Integrative Analysis of Transcriptome and Metabolome Reveals Light Quality-Mediated Regulation of Adventitious Shoot Proliferation in Chinese Fir
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study in this manuscript looks at how different types of light affect the growth, transcriptome, and metabolome of the Chinese Fir, a valuable conifer. The manuscript is of good quality, but some small aspects need to be improved before final publication. Below are my comments on all sections of this article.
The Abstract needs to be significantly shortened before publication; please see author instructions at https://www.mdpi.com/journal/information/instructions; "Abstract: The abstract should be a total of about 200 words maximum...."
The Introduction is well-written and represents the strong overall logic of the authors investigations as well as the scientific background of this study.
Comment:
Line 84: "Aquilaria crassna Pierre. plantlets " remove the redundant period here.
Materials and methods
This section is written in sufficient detail and contains a number of informative details about the research conducted, confirming the adequacy of the research conducted.
Comments:
Line 134-135 - What is the approximate age of the seedlings you used?
Line 166 is empty—please remove it.
2.4. - Which algorithm was used to align raw reads to reference? What reference genome/transcriptome are you using? Please indicate it before line 216.
Results
The results overall appear detailed and compelling, highlighting the authors' key findings in their analysis of plant morphometry, transcriptome, and metabolome.
Comments:
Table 2: Legends to this table and other tables and figures should be self-explanatory, with all abbreviations explained ("Comp", "B", etc). What are letters "a", "b", "c", "d"? In this table, the inscriptions Tr2, Tr3, and Tr4 are shifted relative to Tr1, please correct this. It is also advisable to highlight key statistical indicators and relationships in the table in bold.
Figure 1 A—It's very painful for the eyes to look at this figure, please replace the bright red background with something more neutral, maybe white with black inscriptions and black frames. What are letters "a", "b", "c", "d", etc. Please decipher all the abbreviations used in the legend to the figure. Also, please make for all other figures a deciphering of all abbreviations used.
Line 345-346: "In addition, 7,311 new genes were discovered, of which 2,844 were functionally annotated." Please indicate here or in the materials and methods how exactly and with what tools the prediction and functional annotation of new genes was performed.
Discussion
I am satisfied with the quality of the discussion.
An additional optional question is how consistent are the transcriptomic and metabolomic data with each other? Can you integrate them and generalize them into some systemic picture of what is happening? This could be described in the discussion section.
Conclusions
A small but informative section highlighting the authors' main findings.
Please, before publication, make separate Abbreviations and Data Availability sections. In Data Availability, list the descriptions of all additional tables (Table S1 - Table S10) that you provide in the supplements, and also provide all necessary links to repositories with raw data, such as NCBI BioProject. Please check that your data ID is correct in the text on line 230 "SUB14671786" because it is not currently available on request in BioProject. BioProject references usually start with "PRJ".
Author Response
Dear Reviewer, We, all the authors of the manuscript, would like to express our sincere gratitude for your thorough and insightful reviews. Your comments have been extremely valuable in enhancing the quality of our work. We have carefully considered each of your suggestions and concerns, and have made comprehensive revisions to the manuscript. All the changes we have made are clearly marked in red, making it easy for you to identify the modifications. In response to your comments, we have [list some major areas of improvement, such as clarifying the experimental methods, expanding the discussion of certain results, or adding more relevant references]. We believe these revisions have addressed your concerns effectively and have strengthened the overall integrity of the study. In addition, we have uploaded a detailed Revision Description file. This file elaborates on the specific changes we made for each of your comments, providing a clear explanation of how we have incorporated your feedback. We hope this will further assist you in evaluating the revised manuscript. Once again, thank you for your time and expertise. Best regards, all authors
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsAbstract & Introduction:
The abstract mentions WGCNA analysis, but the introduction doesn't explain why this specific analytical approach was chosen over other possible methods.
The timing and duration of light treatments should be specified in the introduction.
• Consider adding a graphical abstract to help readers quickly grasp the key findings and regulatory pathways identified. • Consider expanding the discussion of species-specific responses to light quality mentioned in lines 57-60. • Lines 92-93 mention specific light intensities (30 μmol m−2 s−1 blue, etc.) - it would be helpful to explain how these optimal values were determined. • The roles of the four key genes identified (PSBS, FTSH, GID1, and GST) could be introduced earlier in the abstract.
The abstract is quite dense and could be more concise while maintaining key information.
Consider breaking up some longer sentences (e.g., lines 78-87) for better readability.
Minor comments
• Line 16: "fast-propagating" should be hyphenated consistently throughout the text
• Line 46: Consider adding "tissue culture" to the keywords
• Lines 65-68: The medicinal uses of Chinese fir could be supported with a reference
• The formatting of in-text citations needs to be standardized
Methodology:
• The experimental design is generally well-structured, but the authors should clarify why they chose 52 μmol m−2 s−1 as the light intensity for all treatments. While they mention it's based on previous research, a brief explanation of the rationale would strengthen the methodology. • The composite light treatment (Tr1) appears to be well-justified through previous research using orthogonal rotation experimental design. However, it would be helpful to include a citation for the referenced "previous research [18]" to allow readers to examine the full justification for these specific ratios. • The morphological measurements are thorough, but the authors should explain why they specifically chose the 2nd and 3rd leaf pairs for leaf area measurements. While they mention these leaves are "fully developed," some discussion of potential variability between different leaf positions would be valuable.
The RNA-seq analysis methodology is robust, with appropriate quality control steps and statistical thresholds. However, the authors should specify:
• The number of reads obtained per sample • The mapping rate to the reference genome • Whether they used any specific methods to account for the high GC content typical in conifer genomes
For the metabolomic analysis, the gradient program for UHPLC separation is mentioned but not detailed. This information would be important for method reproducibility.
Result:
Morphological and Physiological Results:
• The comparison between different light treatments is comprehensive • The authors should consider explaining why shoot height showed no significant differences despite other parameters showing clear responses • The relationship between proliferation coefficient and biomass accumulation could be discussed more deeply
Differential Expression Analysis:
• The PCA analysis shows clear separation between treatments • However, the authors should explain why PC1 and PC2 only account for 58.9% of total variance • More discussion is needed on why red light resulted in the highest number of DEGs (772)
Discussion & Conclusion
• Consider including a schematic diagram illustrating the proposed regulatory network of key genes (PSBS, FTSH, GID1, GST) and their interactions with hormones • The hormone signaling discussion could benefit from more detailed mechanistic explanations of how B-ARR and A-ARR interact under different light conditions • Include statistical power analysis to justify sample sizes • The conclusion could be more concise and focused on the novel findings rather than restating results • Consider discussing potential limitations of the study and their implications • Add specific recommendations for practical applications in commercial tissue culture
Comments on the Quality of English Language
Minor revision
Author Response
Dear Reviewer, We, all the authors of the manuscript, would like to express our sincere gratitude for your thorough and insightful reviews. Your comments have been extremely valuable in enhancing the quality of our work. We have carefully considered each of your suggestions and concerns, and have made comprehensive revisions to the manuscript. All the changes we have made are clearly marked in red, making it easy for you to identify the modifications. In response to your comments, we have [list some major areas of improvement, such as clarifying the experimental methods, expanding the discussion of certain results, or adding more relevant references]. We believe these revisions have addressed your concerns effectively and have strengthened the overall integrity of the study. In addition, we have uploaded a detailed Revision Description file. This file elaborates on the specific changes we made for each of your comments, providing a clear explanation of how we have incorporated your feedback. We hope this will further assist you in evaluating the revised manuscript. Once again, thank you for your time and expertise. Best regards, all authors
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis study investigates the effects of different light qualities on regulating adventitious shoot proliferation and growth in tissue-cultured Chinese fir (Cunninghamia lanceolata) at the seedling stage, using a combined transcriptome and metabolome analysis.
Overall, the research and experiments are clearly described. However, there are significant flaws that need to be addressed.
-The manuscript needs to be checked for grammar and punctuation by a native speaker of the English language and certain sentences need to be rewritten. It is also recommended to shorten some sentences throughout the manuscript. In lines 121-122 a part of the sentence can be replaced with “along with the optimal composite light that was identified in our previous study. These lights were generated using light-emitting diodes (LEDs).” In line 586, “Validation” should be replaced with “validation”. In line 594, “R2” should be replaced with “R2”.
-To validate the transcriptome data (line 585), nine genes were randomly selected from the transcriptome. Why weren't these genes selected based on the different expression levels in RNA sequencing from different treatments?
-- In Figure 1, a better picture is needed for (A) the morphology of new adventitious shoots after 35 days of treatment. I also recommend checking again for potential errors in letters B, C, and D.
- More explanation about the results of principal component analysis and the heatmap of hierarchical clustering should be added based on Figure 2.
Comments on the Quality of English LanguageThe English could be improved to more clearly express the research.
Author Response
Dear Reviewer, We, all the authors of the manuscript, would like to express our sincere gratitude for your thorough and insightful reviews. Your comments have been extremely valuable in enhancing the quality of our work. We have carefully considered each of your suggestions and concerns, and have made comprehensive revisions to the manuscript. All the changes we have made are clearly marked in red, making it easy for you to identify the modifications. In response to your comments, we have [list some major areas of improvement, such as clarifying the experimental methods, expanding the discussion of certain results, or adding more relevant references]. We believe these revisions have addressed your concerns effectively and have strengthened the overall integrity of the study. In addition, we have uploaded a detailed Revision Description file. This file elaborates on the specific changes we made for each of your comments, providing a clear explanation of how we have incorporated your feedback. We hope this will further assist you in evaluating the revised manuscript. Once again, thank you for your time and expertise. Best regards, all authors
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAuthors have sufficiently revised the paper, now it can be accepted.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have adequately addressed all concerns and have made the necessary revisions to the manuscript.
