Physiological Response of Macadamia (Macadamia integrifolia) Seedlings to Drought Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The MS "Physiological Response of Macadamia Seedlings to PEG6000 2 
Induced Drought Stress" describes the results of obviously very extensive research. A lot of work has been done to descrive response of macadamia seedlings to drought stress. 

1. The MS lacks the information why such an extensive research was needed. Was it expected that macadamia response to drought differes from well-known common responses of plants? All dependencies obtained in the MS are described in the text-books on plant physiology. Thus, no news for the theory. What is the practical application of obtained results?
2. Why PEG6000 simulates drought stress particularly in Guizhou Province? It sounds strange. 
3. What is generally known on macadamia drought tolerance? What new knowledge were obtained in thisresearch? 

Minor concerns:

• Lines 16-17 and throughout the MS: round the figures (253 - not 352.63%).
• How do you explain that reductions of palisade and sponge layers by 33% and 29% result in the reduced leaf thickness by 19%?
• Lines 20-21: decrease in Fv/Fm can not be indicated in % as Fv/Fm is a ratio.
• It is not correct to write "method descrived by Wu et al, Wei et al" regardinf RWC and MDA content measurement. You may refer to their works , but methods were not described by these authors. 
• Chl a and b should be italicazed.

 

Comments on the Quality of English Language

Lines 92-93: Some parts of sentences are missing. 

Author Response

Comment 1： The MS lacks the information why such an extensive research was needed. Was it expected that macadamia response to drought differes from well-known common responses of plants? All dependencies obtained in the MS are described in the text-books on plant physiology. Thus, no news for the theory. What is the practical application of obtained results?

Response: Thank you for your comment. (1) The macadamia is an evergreen tree with leathery leaves that have small spines along the edges and a well-developed root system with extensive lateral roots. Introduced to Guizhou, China, a region characterized by typical karst topography and frequent seasonal droughts, it has not only shown excellent adaptability but also brought significant economic benefits, making it an ideal species for controlling rocky desertification. However, current research on the drought resistance of this species is limited, and there may be other mechanisms by which it adapts to arid environments. Therefore, we have studied the phenotype, physiology, biochemistry and cell anatomy. (1) Our results showed that the ABA content of macadamia nuts increased rapidly and stomatal size decreased gradually after drought stress. Macadamia may regulate stomatal size through ABA signal transduction pathway to reduce water loss. At the same time, the activity of antioxidant enzymes and osmotic regulatory substances increased, reducing ROS damage and osmotic stress, and the results of this study can reveal the physiological mechanism of macadamia response to drought stress, and provide a theoretical basis for our next step in the selection of stress-resistant varieties.

Comment 2. Why PEG6000 simulates drought stress particularly in Guizhou Province? It sounds strange.

Response: Thank you for your comment. Polyethylene glycol (PEG-6000) is a macromolecular substance that can cause plants' osmotic stress and is often used to simulate drought stress. In order to quickly obtain a research result, we adopted PEG simulation stress, and next we will conduct soil pot stress test. We rewrote the introduction to make the purpose and meaning of the study clearer.

Comment 3. What is generally known on macadamia drought tolerance? What new knowledge were obtained in thisresearch?

Response: Thank you for your comment. The macadamia inherently adapted to arid environments, employing an isohydric strategy to cope with water stress and possessing a dense root system, which contributes to its strong drought resistance. We have observed that the tree's vessels increase in diameter under drought stress to enhance water transport efficiency, while also accumulating osmotic adjustment substances to maintain osmotic balance and prevent excessive water loss. During this process, the activity of antioxidant enzymes increases to scavenge reactive oxygen species generated within the plant, and hormone levels are regulated to modulate physiological activities. These findings provide a theoretical basis for understanding the drought resistance of macadamia trees. We have also supplemented this information in the introduction.

Comment 4. Commet Lines 16-17 and throughout the MS: round the figures (253 - not 352.63%).

Response: Thank you for your comment. We have rounded the numbers throughout the text, as detailed in the manuscript.

Comment 5. How do you explain that reductions of palisade and sponge layers by 33% and 29% result in the reduced leaf thickness by 19%?

Response: Thank you for your comment. These three values represent the reduction in leaf thickness, palisade tissue thickness, and spongy tissue thickness after 72 hours of drought stress compared to the measurements at 0 hours. They indicate the changing trends of these three indicators under drought stress. Palisade tissue and spongy tissue are only part of the composition of leaves, and other parts of leaves may not change so significantly after water loss, resulting in a less significant reduction in leaf thickness.

Comment 6. Lines 20-21: decrease in Fv/Fm can not be indicated in % as Fv/Fm is a ratio.

Response: Thank you for your comment. We have changed the reduction in Fv/Fm expressed in percentage to 0.23 (In line 25-26 and 276).

Comment 7. It is not correct to write "method descrived by Wu et al, Wei et al" regardinf RWC and MDA content measurement. You may refer to their works, but methods were not described by these authors.

Response: Thank you for your comment. We have revised the manuscript to give a detailed description of the measurement methods of RWC and MDA. (In line 152-159 and 217-224)

Comment 8. Chl a and b should be italicazed.

Response: Thank you for your comment. We have revised the manuscript (In line 181-182).

Comment 9. Lines 92-93: Some parts of sentences are missing.

Response: Thank you for your comment. We have revised the manuscript (In line 50-51).

Finally, thank you for your arduous work and instructive advice. Special thanks to you for your good comments.

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, PEG-6000 was used to simulate drought and examine how the cellular structural features of stems and leaves, as well as the physiological and biochemical processes, change in macadamia seedlings when there is a lack of water. The data collected will assist in developing more efficient methods for growing and breeding macadamia, which is crucial for successfully cultivating this crop in dry conditions.

The strength of this study lies in the use of an integrated approach to examining the effects of different levels of drought on macadamia. A wide range of methods were used to investigate the response of macadamia to drought, providing a comprehensive understanding of the phenomenon. However, one limitation of this research is the absence of molecular genetic studies of the mechanisms of drought tolerance in these plants.

Introduction

It is not clear from the introduction why the cultivar O.C was selected for the study. Is this cultivar drought resistant? I believe this information should be provided in the work.

Materials and methods

Please describe in more detail how you determined IAA, CTK, GA, and AA using HPLC.

 

Line 440. Please delete.

Author Response

Comment 1. It is not clear from the introduction why the cultivar O.C was selected for the study. Is this cultivar drought resistant? I believe this information should be provided in the work.

Response: Thank you for your comment. O.C is the most widely cultivated variety in China, and in our previous studies, it has shown relatively strong drought resistance, stable yield, and quality. Therefore, we chose O.C as the experimental material. This information has been added to the third paragraph of the introduction(In line 87-93).

Comment 2. Please describe in more detail how you determined IAA, CTK, GA, and AA using HPLC.

Response: Thank you for your comment. We have added the method for measuring IAA, CTK, GA, and ABA using HPLC to the experimental methods(In line 204-216).

Comment 3. Line 440. Please delete.

Response: Thank you for your comment. We have revised the manuscript(In line 448-450).

Finally, thank you for your arduous work and instructive advice. Special thanks to you for your good comments.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Editors and Authors,

I read the manuscript entitled "Physiological Response of Macadamia Seedlings to PEG6000 Induced Drought Stress" with interest. In this study, PEG simulated drought stress experiment was carried out in this paper to study the changes in cell anatomical structure, physiology and biochemistry of macadamia nut seedlings, aiming to understand the physiological characteristics of Macadamia nut response to drought stress and provide a reliable reference for planting and breeding of macadamia. The article's subject is essential and relevant to the study area's scientific environment. Therefore, the manuscript needs some adjustments so that it can then be forwarded to the publication process. The manuscript has the potential for publication in the journal Horticulturae and requires the following adjustments:

TITLE
- Delete "PEG6000 Induced".
- Insert the scientific name of the species.

ABSTRACT
- Add an introduction about the study.
You do not need to mention in the objectives that you used PEG-6000. This information should be included in the material and methods.
- What treatments were used?
- What experimental design?
These percentages are calculated incorrectly. Such a significant increase is not possible. Check them throughout the text.
In describing all the results, it is impossible to identify which treatment provided the presented results. This is not clear.
- Replace the keywords that are repeated in the title.

INTRODUCTION

- Line 42: Biological stress? Wouldn't it be biotic stress? Review.
- Line 46: It was mentioned that recent studies have shown that high temperatures...
At the end of the excerpt, only one study was mentioned. Add more studies to complement the excerpt.
- Line 52: Is drought a natural disaster? Confirm the information by inserting a reference.
- Line 53: "Drought has a wide range of environmental, economic and social impacts, and production in agriculture is susceptible to drought and water scarcity as it is directly dependent on precipitation and transpiration".
This information is not clear. Correct and rewrite.
- The second paragraph is too long. I suggest reducing or dividing it.
- Line 87: This paragraph is in the wrong place. The first paragraph was about the species. This information should be close to the first paragraph.
- The last paragraph should contain the research's hypotheses and objective. Add hypotheses and correct the paragraph.

MATERIAL AND METHODS
- Line 109: Add country.
- Line 119: 15 replicates? Was each seedling considered an experimental plot?
- Separate a paragraph to describe the treatments and experimental design used.
- Line 126: It is not sure to record morphological changes. Morphological variables can be used. - Line 174: What does TTCr mean? Add the meaning.
- Were the length and width of the stomata measured using the microscope?
- Line 189: Water use efficiency was not measured; it was calculated. Make this clear.
- Was the CO2 used in the gas exchange analyses controlled or ambient? Cite.
Line 203: The parameters were provided automatically by the device, which is nothing new.
- Line 212: What statistical analyses and tests were performed? This needs to be detailed here. Was only ANOVA used? For what?

RESULTS

- Topic 3. (Line 216) is described as "Results". Correct.
- Line 218: This is the objective. This is not a result.
- Do all the results described in the first paragraph correspond to Figure 1? And the data to confirm these statements? No? We cannot affirm results just by looking at the photos. Analyses should have been performed with a colourimeter to discuss colouration, but this was not done. Review.
- When mentioning a Table or Figure in the text, add it after the first citation. This is a suggestion for the entire text.
- Add in the caption of Figure 3 that the error bars were inserted.
- There is no need to cite all the percentages found for each variable. This becomes very repetitive throughout the text. Review the best way to change this.

DISCUSSION

 

- Much of the information mentioned is a literature review unrelated to the main results found. For example, the explanation about MDA (Line 441). This is nothing new.
- Review this entire section.

CONCLUSIONS

- Line 476: This is not a conclusion. Delete.
- Line 487: What do you mean? What was the theoretical basis? Remove this information.

Author Response

Comment 1. Delete "PEG6000 Induced".
Response: Thank you for your comment. We have removed "PEG6000 Induced" from the title.

Comment 2. Insert the scientific name of the species.

Response: Thank you for your comment. We have added the scientific name of macadamia to the title.

Comment 3. Add an introduction about the study.
Response: Thank you for your comment. We have revised the abstract to explain the background, purpose and significance of this study.

Comment 4. You do not need to mention in the objectives that you used PEG-6000. This information should be included in the material and methods.
- What treatments were used?
- What experimental design?
Response: Thank you for your comment. We have removed the description of PEG-6000 usage from the abstract and added details about the experimental treatments and design.

Comment 5. These percentages are calculated incorrectly. Such a significant increase is not possible. Check them throughout the text.
Response: Thank you for your comment. We have reviewed the calculated data and corrected stomatal length and leaf water potential errors in the calculations. It is possible that the 25% PEG concentration we used was relatively high for macadamia nuts, leading to a rapid response to drought stress, as seen in the case of MDA and Pro.

Commet 6. In describing all the results, it is impossible to identify which treatment provided the presented results. This is not clear.
Response: Thank you for your comment. In our study, we designed five treatments: 0h (CK), 24h, 36h, 48h, and 72h. When describing the results, we consistently compared the changes at 72h to those at 0h (CK) in terms of percentage increase or decrease or absolute reduction.

Comment 7. Replace the keywords that are repeated in the title.

Response: Thank you for your comment. We have changed the keywords (In line40).

Comment 8. Line 42: Biological stress? Wouldn't it be biotic stress? Review.
Response: Thank you for your comment. We have carefully reviewed the original text, and here we wrote "biological and abiotic stresses," not "biological and biotic stresses." Following your suggestion, we have rewritten the introduction, and this description has been removed.

Comment 9. Line 46: It was mentioned that recent studies have shown that high temperatures...
At the end of the excerpt, only one study was mentioned. Add more studies to complement the excerpt.
Response: Thank you for your comment. We have adjusted the content of this section and cited three references to support our argument (In line 77-82).

Comment 10. Line 52: Is drought a natural disaster? Confirm the information by inserting a reference.
Response: Thank you for your comment. We have inserted a reference defining drought in the text (In line 56-60).

Comment 11. Line 53: "Drought has a wide range of environmental, economic and social impacts, and production in agriculture is susceptible to drought and water scarcity as it is directly dependent on precipitation and transpiration".
This information is not clear. Correct and rewrite.
The second paragraph is too long. I suggest reducing or dividing it.

Response: Thank you for your comment. We have rewritten the second paragraph, removing some redundant expressions.

Comment 12. Line 87: This paragraph is in the wrong place. The first paragraph was about the species. This information should be close to the first paragraph.
Response: Thank you for your comment. We have moved this information to the first paragraph.

Comment 13. The last paragraph should contain the research's hypotheses and objective. Add hypotheses and correct the paragraph.

Response: Thank you for your comment. We have rewritten the final paragraph of the introduction, adding current research findings on the drought resistance of macadamia and the questions addressed in our study.

Comment 14. Line 109: Add country.
Response: Thank you for your comment. We apologize for omitting the country in our writing and have now added it to the manuscript.

Comment 15. Line 119: 15 replicates? Was each seedling considered an experimental plot?
Response: Thank you for your comment. In our experiment, five seedlings were treated as one biological replicate, and each treatment was repeated three times biologically, resulting in a total of 15 seedlings per treatment.

Comment 16. Separate a paragraph to describe the treatments and experimental design used.
Response: Thank you for your comment. We divided experimental materials and drought treatment into two sections. (In 106-111).

Comment 17. Line 126: It is not sure to record morphological changes. Morphological variables can be used.

Response: Thank you for your comment. In the experiment, we focused only on the morphological changes of seedlings at different stages and did not statistically analyze morphological variables.

Comment 18. Line 174: What does TTCr mean? Add the meaning.

Response: Thank you for your comment. TTCr, Wr, and T indicate the amount of TTC reduction, root sample weight, reaction time, respectively (In line 176-177).

Comment 19. Were the length and width of the stomata measured using the microscope?

Response: Thank you for your comment. For ease of measurement, we used a microscope to capture images of our observations and then measured the length and width of stomata using Image J 1.53a software(In line 130-132).

Comment 20. Line 189: Water use efficiency was not measured; it was calculated. Make this clear.
Response: Thank you for your comment. We have revised the calculation of WUE to determine water use efficiency based on photosynthetic rate and transpiration efficiency(In line 187-189).

Comment 21. Was the CO2 used in the gas exchange analyses controlled or ambient? Cite.
Response: Thank you for your comment. When measuring photosynthesis during gas exchange analysis, we used ambient CO₂. A reference has been added to the Methods section (In line 188-190).

Comment 22. Line 203: The parameters were provided automatically by the device, which is nothing new.
Response: Thank you for your comment. The implication here is that our data has not been artificially altered, with no other meaning intended.

Commet 23. Line 212: What statistical analyses and tests were performed? This needs to be detailed here. Was only ANOVA used? For what?

Response: Thank you for your comment. To analyze whether the changes in the measured indicators were significant across different drought levels, we used only one-way ANOVA in the statistical analysis. This has been added to the original text (In line 234-235).

 

Comment 24. Topic 3. (Line 216) is described as "Results". Correct.
Response: Thank you for your comment. We have reviewed the manuscript (In line 237).

Comment 25. Line 218: This is the objective. This is not a result.
Do all the results described in the first paragraph correspond to Figure 1? And the data to confirm these statements? No? We cannot affirm results just by looking at the photos. Analyses should have been performed with a colourimeter to discuss colouration, but this was not done. Review.
Response: Thank you for your comment. When describing morphological changes, we provided only an objective description and did not use a colorimeter for measurement.

Comment 26. When mentioning a Table or Figure in the text, add it after the first citation. This is a suggestion for the entire text.
Response: Thank you for your comment. Throughout the text, we have added references to tables or figures upon their first mention.

 

Comment 27. Add in the caption of Figure 3 that the error bars were inserted.
Response: Thank you for your comment. We have added error bars to the description of Figure 3 (In line 237).

Comment 28. There is no need to cite all the percentages found for each variable. This becomes very repetitive throughout the text. Review the best way to change this. 

Response: Thank you for your comment. We have reviewed the original text and revised sections 3.4 and 3.5 to describe the variables as increases or decreases at 72h compared to 0h, consistent with the rest of the text.

Comment 29. Much of the information mentioned is a literature review unrelated to the main results found. For example, the explanation about MDA (Line 441). This is nothing new.
Review this entire section.

Response: Thank you for your comment. Most of the information mentioned in this section is based on proven results. We included it here to demonstrate that our work is well-founded and to help research newcomers quickly understand the meaning of these indicators and our work.

Comment 30. Line 486: This is not a conclusion. Delete.
Response: Thank you for your comment. We have deleted this part. Details are as shown in the text (In line 237).

Comment 31. Line 487: What do you mean? What was the theoretical basis? Remove this information.

Response: Thank you for your comment. We have deleted this part. Details are as shown in the text (In line 237).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The MS has been improved, but unfortunately the main questions are left without convincing answers.

Comment 1. The authors claim that “the results showed that the ABA content of macadamia nuts increased rapidly and stomatal size decreased gradually after drought stress. Macadamia may regulate stomatal size through ABA signal transduction pathway to reduce water loss. At the same time, the activity of antioxidant enzymes and osmotic regulatory substances increased, reducing ROS damage and osmotic stress, and the results of this study can reveal the physiological mechanism of macadamia response to drought stress, and provide a theoretical basis for our next step in the selection of stress-resistant varieties”. The matter of fact that this description of plant response to drought stress corresponds to the one described in Plant Physiology text-books. Most of plants respond to drought stress via such mechanism. Therefore, the novelty of the work should be determined and highlighted. Otherwise, it is quite accurate and extensive work that confirm existing ideas on how plant respond to drought.

The conclusion states that “Under PEG-6000 simulated drought stress, macadamia seedlings alleviated the damage caused by drought stress by changing the cell structure of leaves and stems and activating antioxidant and osmotic regulatory mechanisms”. What was the hypothesis? Did authors expect that nacadamia does not have anatomical and physiological mechanisms to alleviate damages caused by stress?

Comment 5. How do you explain that reductions of palisade and sponge layers by 33% and 29% result in the reduced leaf thickness by 19%?

Response: Thank you for your comment. These three values represent the reduction in leaf thickness, palisade tissue thickness, and spongy tissue thickness after 72 hours of drought stress compared to the measurements at 0 hours. They indicate the changing trends of these three indicators under drought stress. Palisade tissue and spongy tissue are only part of the composition of leaves, and other parts of leaves may not change so significantly after water loss, resulting in a less significant reduction in leaf thickness.

What are other parts of leaves that contribute to leaf thickness?

Author Response

Comment 1. The conclusion states that “Under PEG-6000 simulated drought stress, macadamia seedlings alleviated the damage caused by drought stress by changing the cell structure of leaves and stems and activating antioxidant and osmotic regulatory mechanisms”. What was the hypothesis? Did authors expect that nacadamia does not have anatomical and physiological mechanisms to alleviate damages caused by stress?

Response: Previous studies have demonstrated that macadamia (Macadamia integrifolia) adopts an isohydric strategy, rapidly closing stomata under drought stress to minimize water loss and xylem embolism. Additionally, its leathery leaves and highly lignified xylem contribute to structural resilience. This comprehensive study aimed to determine whether macadamia's oxidative stress responses and osmoregulatory mechanisms align with those of other plant species, beyond its stomatal sensitivity. Our findings revealed that anatomical structures in leaves and stems—including leaf thickness, palisade parenchyma thickness, and xylem cell wall thickness—exhibited delayed responses to drought, with significant changes emerging only after 36 hours. In contrast, the antioxidant system (e.g., SOD, CAT activity) and osmoregulatory mechanisms (e.g., proline accumulation) were activated within hours of stress onset. These traits—stomatal priority, delayed anatomical plasticity, and rapid biochemical defense—may collectively represent macadamia's unique drought adaptation strategy.

Comment 2. What are other parts of leaves that contribute to leaf thickness?

Response: Leaf thickness is composed of epidermal tissue, palisade tissue, spongy tissue, and vascular bundles. The palisade and spongy tissues exhibit higher water content and experience greater proportional reductions under drought stress. However, macadamia (Macadamia integrifolia), characterized by leathery leaves, demonstrates significant structural contributions from other components (e.g., thickened epidermis, lignified vascular bundles). This anatomical specialization likely minimizes drought-induced alterations in epidermal and vascular tissues, resulting in a smaller overall reduction in leaf thickness compared to the palisade and spongy tissues.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been corrected.

Author Response

Comment 1. Delete "PEG6000 Induced".
Response: Thank you for your comment. We have removed "PEG6000 Induced" from the title.

Comment 2. Insert the scientific name of the species.

Response: Thank you for your comment. We have added the scientific name of macadamia to the title.

Comment 3. Add an introduction about the study.
Response: Thank you for your comment. We have revised the abstract to explain the background, purpose and significance of this study.

Comment 4. You do not need to mention in the objectives that you used PEG-6000. This information should be included in the material and methods.
- What treatments were used?
- What experimental design?
Response: Thank you for your comment. We have removed the description of PEG-6000 usage from the abstract and added details about the experimental treatments and design.

Comment 5. These percentages are calculated incorrectly. Such a significant increase is not possible. Check them throughout the text.
Response: Thank you for your comment. We have reviewed the calculated data and corrected stomatal length and leaf water potential errors in the calculations. It is possible that the 25% PEG concentration we used was relatively high for macadamia nuts, leading to a rapid response to drought stress, as seen in the case of MDA and Pro.

Commet 6. In describing all the results, it is impossible to identify which treatment provided the presented results. This is not clear.
Response: Thank you for your comment. In our study, we designed five treatments: 0h (CK), 24h, 36h, 48h, and 72h. When describing the results, we consistently compared the changes at 72h to those at 0h (CK) in terms of percentage increase or decrease or absolute reduction.

Comment 7. Replace the keywords that are repeated in the title.

Response: Thank you for your comment. We have changed the keywords (In line40).

Comment 8. Line 42: Biological stress? Wouldn't it be biotic stress? Review.
Response: Thank you for your comment. We have carefully reviewed the original text, and here we wrote "biological and abiotic stresses," not "biological and biotic stresses." Following your suggestion, we have rewritten the introduction, and this description has been removed.

Comment 9. Line 46: It was mentioned that recent studies have shown that high temperatures...
At the end of the excerpt, only one study was mentioned. Add more studies to complement the excerpt.
Response: Thank you for your comment. We have adjusted the content of this section and cited three references to support our argument (In line 77-82).

Comment 10. Line 52: Is drought a natural disaster? Confirm the information by inserting a reference.
Response: Thank you for your comment. We have inserted a reference defining drought in the text (In line 56-60).

Comment 11. Line 53: "Drought has a wide range of environmental, economic and social impacts, and production in agriculture is susceptible to drought and water scarcity as it is directly dependent on precipitation and transpiration".
This information is not clear. Correct and rewrite.
The second paragraph is too long. I suggest reducing or dividing it.

Response: Thank you for your comment. We have rewritten the second paragraph, removing some redundant expressions.

Comment 12. Line 87: This paragraph is in the wrong place. The first paragraph was about the species. This information should be close to the first paragraph.
Response: Thank you for your comment. We have moved this information to the first paragraph.

Comment 13. The last paragraph should contain the research's hypotheses and objective. Add hypotheses and correct the paragraph.

Response: Thank you for your comment. We have rewritten the final paragraph of the introduction, adding current research findings on the drought resistance of macadamia and the questions addressed in our study.

Comment 14. Line 109: Add country.
Response: Thank you for your comment. We apologize for omitting the country in our writing and have now added it to the manuscript.

Comment 15. Line 119: 15 replicates? Was each seedling considered an experimental plot?
Response: Thank you for your comment. In our experiment, five seedlings were treated as one biological replicate, and each treatment was repeated three times biologically, resulting in a total of 15 seedlings per treatment.

Comment 16. Separate a paragraph to describe the treatments and experimental design used.
Response: Thank you for your comment. We divided experimental materials and drought treatment into two sections. (In 106-111).

Comment 17. Line 126: It is not sure to record morphological changes. Morphological variables can be used.

Response: Thank you for your comment. In the experiment, we focused only on the morphological changes of seedlings at different stages and did not statistically analyze morphological variables.

Comment 18. Line 174: What does TTCr mean? Add the meaning.

Response: Thank you for your comment. TTCr, Wr, and T indicate the amount of TTC reduction, root sample weight, reaction time, respectively (In line 176-177).

Comment 19. Were the length and width of the stomata measured using the microscope?

Response: Thank you for your comment. For ease of measurement, we used a microscope to capture images of our observations and then measured the length and width of stomata using Image J 1.53a software(In line 130-132).

Comment 20. Line 189: Water use efficiency was not measured; it was calculated. Make this clear.
Response: Thank you for your comment. We have revised the calculation of WUE to determine water use efficiency based on photosynthetic rate and transpiration efficiency(In line 187-189).

Comment 21. Was the CO2 used in the gas exchange analyses controlled or ambient? Cite.
Response: Thank you for your comment. When measuring photosynthesis during gas exchange analysis, we used ambient CO₂. A reference has been added to the Methods section (In line 188-190).

Comment 22. Line 203: The parameters were provided automatically by the device, which is nothing new.
Response: Thank you for your comment. The implication here is that our data has not been artificially altered, with no other meaning intended.

Commet 23. Line 212: What statistical analyses and tests were performed? This needs to be detailed here. Was only ANOVA used? For what?

Response: Thank you for your comment. To analyze whether the changes in the measured indicators were significant across different drought levels, we used only one-way ANOVA in the statistical analysis. This has been added to the original text (In line 234-235).

 

Comment 24. Topic 3. (Line 216) is described as "Results". Correct.
Response: Thank you for your comment. We have reviewed the manuscript (In line 237).

Comment 25. Line 218: This is the objective. This is not a result.
Do all the results described in the first paragraph correspond to Figure 1? And the data to confirm these statements? No? We cannot affirm results just by looking at the photos. Analyses should have been performed with a colourimeter to discuss colouration, but this was not done. Review.
Response: Thank you for your comment. When describing morphological changes, we provided only an objective description and did not use a colorimeter for measurement.

Comment 26. When mentioning a Table or Figure in the text, add it after the first citation. This is a suggestion for the entire text.
Response: Thank you for your comment. Throughout the text, we have added references to tables or figures upon their first mention.

 

Comment 27. Add in the caption of Figure 3 that the error bars were inserted.
Response: Thank you for your comment. We have added error bars to the description of Figure 3 (In line 237).

Comment 28. There is no need to cite all the percentages found for each variable. This becomes very repetitive throughout the text. Review the best way to change this. 

Response: Thank you for your comment. We have reviewed the original text and revised sections 3.4 and 3.5 to describe the variables as increases or decreases at 72h compared to 0h, consistent with the rest of the text.

Comment 29. Much of the information mentioned is a literature review unrelated to the main results found. For example, the explanation about MDA (Line 441). This is nothing new.
Review this entire section.

Response: Thank you for your comment. Most of the information mentioned in this section is based on proven results. We included it here to demonstrate that our work is well-founded and to help research newcomers quickly understand the meaning of these indicators and our work.

Comment 30. Line 486: This is not a conclusion. Delete.
Response: Thank you for your comment. We have deleted this part. Details are as shown in the text (In line 237).

Comment 31. Line 487: What do you mean? What was the theoretical basis? Remove this information.

Response: Thank you for your comment. We have deleted this part. Details are as shown in the text (In line 237).