The Effect of Hormonal Priming on Morphological Characteristics and Antioxidant Enzyme Activities in Silage Maize Under Salt Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article determined the effect of different doses of gibberellic acid (GA) and salicylic acid (SA) priming on salt stress, but there are many issues that need to be improved. On this basis, I suggest that authors carefully revise their papers and resubmit them.

1. Some common sense knowledge in the text is suggested to be deleted, such as a in the abstract;
2. There are some detailed writing issues in the text, such as Na+ and Cl- ions in line 36;
3. It is recommended that the indicators in the table be presented in the form of graphs for a more intuitive experience, such as bar charts and box plots.
4. The vertical coordinate scale in Figure 1 is missing;
5. Are there any other indicators regarding the root system, such as surface area and volume?
6. The citation of references does not meet the requirements of the journal, such as whether the journal is abbreviated or not. 7. In the conclusion and abstract, authors need to add more explicit and accurate conclusions, such as the effective hormone treatment concentration.
7. The ruler is missing in Figure 2.
8. In data processing, one-way analysis of variance should be clearly indicated.
9. The unit of enzyme activity is incorrect

Author Response

We are grateful for the valuable inputs and suggestions. We edited entire manuscript based on the inputs, suggestions and edits of all three referees. Please find our responses to each query below and revised version as highlighted sections in the manuscript. The aim of the study were revised and rewritten.

Comment 1: This article determined the effect of different doses of gibberellic acid (GA) and salicylic acid (SA) priming on salt stress, but there are many issues that need to be improved. On this basis, I suggest that authors carefully revise their papers and resubmit them. Some common sense knowledge in the text is suggested to be deleted, such as a in the abstract;

Response 1: Thank you for the suggestion. We carefully revised entire abstract and intrduction and deleted unnecessary parts.

Comment 2: There are some detailed writing issues in the text, such as Na+ and Cl- ions in line 36;

Response 2: Corrected. The notation for the Na+  and Cl− ions on line 36 has been corrected as requested.

Comment 3: It is recommended that the indicators in the table be presented in the form of graphs for a more intuitive experience, such as bar charts and box plots.

Response 3: We appreciate the suggestion for adding more detailed figures; however, we believe that adding 12 individual graphs would lead to redundancy and disrupt the flow of the manuscript. All of the necessary data are already presented comprehensively within the existing tables and combined figures. We have opted to maintain the current visual presentation to ensure the reader remains focused on the main outcomes without becoming overwhelmed by excessive detail.

Comment 4: The vertical coordinate scale in Figure 1 is missing;

Response 4: We have implemented a revision to improve the presentation of the data.

Comment 5: Are there any other indicators regarding the root system, such as surface area and volume?

Response 5: We appreciate the reviewer's interest in the root system morphology. Unfortunately, these specific root indicators, such as surface area and volume, were not measured during the data collection phase of this study and are therefore not available within our current dataset. We agree that these parameters are valuable and will prioritize their inclusion in our future follow-up studies to provide a more comprehensive assessment of the root system response.

Comment 6: The citation of references does not meet the requirements of the journal, such as whether the journal is abbreviated or not. 7. In the conclusion and abstract, authors need to add more explicit and accurate conclusions, such as the effective hormone treatment concentration.

Response 6: Corrected. We have thoroughly checked and revised all citations and the reference list to ensure they strictly adhere to the journal's guidelines, including the proper use of journal abbreviations and overall formatting style

Comment 7: The ruler is missing in Figure 2.

Response 7: We appreciate the observation regarding Figure 2. We recognize that a standard scale (ruler) is missing. However, the intent of Figure 2 is purely qualitative—it is designed to offer a visual comparison and demonstration of the morphological differences between the treatment groups, rather than presenting a measurable data point.

Comment 8: In data processing, one-way analysis of variance should be clearly indicated.

Response 8: We appreciate the reviewer's focus on statistical clarity. Statistical Analysis: We confirm that we have revised the manuscript to clearly and explicitly indicate the use of one-way analysis of variance (ANOVA) in the Data Processing section. This method is now clearly stated in the Materials and Methods section.

Comment 9: The unit of enzyme activity is incorrect

Response 9: We appreciate the reviewer's concern regarding the unit of enzyme activity. We assure the reviewer that the current unit was based on the standard protocol provided by the analytical lab and aligns with several high-impact studies in this field. We have added supporting references to the relevant section to justify our choice. Please find relevant studies below.

• Dewi, R.S., Rahayu, L., Sandhiutami, N.M.D. and Sari, O.P., 2018. Effects of bungur leaves (Lagerstroemia speciose (L.) Pres.) on malondialdehyde and blood glucose levels in hyperglycemic mice. Journal of Young Pharmacists, 10(2s), p.S124.
• Ozensoy, O., Kockar, F., Arslan, O., Isik, S., Supuran, C.T. and Lyon, M., 2006. An evaluation of cytosolic erythrocyte carbonic anhydrase and catalase in carcinoma patients: An elevation of carbonic anhydrase activity. Clinical biochemistry, 39(8), pp.804-809.
• Krishnan, N. and Sehnal, F., 2006. Compartmentalization of oxidative stress and antioxidant defense in the larval gut of Spodoptera littoralis. Archives of Insect Biochemistry and Physiology: Published in Collaboration with the Entomological Society of America, 63(1), pp.1-10.
• Esringü, A., Kotan, R., Bayram, F., Ekinci, M., Yıldırım, E., Nadaroğlu, H., & Katırcıoğlu, H. (2016). Sarımsak yetiştiriciliğinde farklı bakteri biyoformülasyonu uygulamalarının bitki gelişimi parametreleri, verim ve enzim düzeyleri üzerine etkisi. Nevşehir Bilim ve Teknoloji Dergisi, 5, 214-227.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

According to the comments and suggestions, improve your article.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript's English writing is moderate and can be improved with a thorough review. 

Author Response

We are grateful for the valuable inputs and suggestions. We edited entire manuscript based on the inputs, suggestions and edits of all three referees. Please find our responses to each query below and revised version as highlighted sections in the manuscript.

Abstract and keywords

Comment 1: The concentrations of the treatments (NaCl, SA, and GA) are not explicitly stated. Please include these (line 13-14)

Response 1: Thank you for the valuable input. We revised Material and methods section and provided concentrations for NaCl, SA, and GA)

Comment 2: The study design (hydroponic or soil-based) should be clearly mentioned (12-14).

Response 2: We appreciate the feedback. We clarified and added details of the experimental design.

Comment 3: Line 16-17, or 19-20- herein, mention some of the important parameters on which hormones have positive or negative impact. 

Response 3: Thank you for the suggestion. We added relevant information.

Comment 4: In the abstract, you have mention the importance of GA and SA that has positive impact, however, not mention the impact of NaCl. Besides, non-of the concentration of salt stress has been mention does it effect the plant or not, if effected which concentration is?

Response 4: We are grateful for the suggestion. We clarified the section and added below text “….”

Comment 5: Line-12, the abstract mentions molecular traits but does not describe what they are. This information should be included for clarity.

Response 5: We are thankful for the valuable input. We apologize for the typo, Molecular is deleted and antioxidant is added.

Comment 6: In keywords, instead of maize use the scientific name, the term “maize” is already been used in title.

Response 6: Thank you for the suggestion. Revised accordingly.

Introduction

Comment 7: Line 59-61: Please provide the appropriate references. If possible, include a brief mechanistic explanation from previous literature that how GA and SA trigger seed germination at a genomic level.

Response 7: We thank the reviewer for their constructive feedback. We have revised the manuscript by adding the requested citations to lines 59-61. Furthermore, a detailed discussion of the implications can now be found in lines 226-247

Comment 8: The last part of the introduction (lines 55-70) lacks specific literature on the combined effects of GA and SA under salinity stress. The discussion should be more specific and less general.

Response 8: Thank you for raising this point. The relevant discussion is presented in lines 226-247.

 

Comment 9: Line 61-64: This statement is poorly written and needs to be revised for clarity.

Response 9: We agree with the reviewer that this statement was poorly written. We apologize for the lack of clarity. We have completely rewritten this statement/paragraph (lines X-Y) to improve clarity, precision, and readability.

Material and methods

Comment 10: The sub-parts of this section should be restructured with a single, clear sub-heading, such as "Plant Materials and Growth Conditions." Lines 93-118: This entire section needs a separate heading, such as "Measurement of Growth  and Physiological Parameters." First, describe the growth parameters, and then the  physiological or biochemical parameters. The paragraph from lines 119-125 appears to be a  repetition and can be omitted. The statement regarding root scanning (lines 123-124) should be moved to the section on  growth attributes.

Response 10: Thanks for these great suggestions for re-structuring the MM section. We have made all the changes that were asked for to make sure that the methodological structure is clear. We combined the sub-headings into one big heading called "Plant Materials and Growth Conditions." We then made a new main section called "Measurement of Growth and Physiological Parameters." In this section, the content is logically rearranged so that growth parameters come first, followed by physiological/biochemical parameters.

Comment 11: Line 73-76: This statement does not make sense in its current form and should be rephrased or removed.

Response 11: We agree that the statement on lines 73-76 was unclear. We have rephrased this text completely

Comment 12: Line 78: Please clarify the units for the SA treatments. If they are in mg/L, as with the GA treatments, the units should be explicitly stated.

Response 12: Thank you for the suggestion. Revised accordingly.

Comment 13: The abbreviations for salicylic acid (Sa1, Sa2) and gibberellic acid (G1, G2) are inconsistent. For better readability, I suggest using a consistent format such as S1, S2 and G1, G2, or the standard abbreviations SA1, SA2, and GA1, GA2.

Response 13: In accordance with the suggestion, corrections have been implemented throughout the manuscript

Results and discussion

Comment 14: Line 150: The phrase "In the control groups, increases in salt concentration" is confusing. Please clarify what you mean by "control group" and how it was treated with increasing salt concentrations.

Response 14: Thank you for the suggestion. We re-phrased the sentence.

Comment 15: Figure 1: The bar chart lacks standard error bars, which are essential for assessing data variability. Please add them.

Response 15: The figure was re-submitted after the standard errors were corrected

Comment 16: Generally, a good paper contain some high quality figures and a mechanistic approach, that claim to support the data. While in this paper the data is very limitted regarding the topic of the study. If possible, I suggest to add some other physiological or biochemical parameters to provide a more robust mechanistic understanding of how the hormones and salt stress interact.

Response 16: We acknowledge that this manuscript presents preliminary findings and agree that a more detailed mechanistic study would strengthen the conclusions. The current submission utilizes all available data from this phase of the study. We plan to design follow-up experiments that incorporate the physiological and biochemical parameters you suggested to provide the robust mechanistic understanding necessary for a conclusive paper.

Conclusion

Comment 17: The manuscript's English writing is moderate and can be improved with a thorough review. I recommend a major revision to address the significant issues outlined above. The inclusion of more comprehensive data and a clearer mechanistic approach would greatly strengthen the paper and its findings.

Response 17: English Language: We agree the manuscript needed improvement. The entire manuscript has been thoroughly edited for clarity, grammar, and style.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

• Mention the levels of the study factors (GA₃, SA, and NaCl) in the abstract.
• No results for salinity concentrations were reported in the abstract.
• In the abstract, determine the best treatment with the best concentration that gives the best results.
• Line 31: (Salinity stress is one of the most significant problems limiting plant development, growth, survival, yield, and quality). This statement is repetition (same meaning as the previous statement).
• Line 33: (approximately 20% of irrigated agricultural land is affected by salinity [4].). This reference is old (2011), the current percentage has increased a lot. Recent references indicate that 33% of irrigated land worldwide has become saline, and by 2050, approximately 50% of arable land is expected to become saline (https://doi.org/10.1080/01904167.2023.2291022).
• Since the study is about the effect of salinity levels on maize, it is preferable to indicate the salinity threshold for the plant.
• The English name of the plant should be consistent throughout the manuscript. In line 13 (forage maize), in line 42 (corn), line 46 (maize), while line 69 (silage corn).
• Line 67: Add the reference.
• Line 84: (one additional plant was removed after emergence). Rephrase for clarity, you can remove the word (additional)
• Line 85-87: What are the benefits of sterilizing seeds twice with ethanol and sodium hypochlorite? Isn't one treatment enough?
• Line 91: What do you mean by "at regular intervals". Explain specifically
• Several of the statements made in the discussion section are not supported by any reference.
• The discussion should be supported by more references that discuss in depth the potential mechanisms of both gibberellic acid and salicylic acid on salt-stressed plants.

Comments for author File: Comments.pdf

Author Response

We are grateful for the valuable inputs and suggestions. We edited entire manuscript based on the inputs, suggestions and edits of all three referees. Please find our responses to each query below and revised version as highlighted sections in the manuscript. The aim of the study were revised and rewritten.

 

Comment 1- Mention the levels of the study factors (GA₃, SA, and NaCl) in the abstract.

Response 1: We agree with this suggestion. The Abstract has been revised to include the specific levels/concentrations of the study factors (GA₃, SA, and NaCl).

Comment 2- No results for salinity concentrations were reported in the abstract.

Response 2: We have now revised the Abstract to include the specific salinity (NaCl) concentration used in the study, alongside the levels of GA3 and SA.

Comment 3- In the abstract, determine the best treatment with the best concentration that gives the best results.

Response 3: This is an excellent point; we have revised the Abstract to clearly state the optimal treatment

Comment 4- Line 31: (Salinity stress is one of the most significant problems limiting plant development, growth, survival, yield, and quality). This statement is repetition (same meaning as the previous statement). 

Response 4: We agree that the statement on Line 31 was repetitive. We have removed this sentence entirely to make the Introduction more concise

Comment 5- Line 33: (approximately 20% of irrigated agricultural land is affected by salinity [4].). This reference is old (2011), the current percentage has increased a lot. Recent references indicate that 33% of irrigated land worldwide has become saline, and by 2050, approximately 50% of arable land is expected to become saline (https://doi.org/10.1080/01904167.2023.2291022).

Response 5: We agree with the reviewer's comment. We have corrected the statistic in Line 33 to reflect the current, more severe impact of salinity on irrigated lands. We have replaced the outdated citation [4] with the recent reference provided (DOI: 10.1080/01904167.2023.2291022) to support the claim that approximately 33% of irrigated land is now saline, with projections suggesting nearly 50% of arable land will be affected by 2050. This change is implemented in the Introduction on line 33.

Comment 6- Since the study is about the effect of salinity levels on maize, it is preferable to indicate the salinity threshold for the plant.

Response 6: We agree with the reviewer that establishing the salinity tolerance threshold for maize is important context. We have revised the Introduction section to include the generally accepted salinity threshold for maize and added relevant references.

Comment 7- The English name of the plant should be consistent throughout the manuscript. In line 13 (forage maize), in line 42 (corn), line 46 (maize), while line 69 (silage corn).

Response 7: We have corrected all instances as requested. Where appropriate, the terms are specified as 'maize' or 'silage maize'. General mentions use 'maize', while specific references include 'silage'. We also changed all previous instances of 'corn' to 'maize' for consistent terminology throughout the paper. All of the reviewer's specific corrections are marked in red.

Comment 8- Line 67: Add the reference.

Response 8: The reference has been inserted on line 67 as requested.

Comment 9- Line 84: (one additional plant was removed after emergence). Rephrase for clarity, you can remove the word (additional)

Response 9: We have rephrased Line 84 to improve clarity

Comment 10- Line 85-87: What are the benefits of sterilizing seeds twice with ethanol and sodium hypochlorite? Isn't one treatment enough?

Response 10: We appreciate the need for clarification regarding the sterilization protocol. The high level of surface sterilization was essential due to the significant fungal load typically present on field-harvested seeds. In preliminary trials, we observed that fungal contamination severely compromised initial coleoptile and root development during the critical germination phase. Therefore, we performed the most stringent surface sterilization possible to ensure axenic conditions and isolate the true effects of the hormonal and salt treatments from microbial interference.

Comment 11- Line 91: What do you mean by "at regular intervals". Explain specifically

Response 11: Irrigation was performed according to soil moisture levels, using saline water prepared at the specified concentrations. This ensures that the salinity levels remained consistent based on the concentrations outlined in the study design.

Comment 12- Several of the statements made in the discussion section are not supported by any reference.

Response 12: Thank you for this feedback, but the line or section requiring correction is unclear. We are happy to address this point immediately. However, to ensure we make the correct revision, could you please specify the line number, paragraph, or section that needs attention?

Comment 13- The discussion should be supported by more references that discuss in depth the potential mechanisms of both gibberellic acid and salicylic acid on saltstressed plants.

Response 13: To address the reviewer's concern regarding clarity in this section, we have added the necessary explanations/information between lines 226-247

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have carefully responded to and revised the manuscript, but I noticed that the percent match of the revised manuscript is too high at 41%. Please submit the revised manuscript again after further modifications.

Author Response

Dear Reviewer, 

We are thankful for the edits, comments and suggestion. After receiving your comment about the similarity, we carefully checked the Ms. and run additional similarity checks through ithenticate, turnitin and intihal.net. We reailized that, turnitin catches bibliography and references. So, after excluding those sections similarty ratio dropped to 18% in turnitin, 16% ithenticate and 15% in intihal.net. If there is still any concerns about the similarity please let us know, we will re-edit any section mentioned. Thank you again for your support. Regards. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The author has implemented all the comments in the previous review, but there are some minor comments.

• Line 29: Do you mean (and in reducing salt stress)? Or (and with increased salt stress).
• Line 37: Replace dashes with commas.
• The reference to the maize salinity threshold in the introduction section may justify the salinity levels used in the study.

Author Response

Dear Referee, 

 

Thank you for supportive suggestions. We carefully edited relevant sections and made all corrections. 

Regards