Transcriptomic Analysis of Broussonetia papyrifera Fruit Under Manganese Stress and Mining of Flavonoid Synthesis Genes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a valuable and novel study on the transcriptomic analysis of Broussonetia papyrifera under manganese (Mn) stress, highlighting key genes involved in flavonoid biosynthesis. It is one of the few studies addressing the molecular mechanisms of B. papyrifera's stress response, providing insights into its potential for phytoremediation and functional food development.

Minor revisions are suggested to improve the manuscript, including restructuring the abstract, enhancing figure captions, and refining the language for clarity and consistency.

Title:

1. Title: Change "Transcriptome" to "Transcriptomic" for correctness and to align with common scientific terminology.

Abstract:

2. Structure: The abstract requires restructuring. Suggested sequence:
• Begin with 1-2 lines on the significance of Broussonetia papyrifera.
• Discuss the impact of manganese (Mn) on Broussonetia papyrifera.
• Mention flavonoids and their relevance.
• Conclude with your experiment’s importance and key findings.
3. Revise sentences for clarity and conciseness:
• "Flavonoids are secondary metabolites that enhance plant resilience..." → "Flavonoids are vital secondary metabolites that improve plant resilience to environmental stresses."
• "This study provides a basis for future functional gene mining in B. papyrifera." → "This study lays the groundwork for functional gene research in B. papyrifera."
4. Page 1, Line 30: The phrase "provides a basis for future functional gene mining" could be clarified. Suggest rephrasing to "lays a foundation for future studies on functional gene mining."

Keywords:

4. Change "Environmental stress" to "Heavy Metal Stress" for specificity and relevance.

Introduction:

5. Line 35: Replace "heavy metal pollution" with "heavy metal toxicity" for accuracy. Apply this correction throughout the manuscript.
6. Page 2, Line 38: "Plants develop a series of defense strategies, among which the synthesis of secondary metabolites is an important way to regulate environmental stress." Revise to "Plants develop various defense strategies, with the synthesis of secondary metabolites being a key mechanism to regulate environmental stress."
7. Page 2, Line 41: "Under salt stress and drought stress, environmental stress in cotton is closely related to elevated..." Simplify to "Under salt and drought stress, cotton exhibits elevated..."
8. Page 2, Line 47: "PSM has been associated with abiotic stress, plant metabolites, biostimulants, and functional food." Revise to "PSMs are associated with abiotic stress tolerance, plant metabolite production, biostimulants, and functional foods."
9. Paragraph restructuring:
• Lines 63-78: Move to the first paragraph to introduce Broussonetia papyrifera and its importance.
• Lines 35-48: Move to the second paragraph to discuss heavy metal toxicity and its global impact.
• Lines 49-62: Place as the third paragraph to introduce flavonoids and their role in stress tolerance.
• Lines 78-92: Use as the fourth and final paragraph to transition into the experiment's focus and objectives.

Figures:

7. Captions: Expand all figure captions (Figures 1–5) to provide sufficient details for interpretation.
8. Figure 4: Improve figure quality to ensure text and data are clearly visible.

Methods:

9. Line 270: Change heading to "Plant Materials and Soil Analysis" for better alignment with the content.
10. Page 10, Line 287: "The samples were allowed to air-dry naturally, ground, and sieved..." Revise to "Samples were air-dried, ground, and sieved using a 100-mesh sieve."
11. Page 11, Line 303: The phrase "To ensure precise sequence assembly..." could be streamlined to "Sequence assembly precision was ensured by trimming adapters, filtering low-quality reads, and calculating Q20 and Q30 values."

Results and Discussion:

11. Page 3, Line 95: "The correlation between the total flavonoid content of B. papyrifera fruits..." Consider rephrasing to "The relationship between total flavonoid content in B. papyrifera fruits and soil parameters..."
12. Page 4, Line 112: The phrase "The sequencing results demonstrated..." could be more concise. Suggest "Sequencing revealed that green and red fruit samples of B. papyrifera were of high quality and suitable for analysis."
13. Page 5, Line 129: "Two-by-two comparisons of DEGs were performed." Clarify to "Pairwise comparisons of DEGs were conducted."
14. Page 9, Line 236: "These factors may be major contributors to the low flavonoid content..." Revise to "These factors likely contribute significantly to the low flavonoid content..."
15. Page 9, Line 253: "Our findings revealed that several key genes involved in the flavonoid synthesis pathway exhibited a notable downregulation." Simplify to "Key genes in the flavonoid synthesis pathway showed significant downregulation."
16. Page 9, Line 261: "FAOMT has the capacity to methylate flavonoid glycosides..." Clarify by adding a brief example of this process, e.g., "FAOMT methylates flavonoid glycosides at the 3′-OH and 5′-OH positions, promoting flavonoid production."
17. Line 350: Change "transcriptomes" to "transcriptomic analysis of" to reflect the content more accurately.
18. Rephrase long sentences for clarity:
• "These factors may be major contributors to the low flavonoid content observed in B. papyrifera fruits in Mn mining areas." → "These factors likely contribute to the reduced flavonoid content observed in B. papyrifera fruits from Mn mining areas."

Author Response

Comments and Suggestions for Authors:

This manuscript presents a valuable and novel study on the transcriptomic analysis of Broussonetia papyrifera under manganese (Mn) stress, highlighting key genes involved in flavonoid biosynthesis. It is one of the few studies addressing the molecular mechanisms of B. papyrifera's stress response, providing insights into its potential for phytoremediation and functional food development.

Minor revisions are suggested to improve the manuscript, including restructuring the abstract, enhancing figure captions, and refining the language for clarity and consistency.

 

Response:

Dear Reviewer, thank you sincerely for your thoughtful evaluation of our manuscript and your constructive feedback. We deeply appreciate your recognition of the novelty and value of this study. Your suggestions for refining terminology, sentence clarity, and structural organization have been invaluable in enhancing the rigor and readability of the manuscript.

We have carefully revised the manuscript in response to your comments to ensure accurate use of terminology, improve sentence flow, and make the presentation of results and discussion more logical. Below is a point-by-point response to your comments:

 

Title:

Comment 1:

Title: Change "Transcriptome" to "Transcriptomic" for correctness and to align with common scientific terminology.

Response 1:

We sincerely thank the reviewer for this valuable suggestion. In response, we have revised the title to replace "Transcriptome" with "Transcriptomic" to better reflect the nature of the analysis and align with standard scientific terminology. We also checked the entire manuscript for consistency.

 

Abstract:

Comment 2:

Structure: The abstract requires restructuring. Suggested sequence:

Begin with 1-2 lines on the significance of Broussonetia papyrifera.

Discuss the impact of manganese (Mn) on Broussonetia papyrifera.

Mention flavonoids and their relevance.

Conclude with your experiment’s importance and key findings.

Response 2:

We deeply appreciate the reviewer’s constructive feedback on improving the abstract’s clarity and flow. Following this suggestion, we have restructured the abstract to align with the recommended sequence. The revised version now begins by emphasizing the ecological and economic significance of Broussonetia papyrifera as a pioneer species in manganese (Mn)-contaminated soils, followed by its susceptibility to Mn toxicity in fruits. We then highlight flavonoids as critical metabolites for Mn stress resilience and describe our transcriptomic approach to identify key biosynthetic genes. The conclusion underscores the study’s novel insights into flavonoid regulation under Mn stress.

 

The revised abstract:

Broussonetia papyrifera is a deciduous tree with significant economic and medicinal value. It demonstrates notable physiological adaptability to mining areas with severe man-ganese contamination and is a pioneering species in the field of ecological restoration. Flavonoids are vital secondary metabolites that improve plant resilience to environ-mental stresses. Here, immature and mature fruits of B. papyrifera grown under normal and high manganese environments were used as the test materials. B. papyrifera fruit was subjected to transcriptome sequencing via high-throughput sequencing technology to analyze its flavonoid metabolic pathways and related genes. Transcriptome se-quencing identified a total of 46,072 unigenes, with an average length of 1,248 bp and a percentage of Q30 bases ranging from 92.45 to 93.17%. Furthermore, 31,792 unigenes (69% of the total) were annotated using eight databases, including the GO and KEGG. Anal-ysis of KEGG metabolic pathways and flavonoid content trends in B. papyrifera fruits revealed four unigenes with strong links to the flavonoid biosynthesis pathway under manganese stress: flavone 3-hydroxylase, flavonoids 3’,5’-O-methyltransferase and chalcone synthase, and flavonol synthase. These unigenes may play important roles in regulating flavonoid synthesis in B. papyrifera fruits under manganese stress. This study lays the groundwork for functional gene research in B. papyrifera (Line 15).

 

Comment 3:

Revise sentences for clarity and conciseness:

"Flavonoids are secondary metabolites that enhance plant resilience..." → "Flavonoids are vital secondary metabolites that improve plant resilience to environmental stresses."

"This study provides a basis for future functional gene mining in B. papyrifera." → "This study lays the groundwork for functional gene research in B. papyrifera."

Response 3:

Thank you. As you suggested, we have revised both sentences to enhance clarity and conciseness. In the Abstract the sentence now reads: "Flavonoids are vital secondary metabolites that improve plant resilience to environmental stresses" (Line 18).

Last sentence of the Abstract section, has been updated to: "This study lays the groundwork for functional gene research in B. papyrifera" (Line 30). These adjustments align with the reviewer’s recommendations and improve the manuscript’s readability. Thank you for your constructive feedback.

 

Comment 4:

Page 1, Line 30: The phrase "provides a basis for future functional gene mining" could be clarified. Suggest rephrasing to "lays a foundation for future studies on functional gene mining."

Response 4:

We thank the reviewer for this thoughtful suggestion to enhance the clarity of our concluding statement. As recommended, we have revised the sentence in the Abstract to read: "This study lays the groundwork for functional gene research in B. papyrifera" (Line 30).

 

Keywords:

Comment 5:

Change "Environmental stress" to "Heavy Metal Stress" for specificity and relevance.

Response 5:

We sincerely appreciate the reviewer’s valuable suggestion to enhance the precision of our terminology. As recommended, we have replaced "Environmental stress" with "Heavy Metal Stress" in the revised manuscript (Line 34).

 

Introduction:

Comment 6:

Line 35: Replace "heavy metal pollution" with "heavy metal toxicity" for accuracy. Apply this correction throughout the manuscript.

Response 6: We thank the reviewer for this critical observation, which enhances the precision of our terminology. The term "heavy metal pollution" has been revised to "heavy metal toxicity" throughout the manuscript (Line 53).

 

Comment 7:

Page 2, Line 38: "Plants develop a series of defense strategies, among which the synthesis of secondary metabolites is an important way to regulate environmental stress." Revise to "Plants develop various defense strategies, with the synthesis of secondary metabolites being a key mechanism to regulate environmental stress."

Response 7:

We sincerely thank the reviewer for this constructive suggestion to enhance the clarity and scientific tone of the sentence. As recommended, we have revised the sentence in the Introduction to read: "plants develop various defense strategies, with the synthesis of secondary metabolites being a key mechanism to regulate environmental stress" (Line 61).

 

Comment 8:

Page 2, Line 41: "Under salt stress and drought stress, environmental stress in cotton is closely related to elevated..." Simplify to "Under salt and drought stress, cotton exhibits elevated..."

Response 8:

We are grateful to the reviewer for highlighting the need for conciseness in this sentence. As suggested, we have revised the sentence in the Introduction to: "Under salt and drought stress, cotton exhibits elevated callose, chitinase, flavonoid, and phenol contents, along with higher secondary metabolism-related enzyme activities and transcript levels" (Line 64).

 

Comment 9:

Page 2, Line 47: "PSM has been associated with abiotic stress, plant metabolites, biostimulants, and functional food." Revise to "PSMs are associated with abiotic stress tolerance, plant metabolite production, biostimulants, and functional foods."

Response 9:

As recommended, we have revised the sentence in the Introduction (Line 70) to: "PSMs are associated with abiotic stress tolerance, plant metabolite production, biostimulants, and functional foods." Thank you for this valuable feedback.

 

Comment 10:

Paragraph restructuring:

Lines 63-78: Move to the first paragraph to introduce Broussonetia papyrifera and its importance.

Lines 35-48: Move to the second paragraph to discuss heavy metal toxicity and its global impact.

Lines 49-62: Place as the third paragraph to introduce flavonoids and their role in stress tolerance.

Lines 78-92: Use as the fourth and final paragraph to transition into the experiment's focus and objectives.

Response 10:

We sincerely thank the reviewer for this critical suggestion to enhance the logical flow and readability of the Introduction. In response, we have restructured the section as follows (Due to the large number of changes in the paragraphs, they are not highlighted in this revision):

(1) First paragraph: Begins with the ecological, economic, and medicinal significance of Broussonetia papyrifera, emphasizing its role as a pioneer species in heavy metal-contaminated soils.

Line 37: Paper mulberry (Broussonetia papyrifera) is an ecologically, economically, and me-dicinally important tree species belonging to the family Moraceae...

 

(2) Second paragraph: Now discusses the global challenge of heavy metal toxicity, including Mn accumulation risks and the need for sustainable phytoremediation strategies.

Line 53: Soil environmental pollution, particularly heavy metal toxicity, poses a major challenge to global ecosystem health. Heavy metals in soil are toxic to organisms and threaten human health through the food chain and other pathways...

 

(3) Third paragraph: Introduces flavonoids as key stress-tolerance metabolites, detailing their antioxidant functions and prior research gaps in B. papyrifera fruits.

Line 72: Flavonoids are phenolic compounds that are important for plants to resist adverse environments and are functional substances...

 

(4) Fourth paragraph: Concludes with the study’s objectives, focusing on transcriptomic analysis of flavonoid synthesis under Mn stress.

Line 86: Currently, research on flavonoids in B. papyrifera has mainly focused on the leaves [25] and root bark [26], paying less attention to the fruits. Integrative metabolome and transcriptomic analyses of B. papyrifera leaves have identified several key genes regu-lating flavonoid accumulation...

 

This reorganization ensures a clearer progression from the plant’s significance → problem (heavy metals) → solution (flavonoids) → study purpose. We have carefully adjusted transitional phrases and citations to maintain coherence. These changes significantly improve the narrative structure, aligning with the reviewer’s insightful guidance. Thank you for this invaluable contribution to strengthening our manuscript.

 

Figures:

Comment 11:

Captions: Expand all figure captions (Figures 1–5) to provide sufficient details for interpretation.

Response 11:

We thank the reviewer for highlighting the need for more detailed figure captions. In the revised manuscript, we have expanded all figure captions to include essential methodological and interpretive information, ensuring they are self-explanatory. Below are examples of the revised captions:

(1) Revised Figure 1 Caption:

Figure 1. Pearson correlation analysis between soil elements (Mn, TOC, TN, and TP) and total flavonoid content in B. pa-pyrifera fruits. The X-axis denotes various soil elements, while the Y-axis indicates the correlation coefficient.

 

(2) Revised Figure 2 Caption:

Figure 2. Pairwise comparison of DEGs between sample groups. (a) CIF vs. GIF, and CMF vs. GMF. (b) CIF vs. CMF, and GIF vs. GMF. Venn diagrams display overlaps of DEGs between comparisons.

 

(3) Revised Figure 3 Caption:

Figure 3. Validation of transcriptome data by RT-qPCR for five randomly selected genes. (a) Immature fruit; and (b) mature fruit. “Relative expression log2” on the Y-axis represents log2-transformed fold changes normalized to the reference gene Actin and calibrated against the respective control group. Error bars indicate the standard deviation (n = 3 biological replicates).

 

(4) Revised Figure 4 Caption:

Figure 3. Validation of transcriptome data by RT-qPCR for five randomly selected genes. (a) Immature fruit; and (b) mature fruit. “Relative expression log2” on the Y-axis represents log2-transformed fold changes normalized to the reference gene Actin and calibrated against the respective control group. Error bars indicate the standard deviation (n = 3 biological replicates).

 

(5) Revised Figure 5 Caption:

Figure 5. KEGG pathway enrichment analysis of DEGs. (a) CIF vs. GIF. (b) CMF vs. GMF. (c) CIF vs. CMF. (d) GIF vs. GMF. The top 20 enriched pathways are ranked by p-value. Circle size indicates the number of DEGs in each pathway; color intensity represents the enrichment significance.

 

Comment 12:

Figure 4: Improve figure quality to ensure text and data are clearly visible.

Response 12:

Thanks. Figure 4 has been re-exported in high resolution to improve text and data visibility.

 

Methods:

Comment 13:

Line 270: Change heading to "Plant Materials and Soil Analysis" for better alignment with the content.

Response 13:

We sincerely thank the reviewer for this thoughtful suggestion to improve the clarity of the section heading. As recommended, we have revised the heading to "4.1. Plant Materials and Soil Analysis" (Line 320), which more precisely reflects the content describing the collection of B. papyrifera fruits and the methodologies employed for soil parameter assessments.

 

Comment 14:

Page 10, Line 287: "The samples were allowed to air-dry naturally, ground, and sieved..." Revise to "Samples were air-dried, ground, and sieved using a 100-mesh sieve."

Response 14:

As recommended, we have revised the sentence in the Materials and Methods section to: "Samples were air-dried, ground, and sieved using a 100-mesh sieve" (Line 337). This revision enhances methodological precision. We appreciate the reviewer’s attention to detail.

 

Comment 15:

Page 11, Line 303: The phrase "To ensure precise sequence assembly..." could be streamlined to "Sequence assembly precision was ensured by trimming adapters, filtering low-quality reads, and calculating Q20 and Q30 values."

Response 15:

Thank you so much for your advice. Another reviewer asked us to provide more details about the methods used in the "Data assembly and gene function annotation" (Line 354). We've gone ahead and updated this paragraph, and we're happy to report that the issue has been resolved.

 

Results and Discussion:

Comment 16:

Page 3, Line 95: "The correlation between the total flavonoid content of B. papyrifera fruits..." Consider rephrasing to "The relationship between total flavonoid content in B. papyrifera fruits and soil parameters..."

Response 16:

We sincerely thank the reviewer for this thoughtful suggestion to improve the clarity and scientific precision of the sentence. As recommended, we have revised the phrase in the Results section to: "The relationship between total flavonoid content in B. papyrifera fruits and soil parameters..." (Line 107).

 

Comment 17:

Page 4, Line 112: The phrase "The sequencing results demonstrated..." could be more concise. Suggest "Sequencing revealed that green and red fruit samples of B. papyrifera were of high quality and suitable for analysis."

Response 17:

Thank you. As you suggested, we have revised the text in the Results section to: "Sequencing revealed that immature and mature fruit samples of B. papyrifera were of high quality and suitable for analysis" (Line 129).

We'd like to let you know that, at the request of another reviewer, we've changed the name of the sample:

Green fruit → Immature fruit

Red fruit → Mature fruit

 

Comment 18:

Page 5, Line 129: "Two-by-two comparisons of DEGs were performed." Clarify to "Pairwise comparisons of DEGs were conducted."

Response 18:

We sincerely thank the reviewer for highlighting the need for clarity in this methodological description. As suggested, we have revised the sentence in the Results section (Line 156) to: "Pairwise comparisons of DEGs were conducted for immature and mature fruits from Mn mining and control areas." The adjustment aligns with conventional terminology and ensures readers clearly understand the comparative approach. We appreciate this constructive feedback.

 

Comment 19:

Page 9, Line 236: "These factors may be major contributors to the low flavonoid content..." Revise to "These factors likely contribute significantly to the low flavonoid content..."

Response 19:

We really appreciate your comments. We've taken another reviewer's suggestion and reorganized the Discussion section (Line 244). This issue has been resolved.

 

Comment 20:

Page 9, Line 253: "Our findings revealed that several key genes involved in the flavonoid synthesis pathway exhibited a notable downregulation." Simplify to "Key genes in the flavonoid synthesis pathway showed significant downregulation."

Response 20:

We sincerely thank the reviewer for this suggestion to enhance the clarity and conciseness of the statement.

Based on the comments of another reviewer, we have reconstructed this paragraph, and the sentence has now been revised to read “These physiological perturbations are directly correlated with the downregulation of core flavonoid biosynthetic genes (F3H, CHS, FAOMT, and FLS)” (Line 253).

 

Comment 21:

Page 9, Line 261: "FAOMT has the capacity to methylate flavonoid glycosides..." Clarify by adding a brief example of this process, e.g., "FAOMT methylates flavonoid glycosides at the 3′-OH and 5′-OH positions, promoting flavonoid production."

Response 21:

We sincerely thank the reviewer for highlighting the need to clarify FAOMT’s enzymatic role. As suggested, we have revised the sentence in the Discussion (Line 278) to: "Furthermore, Mn-induced dysregulation of FAOMT, which methylates flavonoid gly-cosides at the 3′-OH and 5′-OH positions to promoting generation [42], could acceler-ate flavonoid degradation in fruits from mining areas, thereby compounding the loss of bioactive metabolites." We appreciate this constructive suggestion to enhance the clarity and depth of our discussion.

 

Comment 22:

Line 350: Change "transcriptomes" to "transcriptomic analysis of" to reflect the content more accurately.

Response 22:

We sincerely thank the reviewer for this valuable suggestion to enhance the precision of our terminology. As recommended, we have replacing "transcriptomes" with "transcriptomic analysis of" (Line 421) clarifies that the study focuses on the analytical process rather than merely describing the transcriptome set.

 

Comment 23:

Rephrase long sentences for clarity:

"These factors may be major contributors to the low flavonoid content observed in B. papyrifera fruits in Mn mining areas." → "These factors likely contribute to the reduced flavonoid content observed in B. papyrifera fruits from Mn mining areas."

Response 23:

We sincerely appreciate the reviewer’s suggestion to enhance the clarity and conciseness of this statement. As recommended, we have revised the sentence in the Discussion (Line 259) to: "These factors likely contribute to the reduced flavonoid content observed in B. papyrifera fruits from Mn mining areas." Thank you for this valuable refinement.

 

 

Once again, we deeply appreciate the time and effort you dedicated to reviewing our manuscript. Your insightful comments and constructive suggestions have significantly strengthened the quality of this work. The revisions guided by your expertise have enhanced the clarity, scientific rigor, and logical flow of the paper, particularly in refining the discussion of streamlining methodological descriptions, and improving figure interpretability. Your attention to detail and thoughtful critiques have not only addressed critical gaps but also provided valuable directions for future research. Thank you for your invaluable contribution to advancing this study.

 

Best regards,

Zhenggang Xu

On behalf of all authors

Reviewer 2 Report

Comments and Suggestions for Authors

The study describes transcriptome analyses of fruit of the paper mulberry tree, Broussonetia papyrifera. Four different samples are analyzed and compared, mature and immature fruit, from plants grown either in soil near a mining center and contaminated with heavy metals, versus plants grown in a garden and clean soil.

As usual with genomic studies such as this, it is generally assumed that the researchers did things correctly based on the descriptions in the Methods section. Similarly with bioinformatics analysis, short of repeating the entire analysis from raw sequence reads the accuracy of the results can’t really be critically assessed. There are no particular concerns of that sort in this manuscript, however there are some important technical details that are not adequately described, but can easily be amended. First of all, it is simply stated that the biological samples (fruit) were stored at -80oC prior to RNA preparation. In looking at photos of the fruit online, it seems that the fruit is of some size, not large but not tiny either. RNA tends to quickly degrade once an organism or the cells therein are dead. The authors should clarify exactly how they froze the fruit, presumably in liquid nitrogen but it should be documented. Also, in describing the results of the sequencing, there is no obvious discussion of individual read lengths, just of the lengths of the resulting gene annotated models. The authors should comment as to whether there are many full-length individual reads or whether most reads are shorter than full-length, indicating significant RNA degradation during storage and preparation.

In the Results section, it would help if the section started with some general presentation of the transcriptomes, instead of jumping right into a comparison of expression levels without any comments about the overall data set. 1) Please explain at the beginning of the Results, what the abbreviations MK1, MK2, CK1 and Ck2 mean, and how these abbreviations relate to the terms “red fruit” and “green fruit” used in the text. Do “green” and “red” refer to mature and immature, and if so which is which? To a reader unfamiliar with this species (such as this reviewer), it is not obvious which color mature and immature fruit may be. The abbreviations are defined in the Methods section, but not the meaning of “red” and “green”, and anyway these should be in the Results section also.

In general the Results are a bit confusing to follow. There are two kinds of differential analysis here (at least), immature versus mature fruit, and fruit grown in the mining area hence contaminated soil versus fruit grown in the garden hence presumably uncontaminated soil. It is difficult to follow the presentation of the various analyses from the abbreviations, as to which comparison is being done in each section. It would help a lot if instead of the abbreviations, the various expression comparisons were explained in terms of mature/immature and contaminated/garden. The manuscript emphasizes the identification of genes differentially expressed as a function of the soil, but what about as a function of fruit maturity, that should also be quite interesting; if it is there is is hard to find amid the abbrevations.

2) There does not appear to be any analysis of the transcript reads with respect to the published genome of the species (Peng et al, Molecular Plant 12: 661 (2019). That paper describes a chromosome level assembly of the genome, together with transcriptomics of leaf, stem, root and flower. The present study should do some kind of comparison, for example do all their reads align to the published genome assembly, and if not why not? Could some of the reads come from microbiome genomes, which might be interesting? Were there genes that seemed to be specifically expressed in the fruit, but not in the other tissues analyzed in the Peng paper?

3) In the Peng paper, there is the following statement: “A new hybrid of Paper mulberry with a high crude protein content has been cultivated by our team and planted in almost all provinces of China as part of a national project to resolve the silage deficiency (Shen and Peng, 2017).” Could the authors comment about how the variety that was studied here is or is not related to the hybrid mentioned in the Peng paper?

In the Methods section it is not clearly indicated whether the raw RNA-Seq reads were actually assembled into gene contigs and gene models, or just compared individually to gene annotation databases such as KEGG and others, and the reads counted quantitatively. Were genes actually assembled, and if not, they should be, and the state of completion of the gene models should be assessed by looking manually at a couple of well-known standard genes. Also, in the Peng paper, those authors did a BUSCO analysis and showed that most BUSCO annotated genes are present in their genome assembly. In this paper, the authors annotate their raw reads with respect to several databases, such as KEGG and several others. However, some fraction of the genes they annotate were not found in those databases, as much as 30% which seems odd. Is there any explanation for the un-identified genes? BUSCO genes are supposedly basic housekeeping genes, so they should be mostly present in the transcriptomes of any tissue that is used for sequencing, the authors should do a BUSCO analysis on their genes.

The key software used for the quantitative analysis of the raw reads are the tools RSEM and EBSeq. This reviewer is not familiar with either of these. However, the only citation provided for RSEM is another paper using the tool (Deng et al, Amer J Botany (2024) 111). The Deng paper is very recent, thus this does not appear to be a standard tool used for quantitative analysis. Some more detail is required regarding RSEM therefore, for example the Deng paper has a paragraph describing it (qualitiatively) with a link to the software; a similar paragraph and the link should be provided in the Methods section here. It is difficult to know how reliable RSEM is therefore. It would be a lot of extra work to repeat the whole quantitative analysis using different software, however the reliability of RSEM should at least be commented on here. Concerning EBSeq, there are several papers in PubMed that mention it, however the reference cited in this manuscript is incomplete, it only has author names and the year 2023. The complete reference needs to be provided, but in any case it should not be just some paper that uses EBSeq, but the source citation, which is possibly Leng et al, Bioinformatics 29(8): 1035-1043 (2013).

For the RT-PCR analysis, it is not stated which actin gene was used as a normalization standard. In animal genomes at least, there are many actin genes, some of which are highly regulated and thus not appropriate for use as control genes. More detail should be provided on this experiment in the Methods, and also in the legend to Figure 3, for example it is not clear what is meant by the y-axis label “Relative expression log2”. Presumably for each gene relative to the actin gene? The signal for RNA-Seq is read counts, but for RT-PCR it is just a ratio, so it’s not clear how to interpret the bars in the figure. In the Methods section it should be explained in more detail how the different raw signals were converted to “Relative expression log2”.

The main body of the Results addresses what gene families are differentially expressed in fruits of trees grown in an area with heavy metal contamination. The results, while not unexpected, are certainly interesting.

One point of interest, not directly related to the point of the study, but worth some comment in the introduction or discussion. Given that the species is useful both as a food crop and for phytoremediation, that raises the question whether they might be a problem of the plant accumulating toxic levels of heavy metals in areas where it is also being used as a food source for livestock. How is this potential conflict addressed in practice in the field (if it is).

Overall the study is interesting and potentially useful for those working with this plant. The manuscript needs some additional detail added, and a few more bioinformatic analyses which should be fairly easy to do, to be publishable.

Author Response

Comments and Suggestions for Authors

The study describes transcriptome analyses of fruit of the paper mulberry tree, B. papyrifera. Four different samples are analyzed and compared, mature and immature fruit, from plants grown either in soil near a mining center and contaminated with heavy metals, versus plants grown in a garden and clean soil.

Response:

We would like to express our deepest gratitude for your exceptionally thorough and insightful evaluation of our manuscript. The detailed comments and thoughtful questions reflect not only your expertise in the field, but also your commitment to promoting scientific rigor. the attention to technical nuances, clarity of presentation, and suggestions for additional analyses have greatly enhanced the quality and impact of this work. We sincerely appreciate the time and effort invested in reviewing our study and are honored to have benefited from such a constructive review process.We have carefully revised the manuscript in accordance with your valuable suggestions, and below is a point-by-point response to your comments:

 

Comment 1:

As usual with genomic studies such as this, it is generally assumed that the researchers did things correctly based on the descriptions in the Methods section. Similarly with bioinformatics analysis, short of repeating the entire analysis from raw sequence reads the accuracy of the results can’t really be critically assessed. There are no particular concerns of that sort in this manuscript, however there are some important technical details that are not adequately described, but can easily be amended. First of all, it is simply stated that the biological samples (fruit) were stored at -80℃ prior to RNA preparation. In looking at photos of the fruit online, it seems that the fruit is of some size, not large but not tiny either. RNA tends to quickly degrade once an organism or the cells therein are dead. The authors should clarify exactly how they froze the fruit, presumably in liquid nitrogen but it should be documented. Also, in describing the results of the sequencing, there is no obvious discussion of individual read lengths, just of the lengths of the resulting gene annotated models. The authors should comment as to whether there are many full-length individual reads or whether most reads are shorter than full-length, indicating significant RNA degradation during storage and preparation.

Response 1:

We sincerely appreciate your valuable comments! Below are our detailed responses:

(1) Sample freezing protocol:

In this study, fresh B. papyrifera fruits were immediately frozen in liquid nitrogen after collection to prevent RNA degradation. Specifically, fruits were cut into small pieces (approximately 0.5 cm³) and submerged in liquid nitrogen within 5 minutes post-harvest. The frozen samples were then stored at -80°C until RNA extraction. This protocol ensured RNA integrity, as validated by the high-quality sequencing metrics (Q30 > 92%).

We have added sample preservation details to the manuscript (Line 330).

 

(2) RNA integrity and read length analysis:

To address concerns about RNA degradation, we analyzed the raw sequencing read lengths. The average raw read length was 150 bp (paired-end), with >85% of reads exceeding 100 bp. The N50 of assembled unigenes was 1,248 bp, indicating minimal RNA fragmentation. These results confirm that RNA degradation during storage and preparation was negligible.

We added this conclusion to the manuscript (Line 132), along with a figure entitled "Length distribution of unigenes in the B. papyrifera fruit transcriptome (Figure S1)".

 

Comment 2:

In the Results section, it would help if the section started with some general presentation of the transcriptomes, instead of jumping right into a comparison of expression levels without any comments about the overall data set.

Response 2:

We sincerely appreciate the reviewer’s valuable suggestion to improve the clarity of the Results section. As requested, we have incorporated this paragraph preceding the comparison of expression levels to enhance its logical clarity:

The high annotation rate and sequencing quality indicated robust data integrity, providing a solid foundation for downstream comparative analysis. Leveraging this high-quality transcriptome dataset, we subsequently analyzed differentially expressed genes (DEGs) between the Mn-exposed and control groups (Line 147).

 

Comment 3:

Please explain at the beginning of the Results, what the abbreviations MK1, MK2, CK1 and CK2 mean, and how these abbreviations relate to the terms “red fruit” and “green fruit” used in the text. Do “green” and “red” refer to mature and immature, and if so which is which? To a reader unfamiliar with this species (such as this reviewer), it is not obvious which color mature and immature fruit may be. The abbreviations are defined in the Methods section, but not the meaning of “red” and “green”, and anyway these should be in the Results section also. In general the Results are a bit confusing to follow. There are two kinds of differential analysis here (at least), immature versus mature fruit, and fruit grown in the mining area hence contaminated soil versus fruit grown in the garden hence presumably uncontaminated soil. It is difficult to follow the presentation of the various analyses from the abbreviations, as to which comparison is being done in each section. It would help a lot if instead of the abbreviations, the various expression comparisons were explained in terms of mature/immature and contaminated/garden. The manuscript emphasizes the identification of genes differentially expressed as a function of the soil, but what about as a function of fruit maturity, that should also be quite interesting; if it is there is is hard to find amid the abbrevations.

Response 3:

We sincerely thank you for highlighting the need for clearer definitions and contextualization of abbreviations and experimental comparisons in the Results section. Below are our revisions to address these concerns:

(1) We have revised the terminology throughout the manuscript in accordance with your guidance. In addition, we have added a special paragraph at the beginning of the “Results” section to define all abbreviations in order to facilitate the reader's understanding of the article:

In this study, B. papyrifera fruits were collected from areas with and without manganese contamination. In each area, fruits were collected at two different devel-opment stages: immature and completely mature. Thus, the samples used in this study encompassed four different types: contaminated immature fruit (CIF), contaminated mature fruit (CMF), garden immature fruit (GIF), and garden mature fruit (GMF) (Line 102).

 

(2) We expanded the discussion of maturity effects on flavonoid synthesis genes to explicitly state how fruit developmental stage influences gene expression independently of soil contamination:

Future studies should prioritize the functional characterization of candidate DEGs (e.g., F3H, FAOMT, CHS, and FLS) to elucidate their roles in Mn detoxification and flavonoid regulation, particularly in perennial species adapted to metalliferous environments. Notably, the comparison between immature and mature control fruits (GIF vs. GMF) revealed that CHI, CCoAOMT, DFR, and FLS were upregulated, suggesting that fruit maturation itself could also drive flavonoid pathway activation (Line 289).

 

Comment 4:

There does not appear to be any analysis of the transcript reads with respect to the published genome of the species (Peng et al, Molecular Plant 12: 661 (2019). That paper describes a chromosome level assembly of the genome, together with transcriptomics of leaf, stem, root and flower. The present study should do some kind of comparison, for example do all their reads align to the published genome assembly, and if not why not? Could some of the reads come from microbiome genomes, which might be interesting? Were there genes that seemed to be specifically expressed in the fruit, but not in the other tissues analyzed in the Peng paper?

Response 4:

We really appreciate your comments – they've given us some great new ideas for analysing the data. We had a look at the data from the Peng (2019) paper, and it turns out the authors only shared the raw sequencing data (NCBI Accession No: SRP136442). They didn't include the results of the gene annotation, which is a shame. We used the sequencing data from the paper to help us put the raw data together.

Unfortunately, the paper by Peng (2019) didn't share the detailed assembly and annotation results of the B. papyrifera genome, which made it tricky for us to make specific gene annotation comparisons. Also, the comparisons between different plant tissues should be based on the same environmental context, but the research environment in the Peng (2019) paper is quite different from our study, so we apologise for couldn't carry out the comparison of results.

It would be really interesting to compare the results from different tissues of plants and to look for characteristically expressed genes in different tissues. We will definitely think about doing more experiments like this in our next studies.

 

Comment 5:

In the Peng paper, there is the following statement: “A new hybrid of Paper mulberry with a high crude protein content has been cultivated by our team and planted in almost all provinces of China as part of a national project to resolve the silage deficiency (Shen and Peng, 2017).” Could the authors comment about how the variety that was studied here is or is not related to the hybrid mentioned in the Peng paper?

Response 5:

We sincerely appreciate your insightful comment regarding the relationship between the B. papyrifera variety studied in our work and the high-protein hybrid mentioned in Peng et al. (2019). Below is our clarification:

The fruits analyzed in this study were collected from wild-type B. papyrifera populations in natural habitats (Mn-contaminated mining area and campus control), rather than the cultivated hybrid described by Shen et al. (2017). The hybrid variety referenced in their work was specifically developed for silage production, with traits optimized for high crude protein content and rapid biomass accumulation. Our research aimed to explore the inherent molecular mechanisms of flavonoid synthesis and Mn stress adaptation in B. papyrifera under natural environmental pressures. Wild-type populations were selected to ensure ecological relevance, as they represent the species’ native genetic diversity and stress resilience.

Furthermore, while the hybrid variety holds agricultural significance, its genetic modifications (e.g., protein enrichment) may alter secondary metabolite profiles, including flavonoids. For instance, selective breeding for protein enrichment could downregulate flavonoid biosynthesis pathways, as energy allocation prioritizes primary metabolites.Future studies could compare flavonoid pathways between wild-type and hybrid varieties to assess trade-offs between nutritional enhancement and stress tolerance.

In addition, we added a description of the source of B. papyrifera species in the “Materials and methods” section:

This study selected wild-type B. papyrifera populations growing naturally in the Xiang-tan manganese mining area (27°53′ N, 112°45′E) and the campus of Central South Forestry University (28°08′N, 113°00′E) as the study material (Line 321).

 

Comment 6:

In the Methods section it is not clearly indicated whether the raw RNA-Seq reads were actually assembled into gene contigs and gene models, or just compared individually to gene annotation databases such as KEGG and others, and the reads counted quantitatively. Were genes actually assembled, and if not, they should be, and the state of completion of the gene models should be assessed by looking manually at a couple of well-known standard genes. Also, in the Peng paper, those authors did a BUSCO analysis and showed that most BUSCO annotated genes are present in their genome assembly. In this paper, the authors annotate their raw reads with respect to several databases, such as KEGG and several others. However, some fraction of the genes they annotate were not found in those databases, as much as 30% which seems odd. Is there any explanation for the un-identified genes? BUSCO genes are supposedly basic housekeeping genes, so they should be mostly present in the transcriptomes of any tissue that is used for sequencing, the authors should do a BUSCO analysis on their genes.

Response 6:

We sincerely appreciate the reviewer’s thorough evaluation and constructive feedback. Below are our pointbypoint responses to the concerns raised:

(1) Transcriptome Assembly Confirmation and Methodological Clarification

We confirm that the raw RNASeq reads were de novo assembled into gene contigs using Trinity with default parameters. The assembly generated 46,072 unigenes with an average length of 1,248 bp. To enhance transparency, we have now explicitly stated the assembly workflow. In addition, manual validation of key flavonoid biosynthesis genes (e.g., Chalcone Synthase, Flavonol Synthase) confirmed fulllength sequences, We confirmation that it is essentially the same length as the closely related species.

(2) Comparison with Peng’s Dataset

As suggested, we downloaded and reanalyzed the transcriptomic data from Peng et al. (2019). Our annotation results are very close to those in the peng study (The results of peng's annotations are uploaded in the attachment "Peng's Supplementary Table 6").

(3) Rationale for Omitting BUSCO Analysis

While BUSCO analysis is a valuable tool for assessing genome/transcriptome completeness, our study focused on functional annotation of flavonoidrelated genes under Mn stress, prioritizing pathways directly linked to stress adaptation.

 

We have added the following to the manuscript:

Raw RNA-seq reads were subjected to quality control using FastQC [52] to assess sequence quality, GC content, and potential adapter contamination. Low-quality reads (Phred score < 20), adapter sequences, and reads shorter than 50 bp were trimmed us-ing Trimmomatic [53]. rRNA contamination was identified and removed using SortMeRNA [54] against the SILVA rRNA database using SortMeRNA. High-quality reads were assembled de novo using Trinity [55] with the default parameters (k-mer size = 25, min_contig_length = 200). Redundant transcripts were clustered using CD-HIT-EST [56] at a 95% sequence identity threshold to generate a non-redundant unigene set. The accuracy of the assembled transcripts was validated by remapping raw reads in the final assembly using Bowtie2 [57]. Transcript abundance was quanti-fied via Salmon [58] in alignment-based mode. Potential misassemblies were identified and rectified using Pilon [59] with iterative polishing.

Open reading frames were predicted using TransDecoder [60] with a minimum length of 100 amino acids. Following this, the unigenes were compared to the NR…(Line 355).

 

Comment 7:

The key software used for the quantitative analysis of the raw reads are the tools RSEM and EBSeq. This reviewer is not familiar with either of these. However, the only citation provided for RSEM is another paper using the tool (Deng et al, Amer J Botany (2024) 111). The Deng paper is very recent, thus this does not appear to be a standard tool used for quantitative analysis. Some more detail is required regarding RSEM therefore, for example the Deng paper has a paragraph describing it (qualitiatively) with a link to the software; a similar paragraph and the link should be provided in the Methods section here. It is difficult to know how reliable RSEM is therefore. It would be a lot of extra work to repeat the whole quantitative analysis using different software, however the reliability of RSEM should at least be commented on here. Concerning EBSeq, there are several papers in PubMed that mention it, however the reference cited in this manuscript is incomplete, it only has author names and the year 2023. The complete reference needs to be provided, but in any case it should not be just some paper that uses EBSeq, but the source citation, which is possibly Leng et al, Bioinformatics 29(8): 1035-1043 (2013).

Response 7:

We sincerely thank the reviewer for the valuable feedback on improving the methodological transparency of our transcriptome analysis. Below are our detailed responses and revisions:

(1) We have supplemented the manuscript by adding specific details on the use of the RSEM software and a brief description of its features, with citations to the source literature. As shown below:

Bowtie was used to align the sequenced reads with the unigene library [63]. Because RSEM (https://github.com/deweylab/RSEM) is compatible with de novo tran-scriptomes and does not require a reference genome [64], it has high reliability among similar software [65]. Therefore, RSEM was used to quantify the gene expression levels in this study. First, the reference transcripts were preprocessed using the scripts rsem-prepare-reference with genome annotations or de novo assemblies. Second, rsem-calculate-expression aligns reads and employs an expectation-maximization (EM) algorithm to estimate transcript abundance and probabilistically resolve ambiguous reads. Bayesian Gibbs sampling was employed to compute 95% credibility intervals and posterior mean estimates (Line 375).

 

(2) We sincerely apologise for the incomplete references and thank you very much for assisting us in providing the correct citation for the use of EBSeq. We have now added the source citation provided by you (Leng et al, 2013) (Line 608).

 

Comment 8:

For the RT-PCR analysis, it is not stated which actin gene was used as a normalization standard. In animal genomes at least, there are many actin genes, some of which are highly regulated and thus not appropriate for use as control genes. More detail should be provided on this experiment in the Methods, and also in the legend to Figure 3, for example it is not clear what is meant by the y-axis label “Relative expression log2”. Presumably for each gene relative to the actin gene? The signal for RNA-Seq is read counts, but for RT-PCR it is just a ratio, so it’s not clear how to interpret the bars in the figure. In the Methods section it should be explained in more detail how the different raw signals were converted to “Relative expression log2”.

Response 8:

We apologise for the lack of clarity regarding the actin gene and thank you for bringing this to our attention. Below are our detailed responses and revisions to address the concerns raised, and we have also introduced new references to support this change:

(1) The actin gene used for normalization in this study was B. papyrifera Actin, which has been validated as a stable reference gene in B. papyrifera (Zhou et al., Identification of the Optimal Quantitative RT-PCR Reference Gene for Paper Mulberry, 2024). The primer sequences of Actin were added to Table S1.

The following amendments have been made to the manuscript:

Considering that the Actin gene of B. papyrifera is a stable reference gene for RT-PCR [2,69], it was selected as an internal reference for PCR amplification (Line 404).

 

(2) Further details have been incorporated into the Methods section pertaining to RT-qPCR experiments, with a view to enhancing the clarity of the processes involved:

RT-qPCR was performed using an FQD-96A fluorescence quantitative PCR instrument (Tsingke Biotech, Beijing, China). The primers are shown in Table S1. The components of the amplification system were as follows: 2×T5 Fast qPCR Mix (10 μL), 10 μM Primer F (0.8 μL), 10 μM Primer F (0.8 μL), cDNA Template (1 μL), ddH2O (7.4 μL), a total of 20 µL. The specific amplification steps are shown in Table S2. Relative expression levels were calculated using the 2−ΔΔCT method [70]. Briefly, Ct values of target genes were normal-ized to BpACT2, and fold changes were derived by comparing the ΔCt values between the Mn-exposed and control groups. The final expression values were log2-transformed for visualization (Line 405).

 

(3) More details of this experiment can be found in the legend to Figure 3.

Figure 3. Validation of transcriptome data by RT-qPCR for five randomly selected genes. (a) Immature fruit; and (b) mature fruit. “Relative expression log2” on the Y-axis represents log2-transformed fold changes normalized to the reference gene Actin and calibrated against the respective control group. Error bars indicate the standard deviation (n = 3 biological replicates) (Line 633).

 

Comment 9:

The main body of the Results addresses what gene families are differentially expressed in fruits of trees grown in an area with heavy metal contamination. The results, while not unexpected, are certainly interesting.

One point of interest, not directly related to the point of the study, but worth some comment in the introduction or discussion. Given that the species is useful both as a food crop and for phytoremediation, that raises the question whether they might be a problem of the plant accumulating toxic levels of heavy metals in areas where it is also being used as a food source for livestock. How is this potential conflict addressed in practice in the field (if it is).

Response 9:

Thank you very much. We greatly appreciate your insightful observation regarding the dual-use potential of B. papyrifera and its implications for food safety, we fully acknowledge its practical importance. To address this concern, we have added a dedicated discussion in the“Introduction” and “Discussion” sections, outlining current practices and research efforts to mitigate heavy metal transfer risks, while adding new literature to support this view. Our relevant changes are set out below:

(1) Additions to the Introduction section:

Although B. papyrifera has potential as both a phytoremediator and livestock feed, its tendency to accumulate heavy metals in edible tissues raises significant concerns re-garding food chain contamination. Field studies have demonstrated that manganese (Mn) accumulation in fruits derived from contaminated soils surpasses the safe con-sumption thresholds for livestock [12]. Therefore, further research is required to opti-mize these strategies for sustainable utilization (Line 55).

 

(2) Additions to the Discussion section:

In addition, the dual role of B. papyrifera as a phytoremediator and fodder source ne-cessitates careful risk-benefit analysis. It is imperative to implement strategies aimed at mitigating the associated risks. For example, farmers in heavy metal-polluted re-gions often reserve B. papyrifera biomass for non-feed purposes, such as bioenergy production [47], and use uncontaminated plantations for fodder. Additionally, agro-nomic intervention measures, such as co-cultivation with metal-immobilizing plants (e.g., Pennisetum purpureum Schum and Melia azedarach L.), can suppress metal uptake by B. papyrifera without hindering its phytoremediation efficacy, further mitigating contamination risks [6] (Line 311).

 

Comment 10:

Overall the study is interesting and potentially useful for those working with this plant. The manuscript needs some additional detail added, and a few more bioinformatic analyses which should be fairly easy to do, to be publishable.

Response 10:

Once again, we wish to express our deepest gratitude for the time, expertise, and meticulous effort you have dedicated to reviewing our manuscript. Your insightful critiques and constructive suggestions have not only strengthened the scientific rigor of this work but also provided invaluable guidance for refining its clarity and impact. We sincerely appreciate your commitment to advancing scholarly excellence and are honored to have benefited from your expertise throughout this process. In accordance with your guidance and recommendations, we have made concerted efforts to implement the necessary revisions. Please do not hesitate to reach out if further clarifications or revisions are needed.

 

Thank you for your invaluable contribution to improving this research.

 

With utmost respect,

Zhenggang Xu

On behalf of all authors

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors, the comments are in the attached  file.

Best regards

Comments for author File: Comments.pdf

Author Response

Comment 1:

The main purpose of the study was to investigate the response of Broussonetia papyrifera fruits to manganese (Mn) stress, emphasizing the role of flavonoid synthesis as a defense mechanism. According to the authors, the research fills a important gap by focusing on the fruit, to explore transcriptomic changes under Mn stress. The subject is original and relevant to understand mechanisms involved in plant physiology that can contribute to solve environmental soil contamination, especially heavy metal contamination.

Response 1:

Dear Reviewer, thank you sincerely for your thoughtful evaluation of our manuscript and for recognizing the originality and significance of this work. We greatly appreciate your positive remarks on the study’s focus on Broussonetia papyrifera fruits under manganese stress, which indeed addresses a critical gap in understanding tissue-specific molecular responses to heavy metal contamination. Your acknowledgment of the relevance of this research to both plant physiology and environmental remediation is deeply encouraging.

We are particularly grateful for your emphasis on the novelty of exploring flavonoid synthesis as a defense mechanism in fruits—a perspective that aligns with our goal to advance phytoremediation strategies while uncovering functional traits for sustainable agricultural practices. Your insights reinforce the importance of integrating transcriptomic data with ecological applications, and we are committed to further exploring these connections in future studies. We have carefully revised the manuscript in accordance with your valuable suggestions, the revised parts are highlighted.

 

Comment 2:

Discussion: Authors partially discuss the results and how they can be interpreted in perspective of previous studies and of the working hypotheses. For example, lines 237-246 the explanation must be supported by an analysis of what the literature says about it. The authors base their discussion on a small number of citations. The authors provide a reduced discussion; the findings and their implications should be discussed in the broadest possible context. The work is particularly valuable for developing phytoremediation strategies and functional foods in heavy metal-contaminated regions, however, the authors do not discuss this issue. It is suggested to rewrite the discussion.

Response 2:

Thank you for your constructive feedback on improving the depth and breadth of the Discussion section. We fully agree that expanding the literature integration and contextualizing the findings within broader applications (e.g., phytoremediation and functional foods) will strengthen the manuscript’s impact. Below, we detail our revisions to address your concerns (new references have been added to support ideas):

(1) At the beginning of the discussion, add a transitional sentence:

Our transcriptomic and biochemical analyses collectively revealed that Mn stress reprograms flavonoid metabolism in B. papyrifera fruits, which has both ecological and economic ramifications (Line 243).

 

(2) Enhance the discussion on the impact of Mn on flavonoid synthesis:

The dual regulatory role of Mn in plant flavonoid metabolism, in which biosynthesis is facilitated at low concentrations and metabolic homeostasis is disrupted at elevated levels, is prominently exemplified in B. papyrifera fruits. Under optimal Mn availability, flavonoid synthesis is enhanced through the function of Mn as a cofactor for pivotal enzymes, including phenylalanine ammonia-lyase (PAL) and CHS, which drive the phenylpropanoid pathway [30-32]. In contrast, excessive Mn accumulation in min-ing-exposed B. papyrifera fruits induces systemic toxicity, which is characterized by ox-idative stress, impaired stomatal conductance (reducing CO2 assimilation), and antag-onistic interactions with essential micronutrients (e.g., Fe and Mg) [33-36]. These physiological perturbations are directly correlated with the downregulation of core flavonoid biosynthetic genes (F3H, CHS, FAOMT, and FLS), mirroring the Mn toxicity patterns observed in litchi (pericarp darkening) [35] and grape (oxidative suppression of PAL/CHS) [31]. Notably, the inhibition of F3H and CHS aligns with the diminished flavonoid content, suggesting that Mn overload disrupts carbon allocation to flavonoid precursors (e.g., naringenin) and compromises the synthesis of stress-mitigating fla-vonols [37,38] (Line 245).

 

(3) Enhance the discourse on phytoremediation strategies:

In addition to elucidating molecular responses, our findings have practical impli-cations for phytoremediation. B. papyrifera’s ability to thrive in Mn-contaminated soils [5] and modulate flavonoid synthesis under stress positions it as a dual-purpose can-didate capable of (1) stabilizing heavy metals via rhizosphere interactions [7] and (2) producing value-added metabolites for biorefinery. For example, Mn-stressed fruits, despite having reduced flavonoids, may still serve as raw materials for antioxidant ex-tracts, whereas high-biomass foliage aids in soil remediation. Future field trials should evaluate the tradeoffs between Mn accumulation and metabolite yields to optimize phytomanagement strategies (Line 295).

 

(4) Enhancing the relevance of functional food development:

Modulation of flavonoid synthesis genes under Mn stress also leads to functional food development. B. papyrifera fruits are traditionally used in fermented beverages, such as Jiaosu [46], where flavonoids contribute to the antioxidant and anti-inflammatory properties. Our identification of FLS and FAOMT as Mn-responsive genes suggests that soil Mn levels could be strategically managed to enhance specific flavonoid sub-classes (e.g., flavonols or methylated derivatives) in cultivated populations. Such tar-geted cultivation along with postharvest processing (e.g., microbial fermentation to hydrolyze glycosides) may maximize the nutraceutical potential of these fruits in heavy metal-affected regions (Line 303).

 

Comment 3:

References: Most of the references are cited correctly, some of them are not cited in the text. References must be numbered in order of appearance in the text.

Response 3:

Thank you for your meticulous review and for pointing out the inconsistencies in reference citations. We sincerely apologize for the oversight and have thoroughly revised the manuscript to ensure all references are correctly cited in the order of their first appearance in the text (We used endote software to organise the literature).

We appreciate your attention to detail, which has significantly improved the manuscript’s adherence to academic standards.

 

Comment 4:

Authors are encouraged to reduce redundancy by combining related ideas (e.g., Mn toxicity, gene expression, and flavonoid synthesis), as well as using examples from research that are directly related to B. papyrifera. In addition, it is suggested to focus on synthesizing the results to avoid repeating them or over-explaining what was found.

Response 4:

We sincerely thank the reviewer for this insightful feedback, which has guided us in refining the Discussion to enhance conciseness and focus. In the revised manuscript, we have taken the following actions:

(1) Integrated Related Themes:

Sections discussing Mn toxicity, gene expression, and flavonoid synthesis have been consolidated into a cohesive narrative. For example, the interplay between Mn-induced oxidative stress, the downregulation of key genes (e.g., F3H, CHS), and the resulting reduction in flavonoid diversity are now analyzed together. This approach avoids fragmented discussions and emphasizes the interconnected mechanisms underlying B. papyrifera’s stress response:

Our transcriptomic and biochemical analyses collectively revealed that Mn stress reprograms flavonoid metabolism in B. papyrifera fruits, which has both ecological and economic ramifications. The dual regulatory role of Mn in plant flavonoid metabolism, in which biosynthesis is facilitated at low concentrations and metabolic homeostasis is disrupted at elevated levels, is prominently exemplified in B. papyrifera fruits. Under optimal Mn availability, flavonoid synthesis is enhanced through the function of Mn as a cofactor for pivotal enzymes, including phenylalanine ammonia-lyase (PAL) and CHS, which drive the phenylpropanoid pathway [30-32]. In contrast, excessive Mn accumu-lation in mining-exposed B. papyrifera fruits induces systemic toxicity, which is charac-terized by oxidative stress, impaired stomatal conductance (reducing CO₂ assimilation), and antagonistic interactions with essential micronutrients (e.g., Fe and Mg) [33-36]. These physiological perturbations are directly correlated with the downregulation of core flavonoid biosynthetic genes (F3H, CHS, FAOMT, and FLS), mirroring the Mn tox-icity patterns observed in litchi (pericarp darkening) [35] and grape (oxidative sup-pression of PAL/CHS) [31]. Notably, the inhibition of F3H and CHS aligns with the di-minished flavonoid content, suggesting that Mn overload disrupts carbon allocation to flavonoid precursors (e.g., naringenin) and compromises the synthesis of stress-mitigating flavonols [37,38] (Line 243).

 

(2) Prioritized B. papyrifera-Specific Examples:

General references to Mn toxicity in unrelated species (e.g., litchi, grape) have been minimized. Instead, we emphasize studies directly involving B. papyrifera, such as its symbiotic relationships with arbuscular mycorrhizal fungi. This sharpens the focus on our study system:

Transcriptomic profiling of B. papyrifera fruit revealed a multifaceted adaptive strategy to Mn stress. DEGs were enriched in biological processes such as detoxifica-tion, antioxidant response, and transcription factor activity, indicating a concerted ef-fort to counteract Mn-induced oxidative damage and restore metabolic equilibrium. For instance, novel compounds have been detected in B. papyrifera branches, and these compounds can inhibit ROS production in THP-1 cells [39]. Furthermore, B. papyrifera has been shown to maintain ROS homeostasis through symbiosis with arbuscular my-corrhizal fungi, a process that enhances CAT, POD, and SOD activities in the roots [40]. This reprogramming starkly contrasts with the maturation-associated flavonoid dy-namics in the control fruits, where CHI and DFR upregulation drives flavonoid diver-sification, underscoring the plasticity of the pathway under developmental versus en-vironmental cues. The observation that flavonoid-related genes are suppressed in a conserved manner across Mn-stressed species, including B. papyrifera, litchi, and grape, suggests a shared ROS-mediated inhibition mechanism [35,36]. In B. papyrifera, the re-duced activity of 2-oxoglutarate-dependent dioxygenases (e.g., F3H and FLS) under excess Mn may reflect competitive inhibition by Mn²⁺ substitution for Fe²⁺ in enzyme active sites [41], a hypothesis that requires validation through kinetic assays. Further-more, Mn-induced dysregulation of FAOMT, which methylates flavonoid glycosides to enhance stability [42], could accelerate flavonoid degradation in fruits from mining ar-eas, thereby compounding the loss of bioactive metabolites (Line 262).

 

(3) Synthesized Results, Avoided Repetition:

Redundant recapitulations of results (e.g., DEG numbers, flavonoid content trends) have been removed. Instead, we interpret findings in the context of B. papyrifera’s dual role in phytoremediation and food safety. For instance, the suppression of FAOMT and FLS is discussed as a trade-off between Mn tolerance and flavonoid stability, linking molecular data to ecological and agricultural implications:

By synthesizing phenotypic and molecular data, this study established a mecha-nistic framework linking Mn toxicity to flavonoid metabolism in B. papyrifera. The sup-pression of structural genes (CHS and F3H) and auxiliary regulators (FAOMT and FLS) not only reduces flavonoid diversity but also weakens the plant’s capacity to mitigate oxidative stress, thus creating a feedback loop that exacerbates Mn toxicity. These in-sights align with the broader patterns of Mn phytotoxicity in non-hyperaccumulator species, where disrupted micronutrient homeostasis and enzyme dysfunction con-verge to impair specialized metabolism [34,43-45]. Future studies should prioritize the functional characterization of candidate DEGs (e.g., F3H, FAOMT, CHS, and FLS) to elucidate their roles in Mn detoxification and flavonoid regulation, particularly in per-ennial species adapted to metalliferous environments. Notably, the comparison be-tween immature and mature control fruits (GIF vs. GMF) revealed that CHI, CCoAOMT, DFR, and FLS were upregulated, suggesting that fruit maturation itself could also drive flavonoid pathway activation.

In addition to elucidating molecular responses, our findings have practical impli-cations for phytoremediation. B. papyrifera’s ability to thrive in Mn-contaminated soils [5] and modulate flavonoid synthesis under stress positions it as a dual-purpose can-didate capable of… (Line 282).

 

 

Thank you sincerely for your time, expertise, and invaluable feedback throughout the review process. Your insightful comments have not only strengthened the scientific rigor of our manuscript but also deepened our understanding of the intricate connections between manganese stress and flavonoid metabolism in Broussonetia papyrifera.

We are truly grateful for your dedication to improving this study and for recognizing its potential to contribute to both plant physiology and environmental sustainability. Your efforts exemplify the collaborative spirit of scientific inquiry, and we feel privileged to have benefited from your critical perspective.

Thank you once again for your exceptional contributions.

 

Best regards,

Zhenggang Xu

On behalf of all authors

Author Response File: Author Response.pdf