed strictly for the leaf scale, which may not apply here. The photo in the Appendix shows that this was a rather ‘dense canopy scale’ experiment. The authors note this can explain some of the non-typical observations, but there is no discussion on how to scale from leaf to canopy (or vice versa). The photo clearly indicates no uniformity in conditions and, in turn, in activities. This scaling gap should be addressed, and if it can be overcome, it should be explained in more detail. By the way, it seems there are some publications on branch scale measurements, which can be helpful to compare (likely also Yang et al. 2017 or 2018 who tried to scale between leaf to canopy).
Along these lines, internal concentrations (Ci) are estimated using the leaf-scale equations, and Cm is calculated based on the D34S estimates. The difference between the Ci and Cm is interesting but not defined or discussed, except that very different values are reported for Ci/Ca and Cm/Ca.
Note also that the physiological calculations of conductance, g, based on E and ci, depend on leaf temperature and water vapor saturation assumption. This is tricky in the present study, which uses a dense canopy in a different light and temperature in the chamber. It seems that COS flux, as long as it is based on the assumption of near-zero internal concentrations (i.e., no compensation point, an issue that is ignored in this paper), may offer a simpler alternative to total conductance, which could at least be compared (i.e., As=gCa…).
The LRU estimates are important. However, it is clearly sensitive to the high ambient COS used as COS uptake can generally be linearly related to Ca-cos, and it also seems that some information on this response may be available in the literature. In this case, the effect could be estimated to some extent, and an attempt to correct the LRU for comparison with literature values at ambient COS could be made, and discussed. In fact, a good agreement on the uncorrected LRU does not add confidence, as noted above.