Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the effort put into this manuscript and find the topic both relevant and interesting. Evaluating visible foliar injury (O₃ VFI) in Viburnum lantana L., which is a widely distributed and ozone-sensitive species in Europe, using a controlled fumigation system is a highly meaningful approach that addresses an important ecological question. However, the primary objectives and the overall focus of the study remain somewhat unclear in the current version. Furthermore, the specific scope of this manuscript appears to deviate slightly from the central theme of this Special Issue. Addressing these ambiguities through a major revision will significantly enhance the quality and impact of the manuscript. Additionally, the authors should thoroughly check and revise the References section to strictly comply with the formatting guidelines of the journal Forests. My specific comments and detailed suggestions are provided in the attached PDF file for the authors' convenience.

Comments for author File: Comments.pdf

Author Response

Reviewer Comments to Author

Overall

I appreciate the effort put into this manuscript and find the topic both relevant and interesting. Evaluating visible foliar injury (O3 VFI) in Viburnum lantana L., which is a widely distributed and ozone-sensitive species in Europe, using a controlled fumigation system is a highly meaningful approach that addresses an important ecological question. However, the primary objectives and the overall focus of the study remain somewhat unclear in the current version. Furthermore, the specific scope of this manuscript appears to deviate slightly from the central theme of this Special Issue. Addressing these ambiguities through a major revision will significantly enhance the quality and impact of the manuscript. Additionally, the authors should thoroughly check and revise the References section to strictly comply with the formatting guidelines of the journal Forests. My specific comments and suggestions are detailed below.

We thank the Reviewer for carefully reviewing our manuscript and for the valuable comments and suggestions. We have revised the manuscript accordingly and addressed each point raised by the Reviewer in detail below. We would also like to clarify that the submission to the Special Issue was made at the suggestion of the Journal Editor. The revised Word file, with all changes highlighted in Track Changes mode, was uploaded for the reviewer’s consideration.

 

Title

Line 3-4: The length of the abstract, along with its list-like presentation of results, could be reduced to make it more concise and engaging.

The Abstract has been revised to make it more concise for improving its overall narrative flow.

 

Abstract

Line 29-33: The term "leaf color index" used to define SPAD is inaccurate. SPAD values represent relative chlorophyll content, not chromatic color coordinates. Please correct this terminology throughout the manuscript to maintain scientific accuracy.

We agree that the term “leaf color index” was not scientifically accurate when used to refer to SPAD. In the manuscript, SPAD was otherwise correctly described as an indicator or proxy of relative chlorophyll content. These instances have now been corrected in accordance with the Reviewer’s suggestion.

 

Line 41-43: Please rearrange the keywords in alphabetical order to comply with standard journal formatting.

The keywords are now in alphabetical order, as requested for the standard journal formatting.

 

Line 89-92: In the Introduction, the authors emphasize validating field-based observations as their primary aim. However, this objective is not properly addressed or matched in the Results and Discussion sections, as no direct comparison with natural forest data is presented. I strongly suggest removing this stated aim from the Introduction to avoid creating false expectations for the reader, or the authors must include a dedicated comparative section in the Discussion.

We agree that the previous wording could create the expectation of a direct comparison between our experimental results and natural forest data, which was not the purpose of the present study. We have therefore removed the statement indicating validation of field-based observations as the primary aim.

 

Line 93-95: The current description of the study's objectives is somewhat generic and lacks precision. To enhance readability and scientific rigor, the authors should clearly elaborate on the specific aims of the experiment at the end of the Introduction. Structuring the introduction with distinct objectives and hypotheses will guide the readers more effectively.

We agree that the previous version could be too generic for a scientific overview. According with the suggestion, the end of the Introduction has been revised to clearly define the study's specific objectives and hypotheses.

 

Material and methods

Line 103: In Section 2.1, the authors state that the saplings were grown in "18 cm pots." Please clarify whether "18 cm" refers to the top diameter of the pot to ensure exact consistency with the subsequent description of the 9 L pots, which clearly specifies the diameter as "(24 cm diameter)."

We apologize for the inconsistency. The text has been revised to provide the correct information and to clearly specify the pot size used for plant growth during the experiment, including the pot volume and diameter.

 

Line 108-111: There is a critical contradiction regarding the experimental timeline between Section 2.1 and Section 2.3. In Section 2.1, the authors state that the O3 treatment lasted for 152 days, from 17th May to 16th October 2024. However, in Section 2.3, the final sampling timepoint (T2) on 8th September is described as having only "114 days exposure." Please clarify this discrepancy. Did the O3 exposure continue for another 38 days after the T2 measurements until 16th October? If so, please explain why no physiological measurements were conducted at the actual end of the 152-day exposure period, or correct the timeline throughout the manuscript to ensure exact consistency.

We thank the reviewer for pointing out this lack of clarity. The O₃ exposure continued for an additional 38 days after the T2 measurements, until 16^th October 2024. T2 was selected as the final physiological sampling point because it preceded the onset of leaf senescence, which could have affected the physiological measurements and confounded the interpretation of O₃-related responses. We have clarified this point in the revised manuscript to ensure consistency across Section 2.3.

 

Figure 1: In Figure 1, the seasonal patterns for solar irradiance, precipitation, and mean temperature are plotted across a broad range of the year. Since the actual O3 plant exposure was conducted only during the specific fumigation period, it would be much more effective to truncate the X-axis (DOY) to cover only this active window. Removing the irrelevant data from the non-exposure periods will significantly enhance the readability and focus of the meteorological trends.

Figure 1 has been replotted using only the data from the experimental fumigation period. In addition, new images were added to Figure 1 in response to another reviewer’s suggestion.

 

Line 125-126: The phrasing "with ICP Forests Intercalibration experience" in Section 2.2 sounds somewhat narrative and overly detailed. To make the methodology more concise, the authors may simplify it to focus strictly on the qualifications, such as "two trained and calibrated observers," or move the specific organizational experience to the Acknowledgments if necessary.

The narrative sentence was removed to make the methodology more concise and focused.

 

Line 142-144: As mentioned previously, there is a critical contradiction regarding the experimental timeline. The text here states that the final sampling (T2) took place after 114 days of exposure, whereas Section 2.1 claims a total fumigation period of 152 days. Please clarify this discrepancy.

As commented below, T2 was selected as the final physiological sampling point because it preceded the onset of leaf senescence, which could have affected the physiological measurements and confounded the interpretation of O₃-related responses. We have clarified this point in the revised manuscript to ensure consistency across Section 2.3.

 

Line 163-165: This inquiry is out of pure scientific curiosity rather than a formal comment. Since the LI-6800 is fully capable of measuring gs, what was the rationale or methodological advantage of switching to the LI-600 porometer?

We used the LI-600 porometer because it allowed faster stomatal conductance measurements under variable climatic conditions. This was important to reduce the time required for each measurement and to ensure a large number of measurements. We have included this explanation in Section 2.4.

 

Line 165-167: The statement that "Stomatal conductance was measured on 19 sampling days, scheduled at approximately monthly intervals" contains a fundamental logical contradiction. If measurements were taken monthly over a 114-day (or 152-day) period, the total sampling days should be 4 to 5 days, not 19. Please clarify this severe methodological inconsistency and correct the text.

We apologize for the error in the description. Stomatal conductance measurements were not conducted at monthly intervals. They were performed on 19 sampling days distributed along the experimental period. The text has been corrected to remove the incorrect reference to monthly intervals and to clarify the actual sampling frequency.

 

Line 185-186: The authors used the phrase "with i ranging from 1 to n in the integral" (Line 185), but the corresponding equation utilizes a summation, not an integral. Please correct "integral" to "summation" to ensure exact methodological consistency.

We thank the reviewer for noting this inconsistency. The term “integral” has been corrected to “summation” to match the equation and ensure methodological consistency.

 

Line 211-214: Please provide a proper reference for the "Akaike Information Criterion (AIC)" to support the methodological background of your model selection procedure.

We have revised the manuscript and included two additional references to support the model selection procedure based on the Akaike Information Criterion. Aho et al. (2014) were added to support the general use of AIC as a model-selection criterion, and Hoshika et al. (2024), who applied AIC to compare linear and non-linear dose-response models in a review of O₃ dose-response study.

 

Results and discussion

Table 1: In Table 1, the different letters indicating statistical significance for Asat, g_s, and SPAD are completely missing. Furthermore, since all relevant ANOVA results are already integrated into Table 1, Table S1 appears entirely redundant. Please add the missing statistical letters to Table 1 and remove the unnecessary Table S1.

The missing statistical letters have been added to Table 1, and Table S1 has been removed.

 

Line 286-293: While the dataset for the "g_s model parameterization" in Section 3.3 appears meaningful on its own, its inclusion in the manuscript is questionable since these data are never mentioned or interpreted in the Discussion. If this modeling work is essential to the paper's primary objective, it must be thoroughly discussed. Otherwise, Section 3.3 should be omitted to avoid unnecessary data redundancy.

We thank the Reviewer for highlighting this omission in the discussion. The parameterization of the g_s model is crucial to the primary aim of the study, as it underpins the calculations of POD₁ and the interpretation of the flux-based response functions. Consequently, Section 3.3 has been retained, and the Discussion in Section 4.2 has been expanded. Specifically, we now address the estimated g_max value for V. lantana in comparison with Vaccinium myrtillus, a functionally similar forest shrub species previously examined for O₃ response.

 

Figure 4: In Figure 4, the AOT40 regression model is currently embedded within the POD1 graph, which is highly confusing. Since AOT40 and POD1 represent distinct metric concepts, it would be much more effective to present the AOT40-relative PII regression model as a separate standalone graph. Additionally, there are only two data points (dots) visible per treatment in the regression plots. Please clarify whether the number of biological replicates was limited to only two, or explicitly explain how these data points were derived.

Figure 4 has been replotted to present the AOT40-relative PII regression model as a separate graph. We clarify here that the two visible data points per treatment do not represent only two biological replicates. They are relative values calculated using the AA treatment as the reference. We have clarified this in the revised figure caption.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the article "Ozone flux-based response functions for visible foliar injury and photosynthetic traits in a bioindicator species, Viburnum lantana L."
The article is an up-to-date experimental study on the validation of responses of the bioindicator species Viburnum lantana to ozone using a free-aerosol installation (FO₃X). The work has theoretical and practical value for the development of critical ozone levels (CL) for forest ecosystems. However, the manuscript contains a number of shortcomings that need to be addressed before publication.

1. Lines 45-68. I recommend that you specify the cited literature in more detail. There are 15 citations per 20 lines of text. 
2. In section 2.1, provide a geographical map of the study area. Specify the sampling locations (using the table or on the map). 
3. Section 2.1 states that 3 repeated plots were used for processing, with the placement of "1-2 plants per plot". Thus, the total number of plants for processing is approximately 3-6, which is very low for reliable statistics. Please provide more detailed comments on this issue. 
4. Does Figure 1 refer to the results or the source data? If this is the source data, then specify the sources of its receipt.
5. What programs were used for charting?
6. Does section 2.2 specify exactly how the digital photos were processed to extract the data? 
7. In section 2.3, gas exchange measurements were carried out "for one to two plants per plot per treatment" – again a small sample size. You need to specify the exact number of leaves and plants measured.
8. Did the authors compare the data obtained with the results of the Sentinel-5 satellite satellite survey? This would allow us to find matches and extrapolate the data further. Perhaps these are the prospects for future research.
9. In section 4, compare your results with other regions of the world. Please provide a more detailed literary review. 

Author Response

Review of the article "Ozone flux-based response functions for visible foliar injury and photosynthetic traits in a bioindicator species, Viburnum lantana L."

The article is an up-to-date experimental study on the validation of responses of the bioindicator species Viburnum lantana to ozone using a free-aerosol installation (FO₃X). The work has theoretical and practical value for the development of critical ozone levels (CL) for forest ecosystems. However, the manuscript contains a number of shortcomings that need to be addressed before publication.

We thank the Reviewer for carefully reviewing our manuscript and for the valuable comments and suggestions. We have revised the manuscript accordingly and addressed each point raised by the Reviewer in detail below. The revised Word file, with all changes highlighted in Track Changes mode, was uploaded for the reviewer’s consideration.

 

1. Lines 45-68. I recommend that you specify the cited literature in more detail. There are 15 citations per 20 lines of text.

We thank the Reviewer for this helpful comment. The Introduction has been restructured to make the use of references more detailed and balanced. In particular, grouped citations were reduced, the most relevant references were retained, and the contribution of each cited study to the rationale of the manuscript was specified more clearly.

 

2. In section 2.1, provide a geographical map of the study area. Specify the sampling locations (using the table or on the map).

We have revised Figure 1 to include a geographical map of the study area. Panel A now shows the location of the FO3X-FACE facility in Florence, Italy, with an inset map showing its position within Italy. We also updated Panel B to show the experimental plots and Panel C to show the fumigation plots in detail. In section 2.1, the text was revised to include more detailed information.

 

3. Section 2.1 states that 3 repeated plots were used for processing, with the placement of "1-2 plants per plot". Thus, the total number of plants for processing is approximately 3-6, which is very low for reliable statistics. Please provide more detailed comments on this issue.

We have revised Section 2.1 to specify the total number of plants used in the experiment and their distribution among the replicated plots and O3 treatments. We acknowledge that the number of plants was limited, a constraint imposed by the operational requirements of the FO3X-FACE system in that case. However, the use of replicated plots and repeated measurements over time allowed us to detect consistent treatment responses. To avoid overinterpretation, we clarified this limitation in the revised manuscript and interpreted the results accordingly.

 

4. Does Figure 1 refer to the results or the source data? If this is the source data, then specify the sources of its receipt.

Figure 1 does not represent newly generated results. It summarizes source data obtained from the monitoring stations located in the experimental facility. We have added this information to the text for clarity.

 

5. What programs were used for charting?

We have added the information at the end of section 2.6. Data analysis

 

6. Does section 2.2 specify exactly how the digital photos were processed to extract the data?

We have revised Section 2.2 to describe the digital image processing workflow more clearly

 

7. In section 2.3, gas exchange measurements were carried out "for one to two plants per plot per treatment" – again a small sample size. You need to specify the exact number of leaves and plants measured.

We agree that the original wording was unclear about the exact number of leaves per plant measured for gas exchange analyses. We have revised the methods description to clarify how the raw data were acquired. We understand it looks like a small sample size, but the values were quite stable across individuals, as reported in Table 1 (low standard errors for A_sat and g_s).

 

8. Did the authors compare the data obtained with the results of the Sentinel-5 satellite survey? This would allow us to find matches and extrapolate the data further. Perhaps these are the prospects for future research.

A direct comparison with Sentinel-5P/TROPOMI satellite products was not performed in the present study, because the experiment was conducted at the plant level under controlled open-air exposure conditions, whereas satellite-derived products operate at broader spatial scales. Moreover, Sentinel-5P/TROPOMI products do not directly quantify stomatal O₃ uptake or leaf-level O₃ visible foliar injury, which were the main endpoints of this study. However, we agree that the integration of experimental data, flux-based response functions, and satellite-derived information represents an important perspective for future research, particularly for extrapolating O₃ risk assessment beyond local experimental conditions. Therefore, a paragraph has been added to Section 4.1 to discuss this point.

 

9. In section 4, compare your results with other regions of the world. Please provide a more detailed literary review.

We agree that the Discussion would benefit from a broader comparison with the literature. However, the available species-specific literature is necessarily limited by the natural distribution of Viburnum lantana, which extends from Europe to western Asia. Therefore, we expanded the Discussion by comparing our results with functionally comparable forest shrub species (Vaccinium myrtillus) previously investigated for O₃ response.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank the authors for their thorough and careful revision of the manuscript. They have successfully addressed all of my concerns and clarified the methodological inconsistencies in a highly satisfactory manner. The quality of the manuscript has been significantly improved.

Reviewer 2 Report

Comments and Suggestions for Authors

Accept in present form