Review Reports - Real-Time Partitioning of Diurnal Stem CO<sub>2</sub> Efflux into Local Stem Respiration and Xylem Transport Processes

Round 1

Reviewer 1 Report

In this research, the authors build on a previously established open flow-through system designed for the continuous measurement of stem ES_CO2, which relied on ambient air passing through a specially designed 3D-printed stem chamber. Their revised design incorporates high-precision O₂ and CO₂ CRDS systems, an array of mass flow controllers to ensure a steady airflow through several stem chambers and ambient air reference lines, as well as an automated continuous flow selector valve. This advanced system facilitates ongoing measurements of ES_CO2, net stem O₂ influx (ES_O2), and the apparent respiratory quotient (ARQ) from the stem chambers. By assuming a cellular RQ of 1.0 for the complete oxidation of non-structural carbohydrates, they analyzed ES_CO2 by distinguishing between contributions from local respiratory production and transported CO₂ from stem chambers positioned near the base of a California cherry tree (Prunus ilicifolia). They investigated the temperature sensitivities of these processes and monitored their diurnal and weekly variations. Their results lay out a methodological framework for quantifying the relative roles of local stem respiration and CO₂ transport within the transpiration stream concerning ES_CO2.

While the paper presents innovative findings, there are several aspects that could be improved:

Please investigate whether there is any interaction between CO2 efflux and salt or drought signaling pathways involving reactive oxygen species. If so, please provide an extended discussion on this topic.
Do you have any biochemistry experiments?
Please review the Introduction; I think it is too long.
The quality of the figures needs to be improved for better clarity and presentation.
Please compare your results with similar studies in the literature, if available.
In the concluding remarks, it is essential to explicitly emphasize the novelty of the research findings. Please rewrite the conclusion section and focus on the main findings. Make this adjustment.
The literature survey requires expansion. Consider incorporating 4-6 additional relevant studies from this journal, while minimizing self-citations.

Author Response

R1 Comment 1:

R1 Response 1:Thank you for your thoughtful and detailed feedback! We greatly appreciate your positive assessment of our revised system and methodological approach.

R1 Comment 2:

While the paper presents innovative findings, there are several aspects that could be improved:

R1 Response 2: We appreciate the reviewer’s thoughtful feedback. We have carefully considered all the comments and have updated the manuscript accordingly to improve its clarity and rigor. Specific revisions in response to the concerns raised are detailed below.

R1 Comment 3:

Please investigate whether there is any interaction between CO2 efflux and salt or drought signaling pathways involving reactive oxygen species. If so, please provide an extended discussion on this topic.

R1 Response 3: We have now extended this topic by integrating the following text in the discussion including two new citations from IJPB.

“In other words, there is likely an interaction between CO₂ efflux and drought signaling pathways involving reactive oxygen species (ROS). CO₂ efflux from non-photosynthetic plant tissues—such as stems, roots, and xylem parenchyma—is intricately linked to salt, temperature, and drought stress responses through ROS signaling pathways [60]. Under these abiotic stresses, ROS accumulate in organelles like mitochondria and peroxisomes, modulating respiration by altering mitochondrial electron transport and activating alternative oxidase pathways. This directly affects the rate and pattern of CO₂ release from these tissues [61]. As a result, CO₂ efflux reflects both local respiratory activity and broader oxidative stress signaling under challenging environmental conditions. Collectively, monitoring ARQ and thus estimates of xylem sap transport of CO₂ using our method might be used to follow root-to-shoot molecular signaling processes.”

R1 Comment 4:

Do you have any biochemistry experiments?

R1 Response 4: We do not have any as this is outside the scope of the current manuscript. Nonetheless we now include a detailed description of biochemical measurements in the new Table 2 that could be quantitatively compared with stem CO₂ efflux and O₂ influx observations and added text to describe this in the discussion and conclusion sections.

Table 2. Biochemical measurements that can be quantitatively compared with in vivo stem CO₂ efflux and O₂ influx. The table lists recommended assays for assessing mitochondrial respiration, substrate availability, and redox status, along with the required instruments, measured outputs, and how each measurement links mechanistically and quantitatively to observed gas fluxes. These biochemical metrics provide a framework for integrating physiological gas exchange data with underlying metabolic processes under varying environmental conditions.

Discussion

“Given that stem CO₂ efflux and O₂ influx measurements provide real-time, integrative indicators of respiratory activity in woody tissues, mechanistically interpretation in future studies will be enhanced through targeted biochemical assays. Among the most directly comparable methods are mitochondrial O₂ consumption assays using Clark-type electrodes, isothermal calorimetry to quantify total respiratory heat output, and enzymatic activity measurements of the alternative oxidase (AOX) and cytochrome oxidase (COX) pathways. These methods, along with metabolic profiling of sugars, organic acids, and amino acids, enable mechanistic interpretation of gas exchange patterns in relation to respiratory substrate use and pathway activity under varying environmental conditions. These comparisons summarized in Table 2, enable a systems-level understanding of how respiration responds to environmental conditions such as drought and salinity.”

Conclusions

“When coupled with biochemical, hydraulic, and environmental sensors, this approach can be extended to larger spatial scales and diverse ecosystems to improve mechanistic understanding of the coupling between plant carbon cycling and water use.”

R1 Comment 5:

Please review the Introduction; I think it is too long.

R1 Response 5:

The introduction has been organized and condensed by roughly 75% of the original while keeping the key concepts.

Comment 6:

The quality of the figures needs to be improved for better clarity and presentation.

Please compare your results with similar studies in the literature, if available.

In the concluding remarks, it is essential to explicitly emphasize the novelty of the research findings.

R1 Response 6:

All figures have been provided as high-resolution JPEG files and have been carefully reviewed to ensure clarity and visual quality. We have confirmed that the resolution, contrast, and labeling meet the journal’s standards for publication. Please let us know if any specific adjustments are required.

Comment 7:

Please rewrite the conclusion section and focus on the main findings. Make this adjustment.

R1 Response 7:

We have rewritten the conclusion to sharpen the focus on the main findings and improves clarity and flow.

“This study provides key insights into the dynamic partitioning of stem-respired CO₂ between local emission and upward transport via the transpiration stream, with important implications for understanding whole-tree carbon cycling and improving terrestrial carbon flux models. The observation of ARQ values consistently greater than 1.0 demonstrates that transported CO₂ can be a significant component and even dominate stem efflux, challenging the common assumption that stem CO₂ emissions primarily reflect local respiration and reassimilation. Diurnal patterns revealed that high temperatures can suppress ARQ, likely due to an increase in local respiration relative to transported CO₂, whereas midday ARQ peaks suggest enhanced CO₂ transport during periods of high transpiration. Notably, the apparent temperature sensitivity of transported CO₂ was greater than that of local mitochondrial respiration, indicating that transpiration-driven transport is more responsive to temperature fluctuations. These findings highlight the role of hydraulic processes in modulating respiratory activity and stem CO₂ transport dynamics, and suggest that transported CO₂, if reassimilated by canopy leaves, may help buffer photosynthesis during periods of reduced stomatal conductance, such as warm afternoons with high vapor pressure deficit. While the results are based on detailed measurements from a single tree near the laboratory, the study introduces a novel, real-time method for quantifying apparent respiratory quotient (ARQ). When coupled with biochemical, hydraulic, and environmental sensors, this approach can be extended to larger spatial scales and diverse ecosystems to improve mechanistic understanding of the coupling between plant carbon cycling and water use. Integrating ARQ measurements into carbon and water flux models offers a new framework for capturing the physiological complexity of plant-atmosphere exchange. Further research across forest types and climates will be essential to evaluate the generality of these findings and refine predictions of the global carbon budget.”

R1 Comment 8:

The literature survey requires expansion. Consider incorporating 4-6 additional relevant studies from this journal, while minimizing self-citations.

R1 Response 8:

Thank you. We included 2 references from IJPB in responses above, and have expanded the literature survey to include 2 additional references from IJPB avoiding self-citations.

Reviewer 2 Report

The manuscript investigated the real-time partitioning of diurnal stem CO₂ efflux into local stem respiration and xylem transport processes. It is of great importance for improving our understanding of whole-tree carbon cycling. However, there are still some drawbacks need to be addressed.

The Introduction section should be concise and focused, primarily outlining the current research status and existing challenges in the field or research direction. It should explicitly define the research objectives of this paper or propose a scientific hypothesis.
How does plant photosynthetic capacity affect ARQ? Why didn't the author consider measuring the gas exchange parameters of plants?
Stable isotope techniques can be used to accurately trace carbon utilization and cycling in plants. This study investigates stem carbon respiration and transport using a novel method. When complemented by stable isotope-based validation, the reliability of research results can be significantly enhanced.
The conclusion is too lengthy and not concise enough, which impairs readability. It is necessary to optimize the conclusion by highlighting the main findings and providing a brief summary to make it more succinct.
Incorporate citations of literature published within the last three years to demonstrate the authors' mastery of the research frontiers in this field.

Author Response

R2 Comment 1:

The manuscript investigated the real-time partitioning of diurnal stem CO2 efflux into local stem respiration and xylem transport processes. It is of great importance for improving our understanding of whole-tree carbon cycling. However, there are still some drawbacks need to be addressed.

R2 Response 1:

We thank the reviewer for recognizing the importance of our study in advancing understanding of whole-tree carbon cycling through real-time partitioning of stem CO₂ efflux. We appreciate the reviewer’s thoughtful comments and have carefully addressed all identified drawbacks in the revised manuscript. Below, we provide detailed responses to each specific concern and describe the changes made to improve the clarity, robustness, and broader relevance of the work.

R2 Comment 2:

The Introduction section should be concise and focused, primarily outlining the current research status and existing challenges in the field or research direction. It should explicitly define the research objectives of this paper or propose a scientific hypothesis.

R2 Response 2:

The introduction has been organized and condensed by roughly 75% of the original while keeping the key concepts.

R2 Comment 3:

How does plant photosynthetic capacity affect ARQ? Why didn't the author consider measuring the gas exchange parameters of plants?

R2 Response 3:

We thank the reviewer for raising this insightful question. Plant photosynthetic capacity supplies respiratory substrates (e.g., sugars, amino acids, and organic acids) that are transported via the phloem to the stem, where they fuel local respiration and influence both CO₂ production and O₂ consumption, thereby affecting ARQ if the RQ is not equal to 1.0. This concept has been added to the introduction.

“These findings support the idea that local stem-respired CO₂ may be derive from substrates with low cellular respiratory quotients (RQ) such as organic acids (RQ 0.7-0.9) and lipids (RQ ~0.7), transported away in the transpiration stream, or refixed before emission.”

While we did not include gas exchange measurements at the leaf level, we agree that such data would provide valuable context, particularly for quantifying canopy reassimilation and substrate supply. We have now added this detail in the discussion.

“In addition, gas exchange measurements at the leaf level would provide valuable context, particularly for quantifying canopy re-assimilation and stem respiratory substrate supply.”

R2 Comment 4:

Stable isotope techniques can be used to accurately trace carbon utilization and cycling in plants. This study investigates stem carbon respiration and transport using a novel method. When complemented by stable isotope-based validation, the reliability of research results can be significantly enhanced.

R2 Response 4:

We thank the reviewer for this valuable suggestion. We fully agree that stable isotope techniques offer powerful tools to trace carbon fluxes and validate mechanistic interpretations of CO₂ transport and metabolism in plants. In fact, previous studies have used isotopic labeling to demonstrate xylem transport of root- and stem-respired CO₂ and its reassimilation by canopy tissues.

While the current study focused on establishing and applying a new real-time method for measuring ARQ and partitioning stem CO₂ efflux under natural conditions, we recognize that integrating stable isotope approaches—such as ¹³CO₂ or ¹³C-labeled substrate injection—would greatly enhance the interpretive power of the technique. We have now noted this in the discussion, and we plan to incorporate stable isotope labeling in future experiments to validate and expand upon the results presented here.

“In addition, gas exchange measurements at the leaf level would provide valuable context, particularly for quantifying canopy re-assimilation and stem respiratory substrate supply. Future studies could also consider complementing real-time ARQ measurements with stable isotope techniques, such as ¹³CO₂ or ¹³C-labeled substrate tracing, to directly quantify the sources, transport, and fate of respiratory CO₂ within the plant. Isotopic labeling would provide a powerful means to validate the partitioning of CO₂ between local emission, xylem transport, and potential reassimilation by canopy tissues, thereby strengthening the mechanistic interpretation of ARQ dynamics.”

R2 Comment 5:

The conclusion is too lengthy and not concise enough, which impairs readability. It is necessary to optimize the conclusion by highlighting the main findings and providing a brief summary to make it more succinct.

R2 Response 5:

We have rewritten the conclusion to sharpen the focus on the main findings and improves clarity and flow.

R2 Comment 6:

Incorporate citations of literature published within the last three years to demonstrate the authors' mastery of the research frontiers in this field.

R2 Response 6:

New references added to the Introduction and Discussion sections (recent studies over last 3 years).

Introduction

“Stem respiration contributes approximately a quarter of the global above-ground auto-trophic respiration, with an estimated annual emission of around 11.20 ± 5.88 Pg C, comparable to total anthropogenic emissions [3].”

“Dukat et al. (2024) partitioned stem CO₂ efflux in Pinus sylvestris and found that transport-related fluxes accounted for up to 40% of the total stem CO₂ efflux during peak growing season. They employed a combination of stem chamber measurements, sap flow monitoring, and modeling to distinguish between locally respired CO₂, bark photosynthesis, and CO₂ transported via the xylem.”

Discussion

“Consistent with these observations, in a recent study by Darenova et al. (2024), the authors investigated the effects of reduced water availability on stem CO₂ efflux in oak (Quercus petraea) and hornbeam (Carpinus betulus) coppices. They found that severe summer drought led to a significant decrease in stem CO₂ efflux, with reductions ranging from 43% to 81% during July and August. This decline was closely associated with a concurrent reduction in stem growth, suggesting that drought-induced limitations on growth processes were a primary factor in the observed decrease in stem respiration [54]. While the study did not directly measure turgor pressure, the authors discussed that the suppression of stem CO₂ efflux under drought conditions could be attributed to hydraulic constraints affecting cell expansion and metabolic activity, potentially linked to reduced turgor pressure. These findings highlight the complex interplay between water availability, stem growth, and respiration, emphasizing the sensitivity of stem CO₂ efflux to drought stress.”

Round 2

Reviewer 2 Report

Thanks for the authors' hard work for improving the quality of this manuscript. They have addressed all my concerns, I have no other questions. Therefore, I suggest to accept this paper.