Transcriptome and Metabolome-Based Analysis of Carbon–Nitrogen Co-Application Effects on Fe/Zn Contents in Dendrobium officinale and Its Metabolic Molecular Mechanisms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

-Attached-

Comments for author File: Comments.pdf

Author Response

Introduction: The Introduction has been well drafted, with extensive collection of points related to the study, highlighting the rationale behind it. The following corrections may be carried out for improvement of the manuscript,

Comment  1:  This   section  is  well-written  with   extensive  points   about  crop,  its fertilization and impact on Fe/Zn. Further, its utilization in applied aspect should be provided. How, transcriptome and metabolome techniques addresses your research question? Should be added.

• Response 1:Thank you for your suggestions. We have supplemented the practical value related to the application level and added explanations on how transcriptomic and metabolomic technologies address the core scientific questions of this study; see the highlighted content for details.

Comment 2: Many points in this section is not supported by references. Add to give more support

• Response 2:Thank you very much for raising this out.We have supplemented relevant references in the introduction section to strengthen the argumentation.

Materials and methods: The following corrections may be carried out in the manuscript for its improvement,

Comment 3: More clarity is expected for the treatment used. Is there any standard protocol or previous reports for the imposed treatment. Cite such references.

• Response 3: Thank you very much for your valuable suggestions. We have further clarified the treatment protocol employed in the experiment and cited the relevant reference; see section 1，lines 141-143.

Comment  4:  What  is  the  basis  of selecting  8  days  interval  for  the  collection  of samples, since, AKG is applied 3 days interval, while urea applied 7 days interval. Clarify

• Response 4: Thank you for your suggestion.The reasons have been explained in the manuscript; see Page 4, lines 141-143 and lines 148-154 for details.
• The application intervals of AKG (3 days) and urea (7 days) are designed to maintain effective treatment concentrations
• The sample collection interval was set to 8 days, mainly based on three core considerations:first， officinalegrows slowly, and a period of 7-10 days is required for significant differences to appear in the absorption, metabolism, and accumulation of Fe and Zn;second，The synergistic effect of combined carbon-nitrogen application needs to cover both the urea conversion cycle (7 days) and multiple AKG application cycles (3 days × 2) to ensure that the plants maintain a carbon-nitrogen homeostasis；third, it balances data integrity with experimental practicality.

Comment 5: Hoagland nutrient solution itself contains all essential nutrients. How would you justify its inclusion in this study

• Response 5:Thank you for your constructive suggestions. We have incorporated the rationale for using Hoagland solution into the manuscript.; see Page 4, lines 146-148 for details.

Comment 6: Add references for all indicators estimation used in the study. For section

2.2.4, brief the extraction method

• Response 6:Thank you for your valuable suggestions. We have supplemented the relevant references for the determination methods of all indicators used in the study. Regarding Section 2.2.4, we have added a detailed description of the determination method for citric acid and cited the corresponding references.

Comment 7: It was mentioned that HISAT2 was used for alignment, yet the results present Trinity gene IDs. Further, it was mapped with Homo sapiens, Why? Whether authors used  edgeR  or  DEseq2?  The  authors  should  clarify  their  whole  pipeline,  as  the  current description is inconsistent and indicates huge methodological errors

• Response 7:Thank you for your question. We apologize for the error in the original analytical methodology, which has now been revised; see section 2.5.3 for details.

Results: The following corrections may be carried out in the manuscript for its improvement,

Comment   8:   Correct  treatment  group  labels.  Use  consistently  throughout  the manuscript

• Response 8:Thank you for your suggestion. We have standardized the names of the treatment groups to ensure consistency between the manuscript and the figures.

Comment 9: Repeated points in section 3.2 may be corrected

• Response 9:Thank you for your valuable suggestions. We have revised the repetitive expressions in the manuscript.

Comment 10: The authors claim “synergistic effects” for CT+NT, but the data show antagonism at later stages. This weakens the synergy claim. Combined treatments often show transient or even antagonistic effects. Hence, tone down claims of synergy; acknowledge time-dependent antagonism. Explain otherwise 

• Response 10:Thank you for your valuable suggestions. We have rephrased this section of the results in the manuscript; see 3.1-3.4 for details.

Comment 11: Section 3.5.2 and Fig 2B has several confusions. Is it “differential ions” or “differential metabolites”? Maintain consistency. Further, clarify between fig 2B and 2C, it looks like fig 2B represents all detected features and fig 2C represents differentially regulated features.  Is  it  correct?  In  case  of  significant differences mention  the  statistical  test  and threshold.

• Response 11:Thank you very much for raising this out. We have standardized the terminology to "differentially metabolites", supplemented the table data, and explained the statistical test methods used as well as the judgment thresholds; see 3.5.1-3.5.3 for details.

Comment  12:  In case of metabolomics analysis, Variable importance in projection (VIP) metabolites analysis is widely used. I suggest to include it to identify strong candidates

• Response 12:Thank you for your suggestions. We have incorporated this section into the manuscript; see 3.5.3 for details.

Comment 13: Repeated results in section 3.5.2 and 3.5.3 may be merged

• Response 13:Thank you very much for raising this out. We have merged these two sections into Section 3.5.3 of the manuscript and revised certain sections accordingly.

Comment  14:  Section  3.6.1,  correlation  analysis  indicate high correlation between samples, then how authors mentioned significant difference, mention  statistical test used. Otherwise delete this section

• Response 14:Thank you for your valuable suggestions. We have revised this section and reorganized its presentation format to enhance clarity and logical flow.

Comment  15:  Section  3.6.5, Annotation  pipeline is not clear. How was  functional annotation performed for Trinity IDs.

• Response 15:We have revised the annotations and supplemented them with additional figures for clarification.

Comment  16: Line 728-732, the authors mentioned “combined immunotherapy and the overcoming of chemoresistance through combined chemotherapy” which is not at all relevant  to  plant   studies.   Check  the  whole  manuscript   of  similar  instances   and  infer conclusions   related   to   plant   omics.   qPCR   validation   of   important   genes   are   also recommended

• Response 16:Thank you very much for raising this out. We have screened for and removed content irrelevant to the theme of this study; We fully agree that the qPCR validation of key genes, as proposed in your comments, is highly necessary. However, due to practical constraints, we are unable to complete this experiment within the current revision period. The specific reasons are as follows: All samples used for transcriptome sequencing were collected at specific treatment time points, processed with RNAlater, and stored at -80℃. During the transcriptome library construction and subsequent data analysis, the total RNA from these samples has been completely exhausted. Re-collecting and cultivating samples under the same experimental conditions would require a relatively long growth cycle of D. officinale. We sincerely apologize for the inconvenience caused by our inability to complete the qPCR validation during this revision. We highly appreciate the reviewer for pointing out this important aspect, as your suggestion provides valuable guidance for the improvement of our subsequent research. To address this, we have cited multiple relevant literatures to support the functional relevance of these key genes, thereby ensuring the scientific rigor and reliability of this study.

Comment  17:  Terms like “carbamide” and “urea” are used interchangeably, which may confuse readers. Consistency is important.

• Response 17:Thank you for your valuable suggestions. We have standardized the terminology to "urea" to ensure consistency in its usage throughout the manuscript.

Discussion:

Comment 18: The Discussion lists transcriptome and metabolome findings but does not  connect  them  to  physiological  outcomes  (e.g.,  how  flavonoid  biosynthesis  directly explains Fe chelation). Further, overlap between transcriptome and metabolome pathways (~15–19%) is mentioned, but the biological meaning of this overlap is not discussed.

• Response 18:Thank you very much for your valuable suggestions. We have reorganized and rephrased the content of the Discussion section, and supplemented the relevant information in accordance with your requirements.

Comment 19: Address the inconsistencies exhibited between the synergistic effect of AKG and urea in discussion section. (eg. Why Zn peaks at 16 days but collapses at 32 days). Limitations or negative results of the study also must be justified.

• Response 19:Thank you for pointing out this issue. We have explained the reasons why the Fe and Zn contents reached their peak on 16 d, and supplemented the limitations of this experiment as well as future research directions at the end of the section.

Conclusion: The section needs to be revised. All queries raised in the above sections need to be addressed and modified in this section also.

Abstract: Similar to conclusions

General comment: Grammatical and typographic errors may be checked and corrected

• Response :Thank you for your valuable suggestions. The revised Discussion, Conclusion, and Abstract have been updated simultaneously, and we kindly request the reviewers to review them.

Thank you again for your positive and constructive comments and suggestions on our manuscript. We are open to any further suggestions and look forward to your feedback.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

• The manuscript lacks clearly formulated hypotheses.
  The introduction describes the general aim of the study but does not present a specific, testable hypothesis. Clearly defining the hypothesis would help frame the research questions and guide the interpretation of the results.
• The experimental setup requires clearer explanation.
  Please, correct Table 1 - the frequency of the treatments appears inconsistent. It is unclear how often urea is applied in the NT treatment compared to the combined CT+NT treatment. These frequencies should be identical for proper comparison, and if they are intentionally different, the authors need to clearly justify this choice. Additionally, the rationale for sampling at 0, 8, 16, 24, and 32 days should be explicitly explained.
• Statistical analysis is insufficiently described.
  Very little information is provided about the statistical methods used. It is not stated whether tests for homogeneity of variance were performed, nor is the PCA analysis adequately explained. The authors don’t analyze the treatment × time interaction or the overall temporal trends.
• Conclusions should be drawn more cautiously.
  In several places, the manuscript proposes mechanistic explanations without sufficient experimental evidence. Interpretations that extend beyond the data should be toned down or clearly framed as hypotheses.
• Reference genome issue.
  The manuscript inexplicably states that transcriptomic reads were aligned to the Homo sapiens (GRCh38) reference genome. This is unrelated to Dendrobium officinale and needs to be corrected or properly explained.
• A lack of information on molecular events after day 16 should be addressed.
  Since some physiological changes occur between days 16 and 32. What about that?
• Figures are overcrowded and difficult to read.
  Many graphs contain very small fonts, numerous markers, overlapping colours, and complex layouts. Even when zoomed in, the figures remain visually dense and hard to interpret. Simplifying the graphical presentation would greatly improve clarity.
• “Dendrobium officinale Kimura & Migo is a traditional medicinal plant in China significant health and medicinal value in promoting stomach health, generating body fluids, nourishing yin, clearing heat, and regulating immunity.” The statement relies on traditional Chinese medicine terminology such as “nourishing yin” and “clearing heat”, which are not standard scientific concepts and may be unclear to readers unfamiliar with TCM.
• The manuscript is overly narrative and would benefit from a more structured presentation.
  Results should be presented in a more systematic way, ideally supported by additional tables (only one table is currently included). A clearer and more organized structure would make the findings easier to follow.

Author Response

Comment  1:The manuscript lacks clearly formulated hypotheses.

The introduction describes the general aim of the study but does not present a specific, testable hypothesis. Clearly defining the hypothesis would help frame the research questions and guide the interpretation of the results.

• Response 1: Thank you very much for your valuable suggestions. We have added the research hypothesis in the introduction section; see the red content in Line

Comment  2:The experimental setup requires clearer explanation.

Please, correct Table 1 - the frequency of the treatments appears inconsistent. It is unclear how often urea is applied in the NT treatment compared to the combined CT+NT treatment. These frequencies should be identical for proper comparison, and if they are intentionally different, the authors need to clearly justify this choice. Additionally, the rationale for sampling at 0, 8, 16, 24, and 32 days should be explicitly explained.

• Response 2: Thank you for your suggestion.The reasons have been explained in the manuscript; see Page 4, Lines 141-143 and Lines 148-154 for details.
• The application intervals of AKG (3 days) and urea (7 days) are designed to maintain effective treatment concentrations
• The sample collection interval was set to 8 days, mainly based on three core considerations:first， officinalegrows slowly, and a period of 7-10 days is required for significant differences to appear in the absorption, metabolism, and accumulation of Fe and Zn;second，The synergistic effect of combined carbon-nitrogen application needs to cover both the urea conversion cycle (7 days) and multiple AKG application cycles (3 days × 2) to ensure that the plants maintain a carbon-nitrogen homeostasis；third, it balances data integrity with experimental practicality.

Comment  3:Statistical analysis is insufficiently described.

Very little information is provided about the statistical methods used. It is not stated whether tests for homogeneity of variance were performed, nor is the PCA analysis adequately explained. The authors don’t analyze the treatment × time interaction or the overall temporal trends.

Conclusions should be drawn more cautiously.

• Response 3: Thank you very much for your suggestions. We have supplemented information on various statistical methods in the manuscript, reinterpreted the principal component analysis (PCA), and supplemented and discussed the overall temporal variation trend in the discussion section；see Page 7，5.3 and Page 10，section3.5.2 and Page 16，section 3.6.2 and Page 23，section 4.1 for details.

Comment  4:In several places, the manuscript proposes mechanistic explanations without sufficient experimental evidence. Interpretations that extend beyond the data should be toned down or clearly framed as hypotheses.

• Response 4：Thank you very much for raising this out. We have revised the expressions in the discussion and conclusion sections, and added cluster heatmaps in the results section.

Comment  5:Reference genome issue.

The manuscript inexplicably states that transcriptomic reads were aligned to the Homo sapiens (GRCh38) reference genome. This is unrelated to Dendrobium officinale and needs to be corrected or properly explained.

• Response 5:Thank you for your question. We apologize for the error in the original analytical methodology, which has now been revised; see section 2.5.3 for details.

Comment  6:A lack of information on molecular events after day 16 should be addressed.

Since some physiological changes occur between days 16 and 32. What about that?

• Response 6:Thank you for your question. Based on physiological data, this study found that the contents of Fe and Zn were the highest at 16 days of treatment, thus only sequencing analysis was performed on the samples collected at this time point.

Comment  7:Figures are overcrowded and difficult to read.

Many graphs contain very small fonts, numerous markers, overlapping colours, and complex layouts. Even when zoomed in, the figures remain visually dense and hard to interpret.

• Response 7:Thank you for raising this out.We have improved the clarity of the images to ensure they are clearly visible when enlarged.

Comment  8:Simplifying the graphical presentation would greatly improve clarity.

“Dendrobium officinale Kimura & Migo is a traditional medicinal plant in China significant health and medicinal value in promoting stomach health, generating body fluids, nourishing yin, clearing heat, and regulating immunity.” The statement relies on traditional Chinese medicine terminology such as “nourishing yin” and “clearing heat”, which are not standard scientific concepts and may be unclear to readers unfamiliar with TCM.

• Response 8:Thank you for raising this out.Thank you very much for pointing out the issues. We have now removed the non-standard scientific concepts to make the text accessible and easy to understand.

Comment  9:The manuscript is overly narrative and would benefit from a more structured presentation.

Results should be presented in a more systematic way, ideally supported by additional tables (only one table is currently included). A clearer and more organized structure would make the findings easier to follow.

• Response 9:Thank you very much for your suggestions.We have reorganized the presentation method as requested and added tabular data to support this study; see Tables 2–4 for details.

Thank you again for your positive and constructive comments and suggestions on our manuscript. We are open to any further suggestions and look forward to your feedback.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer’s comments for the author’s response

I have carefully reviewed the revised manuscript and the author’s responses to my comments. I am pleased to report that the authors have adequately addressed my suggestions and made the necessary revisions to improve the clarity and quality of the manuscript. I would suggest one more change to be made to improve the quality of the manuscript.

Comment 1: Please include the pipeline used for transcriptome data analysis, including QC, alignment, featurecount etc.

Author Response

Dear reviewer:

Thank you for your constructive suggestions and comments on our manuscript “Transcriptome and Metabolome-Based Analysis of Carbon-Nitrogen Co-Application Effects on Fe/Zn Contents in Dendrobium officinale and Its Metabolic Molecular Mechanisms” (agriculture-4008002). We have carefully considered the suggestions and made some changes in the manuscript.

Comment 1: Please include the pipeline used for transcriptome data analysis, including QC, alignment, featurecount etc.

Response 1:Thank you very much for your valuable suggestions. We have supplemented the transcriptome data analysis pipeline as required. The detailed content has also been highlighted in Section 2.5.3.

The specific content is as follows：

A reference-free transcriptome analysis strategy was adopted. The raw data generated from sequencing were preprocessed as follows: sequencing adapter sequences were removed using Cutadapt software, and low-quality sequences were filtered out via fqtrim software, ultimately yielding high-quality clean data. Trinity software was utilized for de novo assembly of the clean data to obtain unigene sequences. Functional annotation of the unigenes was performed using the Plant Transcription Factor Database (PlantTFDB, http://planttfdb.gao-lab.org/index.php). With Arabidopsis thaliana as the reference species, sequence alignment methods were employed to identify transcription factor families and their respective members.

Thank you again for your positive and constructive comments and suggestions on our manuscript. We are open to any further suggestions and look forward to your feedback.

Reviewer 2 Report

Comments and Suggestions for Authors

No comment

Author Response

We are grateful for your invaluable feedback provided earlier, as it has greatly contributed to the refinement of this manuscript. Thank you！