Transcriptomic and Metabolomic Profiling of Pleurotus eryngii Cultivated on Olive Mill Solid Waste-Enriched Substrates

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigates how different concentrations of Olive Mill Solid Waste (OMSW) in the substrate affect the growth, gene expression, metabolite composition, and glucan biosynthesis in Pleurotus eryngii. It combines RNA-seq, metabolomics, and biochemical assays to explore nutritional value, metabolic changes, and the potential of OMSW as a recycling resource in mushroom cultivation. I have a few concerns which need attention.

1. The title could be more precise: such as  “Transcriptomic and Metabolomic Profiling of Pleurotus eryngii Cultivated on Olive Mill Waste-Enriched Substrates”

2. In section 3.2–3.4, Consider adding qRT-PCR validation for key DEGs.

3. In section 3.5, the decline in protein/nitrogen is important. Discuss possible physiological reasons (e.g., stress response, carbon/nitrogen balance).

4. Although glucan biosynthesis genes (e.g., β-1,3-glucan synthase) are mentioned, regulatory pathways or upstream transcription factors are not explored. Consider validating other DEGs involved in glucan metabolism or stress response (e.g., oxidative stress markers).

5. Focus on some typos and sentence structure problem in the whole manuscript.

Author Response

We thank the reviewer for the time and effort dedicated to evaluating our manuscript. We believe that the comments and suggestions provided have significantly enhanced the clarity and presentation of our study.

Comment 1. The title could be more precise: such as  “Transcriptomic and Metabolomic Profiling of Pleurotus eryngii Cultivated on Olive Mill Waste-Enriched Substrates”
Answer 1: Thank you. we agree. The Title has been rephrased as suggested. 

Comment 2: In section 3.2–3.4, Consider adding qRT-PCR validation for key DEGs.
Answer 2: Thank you for this valuable comment. Indeed we have performed qRT-PCR for beta-glucan synthase during this research now included in the supplementary section (S2).

Comment 3: In section 3.5, the decline in protein/nitrogen is important. Discuss possible physiological reasons (e.g., stress response, carbon/nitrogen balance).
Answer 3: Thank you for this comment.  We also see this notable reduction as one of the key observations of this study. The discussion regarding this result was further elaborated. 

Comment 4: Although glucan biosynthesis genes (e.g., β-1,3-glucan synthase) are mentioned, regulatory pathways or upstream transcription factors are not explored. Consider validating other DEGs involved in glucan metabolism or stress response (e.g., oxidative stress markers).
Answer 4: We have further expanded the discussion section to address the upregulation of peroxisome-related DEGs involved in oxidative stress response.

Comment 5: Focus on some typos and sentence structure problem in the whole manuscript.
Answer 5: Thank you for pointing this out. We have submitted the revised manuscript for additional English language editing.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The agronomy-3737314 submission represents a solid experimental study investigating the impact of olive mill solid waste (OMSW) concentrations on Pleurotus eryngii cultivation, combining phenotypic, transcriptomic, and metabolomic approaches.

This study integrates multiple analytical techniques (RNA-seq, LC-MS metabolomics, biochemical assays), thus linking phenotypic observations with molecular mechanisms through gene expression and metabolite profiling.

The multi-omics approach to OMSW-mushroom cultivation is relatively novel. However, the basic approach of using OMSW as a growth substrate component for mushroom cultivation is well established. In this respect, the work extends previous findings but does not represent a breakthrough. In principle, the results of the study provide actionable information for mushroom producers, identifying conditions for enhanced bioactive compound production. However, it should be appropriate for the authors to point out in the Discussion section that the results are not generalizable, due to the considerable variability in OMSW composition. Could the Authors show the chemical composition of the OMSW used in this work in the text of subsection 2.1 or in tabular form as a supplementary file?

The study highlights how the proportions of OMSW in the substrate formulation capable of increasing yield differ from those that provide the highest concentrations of potentially bioactive glucans. Regarding cell wall polymers, the authors hypothesize that the lower protein content in the carpophores, obtained with a substrate containing 80% OMSW, may be attributable to a relative increase in chitin levels. Could you make an explicit reference to the comparative transcriptomic analysis results (0 vs 80% OMSW) regarding the genes involved in chitin biosynthesis?

Figure 4 is poorly informative and should, therefore, be either eliminated or commented on more clearly. Similarly, the supplementary Figure S1 is mentioned but not adequately described.

Minor Corrections

X-axis captions of Figs1 and 2: Change "consentration" to "concentration"

Line 144: Change "fruting" to "fruiting"

Line 28: Missing space in citation format

Author Response

We thank the reviewer for the time and effort dedicated to evaluating our manuscript. We believe that the comments and suggestions provided have significantly enhanced the clarity and presentation of our study.

Comment 1: It should be appropriate for the authors to point out in the Discussion section that the results are not generalizable, due to the considerable variability in OMSW composition. Could the Authors show the chemical composition of the OMSW used in this work in the text of subsection 2.1 or in tabular form as a supplementary file?
Answer 1: Thank you for this valuable comment. We very much agree and now added a paragraph in the discussion section which addresses this issue. In addition - OMSW key parameters were added to the materials and method section. 

Comment 2: Could you make an explicit reference to the comparative transcriptomic analysis results (0 vs 80% OMSW) regarding the genes involved in chitin biosynthesis?
Answer 2: Thank you for this important suggestion. A specific reference is now included in the discussion section and a supplemental figure (S3) was added exhibiting DEGs in the amino sugar and nucleotide sugar metabolic pathway

Comment 3: Figure 4 is poorly informative and should, therefore, be either eliminated or commented on more clearly. Similarly, the supplementary Figure S1 is mentioned but not adequately described.
Answer 3: The texts referring to both figures was edited and elaborated. 

Comment 4: Comment  X-axis captions of Figs1 and 2: Change "consentration" to "concentration"
Answer 4: Corrected. 

Comment 5: Line 144: Change "fruting" to "fruiting"
Answer 5: Corrected 

Comment 6: Comment Line 28: Missing space in citation format
Answer 6: Corrected

Reviewer 3 Report

Comments and Suggestions for Authors

General 

This manuscript presents a thoughtful and well-designed study exploring how olive mill solid waste (OMSW) impacts the growth, gene expression, and metabolic composition of Pleurotus eryngii. The integration of physiological measurements, transcriptomics, and metabolomics provides a comprehensive view of how substrate composition shapes fungal development and bioactive compound production.
The work addresses an environmentally relevant topic with clear implications for sustainable agriculture and functional food development. The data are generally well presented, and the methods are sound. However, a few areas require clarification and additional detail to ensure the manuscript meets the standards for publication. These are outlined below.

 Major Comments
1. Omission of intermediate omics data: pp. 5–6 (Results Sections 3.1, 3.3); Figures 3 & 4
The authors focus their transcriptomic and metabolomic analyses on the two extremes of OMSW concentration (0% and 80%), despite the fact that intermediate treatments (33% and 60%) yielded biologically significant phenotypic responses (see Figures 1 and 2, p. 4). The lack of molecular data for these intermediate points limits the interpretability of the dose-dependent effects. It would strengthen the study if the authors could either:
- Provide justification for this omission in the Methods or Discussion, and/or
- Include summary omics data for these treatments as supplementary material, if available.

2. Limited interpretation of key metabolites: p. 6 (Section 3.3; Figure 4)
Several metabolites upregulated in the 80% OMSW condition—such as veronal, vitamin B6, and mesalamine—are listed without any functional context. These could be of significant biological interest, particularly if linked to antioxidant or immunomodulatory activity. The authors are encouraged to briefly discuss what is known (or hypothesized) about these compounds, particularly in the context of mushroom physiology or potential health benefits.

3. Unclear statistical correction methodology:  pp. 3 (Section 2.7); pp. 5–6 (Figure captions for Figures 3 & 4)
The statistical analysis section does not specify whether correction for multiple testing (e.g., False Discovery Rate) was applied in RNA-seq or metabolomic data analysis. While Figure 3 caption notes "FDR < 0.05", this is not supported or explained in the Methods. Please clarify whether FDR or another method was used, and explicitly state the statistical thresholds applied throughout the study.

Minor Comments
1. Inconsistent terminology for glucans:  Throughout (e.g., pp. 1, 4, 7)
The manuscript uses both "alpha-glucan"/"beta-glucan" and the Greek-letter versions "α-glucan"/"β-glucan" interchangeably. For consistency and professional tone, please standardize to one format—ideally, the Greek-letter notation throughout the text and figures.

2. Figure captions and axis labeling: pp. 5–6 (Figures 3 & 4)
Several figure captions would benefit from more complete descriptions. Specifically:
- In Figure 4, please include the percentage of variance explained by PC1 and PC2. 
- Indicate what the red and green dots represent (e.g., upregulated and downregulated metabolites, respectively). These additions would improve clarity for readers unfamiliar with omics-based visualizations.
3. Minor grammar and typographic edits: pp. 2–3, 4
- Line 120 (p. 4): “substrate’s dry weight” → should be revised to “substrate dry weight”
- Line 63 (p. 3): Consider changing to “gas-permeable, autoclavable bags” for smoother phrasing. Please proofread the entire manuscript for similar minor issues in punctuation and formatting.

4. Figure readability: p. 4 (Figure 1); p. 8 (Figure 6)
Figures 1 and 6 would benefit from improved graphical clarity. Labels and bars occasionally overlap, and the spacing or contrast could be adjusted to make values easier to interpret. Consider reformatting for better visual balance.

5. Protein-to-Nitrogen (P/N) ratio:  p. 9 (Lines 230–232)
The authors note a decreasing P/N ratio as OMSW increases, which is an interesting and valuable observation. Including this ratio in a table or supplementary figure would help readers better appreciate its implications for nutrient allocation and stress adaptation.
6. Typo in Figures 1 and 2:  p. 4
In both Figure 1 and Figure 2, there is a typographical error in the x-axis label:
“consentration” should be corrected to “concentration”.
7. The in-text citation style does not appear to conform to the journal’s formatting guidelines. A thorough review and correction of citation formatting is recommended to ensure consistency.

Comments on the Quality of English Language

The English language in the manuscript is generally understandable; however, there are several instances of grammatical errors, awkward phrasing, and inconsistent terminology.

Improving sentence structure, refining scientific tone, and correcting typographical mistakes (e.g., "consentration" instead of "concentration") would enhance clarity and professionalism.

Author Response

We thank the reviewer for the time and effort dedicated to evaluating our manuscript. We believe that the comments and suggestions provided have significantly enhanced the clarity and presentation of our study.

comment 1: Omission of intermediate omics data: pp. 5–6 (Results Sections 3.1, 3.3); Figures 3 & 4
The authors focus their transcriptomic and metabolomic analyses on the two extremes of OMSW concentration (0% and 80%), despite the fact that intermediate treatments (33% and 60%) yielded biologically significant phenotypic responses (see Figures 1 and 2, p. 4). The lack of molecular data for these intermediate points limits the interpretability of the dose-dependent
Answer 1: We agree with the reviewer’s comment. Additional omics analyses for the 33% and 60% treatments could indeed offer insights into the dose-dependent effects of OMSW; however, these were not yet conducted due to the limitations of the current study's scope. We have now clarified the rationale for selecting only the two extreme treatment points in the discussion section and have included a Real-time qPCR analysis demonstrating the expression dynamics of the β-glucan synthase gene in the 33% and 60% treatments.

 

Comment 2:  Several metabolites upregulated in the 80% OMSW condition—such as veronal, vitamin B6, and mesalamine—are listed without any functional context. These could be of significant biological interest, particularly if linked to antioxidant or immunomodulatory activity. The authors are encouraged to briefly discuss what is known (or hypothesized) about these compounds, particularly in the context of mushroom physiology or potential health benefits.
Answer 2: Thank you for the suggestion. We now indicate a possible role for these three metabolites and the medical use of mesalamine which seems to be the most established among the three.

Comment 3: Statistics clarification required 
Answer 3:  Thank you for noticing this. Statistical calculations used have been clarified in more detail in the methods section and in relevant figures. 

Comment 4:  Inconsistent terminology for glucans
Answer 4: Corrected  

Comment 5:  Edit Figure captions and axis labeling
Answer 5: The captions were edited to better describe the figures.

Comment 6: Line 120 (p. 4): “substrate’s dry weight” → should be revised to “substrate dry weight”
- Line 63 (p. 3): Consider changing to “gas-permeable, autoclavable bags” for smoother phrasing.
Answer 6: Corrected 

Comment 7: Adjust figure readability: p. 4 (Figure 1); p. 8 (Figure 6)
Answer 7: Thank you. It seems that the figure's format changes between versions- we will make sure that the final version will have a good visual balance. 

Comment 8: Protein-to-Nitrogen (P/N) ratio:  p. 9 (Lines 230–232)
The authors note a decreasing P/N ratio as OMSW increases, which is an interesting and valuable observation. Including this ratio in a table or supplementary figure would help readers better appreciate its implications for nutrient allocation and stress adaptation.
Answer 8: Thank you for this comment. we have now highlighted this observation in the main text- "Discussion" section. 

Comment 9: Typo in Figures 1 and 2:  p. 4
Answer 9: Corrected  

Comment 10: The in-text citation style does not appear to conform to the journal’s formatting guidelines. A thorough review and correction of citation formatting is recommended to ensure consistency.
Answer 10: Thank you, we will make sure citation formatting is according to the journal's guidelines 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have substantially improved the manuscript based on my previous inputs, however, i feel that the discussion of the revised version still needs to be enriched with pre-existing supporting literature. 

1. For instance, lines 273-275, the authors should add supporting evidence (such as https://doi.org/10.3390/ijms252211903; https://doi.org/10.1016/j.stress.2024.100384) in which they showed integrated transcriptome and metabolome for phenolics regulation in important medicinal plants.
2. The references in-text are totally not fine, please revise the style according to the journals format. 
3. The qRt-PCR figures should be added in the main file not in the supplementary data.

Author Response

We appreciate the reviewer’s time and effort devoted to the further refinement of our manuscript.

Comment 1. The authors have substantially improved the manuscript based on my previous inputs, however, i feel that the discussion of the revised version still needs to be enriched with pre-existing supporting literature.
Response 1. Thank you for this comment. As suggested we have now added supporting literature to our discussion section (lines 251-253). 

Comment 2. The references in-text are totally not fine, please revise the style according to the journals format. 
Response 2. Corrected. 

Comment 3. The qRt-PCR figures should be added in the main file not in the supplementary data.
Response 3. Thank you for commenting this, we agree. The qRT-PCR has been moved to the main text . 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately addressed the comments raised by this reviewer. 

Author Response

We would like to thank the reviewer once again for the time and effort invested in enhancing the quality of the manuscript.