GmRWP-RK1 Enhances Salt Tolerance by Modulating Antioxidant Defense, Ion Homeostasis and Stress-Responsive Pathways in Soybean

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “GmRWP‑RK1 Enhances Salt Tolerance by Modulating Antioxidant Defense, Ions Homeostasis and Stress‑Responsive Pathways in Soybean” presents a valuable contribution to the field of plant stress biology. The authors provide functional evidence for the role of GmRWP‑RK1 in enhancing salt tolerance, supported by cloning, transgenic Arabidopsis assays, and soybean hairy root experiments. The study is timely and relevant, as soil salinity is a major constraint in soybean production, and transcription factors such as RWP‑RK remain underexplored in this context. The overall research design is appropriate, and the methods are adequately described, but several areas require major revision before the manuscript can be accepted. The introduction provides a broad overview of transcription factors and abiotic stress responses, but it can be improved by more clearly highlighting the novelty of GmRWP‑RK1 within the RWP‑RK family and its distinction from previously studied RKD/NLP genes. The results are promising but not always clearly presented. Figures should be improved in resolution and formatting, with complete legends and consistent labeling. For example, Figures S4–S6 require clearer scale bars and annotations, and the miRNA prediction network (Table S4, Fig S5) is underdeveloped; either experimental validation of at least one predicted interaction or a deeper discussion of its biological relevance would strengthen the manuscript. The statistical analysis also needs clarification: specify whether replicates are biological or technical, and state the exact statistical tests used (e.g., ANOVA, t‑test) along with p‑values.

The language throughout the manuscript requires careful revision. Several grammatical errors and awkward phrasings reduce readability, and gene nomenclature is inconsistently applied (e.g., AtRKD5 vs AtRWP‑RK5 vs RKD1). A thorough language edit will improve clarity and scientific tone. In addition, the discussion should be expanded to better integrate the findings with broader literature on RWP‑RK transcription factors in other crops, such as rice and pearl millet, and to acknowledge the limitations of the hairy root system compared to stable soybean transformation. Addressing these points will enhance the manuscript’s impact and reliability.

In summary, the study is scientifically sound and offers novel insights into soybean salt tolerance, but revisions are needed to improve clarity, presentation, and contextual depth. With major revisions addressing the issues outlined above, the manuscript will be suitable for publication in Plants.

 

Author Response

We sincerely thank the reviewer for the thorough evaluation of our manuscript entitled GmRWP-RK1 Enhances Salt Tolerance by Modulating Antioxidant Defense, Ions Homeostasis and Stress Responsive Pathways in Soybean. We appreciate the constructive comments and insightful suggestions, which have greatly helped us to improve the clarity, quality, and scientific depth of the manuscript. All comments have been carefully addressed, and the manuscript has been substantially revised accordingly. Our detailed responses are provided below.

Comment:
The manuscript provides a valuable contribution but requires major revision in clarity, presentation, statistical description, language, and contextual depth.

Response:
We thank the reviewer for recognizing the scientific value and relevance of our study. In response, we have thoroughly revised the manuscript to improve presentation, statistical transparency, language quality, and contextual discussion. Major revisions include restructuring the Introduction and Discussion, improving figure quality and legends, clarifying statistical analyses, and performing a comprehensive language and nomenclature revision.

comment:
The introduction should better highlight the novelty of GmRWP-RK1 within the RWP-RK family and distinguish it from RKD/NLP genes.

Response:
We agree with this important suggestion. The Introduction has been revised to clearly distinguish GmRWP-RK1 from previously characterized RKD and NLP members of the RWP-RK family. We now explicitly highlight the limited functional characterization of RWP-RK genes in soybean abiotic stress responses and emphasize the novelty of identifying GmRWP-RK1 as a positive regulator of salt tolerance.

comment:
Figures require improvement in resolution, formatting, legends, and labeling, particularly Figures S4–S6.

Response:
All figures have been carefully revised. Figure resolution has been increased, font sizes standardized, and legends expanded for clarity. scale bars and annotations have been clearly added, and labeling has been made consistent throughout the manuscript and supplementary materials.

comment:
The miRNA prediction network (Table S4, Fig. S5) is underdeveloped and requires validation or deeper discussion.

Response:
We appreciate this comment. While experimental validation of miRNA target interactions is beyond the scope of the current study, we have substantially expanded the Discussion to explain the biological relevance of the predicted miRNA-GmRWPK1 interactions, particularly in relation to stress signaling and transcriptional regulation. The limitations of in silico predictions are now clearly acknowledged, and future experimental validation is proposed in the discussion.

comment:
Clarify whether replicates are biological or technical and specify statistical tests and p-values.

Response:
This issue has been fully addressed. We now explicitly state that all experiments were performed using biological replicates, with the number of replicates clearly indicated in figure legends. The statistical tests used and corresponding significance thresholds have been added to the Materials and Methods section and figure legends.

comment:
The manuscript contains grammatical errors and inconsistent gene nomenclature.

Response:
The entire manuscript has undergone comprehensive language editing to correct grammatical errors and improve readability and scientific tone. Gene nomenclature has been standardized throughout the text.

comment:
Expand the discussion to integrate findings with studies in other crops and acknowledge limitations of the hairy root system.

Response:
The Discussion has been expanded to include comparisons with RWPRK transcription factors reported in rice, pearl millet, and other crops, highlighting conserved and divergent regulatory roles. We also explicitly discuss the limitations of the soybean hairy root system compared to stable transformation and clarify how it nevertheless provides valuable functional insights.

Closing Statement

We once again thank the reviewer for the insightful and constructive comments. We believe that the extensive revisions have significantly improved the clarity, rigor, and impact of the manuscript. We hope that the revised version now meets the standards for publication in Plants and are grateful for the opportunity to improve our work.

 

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents an extensive experimental study addressing the role of GmRWP-RK1 in salt stress tolerance, combining stable transgenic Arabidopsis with a soybean hairy root system. The topic is elevant to plant stress physiology and crop biotechnology, and the work includes a broad range of phenotypic, physiological, biochemical, and transcriptional analyses. However,, the manuscript presents important methodological inconsistencies, conceptual errors, and overinterpretations that currently limit the robustness of the mechanistic claims and some conclusions. 

1. The manuscript states that phylogenetic analysis reveals a “serine/threonine domain” and suggests that GmRWP-RK1 is evolutionarily linked to the oxidoreductase superfamily, implying a catalytic or biochemical regulatory role. This is conceptually incorrect for a member of the RWP-RK transcription factor family and is not supported by any functional or structural evidence provided in the manuscript. I suggest to remove or correct this statement and properly characterize GmRWP-RK1 as a transcription factor, supported by appropriate domain annotation (e.g., InterPro/Pfam/SMART). Any implication of enzymatic or oxidoreductase activity should be avoided unless directly demonstrated.
2. Several methodological descriptions require clarification or correction. Notably, the primers reported for cloning appear to have identical forward and reverse sequences, which is incompatible with directional cloning using BamHI/HindIII and suggests a likely error in the Methods section. In addition, the manuscript often reports n = 3 without clearly distinguishing between biological and technical replicates.
3. Other points also requiring clarification include duration and conditions of root growth assays on plates (e.g., “after one month”), which is unusual and may introduce confounding effects if not carefully controlled, and whether salt treatments were applied abruptly or gradually across all experiments (procedures appear inconsistent between assays).
4. The use of GeneMANIA and STRING to identify “interactor genes” is acceptable as a hypothesis-generating approach, but the manuscript repeatedly interprets differential expression of these genes as evidence that GmRWP-RK1 acts as a central regulatory hub or directly controls these targets. This interpretation is not justified. GeneMANIA and STRING infer functional associations, not direct physical interactions or transcriptional regulation. qRT-PCR data alone do not demonstrate direct regulation.
5. The Discussion introduces links between GmRWP-RK1, auxin signaling, nodulation, and systemic root-to-shoot communication. However, no direct measurements of hormone levels, canonical marker genes, or signaling assays are presented. In the hairy root system, the shoot is non-transgenic, making causal inference about systemic regulation particularly tenuous. Please, limit interpretations strictly to the data presented, or include targeted experiments (e.g., auxin quantification, marker gene analysis) to support these claims.
6. The frequent use of 200 mM NaCl represents a very severe stress for both Arabidopsis and soybean. While this may amplify phenotypic differences, it also limits physiological and agronomic relevance and may reflect acute toxicity rather than adaptive stress responses. So, justify the choice of salt concentrations and discuss this limitation explicitly. Inclusion of at least one moderate stress condition would strengthen the biological relevance of the conclusions.
7. In some experiments (notably in soybean hairy roots), POD activity does not consistently follow the narrative of a uniformly enhanced antioxidant system in OE lines. These discrepancies are not adequately discussed.

Minor comments

1. Statistical tests, assumptions, and post hoc analyses should be clearly described. The meaning of “n” should be explicitly stated in figure legends and Methods.
2. For both Arabidopsis and soybean hairy roots, the number of independent lines/events should be clarified, and key results should ideally be demonstrated in more than one independent line.
3. Statements suggesting immediate applicability to crop improvement or cultivar development are premature given the experimental scope.
4. The Discussion is overly long and repetitive, with grammatical errors and inconsistent gene nomenclature (e.g., AtRKD5/AtRWP-RK5/AtRKD1). Careful language editing is required.

Author Response

We sincerely thank the reviewer for the detailed, rigorous, and constructive evaluation of our manuscript. We appreciate the reviewer’s careful attention to conceptual accuracy, methodological rigor, and interpretation of results. In response, we have substantially revised the manuscript to correct conceptual errors, clarify methodologies, temper overinterpretations, and improve statistical and linguistic clarity. Below, we address each comment point by point.

comment:
The manuscript addresses an important topic but contains methodological inconsistencies, conceptual errors, and overinterpretations that weaken mechanistic claims.

Response:
We appreciate this critical assessment and fully agree that mechanistic claims must be strictly supported by data. In the revised manuscript, we have corrected conceptual inaccuracies, clarified experimental procedures, removed unsupported interpretations, and explicitly acknowledged limitations. Our conclusions have been carefully refined to align strictly with the presented evidence.

comment:
The manuscript incorrectly suggests that GmRWP-RK1 contains a serine/threonine domain and is evolutionarily linked to the oxidoreductase superfamily.

Response:
We fully agree with this criticism and thank the reviewer for pointing out this conceptual error. The statements implying a serine/threonine domain, oxidoreductase activity, or enzymatic function have been completely removed from the manuscript.

comment:
Forward and reverse primers appear identical, which is incompatible with directional cloning.

Response:
We thank the reviewer for identifying this issue. The primer sequences reported in the original version contained a typographical error. This has now been corrected, and the revised Methods section provides accurate forward and reverse primer sequences, with BamHI and HindIII restriction sites

comment:
The manuscript does not distinguish between biological and technical replicates.

Response:
This has been clarified throughout the manuscript. We now explicitly state that “n” refers to biological replicates unless otherwise specified. This information has been added to the Materials and Methods and figure legends, ensuring transparency and reproducibility.

comment:
The duration of plate-based root assays and salt treatment application appears inconsistent or unclear.

Response:
We agree that these points required clarification. The Methods section has been revised to explicitly define the duration and growth conditions for all root assays. The phrase “after one month” has been corrected to reflect the actual experimental timeframe. We have also clarified whether salt stress was applied abruptly or gradually for each experiment and ensured consistent descriptions across assays. Any unavoidable differences between experimental systems are now clearly explained.

comment:
Functional associations inferred from GeneMANIA and STRING are incorrectly interpreted as evidence of direct regulation.

Response:
We fully agree. The revised manuscript now clearly states that GeneMANIA and STRING analyses are hypothesis-generating tools that infer functional associations rather than direct physical interactions or transcriptional regulation. All statements suggesting that GmRWP-RK1 acts as a central regulatory hub or directly controls these genes have been removed or rephrased. We now explicitly state that qRT-PCR expression changes alone do not demonstrate direct regulation, and future validation experiments are suggested cautiously.

comment:
Claims related to auxin signaling, nodulation, and root-to-shoot communication are not supported by direct evidence.

Response:
We agree with this concern. In the revised manuscript, we have removed or substantially tempered interpretations linking GmRWP-RK1 to auxin signaling, nodulation, and systemic signaling. We explicitly acknowledge that, in the soybean hairy root system, the shoot tissue is non-transgenic, which limits causal inference regarding systemic regulation. Claims are now strictly limited to the observed root-level responses, and proposed signaling roles are framed as speculative and future directions.

comment:
200 mM NaCl represents extremely severe stress and limits agronomic relevance.

Response:

We appreciate this comment and agree that 200 mM NaCl represents extremely severe stress and is not intended to directly represent typical agronomic conditions. We used 200 mM as a stringent screening condition to generate a clear, reproducible phenotype and to facilitate mechanistic analyses (e.g., antioxidant response and ion homeostasis) within a defined and robust stress window.

comment:
POD activity does not consistently support the narrative of enhanced antioxidant capacity.

Response:
We appreciate the reviewer’s comment. In our data, POD activity is significantly higher in the OE lines than in the EV control under NaCl stress, supporting enhanced enzymatic antioxidant capacity. We recognize that antioxidant responses can be dynamic and enzyme-specific, and POD alone should not be interpreted as the only indicator. Therefore, in the revised manuscript we (i) clarified the POD results in the Results section with the exact statistical outcomes, and (ii) toned down the wording to state that the antioxidant capacity is supported by a combined pattern across antioxidant enzymes and oxidative damage markers.

Minor Comments

Response:
All statistical tests are now clearly specified, including assumptions and significance thresholds. The meaning of “n” is explicitly defined in both Methods and figure legends.

Response:
We now clearly state the number of independent Arabidopsis transgenic lines and soybean hairy root events used in each experiment. Where possible, key results are demonstrated using multiple independent lines.

Response:
Statements implying immediate application to cultivar development have been softened. The manuscript now frames potential applications as long-term possibilities requiring further validation.

Response:
The Discussion has been condensed to reduce redundancy, and the entire manuscript has undergone thorough language editing. Gene nomenclature has been standardized throughout the manuscript

Closing Statement

We sincerely thank the reviewer for the insightful and rigorous critique. We believe that the extensive revisions have significantly improved the conceptual accuracy, methodological clarity, and interpretive rigor of the manuscript. We hope that the revised version now meets the standards required for publication.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

1. The use of Arabidopsis as a heterologous system is reasonable, but the manuscript would benefit from a clearer rationale explaining why Arabidopsis was chosen instead of soybean stable lines and how conclusions from Arabidopsis translate to soybean stress biology
2. The authors should provide a stronger rationale for selecting GmRWP-RK1 (Glyma.01G159200) for functional characterization under salt stress conditions.
3. In Figure 1A, the germination rate of the OE4 line is not shown. The authors should explain this omission or include OE4 data to ensure consistency with the other analyses
4. Why did authors choose OE1 for other analysis (Figures 2 to 4)?
5. Why did authors not measure the expression level of GmRWP-RK1 in Arabidopsis EV and OE lines?
6. In the soybean hairy root system, all the roots are not transgenic. Did the authors remove the non-transgenic roots?
7. Did authors remove the main root system after getting hairy roots? It would be a good idea if authors could compare the data among main root systems and hairy roots under salt stress conditions.
8. In the soybean hairy root transformation system, the authors should quantify and report the expression levels of GmRWP-RK1 in both empty vector (EV) and overexpression (OE) roots to confirm successful transgene expression.
9. The manuscript contains numerous spelling, grammatical, and typographical errors
10. Some abbreviations (e.g., RWP-RK1) should be introduced using the full name at first occurrence, followed by the abbreviation in parentheses.
11. Ensure consistent formatting of gene and protein names
12. The authors should mention how many biological and technical replicates used in the experiments in the study
13. The authors should give the gene ID of Actin11, which is used for qRT-PCR expression analysis in soybean. Did they use Actin11 from Arabidopsis? If so, it does not make sense to use the gene in the expression analysis in soybean
14. Supplementary figures were not cited in the text. For example, figures S4 and S5 and table S4
15. Why do authors give miRNA networks? They did not mention in the text
16. Some references are still required, for example, the 2-ΔΔCt method.
17. The reference style is not appropriate for this journal; please check the reference style.

Author Response

We sincerely thank the reviewer for the constructive comments and suggestions, which have helped us improve the clarity, rigor, and presentation of the manuscript. All points raised have been carefully addressed, and the manuscript has been revised accordingly. Our responses are provided point by point below.

comment:
The rationale for using Arabidopsis instead of stable soybean lines should be clarified, and the relevance to soybean stress biology explained.

Response:
We appreciate this suggestion. In the revised manuscript, we have added a clearer rationale explaining that Arabidopsis was chosen due to its well-established genetic transformation system, shorter generation time, and suitability for rapid functional validation of transcription factors. We now explicitly state that Arabidopsis serves as a heterologous validation system, while soybean hairy roots were used to assess gene function in the native species context. The limitations and translational relevance of Arabidopsis findings to soybean stress biology are now discussed more clearly.

comment:
A stronger justification is needed for selecting GmRWP-RK1 for functional characterization.

Response:
We agree. The Introduction has been revised to clarify that GmRWP-RK1 was selected based on its strong salt-induced expression, computational approach within the RWP-RK family, and preferential expression in root tissues, suggesting a potential role in salt stress responses. This rationale is now explicitly stated.

comment:
The germination rate of OE4 is missing.

Response:
We thank the reviewer for noting that the germination rate of OE4 is missing. Although OE1 and OE4 were both stable transgenic lines, we performed the germination assay only with OE1 because the initial experimental setup (including the selection and preparation of Petri plates/seed placement) was not optimal for running multiple independent OE lines in parallel with sufficient replication. To avoid presenting incomplete or potentially biased data for OE4, we restricted the germination analysis to OE1, for which the assay conditions and replication were consistent.

comment:
Why was OE1 chosen for further analyses (Figures 2–4)?

Response:
OE1 was selected because it exhibited stable transgene expression and a representative salt-tolerant phenotype, consistent with other OE lines.

comment:
Not all roots are transgenic; were non-transgenic roots removed?

Response:
Yes. In the soybean hairy root system, non-transgenic roots were carefully removed, and only positive transgenic roots were retained for analysis. This clarification has been added to the Materials and Methods section.

comment:
Were main roots removed, and why not compare main roots and hairy roots?

Response:
The main root system was removed after the establishment of transgenic hairy roots to ensure that physiological and molecular analyses reflected responses from transgenic tissues only. While a comparison between main roots and hairy roots would be informative, it was beyond the scope of the present study. This limitation and future research direction are now acknowledged in the Discussion.

comment:
Expression levels of GmRWP-RK1 should be quantified in EV and OE roots.

Response:
We thank the reviewer for this valuable suggestion. Quantifying GmRWP-RK1 transcript abundance in EV and OE roots would indeed provide an additional quality-control check for the overexpression system. However, we are currently unable to perform this experiment within the revision timeframe due to limited access to fresh transgenic root material and the need to re-generate synchronized EV and OE hairy-root lines under identical growth and salt-treatment conditions to ensure valid comparisons. We have therefore added this point to the Limitations/Future Work section and will include qRT-PCR confirmation of GmRWP-RK1 expression (and, where possible, protein-level verification) in a follow-up study using newly generated independent lines.

 Comment:
The manuscript contains numerous spelling and grammatical errors.

Response:
The manuscript has been thoroughly edited by a native English-speaking colleague to correct spelling, grammar, and typographical errors, thereby improving readability and the overall scientific tone.

Comment:  
Abbreviations should be defined at first use.

Response:
All abbreviations, are now introduced using their full names at first occurrence, followed by abbreviations.

comment:
Ensure consistent formatting.

Response:
Gene and protein nomenclature has been standardized throughout the manuscript according to accepted conventions.

comment:
Clarify the number of replicates used.

Response:
The number of biological and technical replicates is now clearly stated in the Materials and Methods section and in all figure legends.

comment:
Clarify the Actin11 gene ID and species source.
Response:

Thank you for this comment. We have clarified the species source and gene identifiers of the internal control used for RT-qPCR in the revised manuscript and its supplementary materials.

comment:
Supplementary figures and tables were not cited.

Response:
All supplementary figures (e.g., Figures S4, S5) and Table S4 are now properly cited in the main text.

comment:
miRNA networks are shown but not explained.

Response:
We have now added a clear explanation in the Discussion sections describing the purpose of the miRNA network analysis as a predictive, hypothesis-generating approach, along with appropriate limitations.

 

comment:
2-ΔΔCt Method references are missing.

Response:
The appropriate reference for the 2-ΔΔCt method, and other missing citations have now been added.

comment:
The reference style is not appropriate.

Response:
All references have been reformatted according to the journal’s guidelines, ensuring consistency and compliance.

Closing Statement

We sincerely thank the reviewer for the detailed and constructive feedback. We believe that the revisions have significantly improved the clarity, rigor, and overall quality of the manuscript, and we hope it now meets the standards required for publication. We remain happy to address any additional comments to further strengthen the manuscript.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript entitled “GmRWP-RK1 Enhances Salt Tolerance by Modulating Antioxidant Defense, Ion Homeostasis, and Stress-Responsive Pathways in Soybean” is generally well written. However, several major concerns must be addressed before the manuscript can be considered for publication in a reputed journal. Throughout the manuscript, the authors have not applied consistent treatments, datasets, or plant lines, which leads to significant confusion in data interpretation.

A major limitation of the study is the absence of knockdown/knockout lines. The authors have relied exclusively on overexpression lines, whereas loss-of-function approaches are essential to convincingly establish the role of GmRWP-RK1 in salt tolerance and to complete the functional characterization of the gene.

Specific figure and data related concerns are as follows:

Figure 1: The 50 mM NaCl treatment is missing from the germination rate analysis. In addition, the OE4 line is not included in the Arabidopsis seed germination assay.

Figure 2: Data for 50 mM and 100 mM NaCl treatments are missing in the stomatal aperture analysis. Similarly, for stress markers and antioxidant-related parameters, results for 50 mM and 100 mM salt treatments are not presented. Furthermore, ROS data are absent for Arabidopsis plants.

Figure 3: Photosynthetic rate measurements under 50 mM and 100 mM NaCl treatments are missing. In addition, data from only a single overexpression line are shown, which weakens the reliability of the conclusions.

Figure 5: In the salt stress assay of chimeric soybean plants, ROS data are provided; however, corresponding ROS analyses are missing for Arabidopsis plants.

Overall, the inconsistent experimental design, incomplete datasets, and lack of loss-of-function analysis significantly weaken the study and must be thoroughly addressed to strengthen the conclusions.

 

Comments for author File: Comments.pdf

Author Response

We sincerely thank the reviewer for the careful evaluation of our manuscript entitled “GmRWP-RK1 Enhances Salt Tolerance by Modulating Antioxidant Defense, Ion Homeostasis, and Stress-Responsive Pathways in Soybean.” We appreciate the recognition that the manuscript is generally well written, as well as the constructive critique highlighting issues related to experimental consistency, dataset completeness, and functional validation. Below, we address each concern in detail.

comment:
Inconsistent treatments, datasets, and plant lines across experiments lead to confusion in data interpretation.

Response:
We acknowledge this concern and agree that consistency is critical for robust interpretation. In the revised manuscript, we have:

• Clearly defined the experimental design for each assay, specifying which salt concentrations and plant lines were used and why.
• Added explicit explanations where certain treatments were excluded due to experimental constraints (e.g., assay sensitivity, survival thresholds).
• Revised the figure legends and Methods to ensure transparency and reduce confusion.

Where complete uniformity was not feasible, this has now been clearly stated and discussed as a limitation.

comment:
The absence of knockdown/knockout lines is a major limitation; reliance on overexpression alone weakens functional conclusions.

Response:
We fully agree that loss-of-function approaches (CRISPR/Cas9 knockouts or RNAi knockdown) are essential for definitive functional characterization. Due to time and technical constraints associated with generating stable soybean knockout lines, such analyses were not feasible in this study and will be pursued in future work

To address this limitation, we have:

• Carefully tempered our conclusions, avoiding claims that require knockout evidence.
• Framed our findings as gain-of-function evidence supporting a positive regulatory role of GmRWP-RK1 under salt stress.
• Clearly proposed CRISPR/Cas9-based knockout validation as a priority for future research.

comment:
The 50 mM NaCl treatment is missing, and the OE4 line is not included.

Response:
We thank the reviewer for noting that, In preliminary optimization experiments, 50 mM NaCl produced only mild stress and did not generate a clear, reproducible phenotype in either WT or OE plants (i.e. no statistically detectable differences under our assay conditions). Because this concentration was not sufficiently discriminatory for evaluating salt tolerance, we excluded it from the final experimental design and focused on moderate-to-severe NaCl levels that produced robust stress responses and allowed meaningful comparison between genotypes. The germination rate of OE4 is missing. Although OE1 and OE4 were both stable transgenic lines, we performed the germination assay only with OE1 because the initial experimental setup (including the selection and preparation of Petri plates/seed placement) was not optimal for running multiple independent OE lines in parallel with sufficient replication. To avoid presenting incomplete or potentially biased data for OE4, we restricted the germination analysis to OE1, for which the assay conditions and replication were consistent.

comment:
Data for 50 mM and 100 mM NaCl treatments are missing, and ROS data are absent for Arabidopsis.

Response:
We agree that this required clarification. Stomatal aperture and biochemical assays were conducted under high salt stress (200 mM NaCl) to induce clear and measurable physiological responses. Lower salt concentrations produced variable or weak responses in preliminary trials, and thus were not included in the final analysis. We acknowledge that ROS data for Arabidopsis were not included, and this is now clearly stated as a limitation. Importantly, we have avoided extrapolating ROS-related conclusions from soybean hairy roots to Arabidopsis.

comment:
Photosynthetic data under 50 mM and 100 mM NaCl are missing, and only one OE line is shown.

Response:
Photosynthetic parameters were measured under 200 mM NaCl, a condition that produced the most pronounced and reproducible differences between genotypes in our screening. Similar approaches have been used previously to reveal clear physiological separation between transgenic and wild-type Arabidopsis under moderate-to-severe salinity, including photosynthetic rate measurements under high NaCl conditions (Gao et al). We further clarify in the revised manuscript that multiple independent OE lines were initially screened, and OE1 was selected for detailed photosynthetic analysis based on stable transgene expression and consistent phenotypic performance across replicates. (Gao, X., Ren, Z., Zhao, Y., & Zhang, H. Overexpression of SOD2 increases salt tolerance of Arabidopsis. Plant Physiology, 133(4), 1873–1881. https://doi.org/10.1104/pp.103.026062)

comment:
ROS data are provided for soybean but missing for Arabidopsis.

Response:
This is a valid point. Due to technical constraints and sample limitations, ROS staining was performed only in the soybean hairy root system, where transgenic tissue could be clearly identified. We have now:

• Explicitly stated this limitation in the Discussion.
• Removed any generalized statements implying ROS regulation in Arabidopsis based solely on soybean data. In Overall Revision Strategy we have address the reviewer’s overarching concerns,

• Improved methodological transparency and consistency.
• Removed or softened overstated conclusions.
• Clearly separated Arabidopsis and soybean-specific findings.
• Explicitly discussed experimental limitations, particularly the absence of knockout lines.

Closing Statement

We sincerely thank the reviewer for the insightful and rigorous critique. We believe that the revised manuscript now presents a clearer, more cautious, and methodologically transparent study that appropriately supports its conclusions within the defined experimental scope. We hope these revisions address the reviewer’s concerns and improve the overall quality and reliability of the manuscript. We remain happy to address any additional comments to further strengthen the manuscript.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have carefully reviewed the revised manuscript entitled “GmRWP-RK1 Enhances Salt Tolerance by Modulating Antioxidant Defense, Ions Homeostasis and Stress-Responsive Pathways in Soybean.” I am pleased to note that the authors have thoroughly addressed all the points raised during the review process. The revisions have strengthened the manuscript, and the responses provided are satisfactory. Based on the improvements made, I recommend that the manuscript be accepted in its present form.

 

Author Response

I have carefully reviewed the revised manuscript entitled “GmRWP-RK1 Enhances Salt Tolerance by Modulating Antioxidant Defense, Ions Homeostasis and Stress-Responsive Pathways in Soybean.” I am pleased to note that the authors have thoroughly addressed all the points raised during the review process. The revisions have strengthened the manuscript, and the responses provided are satisfactory. Based on the improvements made, I recommend that the manuscript be accepted in its present form.

Response

We sincerely thank you for your careful and thorough evaluation of our revised manuscript. We greatly appreciate your positive assessment and your recognition that all points raised during the review process have been adequately addressed. Your constructive comments and guidance were invaluable in improving the quality, clarity, and scientific rigor of the manuscript. We are especially grateful for your recommendation to accept the manuscript in its present form. Thank you for your time, expertise, and supportive feedback throughout the review process.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version of the manuscript shows improvement in conceptual clarity and methodological description The authors have addressed most of the major concerns raised in the previous review, and the manuscript is more coherent than the first version. However, issues remain insufficiently resolved and should be addressed.

The authors’ response indicated that GeneMANIA and STRING analyses are now treated as hypothesis-generating tools, but the revised manuscript still contains language implying direct regulatory roles. In Sections 2.5 and 2.10, statements describing GmRWP-RK1 as a “central regulatory node” and an “upstream molecular regulator” are not fully supported by the presented data. Expression correlations and network predictions alone do not demonstrate direct regulation. These claims should be further softened, and the limitations of these analyses should be stated more explicitly.

The antioxidant responses are better described, however, the results for POD activity remain inconsistent between Arabidopsis and soybean hairy roots. In Arabidopsis, POD activity is higher in overexpression lines, whereas in soybean roots it is higher in control lines under salt stress. This discrepancy is not sufficiently discussed. 

Some methodological descriptions remain difficult to follow, such as the duration of certain root assays and the combination of abrupt and gradual salt treatments would benefit from further clarification and justification to improve reproducibility.

The use of 200 mM NaCl is justified as a severe screening condition, but the manuscript still relies almost exclusively on this extreme stress level. The authors should further emphasize the limitations of this approach with respect to agronomic relevance and avoid overgeneralizing the conclusions.

Author Response

We sincerely thank the reviewer for the thoughtful and constructive feedback. The manuscript has been carefully revised to address the remaining concerns, and we believe the interpretations are now appropriately supported and clearly framed within the limitations of the study. We appreciate the reviewer’s guidance, which has significantly strengthened the scientific rigor and clarity of the work.

Comment/Suggestion

The revised version of the manuscript shows improvement in conceptual clarity and methodological description The authors have addressed most of the major concerns raised in the previous review, and the manuscript is more coherent than the first version. However, issues remain insufficiently resolved and should be addressed.

Response

We thank the reviewer for the careful re-evaluation of our revised manuscript and for acknowledging the improvements in conceptual clarity, methodological description, and overall coherence. We appreciate the continued constructive feedback.

Comment/Suggestion

The authors’ response indicated that GeneMANIA and STRING analyses are now treated as hypothesis-generating tools, but the revised manuscript still contains language implying direct regulatory roles. In Sections 2.5 and 2.10, statements describing GmRWP-RK1 as a “central regulatory node” and an “upstream molecular regulator” are not fully supported by the presented data. Expression correlations and network predictions alone do not demonstrate direct regulation. These claims should be further softened, and the limitations of these analyses should be stated more explicitly.

Response

Regarding the concerns about the interpretation of the GeneMANIA and STRING analyses, we fully agree that these approaches are hypothesis-generating and do not, on their own, demonstrate direct regulatory relationships. In response, we have further revised Sections 2.5 and 2.10 have been replaced with more cautious wording and we have explicitly stated the limitations of expression-based correlations and network predictions in inferring direct regulatory roles. (With track changes). These revisions clarify that the network analyses are used to generate testable hypotheses rather than to assert causal or hierarchical regulation. We believe these changes address the reviewer’s concern and align the manuscript’s interpretations more closely with the scope of the presented data.

Comment/Suggestion

The antioxidant responses are better described, however, the results for POD activity remain inconsistent between Arabidopsis and soybean hairy roots. In Arabidopsis, POD activity is higher in overexpression lines, whereas in soybean roots it is higher in control lines under salt stress. This discrepancy is not sufficiently discussed.  

Responses

We thank the reviewer for this insightful comment. We agree that the apparent discrepancy in POD activity between Arabidopsis and soybean hairy roots warranted clearer discussion. This point has now been explicitly addressed in the Discussion section of the revised manuscript. For your valuable consideration, the following clarification has been added to the Discussion section (with track changes)

Comment/Suggestion

Some methodological descriptions remain difficult to follow, such as the duration of certain root assays and the combination of abrupt and gradual salt treatments would benefit from further clarification and justification to improve reproducibility.

Response

We thank the reviewer for this helpful suggestion. In response, we have added a clarification in the Methodology section to improve reproducibility and to justify the salt‐treatment strategy. This clarification has been incorporated into the revised Methods section for your consideration with track changes.

Comment/Suggestion

The use of 200 mM NaCl is justified as a severe screening condition, but the manuscript still relies almost exclusively on this extreme stress level. The authors should further emphasize the limitations of this approach with respect to agronomic relevance and avoid overgeneralizing the conclusions.

Response

We thank the reviewer for this important comment. In response, we have revised the Discussion section to explicitly acknowledge that 200 mM NaCl represents a severe screening condition and to clarify that conclusions drawn from this stress level should not be directly extrapolated to agronomically relevant field conditions. We also emphasize that further validation under moderate, field-relevant salinity levels will be necessary. This clarification has now been incorporated with track changes into the manuscript discussion section for the reviewer’s consideration.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

1. The authors did not indicate the line numbers corresponding to the revised text in their response, which makes it very difficult to locate and verify the changes in the manuscript. Please provide  line numbers when revised.

2. It was unable to find a  rationale in the manuscript explaining the use of Arabidopsis instead of stable soybean lines as a primary functional validation system. This justification should be explicitly stated in the Introduction or Discussion.

3. The authors should include the following statement in the manuscript: “OE1 was selected because it exhibited stable transgene expression and a representative salt-tolerant phenotype, consistent with other OE lines.”

4. Although the manuscript has improved, several typographical and grammatical errors remain. 

Author Response

We sincerely thank the reviewer for their time, careful evaluation, and insightful comments throughout the review process. Their constructive guidance has been invaluable in improving the clarity, rigor, and overall quality of the manuscript. We greatly appreciate their continued engagement and thoughtful suggestions, which have strengthened the study and its presentation

Comment/Suggestion

The authors did not indicate the line numbers corresponding to the revised text in their response, which makes it very difficult to locate and verify the changes in the manuscript. Please provide  line numbers when revised.

Response

We sincerely apologize for the inconvenience caused by the omission of line numbers in our previous response. We appreciate the reviewer’s careful attention to detail. In the revised response document, we have now explicitly indicated the corresponding line numbers for all revised or newly added text, allowing the changes to be easily located and verified in the manuscript. We hope this revision facilitates the review process and thank the reviewer for this valuable suggestion.

Comment/Suggestion

It was unable to find a  rationale in the manuscript explaining the use of Arabidopsis instead of stable soybean lines as a primary functional validation system. This justification should be explicitly stated in the Introduction or Discussion.

Response

We thank the reviewer for this important comment. In response, we have now explicitly added a clear rationale in the Discussion section (Lines 455–460) explaining the use of Arabidopsis as a rapid and genetically tractable heterologous system for initial functional validation, given the technical difficulty and extended timelines required to generate stable transgenic soybean lines.

Comment/Suggestion

The authors should include the following statement in the manuscript: “OE1 was selected because it exhibited stable transgene expression and a representative salt-tolerant phenotype, consistent with other OE lines.”

Response

We thank the reviewer for this helpful suggestion. In response, we have incorporated the requested statement into the manuscript to clarify the rationale for selecting the OE1 line. The text now reads: OE1 was selected based on its stable transgene expression and representative salt-tolerant phenotype, consistent with other OE lines. This addition has been included in the revised manuscript in the materials and methods section for the reviewer’s consideration. Line numbers  494 to 499.

 

Comment/Suggestion

Although the manuscript has improved, several typographical and grammatical errors remain. 

Response

We thank the reviewer for this comment. In response, the manuscript has undergone a thorough language revision, including careful correction of remaining typographical and grammatical errors throughout the text. These edits were made to improve clarity, consistency, and overall readability. We believe the revised version now meets the journal’s language and presentation standards.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Authors have tried to add suggestions carefully. However, a few minor corrections remained. 

In line no. 276-281, the text format is different than the whole manuscript text.

In line 372., the discussion section should be properly aligned in the proper row. 

 

Author Response

Comment/Suggestion

Authors have tried to add suggestions carefully. However, a few minor corrections remained. 

Response

We thank the reviewer for their careful assessment and constructive feedback. In response, we have thoroughly reviewed the manuscript once again and corrected the remaining minor issues to improve clarity, consistency, and overall presentation.

Comment/Suggestion

In line no. 276-281, the text format is different than the whole manuscript text.

Response

We thank the reviewer for noting this formatting inconsistency. We have corrected the text formatting in Lines 276–281 to match the style used throughout the rest of the manuscript (font, size, spacing, and alignment), and the revised version is now consistent.

Comment/Suggestion

In line 372., the discussion section should be properly aligned in the proper row. 

Response

We thank the reviewer for pointing out this formatting issue. The Discussion section at Line 372 has now been properly aligned and placed in the correct row to ensure consistency with the manuscript layout.

Author Response File: Author Response.docx