Optimizing Selenium Delivery in Grapevines: Foliar vs. Rhizosphere Fertilization Effects on Photosynthetic Efficiency, Fruit Metabolites, and VOCs of ‘Muscat Hamburg’ Grape (Vitis vinifera L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The topic of the manuscript "Distinct Effects of Foliar versus Rhizosphere Selenium Fertilization on Leaves Photosynthetic Physiology, Fruit Biochemistry, and Volatile Organic Compounds of 'Muscat Hamburg' Grape (Vitis vinifera L.)" is within the scope of the Journal of Horticulturae and is interesting, although greater attention to potential of selenium to enhance tolerance to abiotic stresses—intensified by climate change—would make the study even more compelling.

In my opinion, the manuscript needs a major revision before it can be considered for publication.

General comments:

The vast amount of data in the manuscript highlights the considerable work carried out. However, for many parameters, only the observed data is reported without an analysis of the causes that conditioned them.

A large part of the discussion tends to be a simple comparison between the obtained results and what is available in the literature (which, moreover, for some aspects, is not sufficiently considered in its entirety). It would be useful and interesting to deepen the discussion to enrich knowledge on Selenium’s effects and mechanisms with original insights based on the obtained results, thereby increasing the scientific impact of the work.

In addition, the authors should better highlight (in the discussion/conclusions) the potential applications of the obtained results and emphasize the novel elements of the work compared to existing literature.

In the specific comments, some papers of my knowledge (but others could also be selected) are suggested to complete the state of the art on the Selenium aspects considered in the manuscript and to better support the content of the manuscript; some of them could also be considered to improve/expand the discussion.

Specific comments:

Lines 2-5 – The title is somewhat long and generic: it tries to summarize the measured parameters without clearly stating the purpose of these determinations. Furthermore, by including “Photosynthetic Physiology” in the title, one would expect measurements of photosynthesis itself, not just related parameters. The same applies to “Fruit Biochemistry”.

Line 28 – It is unnecessary to repeat keywords already present in the title (e.g., volatile organic compounds).

Line 37 – their?

Lines 40-41 – Selenium also positively affects pollen germination and fertility (DOI: 10.1016/j.flora.2015.05.009) and growth and vigor of in vitro cultivated explants (DOI: 10.3390/plants10081630).

Line 43 – Also for drought stress (DOI: 10.3389/fpls.2018.01191).

Line 45 – Also in olive trees (DOI: 10.1016/j.scienta.2020.109866).

Line 47 – References 8 and 9 seem not very relevant.

Line 66 – See also DOI: 10.1016/j.jtemb.2016.11.015.

Line 86 – In greenhouses, the results might differ from those obtained outdoors. The authors should discuss this limitation.

Lines 86 and following – If I understood correctly, the climatic data refer to the outside of the greenhouse? And what about the internal conditions?

Line 184 – In the table captions, explain the statistical analysis.

Line 184 – To what can the sometimes considerable differences between the controls of foliar and soil treatments in the same periods be attributed?

Line 184 – The abbreviation CK was not previously introduced.

Lines 352-353 – The sentence seems incomplete.

Line 402 – References 46 and 47 seem not very relevant. In any case, measuring the photosynthetic rate would have provided the authors with more information and stronger evidence of Selenium's impact on photosynthesis. For example, see the papers DOI: 10.1016/j.scienta.2013.09.034. The authors should discuss this.

Lines 409 and following – The paper seems to underestimate the direct and indirect antioxidant capacities that can be exploited in plants to combat abiotic stresses caused by reactive oxygen species produced in excess (ROS). E.g., Selenium can increase the activities of APX, CAT, and GPOX enzymes and reduced the content of malondialdehyde (MDA), which otherwise accumulate during oxidative disturbances. Selenium application can protect plant cells from oxidative damage and thus merits consideration in strategies designed to combat abiotic stress leading to oxidative injuries. About this, see e.g., DOI: 10.1007/s11240-023-02575-7.

Lines 442-459 – Much of the conclusions merely summarizes the results and seems more like an abstract than conclusions. It would be better if the conclusions better developed and highlighted the new insights gained from the study's findings.

Line 461 – Combined application, through foliar and rhizosphere, could be explored to see if it offer additional benefits also possibly reducing the total dose.

Comments on the Quality of English Language

I am not competent to evaluate the correctness of English.

Author Response

General comments:

 

The vast amount of data in the manuscript highlights the considerable work carried out. However, for many parameters, only the observed data is reported without an analysis of the causes that conditioned them.

 

A large part of the discussion tends to be a simple comparison between the obtained results and what is available in the literature (which, moreover, for some aspects, is not sufficiently considered in its entirety). It would be useful and interesting to deepen the discussion to enrich knowledge on Selenium’s effects and mechanisms with original insights based on the obtained results, thereby increasing the scientific impact of the work.

 

In addition, the authors should better highlight (in the discussion/conclusions) the potential applications of the obtained results and emphasize the novel elements of the work compared to existing literature.

 

In the specific comments, some papers of my knowledge (but others could also be selected) are suggested to complete the state of the art on the Selenium aspects considered in the manuscript and to better support the content of the manuscript; some of them could also be considered to improve/expand the discussion.

 

General response:

 

Thank you for taking the time to review our manuscript and for providing us with valuable feedback. We have carefully considered your comments and would like to address them.

We acknowledge the point raised regarding the lack of analysis of the causes conditioning many of the reported parameters. Meanwhile, the discussion section currently focuses mainly on a comparison between our obtained results and existing literature. Regarding these issues, we have undertaken a thorough examination of the data presented in the report and conducted an in-depth analysis of the underlying reasons for the observed phenomena. By drawing comparisons with existing literature and theoretical frameworks, we have enriched our understanding of selenium's role in grape growth, delved into its mechanisms of action, and speculated on the potential applications of our findings. Following our revisions, the discussion section has been significantly expanded and deepened in scope. Throughout this process, we have cited a substantial number of pertinent and significant literature sources, including those recommended by the reviewers. For further details, please refer to the discussion section of the revised article.

 

Specific comments:

 

Comments 1: Lines 2-5 – The title is somewhat long and generic: it tries to summarize the measured parameters without clearly stating the purpose of these determinations. Furthermore, by including “Photosynthetic Physiology” in the title, one would expect measurements of photosynthesis itself, not just related parameters. The same applies to “Fruit Biochemistry”.

Response 1: Thank you for your valuable feedback regarding the title of our manuscript. We agree that the current title does not sufficiently emphasize the study’s primary objectives or the novelty of its findings. We have carefully revised the title as ‘Optimizing Selenium Delivery in Grapevines: Foliar vs. Rhizosphere Fertilization Effects on Photosynthetic Efficiency, Fruit Metabolites, and VOCs of 'Muscat Hamburg' Grape (Vitis vinifera L.)’, which better reflects the purpose of the research and to ensure clarity and conciseness. Please see line 2-5.

 

Comments 2: Line 28 – It is unnecessary to repeat keywords already present in the title (e.g., volatile organic compounds).

Response 2: Thank you for your reminder. The keyword has been replaced by “fatty acid metabolic pathways”. Please see line 28-29.

 

Comments 3: Line 37 – their?

Response 3: Thank you for pointing out the issue. The sentence has been replaced by “…………threaten grape yield and quality” in the revision. Please see line 36.

 

Comments 4: Lines 40-41 – Selenium also positively affects pollen germination and fertility (DOI: 10.1016/j.flora.2015.05.009) and growth and vigor of in vitro cultivated explants (DOI: 10.3390/plants10081630).

Response 4: Thank you for providing these two valuable references, which have significantly contributed to enhancing the quality of the article. We have thoroughly examined the content of the literatures and have included appropriate citations as reference [3]and [6]. Please see line 40-41.

 

Comments 5: Line 43 – Also for drought stress (DOI: 10.3389/fpls.2018.01191).

Response 5: Thank you for providing this literature. It has been cited as reference [7]. Please see line 43.

 

Comments 6: Line 45 – Also in olive trees (DOI: 10.1016/j.scienta.2020.109866).

Response 6: Thank you for providing this literature. It has been cited as reference [8]. These two articles offer abundant case support for the role of selenium in alleviating stress induced by ROS under adverse conditions. Please see line 43.

 

Comments 7: Line 47 – References 8 and 9 seem not very relevant.

Response 7: Thank you for the reviewer's reminder. We have now removed these two references.

 

Comments 8: Line 66 – See also DOI: 10.1016/j.jtemb.2016.11.015.

Response 8: Thank you for providing this literature. This reference has been used to replace the original reference [2]. Please see line 40.

To enhance the clarity of the article and emphasize the purpose of our study, we have thoroughly revised the preface, making substantial improvements to this section and clearly delineating our objectives. Any inappropriate and redundant statements have been revised accordingly. Please see line 66-69.

 

Comments 9: Line 86 – In greenhouses, the results might differ from those obtained outdoors. The authors should discuss this limitation.

Response 9: Thank you for your comment, which we gratefully accept. In this section, we have included additional information about the environmental conditions inside the greenhouse during the experiment, and at the end of the article, we have provided a thorough explanation of the experimental constraints imposed by these conditions. Please see line 89-92 and 455-457 in revision.

In reality, the majority of table grapes in China are cultivated in greenhouses, a practice that guarantees the quality and yield of the grapes while protecting them from natural disasters. For farmers, ensuring a stable income is paramount, and greenhouses offer a reliable means of achieving this. This is the primary reason our experiment was conducted in a greenhouse setting.

Nevertheless, we acknowledge that the application of selenium in grapes should not be restricted to greenhouses; it is also widely utilized in outdoor cultivation. Consequently, in our future research, we intend to investigate outdoor conditions and explore wine grape varieties to expand the range of applications for selenium.

 

Comments 10: Lines 86 and following – If I understood correctly, the climatic data refer to the outside of the greenhouse? And what about the internal conditions?

Response 10: Thank you for your comment again. In the previous paragraph, we explained why we chose to conduct experiments in a greenhouse, which indeed imposes certain limitations on the experimental conclusions. In the revision, we have provided detailed environmental conditions inside the greenhouse, “daytime temperatures below 30 ℃ and nighttime temperatures above 15 ℃, with relative humidity controlled within 40% ~ 70%”. Please see line 89-92.

 

Comments 11: Line 184 – In the table captions, explain the statistical analysis.

Response 11: Thank you for your reminder. The data of all tables in the article are expressed as mean±SE. one-way analysis of variance and Duncan’s Multiple Range Test were used to compare the different treatments. Different letters indicate significant differences (p < 0.05) across Se concentrations in the same period. More detail see line 183-184 in revision.

 

Comments 12: Line 184 – To what can the sometimes considerable differences between the controls of foliar and soil treatments in the same periods be attributed?

Response 12: This is a good question and also what we want to discuss. We observed that even at the same selenium concentration, foliar fertilization and rhizosphere fertilization have significantly different effects on the indicators.

 

Actually, foliar fertilization and root fertilization are two distinct methods of nutrient application, differing primarily in the pathways and efficiency of nutrient uptake. Foliar fertilization involves the direct absorption of nutrients through the stomata or cuticle of leaves, making it suitable for quickly supplying micronutrients or addressing urgent deficiencies. In contrast, root fertilization relies on nutrient uptake by roots from the soil, which is then transported to various parts of the plant, serving as the primary means of nutrient acquisition. Foliar fertilization acts rapidly, often showing effects within hours to days, while root fertilization is slower but provides longer-lasting results. Foliar fertilization is highly efficient for targeted nutrient delivery but is limited in the amount that can be applied at once, whereas root fertilization can supply larger quantities of nutrients, though its efficiency is influenced by soil conditions. 

We provide a more in-depth discussion in the introduction and discussion part in revision. Please see line 49-55 and 349-354.

 

Comments 13: Line 184 – The abbreviation CK was not previously introduced.

Response 13: Thank you for your reminder. We have added the introduction of the abbreviation CK in the method part of revision. Typically, CK denotes the control group, which in the context of this article consists of distilled water without any added selenium serving as the baseline for comparison. Please see line 101.

 

Comments 14: Lines 352-353 – The sentence seems incomplete.

Response 14: Sorry！Thank you for pointing out the omission. The original sentence is ' lies in '. we corrected it in the revision. Please see line 335.

 

Comments 15: Line 402 – References 46 and 47 seem not very relevant. In any case, measuring the photosynthetic rate would have provided the authors with more information and stronger evidence of Selenium's impact on photosynthesis. For example, see the papers DOI: 10.1016/j.scienta.2013.09.034. The authors should discuss this.

Response 15: Thank you for your comment. We have removed the references 46 and 47, but we could not fine the literature with DOI 10.1016/j.scienta.2013.09.034. We have found other relevant literature to replace it.

As is widely recognized, the accumulation of sugar in fruits is intimately linked to the efficiency of photosynthesis. Some literature has reported that the application of appropriate amounts of selenium can enhance photosynthetic efficiency and boost the production of photosynthetic products. In this article, while we did not directly measure the photosynthetic rate using instruments like the LI6400, we did assess fluorescence parameters pertinent to photosystem 2, which lent support to this notion. Indeed, we are currently conducting comprehensive experiments on related photosynthetic indicators, although it will require some time before we can finalize the paper. In the discussion section, we have included pertinent literature, like Zhang et al. 2014 (Ref[45]) and Zhong et al.  2024 (Ref[46]) to substantiate the reliability of our conclusions and the validity of our reasoning. Please see line 389.

 

Comments 16: Lines 409 and following – The paper seems to underestimate the direct and indirect antioxidant capacities that can be exploited in plants to combat abiotic stresses caused by reactive oxygen species produced in excess (ROS). E.g., Selenium can increase the activities of APX, CAT, and GPOX enzymes and reduced the content of malondialdehyde (MDA), which otherwise accumulate during oxidative disturbances. Selenium application can protect plant cells from oxidative damage and thus merits consideration in strategies designed to combat abiotic stress leading to oxidative injuries. About this, see e.g., DOI: 10.1007/s11240-023-02575-7.

Response 16: Thank you for your comment. As highlighted by the reviewer, selenium has the capacity to elevate the activity of APX, CAT, and GPOX enzymes, while also diminishing the levels of malondialdehyde (MDA). The protective effect of selenium on plant cells against oxidative damage has been well-documented in numerous literature sources. In this article, our primary focus is on the influence of selenium on the metabolic quality of grape fruits, encompassing flavonoids, which, alongside grape morphology and sugar and acid content, are crucial determinants of grape quality. Therefore, we have comprehensively discussed the effects of selenium on these metabolites. The intricate issue of how different selenium application methods impact antioxidant enzymatic and non-enzymatic systems warrants a detailed examination, which we intend to delve into in a separate article. To enrich the discussion, we also cited this article with DOI: 10.1007/s11240-023-02575-7 as reference [50]. Please see line 397-401 in revision. 

 

Comments 17: Lines 442-459 – Much of the conclusions merely summarizes the results and seems more like an abstract than conclusions. It would be better if the conclusions better developed and highlighted the new insights gained from the study's findings.

Response 17: Thank you for your comment. We realized that the original conclusion merely summarized our findings and lacked innovative insights and future prospects. Factually, we found that foliar fertilization proved to be more effective in regulating photosynthetic parameters and exerted a moderate influence on fruit size and flavor components. In contrast, rhizosphere fertilization had a more pronounced impact on fruit antioxidant content, overall size, and aroma. These results suggest that the efficacy of selenium (Se) as a growth enhancer and stress mitigator is highly contingent on both its concentration and the method of application.

So, we have made substantial revisions to the conclusion section, fully incorporating the new perspectives gained from this study and offering a glimpse into its future potential. We hope that our new modifications meet your expectations. Please see line 449-455.

 

Comments 18: Line 461 – Combined application, through foliar and rhizosphere, could be explored to see if it offer additional benefits also possibly reducing the total dose.

Response 18: This proposal is highly promising and aligns with our research aspirations and ultimate implementation goals. We also aim to explore the combined use of foliar and rhizosphere fertilization methods in the future, with the objective of reducing chemical inputs and achieving higher yields. Additionally, we have provided an outlook at the end of the article to offer insights into future directions. Please see line 460.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper presents a comparison of foliar and rhizosphere fertilization of grapevine and their impact on several qualitative parameters of the grapevine. This topic has been researched for a long time, and it is already scientifically proven that foliar fertilization has greater advantages over rhizosphere fertilization. Most often, a combination is recommended, where rhizosphere fertilization is applied as the main fertilizer, complemented by foliar fertilization. There are already numerous original scientific papers on this topic.

Considering this aspect, the manuscript does not offer new insights that would represent a step forward in improving grapevine cultivation (improving the quality and quantity of production, which is the ultimate goal of every producer). Also, methodologically, there is no innovation in the analytical part. The only novelty is the statistical processing of the data, where standard statistical tests are presented through new software programs.

However, publication can be considered, but after major revisions:

1. The title is unnecessarily long and overly descriptive. A short title that reflects the content of the paper should be given.
2. The discussion is insufficient. A detailed explanation should be provided on why the results are better for foliar fertilization compared to rhizosphere fertilization (from the physiological aspect of the grapevine).
3. An explanation as in point 2 should also be given for all other results: what accounts for the differences in the parameters (higher or lower content) from a physiological aspect?
4. Comparisons in the discussion should be made specifically with this research, not just as general examples.
5. The conclusion reads more like an abstract and should be completely revised. The conclusion cannot just be a synthesis of already presented data. The most important results should be highlighted, and recommendations and future perspectives should be provided specifically for this research.
6. The nutrition of the grapevine is a specific agronomic measure, and a large number of nutrients (macro and microelements) are required for its development. If the authors have research on the impact of selenium on the dynamics of macro and microelements in the leaves or clusters, it would be good idea to add this data (at least the basic macroelements N, P, K, as well as the microelements Fe, Zn, B), this is recommendation only. 

Author Response

To Review2：

General comments:

The paper presents a comparison of foliar and rhizosphere fertilization of grapevine and their impact on several qualitative parameters of the grapevine. This topic has been researched for a long time, and it is already scientifically proven that foliar fertilization has greater advantages over rhizosphere fertilization. Most often, a combination is recommended, where rhizosphere fertilization is applied as the main fertilizer, complemented by foliar fertilization. There are already numerous original scientific papers on this topic.

Considering this aspect, the manuscript does not offer new insights that would represent a step forward in improving grapevine cultivation (improving the quality and quantity of production, which is the ultimate goal of every producer). Also, methodologically, there is no innovation in the analytical part. The only novelty is the statistical processing of the data, where standard statistical tests are presented through new software programs.

 

General response:

Thank you for providing such constructive feedback on our manuscript. We deeply appreciate the time and effort you have invested in evaluating our work and wholeheartedly agree that advancing scientific knowledge necessitates a blend of rigor and innovation.

We acknowledge that the comparison between foliar and rhizosphere fertilization in grapevine cultivation has indeed been the subject of extensive research. However, our study distinctively focuses on assessing the impact of these two fertilization methods on fruit quality, with a particular emphasis on grape metabolites and aroma, and endeavors to elucidate this from the vantage point of chlorophyll fluorescence. It is worth noting that previous studies have offered limited data on aroma and chlorophyll fluorescence, and we aspire to enrich the existing body of work in this domain.

We have employed a novel statistical method, the Mantel test, to analyze the disparities arising from the two fertilization techniques, and have utilized advanced plotting methods to present the differences between treatments in a more intuitive and visually appealing manner. We are grateful for the reviewer's affirmation of our approach.

Although our manuscript may not introduce a groundbreaking technique, it underscores the pivotal role of combined fertilization (foliar + rhizosphere) in attaining balanced grape quality. Moving forward, we are committed to conducting more in-depth research on this fertilization technology, with the aim of providing valuable insights and serving as a reference for the development of the grape industry in our local area.

 

Specific comments and response:

Comment 1: The title is unnecessarily long and overly descriptive. A short title that reflects the content of the paper should be given.

Response 1: Thank you for the reminder. The title has been replaced by ‘Optimizing Selenium Delivery in Grapevines: Foliar vs. Rhizosphere Fertilization Effects on Photosynthetic Efficiency, Fruit Metabolites, and VOCs of 'Muscat Hamburg' Grape (Vitis vinifera L.)’ to clear our research purpose and content. If you feel it's not suitable, please feel free to provide valuable feedback. Please see line 2.

 

Comment 2: The discussion is insufficient. A detailed explanation should be provided on why the results are better for foliar fertilization compared to rhizosphere fertilization (from the physiological aspect of the grapevine).

Response 2: This is very good comment. In reality, we are also aware of the limitations of our article, particularly in the discussion section where we did not comprehensively compare and elucidate the reasons behind the disparities between foliar fertilization and rhizosphere fertilization. To address this, we conducted extensive literature searches and engaged in discussions with relevant scholars to enhance our analysis of the factors contributing to the differences in fertilization effects between the two methods. Substantial revisions were made to the discussion section to reflect these in-depth modifications. More detail see in line 349-354, line 393-395 and line 412-415.

 

Comment 3: An explanation as in point 2 should also be given for all other results: what accounts for the differences in the parameters (higher or lower content) from a physiological aspect?

Comparisons in the discussion should be made specifically with this research, not just as general examples.

Response 3:  Thank you for the reminder. In the original article, we lacked analysis of data differences. We believe that the main reason for the data discrepancy is the difference between the two fertilization methods. Foliar fertilization quickly supplies nutrients directly through leaves, ideal for urgent deficiencies or micronutrients. Root fertilization, the primary nutrient source, relies on uptake by roots and is slower but longer-lasting. Foliar fertilization is efficient for targeted delivery but limited in quantity, while root fertilization can supply more nutrients, efficiency depending on soil conditions. We have improved the discussion and updated several literature examples to thoroughly elucidate the reasons for the observed differences.

Please see discussion part in revision.

 

Comment 4: The conclusion reads more like an abstract and should be completely revised. The conclusion cannot just be a synthesis of already presented data. The most important results should be highlighted, and recommendations and future perspectives should be provided specifically for this research.

Response 4: Good comments. Many experts have highlighted this shortcoming. So, we have rewritten this part of the content. Not only did it provide a summary of the article's content, but it also synthesized the findings and put forth innovative conclusions. Furthermore, the article concludes by presenting prospects for future work and proposing new research directions.  Please see conclusion part in revision.

 

Comment 5: The nutrition of the grapevine is a specific agronomic measure, and a large number of nutrients (macro and microelements) are required for its development. If the authors have research on the impact of selenium on the dynamics of macro and microelements in the leaves or clusters, it would be good idea to add this data (at least the basic macroelements N, P, K, as well as the microelements Fe, Zn, B), this is recommendation only.

Response 5: Thank you for your suggestion. In our work, we have actually discovered that selenium application not only mitigates ROS-induced stress damage but also enhances the absorption of trace and heavy metal elements, which is clearly crucial for plant growth. We intend to conduct further research to elucidate the mechanism of plant-selenium interactions and develop more production technologies. This was also noted at the end of the article. Please see line 460-462.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have carefully and expertly revised the manuscript based on the comments received. In my opinion the work is interesting e per alcuni aspetti originale, quindi can be published. There is only one comment (below) that I think the authors did not understand.
Regarding comment 12 (line 184), I apologize because I was obviously not clear. If I understood correctly, you used different control plants for foliar fertilization and rhizosphere fertilization. These two groups of control plants, not having undergone any treatment, should give very similar results to each other, instead between the two groups of control plants rather different values ​​emerge for various parameters, denoting a variability that instead should not be there. How can this be explained?

Regarding comment 15, ok, anyway if you are interested in DOI 10.1016/j.scienta.2013.09.034 you can use this link https://www.sciencedirect.com/science/article/pii/S0304423813004937?casa_token=1BbQmTnvgjcAAAAA:Md5VKwrfMebYp8x7PG_kBbekWoA3xDrepB-e5f4UkBflM2a6e-r3KpALj4y90yPcuYCRa1oOCQ

Author Response

Dear Reviewer and Editors,

Thank you for reviewing so carefully. And we apologize for not understanding your meaning correctly in the first response. We sincerely appreciate the opportunity to address this point more comprehensively.

Upon re-examining the data, we agree that the two control groups (for foliar and rhizosphere fertilization experiments) exhibited unexpected variability in certain parameters, despite both being untreated. We recognize that this requires a clear explanation. The foliar and rhizosphere fertilization trials were conducted sequentially under slightly varying environmental conditions (e.g., light intensity, humidity) due to practical constraints. While we maintained standardized growth conditions settings, subtle environment fluctuations may have influenced physiological responses. Additionally, although plants were selected for uniformity, minor differences in developmental stages could have contributed to the discrepancies. In this study, six trees were selected for each treatment, and individual variations often resulted in discrepancies in the final data. These differences might be particularly noticeable during the early stages of development. In future research, we need to maintain a sufficient sample size. This highlights the importance of conducting sufficient repetitions in such experiments to obtain more reliable and accurate data.

Furthermore, we are grateful for the comprehensive information you provided regarding this literature (DOI 10.1016/j.scienta.2013.09.034). Upon thorough examination of the article, we found it immensely beneficial in elucidating the mechanisms of selenium's role in photosynthetic physiology and its antioxidant effects. Consequently, we have diligently cited this literature in place of the original reference.

There are minor revisions in the article, which we have highlighted in yellow. Please see line 390 and 594.

We thank you for raising this critical point, as it has strengthened the rigor of our work. Please let us know if further clarification or data would be helpful.

Sincerely Tian

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article, after corrections, can be presented in its presented form. 

Author Response

Dear Reviewer and Editors,

Thank you for your acknowledgment and appreciation. We confirm that all recommended revisions have been fully addressed in the final version, and the manuscript is now prepared for publication in its current form. It has been a privilege to collaborate with your team throughout this process. The constructive critiques not only strengthened the article’s academic value but also provided us with valuable perspectives for future research. We are grateful for the time and expertise invested by the editorial office and reviewers in elevating the quality of our work.

Thank you once again for your guidance.

Best regards,

Sincerely Tian

Author Response File: Author Response.pdf