Integrative Transcriptomic and Metabolomic Analysis Reveals Regulatory Networks and Metabolite Dynamics in Gastrodia elata Flower Development

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article titled “Integrated Transcriptomic and Metabolomic Studies Reveal Regulatory Networks and Metabolite Dynamics of Flower Development in Gastrodia elata"** combines transcriptomic and metabolomic analyses to investigate the molecular and biochemical mechanisms underlying flower development in Gastrodia elata. Using RNA sequencing (RNA-Seq) and widely targeted metabolomics, the authors examined three distinct stages of flower development, identifying differentially expressed genes (DEGs) and significant changes in metabolite accumulation. The study's objectives are clearly defined, and the results are highly interesting. I genuinely enjoyed reading this manuscript. While I do not have major concerns, I offer the following suggestions for improvement:

The study presents transcriptomic and metabolomic findings separately but does not establish a strong correlation between gene expression and metabolite levels. Strengthening this connection would provide a more integrated understanding of the regulatory mechanisms described in the manuscript.

Although the study identifies transcription factors (TFs) involved in flower development, it does not elaborate on their specific regulatory mechanisms. Several previous studies have explored this aspect, and a more detailed discussion would enhance the mechanistic insights presented.

The manuscript discusses changes in flavonoids, terpenoids, and alkaloids, yet their physiological roles in flower development are not clearly explained. This lack of discussion leaves an unclear link between metabolite accumulation and floral morphology. Providing further insights into these relationships would strengthen the study’s conclusions.

 

While the manuscript is generally well-structured and grammatically sound, several areas could be refined to improve readability. Below, I provide specific comments on writing aspects that may benefit from revision.

For the title, I suggest to replace as “Integrative Transcriptomic and Metabolomic Analysis Reveals Regulatory Networks and Metabolite Dynamics in Gastrodia elata Flower Development”

Lines 19–21: The phrase " FPKM-based analysis revealed substantial transcriptomic shifts throughout G. elata flower development, while widely targeted metabolomics identified key metabolites with significant variations in abundance across developmental stages.” could be revised to “FPKM analysis showed significant transcriptomic changes during G. elata flower development, while targeted metabolomics identified key metabolites with stage-specific variations.”

Lines 21–24: The phrase“This study presents dynamic models of transcriptome and metabolite changes, unveiling crucial regulatory networks underlying G. elata flower growth and development which will enhance knowledge base for G. elata’s applications.” could be revised to “This study proposes dynamic models of transcriptomic and metabolite changes, revealing key regulatory networks that govern G. elata flower development and potential applications.”

Lines 40–43: The phrase “Due to rising demand and habitat loss, wild populations of G. elata have significantly declined, leading to its classification as a category II protected wild plant in China and underscoring the urgent need for conservation efforts.” could be revised to “Increasing demand and habitat loss have led to a significant decline in wild G. elata populations, classifying it as a category II protected species in China and highlighting the urgent need for conservation.”

Lines 87–90: The phrase“Three developmental stages were selected for transcriptome analysis, with representative tissues designated as Stage1 (TMH1), Stage2 (TMH2), and Stage3 (TMH3). Three biological replicates were examined for each sample.” could be revised to “Three flower developmental stages (Stage 1: TMH1, Stage 2: TMH2, Stage 3: TMH3) were selected for transcriptomic analysis. Each stage was analyzed with three biological replicates.”

Lines 180–181: The phrase “Figure 1. Flower and petal phenotypes across three flower developmental stages: Stage1 (TMH1) (A), Stage2 (TMH2) (B), and Stage3 (TMH3) (C).” could be revised to “Figure 1. Flower and petal phenotypes at three developmental stages: Stage 1 (TMH1) (A), Stage 2 (TMH2) (B), and Stage 3 (TMH3) (C).”

Lines 326–329: The phrase “Differential metabolites and their associated indexes were annotated in KEGG pathways (Figure 4A, Figure 4B, Table S6, Table S7), enabling the creation of a network diagram to visually represent connections between metabolites and their related indexes across multiple pathways.” could be revised to “Differential metabolites were annotated in KEGG pathways (Figure 4A, 4B, Tables S6–S7), allowing the visualization of metabolite interactions across pathways.”

Lines 317–320: The phrase “These findings offer valuable insights into the complex regulatory networks governing G. elata flower development, establishing a foundation for future research aimed at advancing the functional genomics of this important medicinal plant through targeted improvements.” could be revised to “These findings provide new insights into G. elata flower development and lay the foundation for future research in functional genomics and medicinal applications.”

Comments on the Quality of English Language

While the manuscript is generally well-structured and grammatically sound, several areas could be refined to improve readability. Below, I provide specific comments on writing aspects that may benefit from revision.

For the title, I suggest to replace as “Integrative Transcriptomic and Metabolomic Analysis Reveals Regulatory Networks and Metabolite Dynamics in Gastrodia elata Flower Development”

Lines 19–21: The phrase " FPKM-based analysis revealed substantial transcriptomic shifts throughout G. elata flower development, while widely targeted metabolomics identified key metabolites with significant variations in abundance across developmental stages.” could be revised to “FPKM analysis showed significant transcriptomic changes during G. elata flower development, while targeted metabolomics identified key metabolites with stage-specific variations.”

Lines 21–24: The phrase“This study presents dynamic models of transcriptome and metabolite changes, unveiling crucial regulatory networks underlying G. elata flower growth and development which will enhance knowledge base for G. elata’s applications.” could be revised to “This study proposes dynamic models of transcriptomic and metabolite changes, revealing key regulatory networks that govern G. elata flower development and potential applications.”

Lines 40–43: The phrase “Due to rising demand and habitat loss, wild populations of G. elata have significantly declined, leading to its classification as a category II protected wild plant in China and underscoring the urgent need for conservation efforts.” could be revised to “Increasing demand and habitat loss have led to a significant decline in wild G. elata populations, classifying it as a category II protected species in China and highlighting the urgent need for conservation.”

Lines 87–90: The phrase“Three developmental stages were selected for transcriptome analysis, with representative tissues designated as Stage1 (TMH1), Stage2 (TMH2), and Stage3 (TMH3). Three biological replicates were examined for each sample.” could be revised to “Three flower developmental stages (Stage 1: TMH1, Stage 2: TMH2, Stage 3: TMH3) were selected for transcriptomic analysis. Each stage was analyzed with three biological replicates.”

Lines 180–181: The phrase “Figure 1. Flower and petal phenotypes across three flower developmental stages: Stage1 (TMH1) (A), Stage2 (TMH2) (B), and Stage3 (TMH3) (C).” could be revised to “Figure 1. Flower and petal phenotypes at three developmental stages: Stage 1 (TMH1) (A), Stage 2 (TMH2) (B), and Stage 3 (TMH3) (C).”

Lines 326–329: The phrase “Differential metabolites and their associated indexes were annotated in KEGG pathways (Figure 4A, Figure 4B, Table S6, Table S7), enabling the creation of a network diagram to visually represent connections between metabolites and their related indexes across multiple pathways.” could be revised to “Differential metabolites were annotated in KEGG pathways (Figure 4A, 4B, Tables S6–S7), allowing the visualization of metabolite interactions across pathways.”

Lines 317–320: The phrase “These findings offer valuable insights into the complex regulatory networks governing G. elata flower development, establishing a foundation for future research aimed at advancing the functional genomics of this important medicinal plant through targeted improvements.” could be revised to “These findings provide new insights into G. elata flower development and lay the foundation for future research in functional genomics and medicinal applications.”

Author Response

Response to Reviewer 1 Comments

 

Summary                    

Thank you for your professional opinion on our manuscript. Your feedback is of great significance to us in improving our manuscript. We have revised and supplemented the manuscript accordingly based on your suggestions. In the following section, we have your comments in italics and numbered the issues identified. Our responses are shown in normal font, while changes and additions made to the manuscript are highlighted in blue for ease of reference. The following modifications are based on the newly submitted manuscript.

 

Point-by-point response to Comments and Suggestions for Authors

Comments 1: The study presents transcriptomic and metabolomic findings separately but does not establish a strong correlation between gene expression and metabolite levels. Strengthening this connection would provide a more integrated understanding of the regulatory mechanisms described in the manuscript.

Response 1: Thank you for your valuable suggestions for our article. In order to better display the connection of the gene expression and metabolite levels, we add three examples of gene-regulated metabolite changes at the results section mark by blue in manuscript-revised to predictions the function of genes. Thank you for making this valuable suggestion. I hope these changes will fulfill your critics.

Comments 2: Although the study identifies transcription factors (TFs) involved in flower development, it does not elaborate on their specific regulatory mechanisms. Several previous studies have explored this aspect, and a more detailed discussion would enhance the mechanistic insights presented.

Response 2: Thank you for your invaluable suggestions for further experimental validation. We highly value your suggestions, particularly the additional verification of the MADS genes in flower development  in G. elata. However, limitations such as the small sample size and lack of gene function research based on genetic engineering, the need for further investigations to validate the identified regulatory networks and pathways of transcription factors (TFs) like MADS. This will be a key aspect of our future research work. We hope discovered the MADS family and other TFs specific regulatory mechanisms flower development G. elata, while inversely responding to other plant. We fully recognize the significance of this suggestion and plan to explore the functions of these genes in flower development and abiotic stress more thoroughly in future research. We have separated results and discussion. We added transcription factors details in discussion section to explore this aspect. These additions will enhance the comprehensiveness of our data. Thank you again for your valuable feedback.

Comments 3: The manuscript discusses changes in flavonoids, terpenoids, and alkaloids, yet their physiological roles in flower development are not clearly explained. This lack of discussion leaves an unclear link between metabolite accumulation and floral morphology. Providing further insights into these relationships would strengthen the study’s conclusions.

Response 3: Thank you for your valuable suggestions for our article. But, detailed experimental evidence was required to explain the comprehensive mechanism of flower development in G. elata. The connection of  metabolite accumulation and floral morphology still can't to prove in our research. We identified DEGs and metabolites involved in key biological pathways by data. This approach aimed to identify functional genes or pathways jointly enriched in metabolites, thereby defining pathways linked to crucial biological phenotypes. Predictions of gene-regulated metabolite changes were also made. The results demonstrate the potential of genome-wide approaches to unravel the molecular mechanisms underlying G. elata development and evolution. We will strive to gradually prove these issues in future research. We sincerely appreciate your suggestions, which greatly enhance future research directions.

Comments 4: While the manuscript is generally well-structured and grammatically sound, several areas could be refined to improve readability. Below, I provide specific comments on writing aspects that may benefit from revision. For the title, I suggest to replace as “Integrative Transcriptomic and Metabolomic Analysis Reveals Regulatory Networks and Metabolite Dynamics in Gastrodia elata Flower Development”

Response 4:  Thank you for your valuable suggestions for our article. We have revised the title of the paper at the title section mark by blue in manuscript-revised.

Comments 5: Lines 19–21: The phrase " FPKM-based analysis revealed substantial transcriptomic shifts throughout G. elata flower development, while widely targeted metabolomics identified key metabolites with significant variations in abundance across developmental stages.” could be revised to “FPKM analysis showed significant transcriptomic changes during G. elata flower development, while targeted metabolomics identified key metabolites with stage-specific variations.”

Response 5:  Thank you for your valuable suggestions for our article. We have revised the phrase of the paper at the abstract section mark by blue in manuscript-revised.

Comments 6: Lines 21–24: The phrase“This study presents dynamic models of transcriptome and metabolite changes, unveiling crucial regulatory networks underlying G. elata flower growth and development which will enhance knowledge base for G. elata’s applications.” could be revised to “This study proposes dynamic models of transcriptomic and metabolite changes, revealing key regulatory networks that govern G. elata flower development and potential applications.”

Response 6:  Thank you for your valuable suggestions for our article. We have revised the phrase of the paper at the abstract section mark by blue in manuscript-revised.

Comments 7: Lines 40–43: The phrase “Due to rising demand and habitat loss, wild populations of G. elata have significantly declined, leading to its classification as a category II protected wild plant in China and underscoring the urgent need for conservation efforts.” could be revised to “Increasing demand and habitat loss have led to a significant decline in wild G. elata populations, classifying it as a category II protected species in China and highlighting the urgent need for conservation.”

Response 7:  Thank you for your valuable suggestions for our article. We have revised the phrase of the paper at the introduction section mark by blue in manuscript-revised.

Comments 8: Lines 87–90: The phrase“Three developmental stages were selected for transcriptome analysis, with representative tissues designated as Stage1 (TMH1), Stage2 (TMH2), and Stage3 (TMH3). Three biological replicates were examined for each sample.” could be revised to “Three flower developmental stages (Stage 1: TMH1, Stage 2: TMH2, Stage 3: TMH3) were selected for transcriptomic analysis. Each stage was analyzed with three biological replicates.”

Response 8:  Thank you for your valuable suggestions for our article. We have revised the phrase of the paper at the materials and methods section mark by blue in manuscript-revised.

Comments 9: Lines 180–181: The phrase “Figure 1. Flower and petal phenotypes across three flower developmental stages: Stage1 (TMH1) (A), Stage2 (TMH2) (B), and Stage3 (TMH3) (C).” could be revised to “Figure 1. Flower and petal phenotypes at three developmental stages: Stage 1 (TMH1) (A), Stage 2 (TMH2) (B), and Stage 3 (TMH3) (C).”

Response 9:  Thank you for your valuable suggestions for our article. We have revised the phrase of the paper at the results and discussion section mark by blue in manuscript-revised.

Comments 10: Lines 326–329: The phrase “Differential metabolites and their associated indexes were annotated in KEGG pathways (Figure 4A, Figure 4B, Table S6, Table S7), enabling the creation of a network diagram to visually represent connections between metabolites and their related indexes across multiple pathways.” could be revised to “Differential metabolites were annotated in KEGG pathways (Figure 4A, 4B, Tables S6–S7), allowing the visualization of metabolite interactions across pathways.”

Response 10:  Thank you for your valuable suggestions for our article. We have revised the phrase of the paper at the results and discussion section mark by blue in manuscript-revised.

Comments 11: Lines 317–320: The phrase “These findings offer valuable insights into the complex regulatory networks governing G. elata flower development, establishing a foundation for future research aimed at advancing the functional genomics of this important medicinal plant through targeted improvements.” could be revised to “These findings provide new insights into G. elata flower development and lay the foundation for future research in functional genomics and medicinal applications.”

Response 11:  Thank you for your valuable suggestions for our article. We have revised the phrase of the paper at the results and discussion section mark by blue in manuscript-revised. Thank you again for your valuable feedback.

 

 

 

 

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewed comments:

In the submitted manuscript (agronomy-3447381), the authors described the good results that would be additional potential insight for unveiling the crucial regulatory networks underlying flower growth and development of G. elata. However, the written manuscript needs major revisions.

Here are my reviewed comments.

*The written language needs to be improved in the entire manuscript.

*Gene names should be italicized.

*The complete names of all abbreviations should be written in complete form during first-time writing.

Abstract

It is written too short and not meaningfully. Please revise the abstract by explaining the adopted methodology as well as innovative results.

Introduction

It is written in short form. The more literature review on the genetic basis studies regulating the dynamics of flower development in different crop plants should be added.

Methodology

RNA-seq method: The entire methodology for conducting the RNA-seq should be mentioned enough to allow other researchers to replicate experiments. Also, add the proper citation and references for adopted methods. Please improve the written methodology in possible concise form, covering each aspect.

I think that the molecular sequencing data needs to be uploaded to the NCBI website, and the project ID should be mentioned in the text of the manuscript.

Results and Discussion

Please follow the journal format. I think that these two sections need to be inserted in the separate form.

Regarding the discussion, there is a strong need to add the significant review on more comparative studies on genetic basis and genes modulating the flower growth and development of different crop plants. That will help to understand the differentiated genetic phenomenon between different crop plants.

Regards,

Reviewer

Comments on the Quality of English Language

The English could be extensively improved to more clearly express the research.

Author Response

Response to Reviewer 2 Comments

 

Summary                    

Thank you for your professional opinion on our article. Your feedback is of great significance to us in improving our manuscript. We have revised and supplemented the manuscript accordingly based on your suggestions. In the following section, we have your comments in italics and numbered the issues identified. Our responses are shown in normal font, while changes and additions made to the manuscript are highlighted in blue for ease of reference. The following modifications are based on the newly submitted manuscript.

 

Point-by-point response to Comments and Suggestions for Authors

Comments 1: *The written language needs to be improved in the entire manuscript. *Gene names should be italicized. *The complete names of all abbreviations should be written in complete form during first-time writing.

Response 1: Thank you for your valuable suggestions for our article. We have improved written language by EASYEDITION. We have revised the gene name by italicized in manuscript-revised. All abbreviations showed at line 517 of the abbreviations section in manuscript. We have meticulously reviewed and rectified the technical errors in the article.

Comments 2: Abstract. It is written too short and not meaningfully. Please revise the abstract by explaining the adopted methodology as well as innovative results.

Response 2: Thank you for your valuable suggestions for our article. We have revised the abstract and mark by blue in manuscript-revised. I hope these changes will fulfill your recommendation.

Comments 3: Introduction. It is written in short form. The more literature review on the genetic basis studies regulating the dynamics of flower development in different crop plants should be added.

Response 3: Thank you for your valuable feedback. We have provided information on flower development at introduction, and mark by blue in manuscript-revised. At the same time, we included the regulatory effects of transcription factors on flower organ development in discussion, and mark by blue in manuscript-revised. We directly add MADS transcription factors to demonstrate the important role of transcription factors in flower development. I hope these changes will fulfill your recommendation.

Comments 4: Methodology. RNA-seq method: The entire methodology for conducting the RNA-seq should be mentioned enough to allow other researchers to replicate experiments. Also, add the proper citation and references for adopted methods. Please improve the written methodology in possible concise form, covering each aspect. I think that the molecular sequencing data needs to be uploaded to the NCBI website, and the project ID should be mentioned in the text of the manuscript.

Response 4: Thank you for your valuable suggestions for our article. In order to better display the methodology, we have added entire methodology for RNA-seq and references, and mark by blue in manuscript-revised. The RNA-seq data should uploaded to the NCBI website, and the project ID should be mentioned in the text of the manuscript. But due to inability to connect to NCBI database, and the reason is still unclear. So the sequencing data can only be uploaded to the CNGBdb database, with the public identification number ‘CNP0006738’ at line 515 of the Data Availability Statement section in manuscript, and the data will be made public on February 1, 2025. The network connection issue has been reported to the NCBI database, but it still cannot be resolved.

Comments 5: Results and Discussion. Please follow the journal format. I think that these two sections need to be inserted in the separate form. Regarding the discussion, there is a strong need to add the significant review on more comparative studies on genetic basis and genes modulating the flower growth and development of different crop plants. That will help to understand the differentiated genetic phenomenon between different crop plants.

Response 5: Thank you for your valuable suggestions for our article. We have separated results and discussion. Thank you again for your valuable feedback. I hope these changes will fulfill your recommendation.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear, 

In my opinion, the authors incorporated the suggested corrections and revised the manuscript in satisfactory form. So, the revised version is accepted in its current format.

Thanks

Comments on the Quality of English Language

The English could be improved to more clearly express the research.