the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 11 Jun 2024
Assessing root-soil interactions in wetland plants: root exudation and radial oxygen loss
Abstract. Plant rhizosphere processes, such as root exudation and root oxygen loss (ROL), could have significant impacts on the dynamics and magnitude of wetland methane fluxes, but are rarely measured directly. Here, we measure root exudation and ROL from Schoenoplectus americanus and Spartina patens, two plants that have had opposite relationships between biomass and methane flux in field experiments. We found contrasting rates of ROL in the two species, with S. americanus releasing orders of magnitude more oxygen (O2) to the soil than S. patens. At the same time, S. patens exudes high amounts of carbon to the soil, with much of that carbon pool reduced compared to exudates from other wetland species. This work suggests that the relative inputs of O2 and carbon to the rhizosphere vary significantly between wetland plant species, potentially with major consequences on methane emissions, and highlights the importance of understanding how plant rhizosphere processes mediate soil biogeochemistry at a community level. As global change drivers continue to impact wetlands, future research should consider how feedbacks from plant rhizosphere processes may exacerbate or mitigate coastal wetland methane emissions.
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RC1: 'Comment on egusphere-2024-1547', Anonymous Referee #1, 01 Jul 2024
In this study, Haviland and Noyce evaluated root exudates and radial oxygen loss (ROL) from wetland plants from a specific marsh. They also conducted an incubation experiment to assess the effect of exudates on CH₄ production from soils colonized by specific species. In my opinion, the topic is highly relevant given the current need to understand plant processes that influence greenhouse gas emissions. I consider that the text is clear, provides sufficient detail, and that the experiments were well conducted.
On the weak side of the study is the poor resolution of the ROL profiles provided. The authors clarified the constraints of using planar optodes, noting the low resolution in 2D and the incomplete detection of O₂ loss from all roots in a plant. The methylene blue images provided are somewhat insufficient to indicate the sites of ROL. I believe a more detailed evaluation of ROL using other methods (e.g., root sleeving electrodes, Ti-citrate) together with anatomical characterization of roots would provide a more accurate quantification of ROL from roots. Nonetheless, as stated by the authors, for comparison purposes the methods used might still be valid.
Some aspects that in my opinion will benefit the MS are:
- Including general information on the effect of higher ROL (oxic zones in the rhizosphere) on CH4 oxidation will benefit the text.
- Redox reactions including all the intermediates (i.e., SO4, Fe, Mn..) should be mentioned in the text. The oxidation of such molecules often proceeds at a much higher rate than CH4 oxidation, therefore leaving less O2 for CH4 oxidation. Special consideration should be given to S cycling because of the high sulfate concentrations in the porewater of many intertidal wetland soils.
Ln 45-48: include more information on how O2 diffusion along and across the roots is affected by factors such as respiration, porosity, root length, tissue density.
Adding subtitles to the methods section will make it easier to follow the different methods and techniques applied
Ln 191: is this referred to as for changes in O2?
Is Table 2 referring to an average of measured SOD, or is it simply a calculation based on soil porosity and microbial activity? In any case, clarifications are needed on the estimations, and standard deviations should be included next to the averaged values
Ln 301-304: The buildup of O2 in darkness appears to contradict with Ln 312-314. In light conditions one should expect higher ROL given photosynthesis (DOI: 10.1007/s11104-015-2382-z). Please clarify.
Citation: https://doi.org/10.5194/egusphere-2024-1547-RC1 -
AC2: 'Reply on RC1', Katherine Haviland, 10 Aug 2024
Dear Reviewer 1,
Thank you for taking the time to review our paper, and for your helpful and supportive suggestions. Our responses are below in bold
In this study, Haviland and Noyce evaluated root exudates and radial oxygen loss (ROL) from wetland plants from a specific marsh. They also conducted an incubation experiment to assess the effect of exudates on CH₄ production from soils colonized by specific species. In my opinion, the topic is highly relevant given the current need to understand plant processes that influence greenhouse gas emissions. I consider that the text is clear, provides sufficient detail, and that the experiments were well conducted.
On the weak side of the study is the poor resolution of the ROL profiles provided. The authors clarified the constraints of using planar optodes, noting the low resolution in 2D and the incomplete detection of O₂ loss from all roots in a plant. The methylene blue images provided are somewhat insufficient to indicate the sites of ROL. I believe a more detailed evaluation of ROL using other methods (e.g., root sleeving electrodes, Ti-citrate) together with anatomical characterization of roots would provide a more accurate quantification of ROL from roots. Nonetheless, as stated by the authors, for comparison purposes the methods used might still be valid.
As you highlighted, we present our ROL data largely for comparison between the plants used in our study and other studies with similar methodology. While further quantification of ROL is outside the scope of this project, we agree that the suggested methods would strengthen the community’s understanding of this topic. We plan to address this by expanding our discussion to include the use of more quantitative and precise methods as an avenue for future research.
Some aspects that in my opinion will benefit the MS are:
- Including general information on the effect of higher ROL (oxic zones in the rhizosphere) on CH4 oxidation will benefit the text.
- Redox reactions including all the intermediates (i.e., SO4, Fe, Mn..) should be mentioned in the text. The oxidation of such molecules often proceeds at a much higher rate than CH4 oxidation, therefore leaving less O2 for CH4 oxidation. Special consideration should be given to S cycling because of the high sulfate concentrations in the porewater of many intertidal wetland soils.
Thank you for identifying these gaps and providing helpful suggestions. We will add information on how oxic zones in the rhizosphere affect CH4 oxidation and discuss intermediate redox reactions, especially S cycling, as suggested.
Ln 45-48: include more information on how O2 diffusion along and across the roots is affected by factors such as respiration, porosity, root length, tissue density.
We will include more information on the effect on root anatomy on ROL in the revised manuscript.
Adding subtitles to the methods section will make it easier to follow the different methods and techniques applied.
Thanks for this suggestion, we will do so.
Ln 191: is this referred to as for changes in O2?
You are correct—we meant to say “O2” rather than “R”. Thank you for catching this typo.
Is Table 2 referring to an average of measured SOD, or is it simply a calculation based on soil porosity and microbial activity? In any case, clarifications are needed on the estimations, and standard deviations should be included next to the averaged values.
Table 2 represents an estimated rate of ROL from the plants, calculated using the rate of soil oxygen demand (SOD) in a given rhizosphere, and the steady state % of oxygen around a given plant over a time period. We will update this table to include standard deviations and clarify our SOD calculations in the methods.
Ln 301-304: The buildup of O2 in darkness appears to contradict with Ln 312-314. In light conditions one should expect higher ROL given photosynthesis (DOI: 10.1007/s11104-015-2382-z). Please clarify.
Our observed buildup of O2 in darkness was driven by a precipitous decrease in sediment oxygen demand during the night that overshadowed the smaller increase in ROL due to photosynthesis during the day. This drop in SOD is due to the 7 °C difference in temperature between the night and the day period. We plan to clarify this mechanism in the revised manuscript.
We thank you again for your supportive and helpful feedback,
Drs. Haviland and Noyce
Citation: https://doi.org/10.5194/egusphere-2024-1547-AC2
-
RC2: 'Comment on egusphere-2024-1547', Anonymous Referee #2, 11 Jul 2024
Katherine A. Haviland and Genevieve Noyce investigated root exudation and root oxygen loss from two (four) marsh plant species to test how root oxygen loss and rates of root exudation affect methane production. Root exudates were collected using a combination of soil and hydroponic methods after growing the plants in rhizoboxes, and these exudates were analyzed for total organic carbon (TOC) and metabolites. A 2-D planar optode imaging system was used to measure root oxygen loss. In a separate incubation study, the authors tested the methane production of soil in response to additions of exudate cocktails. The methods and materials used in the study are state-of-the-art and provide valuable insights into the potential consequences of plant community changes in response to sea level rise on methane production, as well as mechanistic insights into how root activity contributes.
Major points:
Exudate Compounds and Methanogenesis: The study does not specify which compounds in the root exudates are responsible for affecting methanogenesis. While the approach of testing natural root exudate cocktails and observing soil response is interesting, it renders the elaborate metabolome analysis somewhat unnecessary, as only speculation on the contributions of specific compounds is possible. Testing single compounds would strengthen the findings.
Global Change Implications: The manuscript could further explore the implications of global change on CO2 production and carbon storage in soil, particularly the relationship between methanogenesis, CO2 production, and soil organic carbon stock changes. For example, could transitions from Spartina patens to Schoenoplectus americanus lead to reductions in soil organic carbon stocks, even with reduced methane production?
Methodologies and Clarity: The explanation of the methodologies was not always clear (see minor comments). The manuscript should ensure that all methods are clearly explained and justified. Additionally, figure captions and table headings should be comprehensive and self-explanatory, with all abbreviations defined within the captions to ensure clarity.
Minor line comments refer to clarification, providing methodological details, and the improvement of the scientific relevance:
L9: Opposite relationships and directions need to be named.
L11: The term "reduced" should be clarified to specify if it refers to the entire carbon pool.
L13: Details on the processes affecting methane emissions and the relevance of rhizosphere processes should be provided. Can rhizosphere processes be measured on the soil surface?
L26: The reference to carbon allocation should be updated with newer articles and corrected to accurately cite the given reference, which addresses rhizodeposition including dead organic matter. Assuming half of the photosynthesis carbon being allocated is incorrectly cited from the given reference as rhizodeposition including dead organic matter is addressed in the reference.
L27-29: The sentence regarding O2 sinks, their fuel by root exudates, and redox conditions in soil needs clarification.
L38: The term "sediment biogeochemical regimes" should be defined.
L60: Soil C storage should be correctly referred to as a stock, not a rate.
L73: Clarify how well growth chamber measurements reflect field conditions.
L88: Explain the integration of Phragmites australis and Spartina alterniflora in the study, given the hypotheses testing only differences between Schoenoplectus americanus and Spartina patens.
L100: Provide additional details on soil water content and matric potential.
L116: Specify in which cases samples are sterile and in which they are not.
L113: Please display root-associated metabolites in Figure 2.
L133: There seems to be a mismatch regarding the filter usage. Was it 0.22 or 0.45 micrometers?
L155: Please clarify which plants were used. Is it the same as described in chapter 2.2? If taken from the field, how did you make sure to excavate them entirely?
L160: Please clarify, have the samples been on a shaker for two weeks? Please explain why you chose this procedure.
L168: Please add an explanation that justifies the duration of the experiment.
L184: Were these the same plants as described in Chapter 2.2? If not, please add this information.
L339: Methane yield is not presented in Figure 5.
L339-344: Please clarify the results and how they link to the field scale under changing water levels.
Citation: https://doi.org/10.5194/egusphere-2024-1547-RC2 -
AC1: 'Reply on RC2', Katherine Haviland, 10 Aug 2024
Dear Reviewer 2,
Thank you for your thorough and supportive feedback on our paper. Your suggestions are very helpful toward improving our paper. Our responses are below in bold.
Katherine A. Haviland and Genevieve Noyce investigated root exudation and root oxygen loss from two (four) marsh plant species to test how root oxygen loss and rates of root exudation affect methane production. Root exudates were collected using a combination of soil and hydroponic methods after growing the plants in rhizoboxes, and these exudates were analyzed for total organic carbon (TOC) and metabolites. A 2-D planar optode imaging system was used to measure root oxygen loss. In a separate incubation study, the authors tested the methane production of soil in response to additions of exudate cocktails. The methods and materials used in the study are state-of-the-art and provide valuable insights into the potential consequences of plant community changes in response to sea level rise on methane production, as well as mechanistic insights into how root activity contributes.
Major points:
Exudate Compounds and Methanogenesis: The study does not specify which compounds in the root exudates are responsible for affecting methanogenesis. While the approach of testing natural root exudate cocktails and observing soil response is interesting, it renders the elaborate metabolome analysis somewhat unnecessary, as only speculation on the contributions of specific compounds is possible. Testing single compounds would strengthen the findings.
We agree completely that testing specific metabolic compounds would strengthen the findings and be a very interesting avenue for future research. Similar work has been carried out on peat previously (e.g. Girkin et al., 2018). One goal of this project was to test methods using the untargeted sequencing approach; now that we are confident it works we will be able to conduct future target approaches, but unfortunately, it is outside the scope of our work at present to do so.
Global Change Implications: The manuscript could further explore the implications of global change on CO2 production and carbon storage in soil, particularly the relationship between methanogenesis, CO2 production, and soil organic carbon stock changes. For example, could transitions from Spartina patens to Schoenoplectus americanus lead to reductions in soil organic carbon stocks, even with reduced methane production?
Thank you for drawing our attention to this. The biogeochemical effects of global change and the transition of S. patens to S. americanus are certainly important and not sufficiently addressed in the current manuscript. Though we do not have data on changes in soil organic carbon stock, we will expand our discussion section to highlight what our results imply for ecosystem responses to global change.
Methodologies and Clarity: The explanation of the methodologies was not always clear (see minor comments). The manuscript should ensure that all methods are clearly explained and justified. Additionally, figure captions and table headings should be comprehensive and self-explanatory, with all abbreviations defined within the captions to ensure clarity.
We agree that our methods section could use clarifications and expansion, as also suggested by Reviewer 1. We plan to include subheadings in the methods section to help readers follow the methodology of our various experiments and will also improve the clarity of our figure captions and table headings.
Minor line comments refer to clarification, providing methodological details, and the improvement of the scientific relevance:
L9: Opposite relationships and directions need to be named.
Thank you for pointing that out; we will update the abstract to clarify which relationships we’re referring to.
L11: The term "reduced" should be clarified to specify if it refers to the entire carbon pool.
We will revise this line to clarify.
L13: Details on the processes affecting methane emissions and the relevance of rhizosphere processes should be provided. Can rhizosphere processes be measured on the soil surface?
While we cannot expand substantially in the abstract due to space constraints, this is a valid and interesting question, and we will make sure to address this in the discussion section of our paper.
L26: The reference to carbon allocation should be updated with newer articles and corrected to accurately cite the given reference, which addresses rhizodeposition including dead organic matter. Assuming half of the photosynthesis carbon being allocated is incorrectly cited from the given reference as rhizodeposition including dead organic matter is addressed in the reference.
Thank you for bringing this to our attention. We will update the reference to carbon allocation and fix the incorrect citation.
L27-29: The sentence regarding O2 sinks, their fuel by root exudates, and redox conditions in soil needs clarification.
We will revise this section to clarify the relationships between O2 sinks, root exudation, and soil redox conditions.
L38: The term "sediment biogeochemical regimes" should be defined.
Thank you for highlighting that this term is unclear; we will clarify what we mean by “sediment biogeochemical regimes”.
L60: Soil C storage should be correctly referred to as a stock, not a rate.
Thank you for catching that; we will fix this sentence.
L73: Clarify how well growth chamber measurements reflect field conditions.
We agree that this is a limitation of growth chambers, though necessary for certain types of data collection. We plan to add some new text comparing growth chamber experiments to field conditions.
L88: Explain the integration of Phragmites australis and Spartina alterniflora in the study, given the hypotheses testing only differences between Schoenoplectus americanus and Spartina patens.
Phragmites australis an invasive species at Kirkpatrick Marsh that is rapidly becoming more dominant and Spartina alterniflora a common wetland plant species. We included them in this study to provide broader context for the measured ROL of our target species. We will revise the manuscript to include this explanation.
L100: Provide additional details on soil water content and matric potential.
We will add these details into the methods.
L116: Specify in which cases samples are sterile and in which they are not.
No samples or methods were completely sterile. We will revise these lines to clarify this point.
L113: Please display root-associated metabolites in Figure 2.
Thank you for pointing out this incongruence, we will change the language in Figure 2 to refer to “root-associated metabolites” rather than root exudates.
L133: There seems to be a mismatch regarding the filter usage. Was it 0.22 or 0.45 micrometers?
The filters were 0.22 microns. As you noted, we incorrectly state in one location that the filters were 0.45 microns and will correct this.
L155: Please clarify which plants were used. Is it the same as described in chapter 2.2? If taken from the field, how did you make sure to excavate them entirely?
The same cohort of plants was used for all analyses. Rather than excavating full plants from the field, we collected rhizomes from the field and grew the plants in a growth chamber. We will clarify this in the revised methods.
L160: Please clarify, have the samples been on a shaker for two weeks? Please explain why you chose this procedure.
Our protocol did include keeping samples on a shaker at a very low RPM for a 2-week period in order to prevent the settling out of sediment from the slurry, to allow for the distribution of the exudate cocktail among the slurry, and to maintain homogeneity of the samples. We will add this explanation to the revised methods.
L168: Please add an explanation that justifies the duration of the experiment.
We will add this explanation to the revised manuscript. The duration of the experiment was based on previous research where synthetic metabolite cocktails were added to peat (e.g. Girkin et al., 2018).
L184: Were these the same plants as described in Chapter 2.2? If not, please add this information.
The same cohort of plants was used for all analyses. We will clarify this in the revised methods.
L339: Methane yield is not presented in Figure 5.
Thank you for catching this. We will change the manuscript so that the reference here is for Figure 6, rather than Figure 5.
L339-344: Please clarify the results and how they link to the field scale under changing water levels.
In the revised manuscript, we will give more attention to the implications of these experiments on a field scale, and with regard to global change—including increased water level.
Thank you again for taking the time to review our paper, and for your helpful suggestions.
Drs. Haviland and Noyce
Citation: https://doi.org/10.5194/egusphere-2024-1547-AC1
-
AC1: 'Reply on RC2', Katherine Haviland, 10 Aug 2024
Status: closed
-
RC1: 'Comment on egusphere-2024-1547', Anonymous Referee #1, 01 Jul 2024
In this study, Haviland and Noyce evaluated root exudates and radial oxygen loss (ROL) from wetland plants from a specific marsh. They also conducted an incubation experiment to assess the effect of exudates on CH₄ production from soils colonized by specific species. In my opinion, the topic is highly relevant given the current need to understand plant processes that influence greenhouse gas emissions. I consider that the text is clear, provides sufficient detail, and that the experiments were well conducted.
On the weak side of the study is the poor resolution of the ROL profiles provided. The authors clarified the constraints of using planar optodes, noting the low resolution in 2D and the incomplete detection of O₂ loss from all roots in a plant. The methylene blue images provided are somewhat insufficient to indicate the sites of ROL. I believe a more detailed evaluation of ROL using other methods (e.g., root sleeving electrodes, Ti-citrate) together with anatomical characterization of roots would provide a more accurate quantification of ROL from roots. Nonetheless, as stated by the authors, for comparison purposes the methods used might still be valid.
Some aspects that in my opinion will benefit the MS are:
- Including general information on the effect of higher ROL (oxic zones in the rhizosphere) on CH4 oxidation will benefit the text.
- Redox reactions including all the intermediates (i.e., SO4, Fe, Mn..) should be mentioned in the text. The oxidation of such molecules often proceeds at a much higher rate than CH4 oxidation, therefore leaving less O2 for CH4 oxidation. Special consideration should be given to S cycling because of the high sulfate concentrations in the porewater of many intertidal wetland soils.
Ln 45-48: include more information on how O2 diffusion along and across the roots is affected by factors such as respiration, porosity, root length, tissue density.
Adding subtitles to the methods section will make it easier to follow the different methods and techniques applied
Ln 191: is this referred to as for changes in O2?
Is Table 2 referring to an average of measured SOD, or is it simply a calculation based on soil porosity and microbial activity? In any case, clarifications are needed on the estimations, and standard deviations should be included next to the averaged values
Ln 301-304: The buildup of O2 in darkness appears to contradict with Ln 312-314. In light conditions one should expect higher ROL given photosynthesis (DOI: 10.1007/s11104-015-2382-z). Please clarify.
Citation: https://doi.org/10.5194/egusphere-2024-1547-RC1 -
AC2: 'Reply on RC1', Katherine Haviland, 10 Aug 2024
Dear Reviewer 1,
Thank you for taking the time to review our paper, and for your helpful and supportive suggestions. Our responses are below in bold
In this study, Haviland and Noyce evaluated root exudates and radial oxygen loss (ROL) from wetland plants from a specific marsh. They also conducted an incubation experiment to assess the effect of exudates on CH₄ production from soils colonized by specific species. In my opinion, the topic is highly relevant given the current need to understand plant processes that influence greenhouse gas emissions. I consider that the text is clear, provides sufficient detail, and that the experiments were well conducted.
On the weak side of the study is the poor resolution of the ROL profiles provided. The authors clarified the constraints of using planar optodes, noting the low resolution in 2D and the incomplete detection of O₂ loss from all roots in a plant. The methylene blue images provided are somewhat insufficient to indicate the sites of ROL. I believe a more detailed evaluation of ROL using other methods (e.g., root sleeving electrodes, Ti-citrate) together with anatomical characterization of roots would provide a more accurate quantification of ROL from roots. Nonetheless, as stated by the authors, for comparison purposes the methods used might still be valid.
As you highlighted, we present our ROL data largely for comparison between the plants used in our study and other studies with similar methodology. While further quantification of ROL is outside the scope of this project, we agree that the suggested methods would strengthen the community’s understanding of this topic. We plan to address this by expanding our discussion to include the use of more quantitative and precise methods as an avenue for future research.
Some aspects that in my opinion will benefit the MS are:
- Including general information on the effect of higher ROL (oxic zones in the rhizosphere) on CH4 oxidation will benefit the text.
- Redox reactions including all the intermediates (i.e., SO4, Fe, Mn..) should be mentioned in the text. The oxidation of such molecules often proceeds at a much higher rate than CH4 oxidation, therefore leaving less O2 for CH4 oxidation. Special consideration should be given to S cycling because of the high sulfate concentrations in the porewater of many intertidal wetland soils.
Thank you for identifying these gaps and providing helpful suggestions. We will add information on how oxic zones in the rhizosphere affect CH4 oxidation and discuss intermediate redox reactions, especially S cycling, as suggested.
Ln 45-48: include more information on how O2 diffusion along and across the roots is affected by factors such as respiration, porosity, root length, tissue density.
We will include more information on the effect on root anatomy on ROL in the revised manuscript.
Adding subtitles to the methods section will make it easier to follow the different methods and techniques applied.
Thanks for this suggestion, we will do so.
Ln 191: is this referred to as for changes in O2?
You are correct—we meant to say “O2” rather than “R”. Thank you for catching this typo.
Is Table 2 referring to an average of measured SOD, or is it simply a calculation based on soil porosity and microbial activity? In any case, clarifications are needed on the estimations, and standard deviations should be included next to the averaged values.
Table 2 represents an estimated rate of ROL from the plants, calculated using the rate of soil oxygen demand (SOD) in a given rhizosphere, and the steady state % of oxygen around a given plant over a time period. We will update this table to include standard deviations and clarify our SOD calculations in the methods.
Ln 301-304: The buildup of O2 in darkness appears to contradict with Ln 312-314. In light conditions one should expect higher ROL given photosynthesis (DOI: 10.1007/s11104-015-2382-z). Please clarify.
Our observed buildup of O2 in darkness was driven by a precipitous decrease in sediment oxygen demand during the night that overshadowed the smaller increase in ROL due to photosynthesis during the day. This drop in SOD is due to the 7 °C difference in temperature between the night and the day period. We plan to clarify this mechanism in the revised manuscript.
We thank you again for your supportive and helpful feedback,
Drs. Haviland and Noyce
Citation: https://doi.org/10.5194/egusphere-2024-1547-AC2
-
RC2: 'Comment on egusphere-2024-1547', Anonymous Referee #2, 11 Jul 2024
Katherine A. Haviland and Genevieve Noyce investigated root exudation and root oxygen loss from two (four) marsh plant species to test how root oxygen loss and rates of root exudation affect methane production. Root exudates were collected using a combination of soil and hydroponic methods after growing the plants in rhizoboxes, and these exudates were analyzed for total organic carbon (TOC) and metabolites. A 2-D planar optode imaging system was used to measure root oxygen loss. In a separate incubation study, the authors tested the methane production of soil in response to additions of exudate cocktails. The methods and materials used in the study are state-of-the-art and provide valuable insights into the potential consequences of plant community changes in response to sea level rise on methane production, as well as mechanistic insights into how root activity contributes.
Major points:
Exudate Compounds and Methanogenesis: The study does not specify which compounds in the root exudates are responsible for affecting methanogenesis. While the approach of testing natural root exudate cocktails and observing soil response is interesting, it renders the elaborate metabolome analysis somewhat unnecessary, as only speculation on the contributions of specific compounds is possible. Testing single compounds would strengthen the findings.
Global Change Implications: The manuscript could further explore the implications of global change on CO2 production and carbon storage in soil, particularly the relationship between methanogenesis, CO2 production, and soil organic carbon stock changes. For example, could transitions from Spartina patens to Schoenoplectus americanus lead to reductions in soil organic carbon stocks, even with reduced methane production?
Methodologies and Clarity: The explanation of the methodologies was not always clear (see minor comments). The manuscript should ensure that all methods are clearly explained and justified. Additionally, figure captions and table headings should be comprehensive and self-explanatory, with all abbreviations defined within the captions to ensure clarity.
Minor line comments refer to clarification, providing methodological details, and the improvement of the scientific relevance:
L9: Opposite relationships and directions need to be named.
L11: The term "reduced" should be clarified to specify if it refers to the entire carbon pool.
L13: Details on the processes affecting methane emissions and the relevance of rhizosphere processes should be provided. Can rhizosphere processes be measured on the soil surface?
L26: The reference to carbon allocation should be updated with newer articles and corrected to accurately cite the given reference, which addresses rhizodeposition including dead organic matter. Assuming half of the photosynthesis carbon being allocated is incorrectly cited from the given reference as rhizodeposition including dead organic matter is addressed in the reference.
L27-29: The sentence regarding O2 sinks, their fuel by root exudates, and redox conditions in soil needs clarification.
L38: The term "sediment biogeochemical regimes" should be defined.
L60: Soil C storage should be correctly referred to as a stock, not a rate.
L73: Clarify how well growth chamber measurements reflect field conditions.
L88: Explain the integration of Phragmites australis and Spartina alterniflora in the study, given the hypotheses testing only differences between Schoenoplectus americanus and Spartina patens.
L100: Provide additional details on soil water content and matric potential.
L116: Specify in which cases samples are sterile and in which they are not.
L113: Please display root-associated metabolites in Figure 2.
L133: There seems to be a mismatch regarding the filter usage. Was it 0.22 or 0.45 micrometers?
L155: Please clarify which plants were used. Is it the same as described in chapter 2.2? If taken from the field, how did you make sure to excavate them entirely?
L160: Please clarify, have the samples been on a shaker for two weeks? Please explain why you chose this procedure.
L168: Please add an explanation that justifies the duration of the experiment.
L184: Were these the same plants as described in Chapter 2.2? If not, please add this information.
L339: Methane yield is not presented in Figure 5.
L339-344: Please clarify the results and how they link to the field scale under changing water levels.
Citation: https://doi.org/10.5194/egusphere-2024-1547-RC2 -
AC1: 'Reply on RC2', Katherine Haviland, 10 Aug 2024
Dear Reviewer 2,
Thank you for your thorough and supportive feedback on our paper. Your suggestions are very helpful toward improving our paper. Our responses are below in bold.
Katherine A. Haviland and Genevieve Noyce investigated root exudation and root oxygen loss from two (four) marsh plant species to test how root oxygen loss and rates of root exudation affect methane production. Root exudates were collected using a combination of soil and hydroponic methods after growing the plants in rhizoboxes, and these exudates were analyzed for total organic carbon (TOC) and metabolites. A 2-D planar optode imaging system was used to measure root oxygen loss. In a separate incubation study, the authors tested the methane production of soil in response to additions of exudate cocktails. The methods and materials used in the study are state-of-the-art and provide valuable insights into the potential consequences of plant community changes in response to sea level rise on methane production, as well as mechanistic insights into how root activity contributes.
Major points:
Exudate Compounds and Methanogenesis: The study does not specify which compounds in the root exudates are responsible for affecting methanogenesis. While the approach of testing natural root exudate cocktails and observing soil response is interesting, it renders the elaborate metabolome analysis somewhat unnecessary, as only speculation on the contributions of specific compounds is possible. Testing single compounds would strengthen the findings.
We agree completely that testing specific metabolic compounds would strengthen the findings and be a very interesting avenue for future research. Similar work has been carried out on peat previously (e.g. Girkin et al., 2018). One goal of this project was to test methods using the untargeted sequencing approach; now that we are confident it works we will be able to conduct future target approaches, but unfortunately, it is outside the scope of our work at present to do so.
Global Change Implications: The manuscript could further explore the implications of global change on CO2 production and carbon storage in soil, particularly the relationship between methanogenesis, CO2 production, and soil organic carbon stock changes. For example, could transitions from Spartina patens to Schoenoplectus americanus lead to reductions in soil organic carbon stocks, even with reduced methane production?
Thank you for drawing our attention to this. The biogeochemical effects of global change and the transition of S. patens to S. americanus are certainly important and not sufficiently addressed in the current manuscript. Though we do not have data on changes in soil organic carbon stock, we will expand our discussion section to highlight what our results imply for ecosystem responses to global change.
Methodologies and Clarity: The explanation of the methodologies was not always clear (see minor comments). The manuscript should ensure that all methods are clearly explained and justified. Additionally, figure captions and table headings should be comprehensive and self-explanatory, with all abbreviations defined within the captions to ensure clarity.
We agree that our methods section could use clarifications and expansion, as also suggested by Reviewer 1. We plan to include subheadings in the methods section to help readers follow the methodology of our various experiments and will also improve the clarity of our figure captions and table headings.
Minor line comments refer to clarification, providing methodological details, and the improvement of the scientific relevance:
L9: Opposite relationships and directions need to be named.
Thank you for pointing that out; we will update the abstract to clarify which relationships we’re referring to.
L11: The term "reduced" should be clarified to specify if it refers to the entire carbon pool.
We will revise this line to clarify.
L13: Details on the processes affecting methane emissions and the relevance of rhizosphere processes should be provided. Can rhizosphere processes be measured on the soil surface?
While we cannot expand substantially in the abstract due to space constraints, this is a valid and interesting question, and we will make sure to address this in the discussion section of our paper.
L26: The reference to carbon allocation should be updated with newer articles and corrected to accurately cite the given reference, which addresses rhizodeposition including dead organic matter. Assuming half of the photosynthesis carbon being allocated is incorrectly cited from the given reference as rhizodeposition including dead organic matter is addressed in the reference.
Thank you for bringing this to our attention. We will update the reference to carbon allocation and fix the incorrect citation.
L27-29: The sentence regarding O2 sinks, their fuel by root exudates, and redox conditions in soil needs clarification.
We will revise this section to clarify the relationships between O2 sinks, root exudation, and soil redox conditions.
L38: The term "sediment biogeochemical regimes" should be defined.
Thank you for highlighting that this term is unclear; we will clarify what we mean by “sediment biogeochemical regimes”.
L60: Soil C storage should be correctly referred to as a stock, not a rate.
Thank you for catching that; we will fix this sentence.
L73: Clarify how well growth chamber measurements reflect field conditions.
We agree that this is a limitation of growth chambers, though necessary for certain types of data collection. We plan to add some new text comparing growth chamber experiments to field conditions.
L88: Explain the integration of Phragmites australis and Spartina alterniflora in the study, given the hypotheses testing only differences between Schoenoplectus americanus and Spartina patens.
Phragmites australis an invasive species at Kirkpatrick Marsh that is rapidly becoming more dominant and Spartina alterniflora a common wetland plant species. We included them in this study to provide broader context for the measured ROL of our target species. We will revise the manuscript to include this explanation.
L100: Provide additional details on soil water content and matric potential.
We will add these details into the methods.
L116: Specify in which cases samples are sterile and in which they are not.
No samples or methods were completely sterile. We will revise these lines to clarify this point.
L113: Please display root-associated metabolites in Figure 2.
Thank you for pointing out this incongruence, we will change the language in Figure 2 to refer to “root-associated metabolites” rather than root exudates.
L133: There seems to be a mismatch regarding the filter usage. Was it 0.22 or 0.45 micrometers?
The filters were 0.22 microns. As you noted, we incorrectly state in one location that the filters were 0.45 microns and will correct this.
L155: Please clarify which plants were used. Is it the same as described in chapter 2.2? If taken from the field, how did you make sure to excavate them entirely?
The same cohort of plants was used for all analyses. Rather than excavating full plants from the field, we collected rhizomes from the field and grew the plants in a growth chamber. We will clarify this in the revised methods.
L160: Please clarify, have the samples been on a shaker for two weeks? Please explain why you chose this procedure.
Our protocol did include keeping samples on a shaker at a very low RPM for a 2-week period in order to prevent the settling out of sediment from the slurry, to allow for the distribution of the exudate cocktail among the slurry, and to maintain homogeneity of the samples. We will add this explanation to the revised methods.
L168: Please add an explanation that justifies the duration of the experiment.
We will add this explanation to the revised manuscript. The duration of the experiment was based on previous research where synthetic metabolite cocktails were added to peat (e.g. Girkin et al., 2018).
L184: Were these the same plants as described in Chapter 2.2? If not, please add this information.
The same cohort of plants was used for all analyses. We will clarify this in the revised methods.
L339: Methane yield is not presented in Figure 5.
Thank you for catching this. We will change the manuscript so that the reference here is for Figure 6, rather than Figure 5.
L339-344: Please clarify the results and how they link to the field scale under changing water levels.
In the revised manuscript, we will give more attention to the implications of these experiments on a field scale, and with regard to global change—including increased water level.
Thank you again for taking the time to review our paper, and for your helpful suggestions.
Drs. Haviland and Noyce
Citation: https://doi.org/10.5194/egusphere-2024-1547-AC1
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AC1: 'Reply on RC2', Katherine Haviland, 10 Aug 2024
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