Cross-Stressful Adaptation to Drought and High Salinity Is Related to Variable Antioxidant Defense, Proline Metabolism, and Dehydrin b Expression in White Clover
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors
I have reviewed the manuscript entitiled "Cross-stressful adaptation to drought and high salinity is related to variable antioxidant defense, proline metabolism, and dehydrin b expression in white clover". With rapid increasing of climatic change, such studies play a significant role.
The manuscript is well presented and nicely explained.
Author Response
Comments and Suggestions for Authors
I have reviewed the manuscript entitiled "Cross-stressful adaptation to drought and high salinity is related to variable antioxidant defense, proline metabolism, and dehydrin b expression in white clover". With rapid increasing of climatic change, such studies play a significant role.
The manuscript is well presented and nicely explained.
Response: Thank you very much for professional review.
Reviewer 2 Report
Comments and Suggestions for AuthorsReview Report
Title: Cross-stressful adaptation to drought and high salinity is related to variable antioxidant defense, proline metabolism, and dehydrin b expression in white clover
· The abstract is dense with technical terms and may not be easily understood by a general audience. Simplify the language to improve accessibility. For example, briefly explain terms like "cross tolerance" and "dehydrin b expression" for non-specialist readers.
· The introduction lacks a clear articulation of the specific knowledge gaps this study addresses. Highlight what is novel about this research compared to prior studies on stress priming in white clover.
· In order to fill the existing research gap, a more detailed analysis of previous research is recommended. I suggest some articles that may help you fill the existing research gap such as, https://link.springer.com/article/10.1186/s40529-024-00440-0; https://link.springer.com/article/10.1007/s10661-023-11982-4
· The study mentions controlled growth conditions, specifics about potential environmental variations during the experimental period (light intensity, temperature fluctuations) are not detailed. Providing a more comprehensive account of the environmental conditions during the experiment would enhance reproducibility and contextual understanding.
· Provide justifications for the chosen stress levels (e.g., 250 mM NaCl for salt stress).
· Include a rationale for selecting white clover as the model organism beyond its agricultural importance.
· Clarify how "biological replicates" were handled to ensure statistical reliability
· Although the results indicate changes in enzyme activities and proline metabolism, the discussion lacks depth regarding the underlying biochemical pathways that facilitate these adaptations. In results the narrative could be made more engaging by highlighting key findings in the text rather than listing them sequentially.
· Provide biological interpretations of results, such as why SP was more effective than DP in mitigating drought stress.
· Some figures are overloaded with information (e.g., Figures 4 and 5). Consider splitting them for clarity. Some figures (e.g., growth metrics) could be more clearly labeled and presented. A clearer explanation of the data would aid in the interpretation of results.
· The discussion is too lengthy and occasionally repetitive. Condense it to focus on key insights.
· Expand on the ecological and practical implications of SP being more effective than DP for drought tolerance in white clover.
· Address potential limitations of the study, such as the short duration of stress exposure or the exclusion of field trials.
· The study focuses solely on white clover. Conclusions may be applicable to similar species, broader implications for other crops under varying abiotic stresses are not addressed.
Author Response
Comments and Suggestions for Authors
- The abstract is dense with technical terms and may not be easily understood by a general audience. Simplify the language to improve accessibility. For example, briefly explain terms like "cross tolerance" and "dehydrin b expression" for non-specialist readers.
Response: Thanks. We have briefly explained terms "cross tolerance" and "dehydrin b expression" in the Abstract according to your suggestions.
- The introduction lacks a clear articulation of the specific knowledge gaps this study addresses. Highlight what is novel about this research compared to prior studies on stress priming in white clover.
Response: Thanks for good suggestion. Yes, we have well explained what is novel about this research compared to prior studies on stress priming in white clover (line 89-92; line 116-119; line 120-127).
- In order to fill the existing research gap, a more detailed analysis of previous research is recommended. I suggest some articles that may help you fill the existing research gap such as, https://link.springer.com/article/10.1186/s40529-024-00440-0; https://link.springer.com/article/10.1007/s10661-023-11982-4
Response: Thank you very much for careful review and good suggestion. These references have been discussed and cited in revised manuscript according to suggestions (line 60-63).
- The study mentions controlled growth conditions, specifics about potential environmental variations during the experimental period (light intensity, temperature fluctuations) are not detailed. Providing a more comprehensive account of the environmental conditions during the experiment would enhance reproducibility and contextual understanding.
Response: Thanks. Potential environmental variations during the experimental period including light intensity, temperature fluctuations, and humidity have been clearly demonstrated in the manuscript (line 141-142).
- Provide justifications for the chosen stress levels (e.g., 250 mM NaCl for salt stress).
Response: Thanks for your insightful comments. In this experiment, different concentrations of NaCl solutions were applied to induce salt stress (line 146-148): For the second treatment (NP-S), plants were subjected to 100 mM NaCl solution for the first day, 150 mM NaCl solution for the second day, 200 mM NaCl solution for the third day, and 250 mM NaCl for subsequent 7 days. Subsequently, we chose final 250 mM concentration based on the following reasons:
- In previous study,final concentration of 250 mM NaCl has been identified as a effective dose to screen different white clover genotypes with different salt tolerance (Li et al., 2022).
Li, Z., Geng, W., Tan, M., Ling, Y., Zhang, Y., Zhang, L., & Peng, Y. (2022). Differential responses to salt stress in four white clover genotypes associated with root growth, endogenous polyamines metabolism, and sodium/potassium accumulation and transport. Frontiers in Plant Science, 13, 896436.
- In nature, the salt concentrationin saline-alkali land areas can even exceed 1.6% (274 mM).
- Include a rationale for selecting white clover as the model organism beyond its agricultural importance.
Response: Thanks. White clover is not a model organism. We study on the stress tolerance of white clover because of its agricultural importance: White clover is a perennial forage with high protein content and also used as an important ground cover plant for urban greening. Drought and salt stress are the primary limiting factors for white clover growth and yield in agricultural system and grazing ecosystem (line 120-127).
- Clarify how "biological replicates" were handled to ensure statistical reliability
Response: Thanks. Three independent biological replicates have been well clarified in the manuscript: Each treatment included three independent biological replications (three containers), and each container had 21 plants (line 159-160).
- Although the results indicate changes in enzyme activities and proline metabolism, the discussion lacks depth regarding the underlying biochemical pathways that facilitate these adaptations. In results the narrative could be made more engaging by highlighting key findings in the text rather than listing them sequentially.
Response: Thanks. In the section of DISCUSSION, we have discussed DP- or SP-induced underlying biochemical pathways including antioxidant defense, proline metabolism, and expression of dehydrin b contributing to subsequent cross tolerance. As you known, for a scientific paper, the section of results should clearly present the main findings without excessive description.Yes, we only highlighted key findings in the section of RESULTS.
- Provide biological interpretations of results, such as why SP was more effective than DP in mitigating drought stress.
Response: Thanks for good question. In current study, we found that DP-regulated salt tolerance and SP-induced drought tolerance were related to some common pathways such as enhanced enzymatic antioxidant system and proline biosynthesis as well as some different mediations of growth and dehydrin b expression. However, SP could significantly up-regulated the expression of dehydrin b under later drought stress, but DP failed to induce the expression of dehydrin b in response to subsequent salt stress. This could be a main reason why SP was more effective than DP in mitigating subsequent stress. We have clearly explained these findings in the last paragraph of Discussion (line 498-518) and the section of Conclusions (line 520-544).
- Some figures are overloaded with information (e.g., Figures 4 and 5). Consider splitting them for clarity. Some figures (e.g., growth metrics) could be more clearly labeled and presented. A clearer explanation of the data would aid in the interpretation of results.
Response: All figures have been clearly labeled to present results. Figure 4 demonstrates those parameters related to oxidative damage and cell membrane stability. Figure 5 presents three main antioxidant enzyme activities in plants. Figure 6 demonstrates four key enzyme activities involved in ascorbate-glutathione cycle.
- The discussion is too lengthy and occasionally repetitive. Condense it to focus on key insights.
Response: Thanks. For a complete research paper, the discussion is not too lengthy in this manuscript. Yes, the discussion in the manuscript focuses on key insights. As you can see, the discussion only includes four paragraphs. First paragraph discussed growth. Second paragraph discussed antioxidant defense. Third paragraph discussed proline metabolism. Fourth paragraph discussed the importance of dehydrins.
- Expand on the ecological and practical implications of SP being more effective than DP for drought tolerance in white clover.
Response: Thanks for good question. In current study, it is not SP being more effective than DP for drought tolerance in white clover, but current findings proved that the pre-exposure of white clover plants to DP or SP could effectively mitigate negative effects of subsequent salt stress or drought related to some common pathways such as enhanced enzymatic antioxidant system and proline biosynthesis as well as some different mediations of growth and dehydrin b expression (line 531-538). We have added a perspective of the ecological and practical implications of SP- and DP-regulated cross tolerance according to your suggestion (line 538-544).
- Address potential limitations of the study, such as the short duration of stress exposure or the exclusion of field trials.
Response: Thank you very much for good suggestion. Potential limitations of the study has been added in the section of Conclusion (line 540-544).
- The study focuses solely on white clover. Conclusions may be applicable to similar species, broader implications for other crops under varying abiotic stresses are not addressed.
Response: Thank you very much for good suggestion. Yes, we totally agree with your point of view. Conclusions may be applicable to similar species. Broader implications for other crops under varying abiotic stresses has been added in the section of Conclusion according your suggestion (line 538-544).
Reviewer 3 Report
Comments and Suggestions for AuthorsIntroduction
-Please include other research works for white clover, or any other legume, regarding their antioxidant activity.
-Please add information and relevant references for the gene you study. Where is it involved? What is its regulation? Are there any other studies of that gene in legumes?
M&M
-Please include in that section clearly the different time points you did for the different measurements you conducted. (some data are on day 0 and day 10 and others on days 0, 5, and 10- Figures 5&6).
--Please add the official gene ID of the genes that you used. (for e.g.NCBI LOC ID).
-How did you design the genes' primers? please add information.
- Also in the results you present the expression levels of the gene in 3 different days. please add the time points of sampling.
-please add the version of spps that you used and cite it.
Results
-Please clarify better which results refer to day 0 and day 10.
-Did you check any interactions among the different drought/salinity environments for all of your tested characteristics (plant growth, antioxidant capacity, gene expression, etc)? It would be interesting and useful for your research to present the interactions, if any.
The authors could clarify the results highlighting the differences noticed between the different dates. (0 &10 days).
Conclusions
An overall conclusion, highlighting the importance of the present findings could be helpful for the manuscript's scientific value. Add information about why white clover is an important species to study and how these results can contribute and add value to the scientific community.
Comments for author File: 
Comments.pdf
The authors are encouraged to revise the manuscript to improve its English language quality, possibly with the assistance of a native speaker or professional language editing service.
Author Response
Comments and Suggestions for Authors
Introduction
- Please include other research works for white clover, or any other legume, regarding their antioxidant activity.
Response: Thank you very much for good suggestion. Other research works for white clover regarding their antioxidant activity have been added in revised manuscript (line 74-83).
- Please add information and relevant references for the gene you study. Where is it involved? What is its regulation? Are there any other studies of that gene in legumes?
Response: Thanks. Relevant information and references about the gene have been added in revised manuscript according your suggestion (line 131-132). In addition, we have added the gene ID in the M&M (line 244). Other studies of the gene have been also well discussed in the section of Discussion (line 498-518).
M&M
- Please include in that section clearly the different time points you did for the different measurements you conducted. (some data are on day 0 and day 10 and others on days 0, 5, and 10- Figures 5&6).
Response: Thanks for good suggestion. We have added relevant explanation about different time points we did for the different measurements we conducted in the M&M (line 160-167).
- Please add the official gene ID of the genes that you used. (for e.g.NCBI LOC ID).
Response: Thanks. The gene ID of dehydrin b has been added in revised manuscript (line 243-252).
- How did you design the genes' primers? please add information.
Response: Thanks. The information has been added in revised manuscript (line 251).
- Also in the results you present the expression levels of the gene in 3 different days. please add the time points of sampling.
Response: Thanks. The information has been added in revised manuscript according to your suggestion (line 167).
- please add the version of spss that you used and cite it.
Response: The SPSS 20.0 was used, and we have cited relevant reference in revised manuscript according to the suggestion (line 258).
Results
- Please clarify better which results refer to day 0 and day 10.
Response: Yes, results were clarified referring to day 0 and day 10 in revised manuscript according to the suggestion (line 261-410).
- Did you check any interactions among the different drought/salinity environments for all of your tested characteristics (plant growth, antioxidant capacity, gene expression, etc)? It would be interesting and useful for your research to present the interactions, if any.
Response: Thanks. Current findings proved that the pre-exposure of white clover plants to DP or SP could effectively mitigate negative effects of subsequent salt stress or drought related to some common pathways such as enhanced enzymatic antioxidant system and proline biosynthesis as well as some different mediations of growth and dehydrin b expression. Possible interactions have been well discussed in the section of Discussion and Conclusion.
- The authors could clarify the results highlighting the differences noticed between the different dates. (0 &10 days).
Response: Yes, results were clarified referring to day 0 and day 10 in revised manuscript according to the suggestion (line 261-410).
Conclusions
- An overall conclusion, highlighting the importance of the present findings could be helpful for the manuscript's scientific value. Add information about why white clover is an important species to study and how these results can contribute and add value to the scientific community.
Response: Thank you very much for good suggestion. The reason why white clover is an important species to study has been clearly explained in the section of Introduction (line 120-127). We also added how these results can contribute and be value to the scientific community in the section of Conclusion (line 537-540) according to your suggestion.
Comments on the Quality of English Language
- The authors are encouraged to revise the manuscript to improve its English language quality, possibly with the assistance of a native speaker or professional language editing service.
Response: Thanks. English language in the manuscript has been checked and improved by a native speaker before submitting. Our research group has published more than 100 peer-reviewed scientific papers since 2010.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI have thoroughly reviewed the revised version of the manuscript. I am pleased to confirm that the authors have adequately addressed all the comments and suggestions provided in the previous review. The revisions have significantly improved the manuscript, and the study now provides valuable insights that are both scientifically sound and well-presented.
In light of this, I am satisfied with the revised version and would like to recommend the manuscript for publication.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe author's revision was based on the proposed suggestions and clarified important points.
Comments on the Quality of English LanguageAlthough the quality of the English language did not prevent me from understanding the whole idea of the research, I still recommend that some changes and language modifications would improve the quality of the manuscript.
