Drought Stress and High Temperature Affect the Antioxidant Metabolism of Cotton (Gossypium hirsutum L.) Anthers and Reduce Pollen Fertility
Round 1
Reviewer 1 Report
attach.
Comments for author File: 
Comments.pdf
The text is difficult to read due to long sentences. Many issues in one sentence at once. The text needs to be reworded and in my opinion its quality/clarity will improve.
Author Response
Response to the referees’ comments for the manuscript of agronomy-2620355:
We are grateful to editors and reviewers for their valuable comments and useful suggestions on our paper “Drought stress and high temperature affect the antioxidant metabolism of cotton (Gossypium hirsutum L.) anthers to reduce pollen fertility”. In the following, we will respond to the comments one by one, and the improvements are showed in red in the revised manuscript.
Editor-in-Chief:
General remarks. Manuscript deals with an abiotic stresses such as drought, high
temperature and combined effect of both in relations to anthers antioxidant metabolism. The model plant was cotton. In the light of ongoing climate change, the problem of drought and high temperatures increasingly affects plant production. So this issue is important and worth consideration. Unfortunately, the text is difficult to read due to the style of language. Long and multi-threaded sentences make the text unclear and the results are difficult to follow (for an examples plese see lines: 143-148, 268-272, 370-375, 396-400, 403-410). The authors performed a statistical analysis that shows the differences between the combinations/treatments and commenting on the results should be based on it. Showing differences in percentages or so colled fold-variations should be limited, especially in the context of this study and presenting data on charts. It is difficult to follow them due to the lack of particular data for their calculation as is in case of tables. Besides, data presented in percentages showing the size of differences between years, for example, do not have to have so many decimal places, which only worsens their perception. Sometimes large differences in the results between 2019 and 2020 were presented, but there is no comment on this issue in the discussion.
Response: Thank you for your suggestion. This has been reviewed.
Further points to consider and improve the text.
Abstract. The abstract sounds like the 'Results' section was rewritten here. Please read the instructions for authors.
Response: Thank you for your suggestion. The authors believe that this kind of summary is necessary.
Key words: Most keywords are already in the title of the manuscript, which is generally avoided.
Response: Thank you for your suggestion. The authors think that the current title of the manuscript can better point out the content of the research.
Mat&Met. The information in the materials and methods should be more precise, because sometimes we can find it only by going to the results, e.g.:
- how long the stressful conditions lasted before taking samples for analyses;
Response: Thank you for your suggestion. Already added to lines 146-147.
- water potential was measured every 3 days but the total time interval is not indicated (start and end points);
Response: Thank you for your suggestion. Already added to lines 154-155.
- no explanation why pollen viability was measured in consecutive years using different methods. There is also no reference (Tab/Fig) in the text where these results are.
Response: Thank you for your suggestion. The explanation were added to lines 327-332. The pollen viability results (Fig. S2 & Table S2) were shown in the attachment. The authors consider this part of the results to serve as a supplementary explanation and do not need to appear in the main text.
There is no consistency in the description of the methods. If the assay was performed based on the supplier's protocol, at least the final technique/final conditions should be demonstrated. It is important for the reader whether it was U/HPLC, a spectrophotometer, etc. On this information sombody may assess whether this method is precise, accessible in their lab or interesting for him to use.
Response: Thank you for your suggestion. Specific instrument/measurement wavelength has been added.
- 106. Which publication is finally cited here?
Response: Thank you for your suggestion. This error has been modified.
L.118. Is the publication cited here actually about rice? Please check the reference to this work also in the line 123.
Response: Thank you for your suggestion. The purpose of this part is to evaluate the effects of stress on the AsA-GSH cycle of different crops, so these references are cited.
L.239. Table 1 does not appear in the main manuscript body but in the S. material, so it should be Table S1 rather. Moreover, I do not see that in the text the authors refer to Figure 2S and Table 2S included in the supplemental material. Please check this.
Response: Thank you for your suggestion. The figures and tables in the attachment have been renumbered in the order in which they appear in the manuscript, and the pollen viability result as a reference does not appear in the text results, but it is indicated in the Mat&Met.
Figures:
An explanation of enzyme abbreviations should be added to the legends of Figs 5 and 7.
Response: Thank you for your suggestion. Already added to image annotation.
Conclusion. The conclusions should be more generalized and not repeat the obtained results. In this paragrapf authors also should back to the last paragraph of the introduction section (L. 143-148). What new information has been obtained on this topic, what does it mean for further use and how? What mechanisms dominate at what stress? Will the ascorbateglutathione cycle and enzymes related to them, looking at the concentration of ascorbate compared to glutathione, be of key importance in cotton anthers antioxidant metabolism and so on.
Response: Thank you for your suggestion. Already added to L. 425-433.
References should be checked carefully, for example: positions 34 and 36 please see reference titles, position 48 – lack of dot, positions 50-53 – confusion in the references order.
Response: Thank you for your suggestion.Position 34, 36 and 48 references have the correct title. The references have been reordered.
Overall, we thank the editors and reviewers again for your help in improving the manuscript. We also checked the format and grammar again and the improvements are showed in red in the revised manuscript. We hope that the editor and reviewers will be satisfied with our responses.
Yours Sincerely,
Wei Hu
College of Agriculture
Nanjing Agricultural University
Author Response File: 
Author Response.docx
Reviewer 2 Report
1, The manuscript exhibits grammatical errors that necessitate further refinement.
2, In the 'Introduction' section, the current portrayal of ROS metabolism that impacts on pollen fertility under conditions of drought and high temperature is notably general. It is imperative to emphasize their pivotal roles in the context of reproductive development and incorporate more pertinent examples.
3, The utilization of I2-KI and TCC for the assessment of pollen viability in 2019 and 2020, respectively. Why?
4, Although the experiment was conducted in both 2019 and 2020, certain parameters (e.g., peroxides labeling, gene expression) were assessed solely in one of these years. It is advisable to replicate the measurement of these indicators across different years to enhance the robustness of the findings.
5, The author contends that there was no statistically significant difference in H2O2 content between the HT and DS+HT treatments in 2020, as depicted in Figure 3. However, the graphical representation suggests otherwise. A clarification of this inconsistency is warranted.
6, Could the authors elucidate the underlying rationale for the observed increase in H2O2 content in 2020 compared to 2019?
7, In Line 106, the reference to "Fu et al. (Krishna et al. 2023)" appears to be incorrect and requires correction to accurately reflect the intended source.
8, In Lines 128, 202, 220, 379, 390, and 391, incorrect citation format for references, please rectify this error and adhere to the prescribed citation style.
9，References contain many errors.
English language requires further refinement.
Author Response
Response to the referees’ comments for the manuscript of agronomy-2620355:
We are grateful to editors and reviewers for their valuable comments and useful suggestions on our paper “Drought stress and high temperature affect the antioxidant metabolism of cotton (Gossypium hirsutum L.) anthers to reduce pollen fertility”. In the following, we will respond to the comments one by one, and the improvements are showed in red in the revised manuscript.
Editor-in-Chief:
1, The manuscript exhibits grammatical errors that necessitate further refinement.
Response: Thank you for your suggestion. This has been reviewed.
2, In the 'Introduction' section, the current portrayal of ROS metabolism that impacts on pollen fertility under conditions of drought and high temperature is notably general. It is imperative to emphasize their pivotal roles in the context of reproductive development and incorporate more pertinent examples.
Response: Thank you for your suggestion. At present, there is a lack of studies on antioxidant metabolism affecting pollen fertility under the combined conditions of drought and high temperature. So the relevant examples have not been found.
3, The utilization of I2-KI and TCC for the assessment of pollen viability in 2019 and 2020, respectively. Why?
Response: Thank you for your suggestion. The explanation were added to lines 327-332. The aim of this study was to verify whether different methods of pollen viability detection are affected by annual differences. The I2-KI method is used to measure pollen viability by measuring starch content. And the TTC method is used to detect pollen viability by measuring respiration intensity. These two methods have been shown to be good measures of pollen viability. And it's not affected by year-to-year differences.
4, Although the experiment was conducted in both 2019 and 2020, certain parameters (e.g., peroxides labeling, gene expression) were assessed solely in one of these years. It is advisable to replicate the measurement of these indicators across different years to enhance the robustness of the findings.
Response: Thank you for your suggestion. We agree that more study or more data would be useful to explain the effects of combined stress on pollen fertility. In the future, we will continue to carry out this type of research.
5, The author contends that there was no statistically significant difference in H2O2 content between the HT and DS+HT treatments in 2020, as depicted in Figure 3. However, the graphical representation suggests otherwise. A clarification of this inconsistency is warranted.
Response: Thank you for your suggestion. Due to the visual difference, the y axis below 0.70 is omitted, so there seems to be a difference between HT and DS+HT. In fact, there is no significant difference between them by LSD method at the level of 0.05.
6, Could the authors elucidate the underlying rationale for the observed increase in H2O2 content in 2020 compared to 2019?
Response: Thank you for your suggestion. Although the treatment conditions of abiotic stress in 2019 and 2020 were basically the same, due to the differences in microclimate of the ponds, the midday leaf water potential of cotton in 2020 was generally lower than that in 2019, and the water stress in cotton was aggravated, resulting in more H2O2 accumulation.
7, In Line 106, the reference to "Fu et al. (Krishna et al. 2023)" appears to be incorrect and requires correction to accurately reflect the intended source.
Response: Thank you for your suggestion. This error has been modified.
8, In Lines 128, 202, 220, 379, 390, and 391, incorrect citation format for references, please rectify this error and adhere to the prescribed citation style.
Response: Thank you for your suggestion. This error has been modified.
9，References contain many errors.
Response: Thank you for your suggestion. This error has been modified.
Overall, we thank the editors and reviewers again for your help in improving the manuscript. We also checked the format and grammar again and the improvements are showed in red in the revised manuscript. We hope that the editor and reviewers will be satisfied with our responses.
Yours Sincerely,
Wei Hu
College of Agriculture
Nanjing Agricultural University
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Lines 425-454, two "Conclusion".
The manuscript exhibits grammatical errors that necessitate further refinement.
Author Response
Response to the referees’ comments for the manuscript of agronomy-2620355:
We are grateful to you for your valuable comments and useful suggestions on our paper “Drought stress and high temperature affect the antioxidant metabolism of cotton (Gossypium hirsutum L.) anthers to reduce pollen fertility”. In the following, we will respond to the comments one by one, and the improvements are showed in red in the revised manuscript.
Editor-in-Chief:
Lines 425-454, two "Conclusion".
Response: Thank you for your suggestion. The last paragraph of the discussion (L. 425-433) has been deleted and the Conclusion (L. 435-454) has been integrated.
The manuscript exhibits grammatical errors that necessitate further refinement.
Response: Thank you for your suggestion. This error has been modified.
Overall, we thank the editors and reviewers again for your help in improving the manuscript. We also checked the format and grammar again and the improvements are showed in red in the revised manuscript. We hope that the editor and reviewers will be satisfied with our responses.
Yours Sincerely,
Wei Hu
College of Agriculture
Nanjing Agricultural University
Author Response File: Author Response.docx
