Combined Pretreatment with Bioequivalent Doses of Plant Growth Regulators Alleviates Dehydration Stress in Lactuca sativa
, Iskren Sergiev 1, Dessislava Todorova 1, Martynas Urbutis 2, Giedrė Samuolienė 2 and Lyudmila Simova-Stoilova 1Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn this study, different plant growth regulators (ABA, NAA, ACC and kinetin) was applied in individually or in combination to evaluate the biometric and biochemical response of lettuce to drought stress. The results have important practical significance.However, there are some concerns that should be addressed. Some specific comments can be found below.
1. Abstract section: the “PGR” should be written in its full name when first appears. Too many keywords should delete a few of them.
2. Introduction section: line 54-64 should be placed in the last paragraph and rewritten.
3. Materials and Methods section: why the authors select these 7 PGR combinations? Why not choose the rest, for example ACC and NAA, or NAA, ACC and KIN.
4. Results section: This physiological index of ACC-only-treated plants was inconsistent with that of other treatments, and the author should explain and discuss it.
5. Figure 3b should be best represented as Figure 3a, 3c
6. Conclusions section: The conclusion should be clearly written and consolidated into a single paragraph.
Author Response
We would like to thank Reviewer 1 for the helpful comments. In the revised version of the manuscript, we tried to address the requested changes in the text of the manuscript. Please find below the answers to each of the remarks.
- “Abstract section: the “PGR” should be written in its full name when first appears. Too many keywords should delete a few of them.
Answer: This remark has been addressed in the revised version of the manuscript. We reduced the number of the keywords as suggested by the reviewer.
- “Introduction section: line 54-64 should be placed in the last paragraph and rewritten.”
Answer: We revised the text according to the reviewer’s remark.
- “Materials and Methods section: why the authors select these 7 PGR combinations? Why not choose the rest, for example ACC and NAA, or NAA, ACC and KIN.”
Answer: The main research idea was to check whether the drought-protective effect of exogenous ABA reported previously by many authors (see for summary Jiang et al. 2022 in MDPI Sustainability) will be strengthened by adding other hormonal components that are known to regulate growth and stress response. ABA was considered the central component of the mixes, as it regulates stomata movement, a particularly crucial process controlling transpiration during water-limited conditions. Therefore it was included in most of the tested mixes (except in the KIN+NAA blend which could be regarded as some sort of a “negative control” for the ABA exogenous effect). The ethylene precursor ACC was tested in the context of its proven effect to enhance root-surface area by crosstalking with auxins and cytokinins (the core modulators of root branching and root hair formation). We presumed that this feature could facilitate the better plant performance under water scarcity. We added some additional lines in the text of “Material and Methods” that reflect on the choice of the tested PGR combinations.
- “Results section: This physiological index of ACC-only-treated plants was inconsistent with that of other treatments, and the author should explain and discuss it.”
Answer: In the “Results and Discussion” section of the manuscript, we refer to a recent paper (Brenya et. al 2023) that provides evidence on the properties of exogenous ethylene as a promoter of growth that predispose to a better performance under stress. Our results confirm this ethylene-related effect (i.e. the higher dry weight of the aboveground part of the plants that have received ACC doses before dehydration). Ethylene (respectively its precursor ACC) promotes the expansion of root surface area and biomass by manipulating local auxin and cytokinin biosynthesis and transport. We made sure to address the observed ACC effect in the text related to Figure 1 as suggested by the reviewer. Related to this additional text we included some extra references (Ruzichka et al. 2007; Zdraska et al. 2019 and Moore et al. 2024).
- “Figure 3b should be best represented as Figure 3a, 3c”
Answer: The articulation of the remark was not very clear to us. We tried to address the criticism by rearranging the panels of Figure 3: former panel 3b (H2O2 levels) is 3a in the revised version of the manuscript, and the SOD panel (former 3a) is now 3b. We hope that this corresponds to the expressed concern in regard to Figure 3.
- “Conclusions section: The conclusion should be clearly written and consolidated into a single paragraph.”
Answer: The text concluding the findings of the presented study has been revised following the recommendations of the reviewers.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe author has conducted valuable research, the content of which is substantial, and the results hold significance. My main suggestions are as follows
1. The term "bioequivalent doses" in the title may not be entirely accurate, as it appears to conflict with the experimental methods.
2. Some information in the introduction appears to have low correlation with the paper's focus. I suggest revising the Introduction section according to the following structure: 1st paragraph: Problem statement. 2nd paragraph: Current ongoing solutions. 3rd paragraph: Proposed solution in this work. 4th paragraph: Summary of the research novelty and objectives.
3. To enhance clarity, consider presenting the combinations of PGRs used in the experiment in a table format.
4. The description of the experimental methods requires further clarification to eliminate ambiguities.
5. If "Plant Growth Regulators" are used, avoid using "hormonal applications" in your paper. Choose one term for consistency.
6. In section 2.1.2 (line 146), using both "via foliar spray" and "through the roots" simultaneously may not be permissible. It's recommended to use only one treatment method in an experiment for result accuracy.
7. Provide references for the determination method of physiological and biochemical indicators.
8. Consider using descriptive subheadings for methods rather than simply listing technology names (e.g., "2.3 Immunoblot analyses, 2.4 RT qPCR Analysis").
9. Thoroughly remove method descriptions from the results and discussion sections, as this is not standard academic paper writing practice.
10. Explain briefly in the introduction why these genes were analyzed.
11. In the "Results and Discussion" section, present and analyze the results first, followed by discussion.
12. Include significance analysis results for all data.
13. Explain and discuss the logical relationship between different research contents, and consider reorganizing the overall structure of the paper to strengthen transitions and connections between sections.
14. Rewrite the conclusions, indicating future directions for research in the investigated field.
Author Response
We would like to thank Reviewer 2 for the very helpful comments. In the revised version of the manuscript, we have introduced most of the requested changes in the text. Please find below the answers to each of the points made in the review.
- “The term "bioequivalent doses" in the title may not be entirely accurate, as it appears to conflict with the experimental methods.”
Answer: “Bioequivalent doses" is indeed a “borrowed” term (in pharmacokinetics it defines the biochemical similarity of compounds that share the same active ingredients and desired outcomes). We use it to reflect as concisely as possible the main research idea of the study (e.g. PGRs applied in naturally occurring concentrations that are capable of promoting desired trait). Considering that Reviewer 1, Reviewer 3 and the Guest Editors, in their comments did not express any concerns regarding the title, we prefer to keep the term "bioequivalent doses".
- “Some information in the introduction appears to have low correlation with the paper's focus. I suggest revising the Introduction section according to the following structure: 1st paragraph: Problem statement. 2nd paragraph: Current ongoing solutions. 3rd paragraph: Proposed solution in this work. 4th paragraph: Summary of the research novelty and objectives.”
Answer: The text of section “Introduction” has been revised according to the suggestions and the critical remarks expressed by the reviewers.
- .”To enhance clarity, consider presenting the combinations of PGRs used in the experiment in a table format.”
Answer: Following the reviewer’s recommendation we included in the section “Materials and methods” a table that presents the composition of the tested PGR combinations (new Table 1).
- “The description of the experimental methods requires further clarification to eliminate ambiguities”.
Answer: To address the expressed general concern on the clarity of the methods’ description, we revisited and corrected the text of this part of the manuscript.
5.”If "Plant Growth Regulators" are used, avoid using "hormonal applications" in your paper. Choose one term for consistency.”
Answer: We have considered this remark in the revised text of the manuscript.
6.” In section 2.1.2 (line 146), using both "via foliar spray" and "through the roots" simultaneously may not be permissible. It's recommended to use only one treatment method in an experiment for result accuracy.”
Answer: We respectfully disagree with the point made by the reviewer. The proven protective property of a certain preparation through various application methods provides practical flexibility catered to particular demands. The consistent beneficial effect of the pretreatment with the blend applied through different methods (leaf spray and root treatment) could be regarded as a convincing argument for its physiological relevance.
- “Provide references for the determination method of physiological and biochemical indicators.”
Answer: The references related to the methods we have used in the study are provided in the list of “References”.
- “Consider using descriptive subheadings for methods rather than simply listing technology names (e.g., "2.3 Immunoblot analyses, 2.4 RT qPCR Analysis")”.
Answer: We revised the subheadings according to the reviewer’s recommendation.
- “Thoroughly remove method descriptions from the results and discussion sections, as this is not standard academic paper writing practice.”
Answer: The text of the section “Results and Discussion” was carefully revisited and changed in a relevant way according to the remark.
- “ Explain briefly in the introduction why these genes were analyzed.”
Answer: The reviewer’s comment was addressed in the revised version of the manuscript by adding the following sentence in introduction: “Often exogenously applied PGRs exert their protective effects via upregulation of genes coding for antioxidant enzymes [18]. Therefore the analyses of the changes in gene expression of elements from the plant defence system could be helpful for elucidating the antioxidant protective potential of exogenously applied compounds.”
- “In the "Results and Discussion" section, present and analyze the results first, followed by discussion.”
Answer: We introduced this manner of presenting and discussing the results whenever appropriate.
12.” Include significance analysis results for all data.”
Answer: As already stated in “Material and methods”, all the data were subjected to one-way ANOVA with Duncan’s multiple range test and Students’s t-test. In Figure 1 we choose to display one-way ANOVA with Students’s t-test as it was more relevant to outline the statistically different results in comparison to the “Mock”-treated PEG-stressed plants. Duncan’s multiple range analyses are also available but if displayed on the figure, there will be multiple “small letter” labels on some of the bars (often more than 6). This makes the graph quite “messy” and it does not bring any added informative value on the comparison of the changes in FW, DW and shoot area resulting from the different treatments. Following the reviewer’s recommendation, we displayed Duncan’s multiple-range analysis of the data related to Figure 6, as it reads well on the bar charts.
“13. Explain and discuss the logical relationship between different research contents, and consider reorganizing the overall structure of the paper to strengthen transitions and connections between sections.”
Answer: We hope that the structural improvements made in the revised version of the manuscript meet the requested changes by Reviewer 2.
“14. Rewrite the conclusions, indicating future directions for research in the investigated field.”
Answer: The “Conclusions” paragraph has been revised according to the remarks made by the reviewers and the SI Guest Editors.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript describe the effects of kinetin, NAA, ABA, and ACC on mitigating dehydration in llettuce grown in 12% polyethylene glycol. The combination of these hormones showed the best protective effect, increasing antioxidative enzyme activities in leaves. Higher levels of the enzyme for proline biosynthesis and greater proline content were detected, along with specific dehydrins. This hormonal mix, applied via spraying or roots, showed similar stress-mitigating properties in soil-grown lettuce under moderate drought.
The authors present a manuscript in very good condition and I consider that it can be accepted for publication.
Author Response
We would like to thank Reviewer 3 for the positive evaluation of our work.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe author made in-depth revisions to the manuscript, and the current version can basically meet the publishing requirements. I have no further comments.
