Glutathione Reductase Affects Hyphal Growth and Fruiting Body Development by Regulating Intracellular ROS Levels in Hypsizygus marmoreus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In general the manuscript is very well written, and the experiments are adequate to answer the scientific question. 

I only have minor observations 

 

LINES 211-213: “Anti-superoxide anion activity…” give a reference about this statement

Instead of using indirect ROS measures, like anti-superoxide anion activity. The authors need to measure with a specific ROS (superoxide anion) probe.

The TEM images are very illustrative, and it appears that the amount of mitochondria is decreased in the GR silencing strains. The authors need to quantify the mitochondria amount.

The authors need to demonstrate whether or not Vc increase the of activities of four key antioxidant enzymes: CAT, SOD, etc.

Could the overexpression of the GR gene have an impact on the fungus growth?

Author Response

# Reviewer 1

Comments and Suggestions for Authors

In general the manuscript is very well written, and the experiments are adequate to answer the scientific question.

I only have minor observations

     1. LINES 211-213: “Anti-superoxide anion activity…” give a reference about this statement.

Re: Thank you for your precious time in reviewing our manuscript. According to your suggestion, we have added the references in the corresponding places (Lines 210-212).

2. Instead of using indirect ROS measures, like anti-superoxide anion activity. The authors need to measure with a specific ROS (superoxide anion) probe.

Re: Thank you for your suggestion. The aim of our study was to investigate the changes in intracellular ROS following gr gene silencing in Hypsizygus marmoreus. We began by measuring the level of intracellular ROS (H₂O₂). As for why we did not use a specific probe to directly label superoxide anions, it is because we employed the DCFH-DA probe to quantify the overall intracellular ROS, which inherently includes superoxide anions.  Consequently, instead of directly detecting superoxide anions with probes, we used the ability to resist superoxide anions to indirectly indicate changes in cellular superoxide anions—a well-established method in ROS research in both plant and animal cells. Most importantly, the results of the superoxide anion scavenging activity support our conclusion that gr gene silencing leads to increased intracellular ROS. We hope this explanation addresses your concerns.

3. The TEM images are very illustrative, and it appears that the amount of mitochondria is decreased in the GR silencing strains. The authors need to quantify the mitochondria amount.

Re: Thank you very much for the instructive comments. We have conducted quantitative statistics on the number of mitochondria and added the corresponding description in the revised manuscript (lines: 244-246).

4. The authors need to demonstrate whether or not Vc increase the of activities of four key antioxidant enzymes: CAT, SOD, etc.

Re: Thank you for your suggestion. Your question was addressed in our previous study [1], which showed that: "Exogenous application of 1 mM L-ascorbic acid (Vc) had no significant effect on the activities of SOD, GPX, and GR, but increased the activity of CAT. Additionally, we found that Vc mainly alleviates ROS generated from mechanical damage and promotes hyphal recovery and regeneration." We also discussed this in the discussion section. We hope this explanation addresses your concern (lines: 507-509).

[1] Chen, H.; Hao, H.; Han, C.; Wang, H.; Wang, Q.; Chen, M.; et al. Exogenous l-ascorbic acid regulates the antioxidant system to increase the regeneration of damaged mycelia and induce the development of fruiting bodies in Hypsizygus marmoreus. Fungal Biol. 2020, 124, 551-61.

5. Could the overexpression of the GR gene have an impact on the fungus growth?

Re:  Thank you for your question. Our main research focuses on whether the gr gene in Hypsizygus marmoreus affects the redox levels within cells and its impact on the growth and development of the fruiting body. Although we used RNA interference technology to construct gr gene-silenced strains and systematically investigated the effects of the gr gene on the growth and development of Hypsizygus marmoreus, the effects of overexpressing the gr gene on Hypsizygus marmoreus have not been thoroughly studied. This is a limitation of our research. Since constructing overexpression gr gene strains requires a relatively long time, in the discussion section of the manuscript, we outlined the limitations and future prospects of our study and suggested using the overexpression of the gr gene as a direction for our future research on intracellular redox levels. We hope our explanation will be acceptable to you (lines: 510-513).

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is good and well-founded scientifically, but it needs adjustments. I invite the authors to accept, if possible, the suggestions of this reviewer.

- We recommend that authors use keywords that are different from the words in the manuscript title.

- The mushroom Hypsizygus marmoreus is important as a nutritional source in Asia. What is its estimated annual production?
What is its consumption? It would be interesting for the authors to cite information related to the importance of this mushroom, since the research is being conducted with it.

- Although the isolate H. marmoreus is in a type culture collection, is it recognized as a GenBank deposit? Or has it already been cited in another paper with a GenBank number? Please cite this in the manuscript.

- In item 2.3, what is the storage temperature for colonies to perform mycelial growth measurements?

- All figures and tables should be self-explanatory, so readers should not need to look up the meaning of abbreviations in the manuscript, even if their meaning is obvious. Therefore, I suggest that the authors of the manuscript review the 
titles of all figures, tables, and graphs, and describe what each abbreviation in the description or present in the figures  or tables means. Ex: WT??? wild-type (WT).

- The discussions are weak in relation to the volume of data obtained in these experiments. I invite the authors to explore  their results further, giving more substance to the discussion, which is basically only partially contained in the manuscript's introduction.

- The manuscript's conclusions are basically presented in the last paragraph of the paper (this study reveals that the GR-mediated redox balance regulates fruiting body development, enhancing our understanding of fungal stress physiology and metabolic regulation). 
I invite the authors to be concise in this item and cite the results instead of explaining them. Explaining the results is done in the discussions.

Author Response

# Reviewer 2

Comments and Suggestions for Authors

The manuscript is good and well-founded scientifically, but it needs adjustments. I invite the authors to accept, if possible, the suggestions of this reviewer.

 

1. We recommend that authors use keywords that are different from the words in the manuscript title.

Re: Thank you for your suggestion, we have updated the keywords (lines: 23-24).

 

2. The mushroom Hypsizygus marmoreus is important as a nutritional source in Asia. What is its estimated annual production? What is its consumption? It would be interesting for the authors to cite information related to the importance of this mushroom, since the research is being conducted with it.

Re:  Thank you for the suggestion. We have reviewed the literature and, although precise data for Asia as a whole is unavailable, we found that China—the largest producer of Hypsizygus marmoreus in Asia—has relevant data. We hope this information addresses your feedback satisfactorily (lines: 29-31).

 

3. Although the isolate H. marmoreus is in a type culture collection, is it recognized as a GenBank deposit? Or has it already been cited in another paper with a GenBank number? Please cite this in the manuscript.

Re: We have added the GenBank number in detail, as described below:“The nucleotide sequence of its internal transcribed spacer 1 (ITS1) region has been deposited in the GenBank database under the accession number: FJ609271.1.”(lines: 78-79 ).

 

4. In item 2.3, what is the storage temperature for colonies to perform mycelial growth measurements?

Re: Thank you for your suggestion, revisions have been made to the manuscript (line: 108).

 

5. All figures and tables should be self-explanatory, so readers should not need to look up the meaning of abbreviations in the manuscript, even if their meaning is obvious. Therefore, I suggest that the authors of the manuscript review the titles of all figures, tables, and graphs, and describe what each abbreviation in the description or present in the figures or tables means. Ex: WT??? wild-type (WT).

Re: Thank you for your suggestion. We have made changes to all the abbreviations. (lines: 228-230; 267; 459)

 

6. The discussions are weak in relation to the volume of data obtained in these experiments. I invite the authors to explore their results further, giving more substance to the discussion, which is basically only partially contained in the manuscript's introduction.

Re:  Thank you for your suggestion. We have further discussed the manuscript in detail (lines: 485-489; 485-498; 507-513).

 

7. The manuscript's conclusions are basically presented in the last paragraph of the paper (this study reveals that the GR-mediated redox balance regulates fruiting body development, enhancing our understanding of fungal stress physiology and metabolic regulation). I invite the authors to be concise in this item and cite the results instead of explaining them. Explaining the results is done in the discussions.

Re: Thank you for your suggestion. We have revised the conclusion section of the manuscript (lines: 531-536).

 

Reviewer 3 Report

Comments and Suggestions for Authors

General comment

The manuscript provides a clear, well-supported analysis of the role of glutathione reductase (GR) in Hypsizygus marmoreus biology, specifically concerning ROS homeostasis and fruiting body development. The extensive supporting data, including successful RNAi knockdown, enzyme activity assays, and the reproducible primer list (Table S1), confirm the high scientific quality and thoroughness of the experimental work.

Additional comments

The summary is informative but overly detailed with methodological descriptions such as assay procedures and vector construction. It should be condensed to highlight only the key findings, concluding with a sentence that underscores the practical implications for mushroom cultivation, for example: “These findings provide new insights for enhancing industrial mushroom production through antioxidant regulation

The introduction presents a thorough literature review, but it is lengthy and somewhat repetitive, especially regarding ROS metabolism and antioxidant enzyme systems. Redundancies about GSH, GPX, GR, and the ROS cycle should be streamlined to enhance clarity. Critically, the last paragraph lacks a clear statement of research gaps and hypotheses. It should explicitly define what is unknown about GR’s role in Hypsizygus marmoreus and how this study aims to fill that gap.

In the Materials and Methods (sections 2.1-2.9), clarity is needed on several methodological points. The number of biological and technical replicates for assays like ROS quantification, mitochondrial observations, qRT-PCR, and enzyme activity should be stated consistently in the respective subsections (e.g., lines ~105, ~121, ~138).

The qRT-PCR section should reference Supplementary Table S1 explicitly and specify that 18S rRNA served as the internal control for expression normalization.

Details regarding fluorophores, imaging parameters, and threshold criteria used in ROS imaging and mitochondrial morphology assays must be included to enable reproducibility.

In section 2.9 Statistical Analysis, indicate whether data are expressed as mean ± SD or SEM, and confirm that tests for normality and homogeneity of variance were conducted before applying Duncan’s multiple range test.

Minor technical corrections are needed in the Results section, particularly Figures 1  and 2. Fluorescence microscopy and TEM images lack scale bars and magnification details; these should be added to Figures 1D and 2A. Some figure captions are overly detailed and could be shortened by moving descriptive content to the text body.

The Discussion (~lines 463-505) effectively situates the results but repeats previously published findings too often rather than focusing on interpretation and broader implications. This section should be condensed to minimize repetition. Also, limitations of the study are absent; around lines ~500-510, a brief paragraph should acknowledge potential limitations, such as lack of GR-overexpressing strains and possible off-target effects of RNAi.

In the concluding part (~lines 510-520), add 1-2 sentences on the application of these findings in edible mushroom biotechnology or commercial cultivation, e.g., antioxidant supplements to improve yield.

Finally, several references are over 10 years old; consider adding recent literature (2022-2024) on ROS signaling in fungi and fruiting body regulation. Ensure all figures, supplementary materials (e.g., Supplementary Table S1), and file references are cited where they first appear in the main text.

Author Response

# Reviewer 3

Comments and Suggestions for Authors

General comment

The manuscript provides a clear, well-supported analysis of the role of glutathione reductase (GR) in Hypsizygus marmoreus biology, specifically concerning ROS homeostasis and fruiting body development. The extensive supporting data, including successful RNAi knockdown, enzyme activity assays, and the reproducible primer list (Table S1), confirm the high scientific quality and thoroughness of the experimental work.

Additional comments

1. The summary is informative but overly detailed with methodological descriptions such as assay procedures and vector construction. It should be condensed to highlight only the key findings, concluding with a sentence that underscores the practical implications for mushroom cultivation, for example: “These findings provide new insights for enhancing industrial mushroom production through antioxidant regulation.

Re: Thank you for this valuable comment. Accordingly, we have revised the abstract to focus on the key results and biological significance, while minimizing methodological descriptions. The revised version concludes with a sentence highlighting the potential application of our findings in industrial mushroom production (lines: 10-22).

 

2. The introduction presents a thorough literature review, but it is lengthy and somewhat repetitive, especially regarding ROS metabolism and antioxidant enzyme systems. Redundancies about GSH, GPX, GR, and the ROS cycle should be streamlined to enhance clarity. Critically, the last paragraph lacks a clear statement of research gaps and hypotheses. It should explicitly define what is unknown about GR’s role in Hypsizygus marmoreus and how this study aims to fill that gap.

Re: We appreciate the reviewer’s constructive suggestion. Accordingly, we have shortened the introduction by deleting redundant information about ROS, GSH, GPX, and GR, and reorganized the text for better logical flow. In addition, the final paragraph has been rewritten to clearly define the research gap and state the hypotheses regarding the role of GR in Hypsizygus marmoreus (lines:44-56).

 

3. In the Materials and Methods (sections 2.1-2.9), clarity is needed on several methodological points. The number of biological and technical replicates for assays like ROS quantification, mitochondrial observations, qRT-PCR, and enzyme activity should be stated consistently in the respective subsections (e.g., lines ~105, ~121, ~138).

Re: We thank the reviewer for the insightful comment. We have revised Sections 2.1–2.9 to specify the biological and technical replicates for all relevant assays, including ROS, mitochondria, qRT-PCR, and enzyme activity measurements, thereby improving clarity and consistency (lines: 136-138; 149-150; 160-163; 188-189).

 

4. The qRT-PCR section should reference Supplementary Table S1 explicitly and specify that 18S rRNA served as the internal control for expression normalization.

Re: We thank the reviewer for the helpful suggestion. The qRT-PCR section has been revised to explicitly reference Supplementary Table S1 and to indicate that 18S rRNA was used as the internal control for expression normalization. (lines: 186-187).

 

5. Details regarding fluorophores, imaging parameters, and threshold criteria used in ROS imaging and mitochondrial morphology assays must be included to enable reproducibility.

Re: We thank the reviewer for the suggestion. Details on fluorophores, imaging settings, and threshold criteria for ROS and mitochondrial assays have been added to the Section 2.3 and 2.4 to ensure reproducibility (line: 116-119; 125-132).

 

6. In section 2.9 Statistical Analysis, indicate whether data are expressed as mean ± SD or SEM, and confirm that tests for normality and homogeneity of variance were conducted before applying Duncan’s multiple range test.

Re: In accordance with the reviewer’s recommendation, we have revised the Statistical Analysis section to indicate that results are expressed as mean ± SD and that assumptions of normality and homogeneity of variance were verified before using Duncan’s multiple range test (lines: 192-196).

 

7. Minor technical corrections are needed in the Results section, particularly Figures 1 and 2. Fluorescence microscopy and TEM images lack scale bars and magnification details; these should be added to Figures 1D and 2A. Some figure captions are overly detailed and could be shortened by moving descriptive content to the text body.

Re: We thank the reviewer for the careful evaluation and constructive suggestions. In response, scale bars and magnification information have been added to Figures 1D and 2A to improve clarity and reproducibility. Additionally, figure captions have been revised to remove excessive detail, with descriptive content moved to the main text where appropriate, in order to enhance readability (lines: 224; 263).

 

8. The Discussion (~lines 463-505) effectively situates the results but repeats previously published findings too often rather than focusing on interpretation and broader implications. This section should be condensed to minimize repetition. Also, limitations of the study are absent; around lines ~500-510, a brief paragraph should acknowledge potential limitations, such as lack of GR-overexpressing strains and possible off-target effects of RNAi.

Re: We thank the reviewer for the constructive suggestion. In response, the Discussion has been revised to reduce repetition of previously published findings while retaining the description of our key results. A brief paragraph acknowledging the study limitations has been added, including the absence of GR-overexpressing strains and the potential off-target effects of RNAi. These changes improve clarity and emphasize the interpretation and broader implications of our findings (lines: 510-513).

 

9. In the concluding part (~lines 510-520), add 1-2 sentences on the application of these findings in edible mushroom biotechnology or commercial cultivation, e.g., antioxidant supplements to improve yield.

Re: We thank the reviewer for the helpful suggestion. We revised this sentence in the conclusion to restate its meaning and potential applications (lines: 536-539).

 

10. Finally, several references are over 10 years old; consider adding recent literature (2022-2024) on ROS signaling in fungi and fruiting body regulation. Ensure all figures, supplementary materials (e.g., Supplementary Table S1), and file references are cited where they first appear in the main text.

Re: We thank the reviewer for the suggestion. In response, we have updated the reference list to include recent literature (2022–2024) on ROS signaling in fungi and fruiting body regulation. In addition, all figures, supplementary materials (including Supplementary Table S1), and file references have been carefully checked and cited at their first mention in the main text to ensure consistency and completeness (lines: 476; 483).

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I believe the requests for improvement to this manuscript have been met. Therefore, I am satisfied.

Author Response

We extend our sincere thanks for your previous review comments. Your suggestions have been instrumental in enhancing the scholarly rigor of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have submitted a thoroughly revised version of the manuscript, and all previously identified weaknesses, concerns, and improvement requests have been fully addressed. The revised manuscript demonstrates substantial improvements in clarity, methodological rigor, and presentation quality. The authors’ responses and modifications clearly indicate a careful and thoughtful revision process.

Author Response

We are pleased that our revised manuscript has been acknowledged. We remain deeply grateful for the feedback you provided earlier; it was instrumental in elevating the quality and rigor of our paper.