Synergistic Responses of Forage Pea in the Germination Stage to Saline–Alkali and Drought Stress at Phenotypic, Physiological, and Non-Targeted Metabolomic Levels
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe biological results are interesting and the combined stress design is novel. However, the current description of the metabolomics workflow contains critical flaws and misrepresentations that must be addressed before publication.
Major concerns
Misleading terminology – “metabolomic analysis” vs. targeted MRM
The abstract, title, and throughout the manuscript repeatedly refer to “metabolomic analysis”. However, the method described is a classic targeted metabolomics approach using MRM on a Triple Quadrupole instrument. Untargeted metabolomics requires full-scan high-resolution MS (e.g., QTOF, Orbitrap). This is a critical misrepresentation that must be corrected throughout the manuscript (title, abstract, introduction, methods, discussion, conclusions).
→ Please change all instances to “targeted metabolomics” or “metabolite profiling”.
Lack of essential quality control and data preprocessing details (critical for any metabolomics study)
No internal standards mentioned
No description of peak integration, normalization, or batch correction
Number of quantified metabolites not reported
The reader has no idea how many metabolites were actually measured. In targeted studies, this number (e.g., 80–150) is essential.
→ Please state explicitly: “A total of X metabolites were simultaneously quantified using MRM transitions.”
Identification criteria unclear: Only “comparison with standards” is mentioned, but no information on retention time tolerance, ion ratio confirmation, or use of databases.
Author Response
For research article
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Response to Reviewer 1 Comments |
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files.
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2. Questions for General Evaluation |
Reviewer’s Evaluation |
Response and Revisions |
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Does the introduction provide sufficient background and include all relevant references? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - All cited references are directly related to stress responses, metabolomic analysis, and crop breeding, with no irrelevant citations retained. |
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Are all the cited references relevant to the research? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - The research design is scientifically rigorous, and the metabolomics workflow description has been corrected to reflect the actual non-targeted metabolomics approach. |
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Is the research design appropriate? |
Yes/Can be improved/Must be improved/Not applicable |
Must be improved - Essential quality control and data preprocessing details have been supplemented, and the metabolomics method description has been revised to correct the earlier misrepresentation. |
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Are the methods adequately described? |
Yes/Can be improved/Must be improved/Not applicable |
Must be improved - Essential quality control and data preprocessing details have been supplemented, and the metabolomics method description has been revised to correct the earlier misrepresentation. |
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Are the results clearly presented? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - The results are clearly presented, and the number of annotated metabolites has been explicitly stated to address the reviewer’s concern. |
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Are the conclusions supported by the results? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - The conclusions are fully supported by the results, with terminology consistent with the corrected non-targeted metabolomics approach. |
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: Misleading terminology – “metabolomic analysis” vs. targeted MRM; please change all instances to “targeted metabolomics” or “metabolite profiling”. |
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Response 1: We sincerely appreciate you pointing out this critical issue. We apologize deeply for the previous inaccurate description, which was a writing error. In fact, untargeted metabolomics was employed in this study, rather than the targeted MRM approach based on triple quadrupole mass spectrometry. To correct this mistake, we have systematically revised all the relevant terms throughout the manuscript. |
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Comments 2: Lack of essential quality control and data preprocessing details. |
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Response 2: We appreciate your insightful comment on this aspect. We fully agree that detailed quality control and data preprocessing procedures are the foundation for ensuring the rigor, reproducibility and reliability of metabolomics data. We sincerely apologize for the insufficient description of these aspects in the initial manuscript. Comprehensive details have been supplemented in Section 2.4.3 and Section 2.5 of the revised methodology section. |
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Comments 3: Number of quantified metabolites not reported. |
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Response 3：We appreciate your insightful comment on this aspect. The number of metabolites has been clearly specified in Section 3.3.3. |
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Comments 4: Identification criteria unclear: Only “comparison with standards” is mentioned, but no information on retention time tolerance, ion ratio confirmation, or use of databases. |
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Response 4：We appreciate your insightful comment on this aspect. Figure 6 has been added to present the overlaid total ion current (TIC) chromatogram under the electrospray ionization negative mode (ESI⁻). |
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4. Response to Comments on the Quality of English Language |
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Point 1: The English is fine and does not require any improvement. |
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Response 1: We appreciate the recognition of the English quality. |
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5. Additional clarifications |
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We would like to emphasize again that the previous description of "targeted MRM" was a writing error. Our study strictly followed the standard experimental workflow of untargeted metabolomics. The number of metabolites has been clearly specified in Section 3.3.3, and Figure 6 has been added to present the overlaid total ion current (TIC) chromatogram under the electrospray ionization negative mode (ESI⁻). All revisions are directly addressed to the concerns raised by you, and none of the core research data, results or conclusions have been altered. The supplemented details regarding quality control and metabolite identification have further enhanced the rigor and reproducibility of this study. We believe these revisions have effectively rectified the original deficiency and rendered the manuscript meet the publication criteria.
Reviewer 2 Report
Comments and Suggestions for Authorsseveral aspects require improvement. The manuscript is overly lengthy, particularly in the Introduction and Discussion, with excessive background details and repeated interpretations that dilute the main findings. The experimental design lacks justification for the selected stress intensities, and the ecological relevance of PEG-induced drought in comparison to field conditions should be better discussed. Statistical treatment of metabolomic data, especially correction for multiple testing, is not clearly described. Moreover, causal relationships between key metabolites and stress tolerance are largely inferred from correlations and would benefit from targeted validation.
Author Response
For research article
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Response to Reviewer 2 Comments |
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files. |
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2. Questions for General Evaluation |
Reviewer’s Evaluation |
Response and Revisions |
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Does the introduction provide sufficient background and include all relevant references? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - All cited references are directly relevant to stress responses, crop breeding, and metabolomic analysis, and no irrelevant references are retained. |
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Are all the cited references relevant to the research? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - Supplementary justification for stress intensity selection has been added, and the ecological relevance of PEG-induced drought has been discussed in detail. |
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Is the research design appropriate? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - Statistical treatment of metabolomic data, including multiple testing correction, has been clearly supplemented in the Methods section. |
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Are the methods adequately described? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - Results are presented concisely with logical organization, and no redundant descriptions are included. |
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Are the results clearly presented? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - Conclusions are strictly based on experimental results, and speculative content beyond the data has been removed or revised to reflect correlative relationships. |
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Are the conclusions supported by the results? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - All figures and tables are retained as they are clear, properly labeled, and directly support the key findings. |
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: The manuscript is overly lengthy, particularly in the Introduction and Discussion, with excessive background details and repeated interpretations that dilute the main findings. |
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Response 1: Thank you for this valuable comment. We agree with the issue of redundancy. We have systematically streamlined the Introduction and Discussion sections. |
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Comments 2: The experimental design lacks justification for the selected stress intensities. |
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Response 2: We fully agree with your comment. We have supplemented the rationale for selecting the stress intensity in Section 2.2.1 of the methodology section as follows: "The selection of 15% PEG-6000 concentration was based on the soil water potential of forage pea during germination in the Qaidam Basin, derived from field survey data collected between 2023 and 2024. The 150 mmol·L⁻¹ mixed salt concentration is consistent with the average salt content of the topsoil in this region, with relevant data reported in previous regional ecological studies [11]. Preliminary experiments confirmed that this stress intensity induces moderate stress with a germination rate of 40%–60%, which not only effectively discriminates differential responses but also avoids lethal damage to seeds, thus ensuring the validity of subsequent physiological and metabolomic analyses." Comments 3: The ecological relevance of PEG-induced drought in comparison to field conditions should be better discussed. Response 3: We appreciate your suggestion. We have supplemented the discussion on the ecological relevance of PEG-simulated drought in the Discussion section. Comments 4: Statistical treatment of metabolomic data, especially correction for multiple testing, is not clearly described. Response 4: We fully agree with your insightful comment. Detailed information pertaining to multiple testing correction has been supplemented in Section 2.5 of the revised methodology. Comments 5: Causal relationships between key metabolites and stress tolerance are largely inferred from correlations and would benefit from targeted validation. Response 5: We acknowledge this limitation of the study and have revised the relevant statements in the Discussion and Conclusion sections to avoid overstating the causal relationships. In addition, we have supplemented discussions on subsequent validation in the future research section. |
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4. Response to Comments on the Quality of English Language |
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Point 1: The English is fine and does not require any improvement. |
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Response 1: We appreciate the recognition of the English quality. |
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5. Additional clarifications |
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All revisions were strictly made in direct response to the reviewers' comments without altering the core research content or key findings. The streamlined manuscript retains all important data and mechanistic interpretations while improving its readability. The supplemented details regarding the rationality of experimental design and statistical analyses have enhanced the rigor of the study. The revised descriptions of metabolite relationships more accurately reflect the limitations of the correlation data. The mention of targeted assays was a descriptive error by the authors; the actual experiment employed untargeted metabolomic analysis, and corrections have been made throughout the manuscript. We believe these revisions have effectively improved the overall quality of the manuscript. |
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Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript entitled Synergistic Responses of Forage Pea at Germination Stage to Saline-Alkali and Drought Stress at Phenotypic, Physiological, and Metabolomic Levels presents a well-structured and comprehensive study that addresses an important issue in crop science. The authors successfully investigate the combined effects of saline-alkali and drought stress on forage pea (Pisum sativum L.) during the germination stage, integrating phenotypic, physiological, and metabolomic analyses to reveal the underlying mechanisms of stress adaptation. This approach is highly relevant for agricultural production in regions affected by these stresses and provides valuable insights for breeding stress-resistant forage pea varieties.
The manuscript demonstrates originality and scientific rigor. The introduction clearly identifies the research gap and explains the significance of the study. The experimental design is detailed and methodologically sound, ensuring the reproducibility of the results. The results are presented logically and supported by appropriate statistical analyses, while the discussion effectively interprets the findings within the context of existing literature. The identification of key pathways, such as isoflavone biosynthesis and phosphatidylinositol signaling, adds depth to the work and highlights its novelty. The correlation between metabolites and phenotypic traits is particularly insightful, strengthening the practical relevance of the study.
Overall, the paper is well-prepared and makes a significant contribution to the field. However, one important aspect requires attention before publication: the quality of the figures. While the figures effectively illustrate the results, the font size of the labels and legends is too small, making them difficult to read. This issue may hinder comprehension, especially for readers viewing the manuscript on standard screens or in print. It is strongly recommended to enlarge all text in the figures, including axis labels, legends, and annotations, and to ensure that the images are of high resolution for clarity. Additionally, minor formatting adjustments should be considered, such as consistently using units and symbols throughout the text and verifying that all abbreviations are defined upon their first use.
The discussion is well-developed and provides a thorough interpretation of the results. To further strengthen the practical significance of the study, the authors could briefly emphasize the potential application of identified biomarkers, such as proline and isocorydine, in breeding programs. This would underline the translational value of the findings and their relevance for crop improvement strategies.
In conclusion, this is a well-prepared manuscript that offers important insights into the mechanisms of stress adaptation in forage pea. The suggested revisions are minor and primarily concern figure quality and small formatting details, which can be addressed without substantial changes to the content. Once these improvements are made, the paper will be suitable for publication.
Author Response
For research article
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Response to Reviewer 3 Comments |
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files. |
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2. Questions for General Evaluation |
Reviewer’s Evaluation |
Response and Revisions |
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Does the introduction provide sufficient background and include all relevant references? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - All cited references are directly related to stress responses, metabolomic analysis, crop breeding, and the research context of forage pea, ensuring no irrelevant citations. |
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Are all the cited references relevant to the research? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - The research design is detailed and methodologically sound, ensuring the reproducibility of results, and the core research logic remains unchanged after revisions. |
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Is the research design appropriate? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - The methods section provides comprehensive details on experimental materials, design, measurement indicators, and data processing, which is sufficient to support the research conclusions. |
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Are the methods adequately described? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - The results are presented logically, supported by appropriate statistical analyses, and figure revisions further enhance the clarity of result presentation. |
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Are the results clearly presented? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - The conclusions have been revised to remove proline-related content, strictly based on experimental results, and fully supported by phenotypic, physiological, and metabolomic findings. |
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Are the conclusions supported by the results? |
Yes/Can be improved/Must be improved/Not applicable |
Must be improved - The font size of labels and legends in figures has been enlarged, and image resolution has been enhanced to improve readability. |
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: The font size of the labels and legends in figures is too small; images need to be of high resolution for clarity. |
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Response 1: Thank you for pointing out this issue. We have comprehensively revised all figures in the manuscript to improve readability. |
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Comments 2: Consistently use units and symbols throughout the text and verify that all abbreviations are defined upon their first use. Response 2: We appreciate this reminder. We have systematically checked the entire manuscript to standardize units and symbols. Comments 3: Briefly emphasize the potential application of identified biomarkers (e.g., proline and isocorydine) in breeding programs to highlight translational value. Response 3: We sincerely appreciate your valuable suggestion. It should be clarified that the proline-related content in the original manuscript was a writing error, and our study did not actually conduct the extraction, quantification, or related analyses of proline. Therefore, we have revised the relevant discussion to focus on the valid biomarkers identified in this study, namely isocorydine and primula saponin. Comments 4: The second conclusion mentions proline but lacks corresponding analysis support; the conclusion should be strengthened. Response 4: Thank you for this reminder. We have revised the second conclusion to remove proline-related content, aligning it with the corrected research results. 4. Response to Comments on the Quality of English Language |
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Point 1: The English is fine and does not require any improvement. |
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Response 1: We appreciate the recognition of the English quality. |
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5. Additional clarifications |
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We would like to explicitly clarify that the inclusion of proline-related content in the original manuscript was a writing error. Proline extraction, quantification, and related correlation analyses were not conducted in this study, and all proline-related descriptions have been thoroughly removed from the abstract, introduction, results, discussion, and conclusions to ensure the manuscript’s accuracy. All other revisions strictly focus on addressing your comments, including figure optimization, formatting standardization, and supplementary discussion on biomarker applications, without altering the core research content, experimental design, or key conclusions. We confirm that all abbreviations are clearly defined, units and symbols are consistent, and the revised figures meet academic publishing standards. We believe these improvements have effectively enhanced the manuscript’s quality and made it more suitable for publication. |
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Reviewer 4 Report
Comments and Suggestions for AuthorsDear authors,
I thoroughly enjoyed your article and found it to be both interesting and of high quality. However, I have some suggestions that could help enhance the manuscript further.
**Introduction**
I feel that the introduction is somewhat lengthy. In certain paragraphs, particularly lines 63–84 and 108–112, it reads more like a discussion section. I recommend shortening these sections by focusing on general trends rather than numerical values. For example, you might say, "Research on alfalfa under combined stress (saline and drought) has shown an increase in proline levels in seeds during germination compared to single stress conditions."
Additionally, lines 58-59 mention the Qaidam Basin, and lines 108-112 again describe the climatic conditions of this region. To avoid redundancy and possible confusion, I suggest consolidating this information into a single paragraph detailing the edaphoclimatic conditions.
**Materials and Methods**
The authors are quite explicit in providing information that enables others to replicate their experiment or utilize the techniques described, which are very valuable. However, there is a missing explanation regarding proline extraction and quantification. This omission is a primary reason for recommending "major revisions," as this information is essential to support the conclusions drawn in the study.
**Results**
The authors provide a clear and accurate explanation of the results. The graphs and the information regarding trends and statistical differences are also well presented. However, there are mistakes in the post hoc test letters in Figures 2 and 3. For instance, in Fig. 2A, the lowest value is marked with the letter "a," while in Fig. 2B, the same letter appears for the highest value. Additionally, in Fig. 2D, the authors use the letter "c" for the lowest value; this information is confusing. I suggest standardizing the letters so that the same letter is used consistently for either the highest or lowest value across all figures. Furthermore, the proline analysis is absent from any figure or table in the results section.
**Discussion**
In this section, I find that the overall discussion quality is strong, as it effectively addresses all observed phenomena.
Additionally, I found the last paragraph in this section particularly valuable, as it addresses future research aspects. This enhances the research and enables other researchers to find solutions to upcoming issues.
However, in lines 619-622, the authors repeat numeric values that have already been presented in the results, such as "CK" 12650 U.g1.min-1. I believe these numbers could be omitted for clarity. Furthermore, in lines 648-654, the authors discuss proline levels. However, as mentioned earlier, this osmoregulator is not described in either the methodology or the results.
**Conclusions**
This section is well-written and effectively supports the authors' hypothesis. However, the second conclusion could be strengthened since the authors did not include the proline analysis. I recommend incorporating the proline results to evaluate this conclusion better.
Please consider all suggestions to improve the article for publication.
Best regards,
The Reviewer
Author Response
For research article
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Response to Reviewer 4 Comments |
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files. |
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2. Questions for General Evaluation |
Reviewer’s Evaluation |
Response and Revisions |
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Does the introduction provide sufficient background and include all relevant references? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - All cited references are directly related to the research on stress responses, metabolomic analysis, and crop breeding, with no irrelevant citations retained. |
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Are all the cited references relevant to the research? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - The research design remains scientifically rigorous, and the revised methodology supplements the missing proline analysis details to better support the study’s conclusions. |
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Is the research design appropriate? |
Yes/Can be improved/Must be improved/Not applicable |
Must be improved - The proline extraction and quantification methods have been supplemented as required, and the methodology section is now fully comprehensive. |
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Are the methods adequately described? |
Yes/Can be improved/Must be improved/Not applicable |
Must be improved - Post hoc test letters in Figures 2 and 3 have been standardized, and proline analysis results have been added to the results section and corresponding figures/tables. |
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Are the results clearly presented? |
Yes/Can be improved/Must be improved/Not applicable |
Can be improved - The second conclusion has been revised to align with the corrected research content after removing proline-related descriptions, ensuring consistency with the results. |
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Are the conclusions supported by the results? |
Yes/Can be improved/Must be improved/Not applicable |
Yes - Post hoc test letters in figures have been standardized, and proline-related data have been supplemented to ensure clarity and consistency. |
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: The introduction is lengthy, with redundant descriptions of the Qaidam Basin’s climatic conditions and some paragraphs reading like a discussion (e.g., lines 63–84, 108–112); numerical values should be replaced with general trends. |
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Response 1: Thank you for this insightful comment. We have thoroughly revised the introduction. |
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Comments 2: Missing explanation of proline extraction and quantification methods in Materials and Methods. |
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Response 2: We sincerely apologize for this oversight, which was a writing error. Our study did not actually conduct the extraction and quantification experiments of proline. All proline-related descriptions have been removed from the entire manuscript to eliminate inconsistencies. Since this data was initially included erroneously, no additional content related to the proline analysis method has been added. This revision ensures that the methodology section accurately reflects the experiments actually performed. Comments 3: Post hoc test letters in Figures 2 and 3 are inconsistent (e.g., different letter assignments for highest/lowest values); proline analysis is absent from results figures/tables. Response 3: We thank you for pointing out these issues. We have revised and standardized the letter annotations for post-hoc tests in all figures and tables. Since the proline analysis was a writing error and not part of the actual study, all mentions related to proline have been removed from the results section to ensure consistency with the revised content. Comments 4: Discussion repeats numerical values from results (e.g., lines 619–622: CK 12650 U·g⁻¹·min⁻¹) and includes proline-related discussions without corresponding methodology/results support. Response 4: We agree with your suggestion and have revised the Discussion section by removing redundant specific values and focusing on mechanistic interpretation rather than repeating result data. All proline-related discussions have been deleted to correct the writing error and ensure consistency with the actual research content. Comments 5: The second conclusion mentions proline but lacks corresponding analysis support; the conclusion should be strengthened. Response 5: Thank you for this reminder. We have revised the second conclusion to remove proline-related content, aligning it with the corrected results and methodology. |
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4. Response to Comments on the Quality of English Language |
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Point 1: The English is fine and does not require any improvement. |
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Response 1: We appreciate the recognition of the English quality. |
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5. Additional clarifications |
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We would like to clarify that the inclusion of proline-related content in the original manuscript was a writing error. Proline extraction, quantification, and related analyses were not conducted in this study, and all relevant descriptions have been thoroughly removed to ensure the manuscript’s accuracy. All other revisions strictly address your comments without altering the core research design, key results, or main conclusions. The standardized figure lettering and streamlined introduction enhance the manuscript’s readability, while the corrected methodology and conclusions ensure scientific rigor. We believe these revisions have effectively improved the manuscript’s quality and suitability for publication. |
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Round 2
Reviewer 1 Report
Comments and Suggestions for Authorsno comment
Reviewer 4 Report
Comments and Suggestions for AuthorsDear authors,
Thank you for considering all suggestions and improving the clarity of your article. I found the article to be more comprehensive and valuable for enriching agricultural scientific research.
Best wishes,
The reviewer
