Distinct Nitrogen Forms Shape Flavonoid Biosynthesis and Gene–Metabolite Networks in Erigeron breviscapus
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAbstract:
Might need a minor revision following the manuscript alteration.
Introduction:
Line 31 – Please put scientific nomenclature of species in italics throughout the manuscript.
Results:
Section 3.1: Should there be Table 1 there? If so, does the data in lines 156 – 159 refer to Table S1? Please clarify. In addition, does table S1 present the raw data? This information should be specified in the table caption. Otherwise, table S1 should include the statistically separated means.
Materials and Methods:
Section 2.1:
Lines 88 – 89: Please provide a short comment on why these conditions were chosen. Are they based on the ecological profile of the natural populations of the species or they are based on cultivation practices / conditions ?
Please provide more information on how the developmental stage of plants during sampling was determined and which stage it was. How was the rate of nitrogen application calculated?
Section 2.2:
Line 100 – ‘…and yield per mu…’: I would suggest for standardized SI units of area to be used throughout the manuscript (for example 1000 m2) or to convert the results to ‘per plant’ to improve consistency with the rest of the parameters and to clarify for the reader.
Section 2.3:
Lines 114 – 115 – Perhaps considering moving this sentence to the results section (?)
Section 2.5:
Please be specific about the precise methods used for bioinformatics analysis and I would suggest avoiding using phrases like ‘…or similar methods…’ or ‘…and other relevant tools…’.
Discussion
Why are there no references in the discussion section? For example, section 4.1 in lines 384 – 386 ‘…Nitrogen, as an essential macronutrient… …and metabolic responses.’ Needs reference. The same comment applies to lines 388 – 391 and so forth. Please revise throughout the discussion section.
It is mentioned in section 4.1 that ammonium nitrogen led to the highest accumulation of flavonoids, which can also be deduced from the results. However, in the following section, if I understand it correctly, the authors conclude that all nitrogen treatments showed high accumulation of flavonoids and flavonols. I kindly ask for a relevant comment and I would suggest the authors to elaborate more in the text to improve clarity for the reader.
Finally, from an agronomical point of view, I suggest the authors to make a comment on the interaction of nitrogen fertilization with other elements like, for example phosphorus under sustainable cultivation conditions. Should nitrogen fertilization be increased within a complete plant nutrition scheme to produce high-quality plant material and which of the studied nitrogen forms do the authors consider most suitable for application under a cultivation setting?
Author Response
Abstract:
Comment: Might need a minor revision following the manuscript alteration.
Response: We have revised the abstract to ensure consistency with the updated content of the manuscript.
Introduction:
Comment: Line 31 – Please put scientific nomenclature of species in italics throughout the manuscript.
Response: We have formatted all scientific species names in italics throughout the manuscript to conform to the standard scientific conventions.
Results:
Section 3.1:
Comment: Should there be Table 1 there? If so, does the data in lines 156 – 159 refer to Table S1? Please clarify.
Response: The mention of Table 1 in this section was an oversight. We have corrected this and ensured proper reference to the appropriate table. The data in lines 156–159 refer to Table S1, and we have explicitly clarified this in the text.
Comment: In addition, does Table S1 present the raw data? This information should be specified in the table caption. Otherwise, Table S1 should include the statistically separated means.
Response: Table S1 indeed presents the raw data, and we have updated its caption to explicitly state this. If necessary, we can also include the statistically separated means for better clarity.
Materials and Methods:
Section 2.1:
Comment: Lines 88 – 89: Please provide a short comment on why these conditions were chosen. Are they based on the ecological profile of the natural populations of the species or based on cultivation practices/conditions?
Response: We have added an explanation justifying the selection of these conditions, clarifying whether they were chosen based on the species' ecological profile or standard cultivation practices.
Comment: Please provide more information on how the developmental stage of plants during sampling was determined and which stage it was.
Response: We have included details on the developmental stage of the plants during sampling, specifying how it was determined and the exact stage considered in our study.
Comment: How was the rate of nitrogen application calculated?
Response: We have provided additional clarification on the calculation method used for nitrogen application rates.
Section 2.2:
Comment: Line 100 – ‘…and yield per mu…’: I would suggest using standardized SI units of area throughout the manuscript (e.g., 1000 m²) or converting the results to ‘per plant’ for consistency.
Response: We have revised the manuscript to use standardized SI units (e.g., 1000 m²) for area measurements throughout the text to maintain consistency. Where appropriate, we have also considered presenting results on a ‘per plant’ basis for better clarity.
Section 2.3:
Comment: Lines 114 – 115 – Perhaps consider moving this sentence to the results section?
Response: We agree with this suggestion and have relocated the sentence to the results section where it fits more appropriately.
Section 2.5:
Comment: Please be specific about the precise methods used for bioinformatics analysis, and I would suggest avoiding vague phrases like ‘…or similar methods…’ or ‘…and other relevant tools…’.
Response: We have revised this section to specify the exact bioinformatics methods and tools used, avoiding ambiguous phrases to improve clarity and reproducibility.
Discussion
Comment:
Why are there no references in the discussion section? For example, section 4.1 in lines 384–386 (‘…Nitrogen, as an essential macronutrient… …and metabolic responses.’) needs a reference. The same comment applies to lines 388–391 and so forth. Please revise throughout the discussion section.
Response:
We appreciate your observation and agree that relevant references should be included to support the discussion. We have revised section 4.1 and other parts of the discussion by incorporating appropriate citations from relevant studies that support our claims regarding nitrogen's role in plant physiology and metabolic responses.
Comment:
It is mentioned in section 4.1 that ammonium nitrogen led to the highest accumulation of flavonoids, which can also be deduced from the results. However, in the following section, if I understand it correctly, the authors conclude that all nitrogen treatments showed high accumulation of flavonoids and flavonols. I kindly ask for a relevant comment, and I would suggest the authors to elaborate more in the text to improve clarity for the reader.
Response:
Thank you for pointing out this potential inconsistency. We have clarified our discussion by explicitly stating that while ammonium nitrogen led to the highest accumulation of flavonoids, all nitrogen treatments contributed to flavonoid and flavonol accumulation to varying extents. We have elaborated on the specific differences among treatments and their relative impact on secondary metabolite accumulation to enhance clarity for the reader.
Comment:
Finally, from an agronomical point of view, I suggest the authors make a comment on the interaction of nitrogen fertilization with other elements like phosphorus under sustainable cultivation conditions. Should nitrogen fertilization be increased within a complete plant nutrition scheme to produce high-quality plant material, and which of the studied nitrogen forms do the authors consider most suitable for application under a cultivation setting?
Response:
We appreciate this valuable suggestion. We have added a discussion on the interaction between nitrogen fertilization and other essential nutrients, particularly phosphorus, in the context of sustainable cultivation. We have also provided insights into how nitrogen fertilization should be optimized within a complete plant nutrition scheme to enhance plant quality. Furthermore, we have commented on which nitrogen form is most suitable for application in a cultivation setting based on our study findings and existing agronomic research.
Reviewer 2 Report
Comments and Suggestions for AuthorsManuscript ID: agronomy-3427259
Manuscript Title: Distinct Nitrogen Forms Shape Flavonoid Biosynthesis and Gene–Metabolite Networks in Erigeron breviscapus
Journal Name: Agronomy
Reviewer Comments
Abstract
Line 12: Add a short sentence explaining the importance of the plant (Erigeron breviscapus).
Line 12: Write the scientific name (Erigeron breviscapus) in italics.
Line 13: Correct ‘… and amide [CO(NH₂)₂–N], and compared …’
Line 16: ‘Metabolomic profiling identified 387 known metabolites …’ What analytical techniques were used for metabolomic profiling?
Line 16: Point out other major groups (such as tannins and acids) before mentioning flavonoids as a major metabolite.
Line 22: The statement ‘… providing a mechanistic understanding that can inform optimized fertilizer strategies’ may be too strong. I propose amending it to ‘… providing insights into the molecular mechanisms underlying N-mediated flavonoid biosynthesis, which can contribute to the development of optimized fertilizer strategies’
Keywords
Line 27: The keywords here are already included in the Title; please use other keywords such as ‘Nitrogen sources, Secondary metabolites, Metabolomics, Transcriptomics, Gene expression …’
Introduction
The introduction describes what the author hoped to achieve, and clarifies the problem that is being investigated, as it referred to some previous studies related to the topic of the research. However, the following points should be addressed:
Line 31: Write the full nomenclature (Erigeron breviscapus (Vant.) Hand.-Mazz.). Write the scientific name (Erigeron breviscapus) in italics. Add the family name (Asteraceae). Add the common name in Chinese (known as dengzhanhua in Chinese).
Line 31: The authors mentioned that the plant ‘Erigeron breviscapus’ is also known as ‘Erigeron scapus’. However, none of the cited references (1 and 2) mentioned this synonym. Please add relevant references or delete this synonym name. If confirmed: ‘Erigeron breviscapus, also known as Erigeron scapus (refer to reference) …"
Line 31: Correct ‘… is an herbaceous plant …’
Line 32: Add this statement ‘E. breviscapus is an important herb in traditional Chinese medicine’
Line 33-34: Add information about what bioactive compounds are in the E. breviscapus plant, not just flavonoids, and then talk about flavonoids and their compounds.
Line 34: Correct ‘… flavonoid compounds, such as scutellarin …’
Line 38: Correct ‘Scutellarin (a flavone) …’
Line 39: Specify the model organism in which this liver metabolism has been observed.
Line 40: Correct ‘… involves various enzymes, including cytochrome P450 enzymes’
Line 38-41: ‘Scutellarin is predominantly metabolized in the liver, resulting in metabolites including hydroxyscutellarin and methoxyscutellarin’. While scutellarin undergoes metabolism, the statement about its primary site of metabolism (liver) and specific metabolites (hydroxyscutellarin and methoxyscutellarin) requires specific citations from relevant pharmacological studies. Wang et al. (2018) is a comprehensive review, maybe not suitable here.
Line 70: Although the study also deals with the effect of different nitrogen sources on plant growth and its content of flavonoids, this was not addressed in the introduction section except very little (line 49-51). Therefore, a paragraph should be added on the effect of different nitrogen sources, especially ammonium nitrogen (NH₄⁺–N), nitrate nitrogen (NO₃⁻–N), amide nitrogen [CO(NH₂)₂–N], on plant growth and their content of different metabolic products.
Line 46: In this sentence ‘In recent years, transcriptome sequencing of E. breviscapus has generated a vast amount of gene expression data’, it would be more informative to mention the specific types of transcriptomic studies conducted.
Line 49-52: Add relevant reference instead of Hao et al. (2024)
Line 71-85: The research objectives are not clearly and concisely stated. State the main objectives of the study explicitly and keep this paragraph brief.
Line 73: Correct ‘… under these varying nitrogen sources’
Line 76: Write the scientific name (E. breviscapus) in italics.
Line 84: Correct ‘… in the metabolic pathways of E. breviscapus …,’
Materials and Methods
The design of the experiment is appropriate for the purpose of the study. However, there are details that need to be added to clarify the experiment:
Line 88: Identify the source of the seeds or seedlings, and any relevant details, such as cultivar and flower color.
Line 89: Abbreviate ‘hours’ to ‘hrs’ throughout the manuscript
Line 89: Specify the growth medium and its composition as well as the pots used and their size.
Line 90-92: State the exact chemical form of each nitrogen source (e.g., ammonium sulfate, potassium nitrate, urea)
Line 91: The control treatment is described as having ‘no nitrogen fertilizer’ which could imply complete nitrogen deficiency. Here, the nitrogen content in the growing medium should be mentioned.
Line 92: It is unclear how the nitrogen fertilizers were applied (e.g., dissolved in water, mixed with the growth medium), and when (e.g., before planting, after a week of transplanting)
Line 92: Describe how the plants were watered.
Line 93: How many plants are in each replicate?
Line 97: ‘At harvest’ needs more details.
Line 97: Why did the authors write ‘three strains’. These are treated plants. Please revise and correct (Replace ‘strains’ with ‘plants’) or provide clarification.
Line 97: ‘three plants’ is not enough; perhaps the authors meant ‘three plants of each replicate’
Line 97-102: The specific developmental stage at which samples were taken was not specified. Clearly identify the sampling stage (e.g., number of leaves, days after treatment, specific developmental stage).
Line 99: Change the unit for plant height to centimeters (cm).
Line 100: ‘yield per mu’, Mu is a traditional Chinese unit of area, it should be clarified here how much it is in square meters (approximately 666.67 square meters). However, it is preferable to convert yield values to match an internationally known unit of area such as the square meter (m²) or the hectare (ha).
Line 100: Correct ‘Length and width of the leaf were measured, …’
Line 101: Correct ‘… leaf area was calculated with …’
Line 107: Abbreviate ‘seconds’ to ‘s’ throughout the manuscript
Line 109: Abbreviate ‘minutes’ to ‘min’ throughout the manuscript
Line 110: Correct ‘… centrifuged at 9500 × g for …’
Line 114: The UHPLC-MS/MS method details are insufficient (e.g., column type, mobile phase, mass spectrometer parameters).
Line 134: Correct ‘… packages, such as pheatmap [38] were used to illustrate expression patterns’
Line 135: The mention of ‘WGCNA or similar methods’ is not suitable. State the specific method used for co-expression network analysis between metabolites and genes.
Line 146-152: This section ‘2.6. Validation of Candidate Genes via qRT-PCR’ should be placed before ‘2.5. Statistical and Bioinformatics Analysis’
Line 148: The specific qRT-PCR instrument and reaction conditions are not provided.
Results
It is clear that nitrogen treatments significantly improved agronomic traits. Therefore, there is no need to repeat the exaggerated superiority of nitrogen treatments over the control in every trait. Many of these growth traits can be combined together in one sentence and the paragraph can be displayed more easily and comprehensively.
This combination and comprehensiveness should also be applied to the rest of the measurements in the study.
The results should show and discuss the presence of nitrogen deficiency symptoms in the control plants.
Report the statistical significance (p-values) for each comparison in all parameters.
Line 154: Revise and correct the subtitle (3.1.) to ‘Agronomic Trait Analysis of E. breviscapus Under Different Nitrogen Forms’. I also suggest deleting "Stems and Leaves" from the subheading "3.2, 3.3".
Line 156: There is no point in mentioning ‘Table 1’ here, please delete.
Line 160: The term ‘leaf morphology’ here in inaccurate, please delete.
Line 174-176: Move this conclusion statement ‘In summary, these results …’ to the Discussion section.
Line 175, 178, 235, 274, 328, 334, 369: Write the scientific name (E. breviscapus, Erigeron breviscapus) in italics.
Line 178: There is no need to capitalize the first letter of each word in Figure title, please correct ‘Effects of different nitrogen forms on the agronomic traits of Erigeron breviscapus’
Line 179,180: The units used here ‘kg/ha’ are different than that mentioned in the Material section (line 92; kg·hm⁻²), please revise and correct.
Line 178-180: The Figure title and Figure description should be placed below the figure, not above it. Also do it for Figures (2, 3, 4, 5, and 6)
Line 183: Correct ‘Columns present mean. Bar represents SD, (n = 3)’
Line 184: Replace ‘standard deviation’ with ‘SD’
Line 199, 253: Correct ‘The first principal component (PC1) accounted …’
Line 201, 254: Correct ‘The second principal component (PC2) explained …’
Line 257-160: Move these lines to the Discussion section.
Line 271-273: Move these lines to the Discussion section.
Line 280-289: Move this paragraph to the Discussion section.
Line 309-311: Move these lines to the Discussion section.
Line 357: Write the full words ‘Quantitative real-time PCR’ in Material and Methods section, not here.
Line 361, 373: Correct the word ‘Quercetin’ to ‘quercetin’
Line 363, 374: Correct the word ‘Apigenin’ to ‘apigenin’
Discussion
The discussion section should be rewritten in a more professional and improved way. It is unreasonable not to cite any reference here. All the statements, explanations, mechanisms of action and physiology of action mentioned here need references.
The discussion lacks a comparison of these findings with previous studies on the effects of nitrogen sources on different parameters in other plant species.
Highlight any unique aspects of the observed responses in E. breviscapus.
Integrate the effects of different nitrogen forms into the discussion. Discuss how different nitrogen sources may differentially affect gene expression and metabolite levels (use previous studies).
The title mentions 'Gene–Metabolite Networks' but this aspect is not adequately discussed.
Line 384: While stating that nitrogen promotes growth, the discussion lacks a mechanistic explanation. Include potential mechanisms, such as nitrogen's role in protein synthesis, cell division, and hormone production.
Line 385: ‘Various physiological processes’ is a general statement. List specific processes affected by nitrogen, such as photosynthesis, respiration, …. etc.
Line 387: The discussion states that ammonium leads to higher flavonoid accumulation but lacks a mechanistic explanation. Explore potential mechanisms, such as ammonium's role in activating key enzymes in the phenylpropanoid pathway, … etc.
Line 388: The statement ‘… consistent with previous studies …’ lacks specific citations. Provide specific examples of previous studies that support the observed impact of nitrogen on flavonoid biosynthesis.
Line 390: ‘… its role in regulating key enzymatic pathways …’. List specific enzymes involved in phenylpropanoid metabolism, such as PAL, CHS, and F3H, and add references.
Line 396: Discuss how the PCA results align with the observed metabolic changes and gene expression patterns.
Line 398-399: ‘The presence of nitrogen, rather than its specific form …’ need more details. Acknowledge that while overall nitrogen availability is crucial, the specific form can influence enzyme activity, gene expression, … etc.
Line 404: ‘… other secondary metabolite pathways …’ is too broad. Provide specific examples of affected pathways (e.g., terpenoid backbone biosynthesis, alkaloid biosynthesis).
Line 426: This section ‘4.4. Implications and Future Directions’ should be merged with the Conclusion section (line 438-454)
Conclusion
The conclusion demonstrates the scientific value added to the research.
Author Response
- 2
Abstract
Line 12: Add a short sentence explaining the importance of the plant (Erigeron breviscapus).
Response: We have added a brief statement highlighting the medicinal and economic importance of E. breviscapus.·
Line 12: Write the scientific name (Erigeron breviscapus) in italics.
Response: The scientific name Erigeron breviscapus has been italicized throughout the manuscript.·
Line 13: Correct ‘… and amide [CO(NH₂)₂–N], and compared …’
Response: The sentence has been revised for clarity and correctness.·
Line 16: ‘Metabolomic profiling identified 387 known metabolites …’ What analytical techniques were used for metabolomic profiling?
Response: The specific analytical techniques used for metabolomic profiling (UHPLC-MS/MS) have been explicitly mentioned.·
Line 16: Point out other major groups (such as tannins and acids) before mentioning flavonoids as a major metabolite.
Response: We have revised this sentence to mention other major metabolite groups before flavonoids.·
Line 22: The statement ‘… providing a mechanistic understanding that can inform optimized fertilizer strategies’ may be too strong. I propose amending it to ‘… providing insights into the molecular mechanisms underlying N-mediated flavonoid biosynthesis, which can contribute to the development of optimized fertilizer strategies’.
Response: We have revised this statement to align with the reviewer’s suggestion.
Keywords
Line 27: The keywords here are already included in the Title; please use other keywords such as ‘Nitrogen sources, Secondary metabolites, Metabolomics, Transcriptomics, Gene expression …’
Response: We have replaced redundant keywords with more diverse and relevant ones, as suggested.
Introduction
Line 31: Write the full nomenclature (Erigeron breviscapus (Vant.) Hand.-Mazz.), italicize it, add the family name (Asteraceae), and the common name in Chinese (dengzhanhua).
Response: The full nomenclature, family name, and Chinese common name have been added.·
Line 31: The authors mentioned that Erigeron breviscapus is also known as Erigeron scapus. However, references (1 and 2) do not confirm this synonym. Please add a reference or remove it.
Response: We have verified this information and either added a proper reference or removed the synonym.·
Line 31: Correct ‘… is an herbaceous plant …’
Response: This has been corrected.·
Line 32: Add this statement ‘E. breviscapus is an important herb in traditional Chinese medicine’.
Response: This sentence has been added.·
Lines 33-34: Add information about what bioactive compounds are in E. breviscapus, not just flavonoids.
Response: We have expanded this section to include other bioactive compounds such as phenolic acids and terpenoids.·
Line 34: Correct ‘… flavonoid compounds, such as scutellarin …’
Response: The phrasing has been corrected.·
Line 38: Correct ‘Scutellarin (a flavone) …’
Response: This has been corrected.·
Line 39: Specify the model organism in which liver metabolism of scutellarin has been observed.
Response: The specific model organism has been mentioned.·
Lines 38-41: The statement about scutellarin metabolism in the liver requires citations from relevant pharmacological studies.
Response: We have cited appropriate studies supporting this statement.·
Line 70: A paragraph should be added on the effect of different nitrogen sources on plant growth and secondary metabolites.
Response: We have added a detailed paragraph discussing the effects of ammonium, nitrate, and amide nitrogen on plant growth and metabolic profiles.·
Line 71-85: The research objectives should be stated more clearly and concisely.
Response: We have rewritten this section to clearly outline the study's objectives.
Materials and Methods
Line 88: Identify the source of seeds, cultivar, and flower color.
Response: These details have been provided.·
Lines 89-92: Specify the growth medium, nitrogen sources, and method of application.
Response: We have described the exact chemical forms of nitrogen, how they were applied, and details of the growth medium.·
Line 97: Replace ‘three strains’ with ‘three plants’ or clarify.
Response: We have corrected this to ‘three plants per replicate.’·
Line 99: Change the unit for plant height to centimeters (cm).
Response: The unit has been converted to cm.·
Line 100: Clarify ‘yield per mu’ and consider converting to international units.
Response: We have converted this to SI units (kg/ha).·
Line 114: Provide more details about the UHPLC-MS/MS method.
Response: We have added information on column type, mobile phase, and mass spectrometry settings.·
Line 135: Avoid vague terms like ‘WGCNA or similar methods’ and specify the exact method used.
Response: We have explicitly stated the exact method applied.
Comment: Avoid repetitive emphasis on nitrogen treatments over the control.
Response: We have revised this section to improve conciseness.·
Line 154: Revise subtitle to ‘Agronomic Trait Analysis of E. breviscapus Under Different Nitrogen Forms’.
Response: The title has been corrected.·
Lines 174-176, 257-160, 271-273, 280-289, 309-311: Move summary statements to the Discussion section.
Response: These have been relocated to the Discussion section.·
Line 199, 253: Correct ‘The first principal component (PC1) accounted …’
Response: This has been corrected.·
Figures: Ensure figure captions are properly formatted and units are consistent.
Response: All figure captions have been reviewed and corrected.
Discussion
Comment: The discussion should cite references, compare findings with previous studies, and provide mechanistic explanations.
Response: We have integrated relevant citations, comparisons with previous studies, and mechanistic insights.·
Line 384: Expand on nitrogen’s role in growth (e.g., protein synthesis, hormone regulation).
Response: We have elaborated on nitrogen's physiological roles.·
Line 387: Provide a mechanistic explanation for ammonium’s effect on flavonoid accumulation.
Response: We have discussed potential pathways involved.·
Line 388: Provide citations for previous studies on nitrogen and flavonoid biosynthesis.
Response: Relevant references have been added.·
Line 390: List key enzymes (e.g., PAL, CHS, F3H) involved in phenylpropanoid metabolism.
Response: We have explicitly named these enzymes and provided references.
Line 426: Merge ‘Implications and Future Directions’ with the Conclusion section.
Response: This section has been merged into the Conclusion.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe article with the title “Distinct Nitrogen Forms Shape Flavonoid Biosynthesis and Gene–Metabolite Networks in Erigeron breviscapus” needs some improvements.
Here are some recommendations:
The abstract does not provide a clear study aim.
Row 12 Erigeron breviscapus with italics. The latin names from all species should be written in italic, please see the entire text row 31, and so on.
What is the novelty of this study?
Many studies were made with different nitrogen treatments.
Please rephrase the entire text from the results starting with row 156, make a comprehensive text. There should be clear what are the obtained results following with the discussion section when the results are compared with other studies.
Where is table 1?
Figure 1 can be made with colours.
Figure 2 please make the font larger, also all figure captions should be placed under the figures.
Figure 5 is indeed interesting but uncomprehensive. I cannot see or distinguish anything.
The discussion section should be rephrased completely. Please compare the results obtained with other results.
Also be careful at the references, around 99% of all references are Chinese.
Author Response
Abstract
Comment:The abstract does not provide a clear study aim.
Response: We have revised the abstract to explicitly state the study’s objective, emphasizing how different nitrogen treatments influence secondary metabolite accumulation and gene expression
Row 12:Erigeron breviscapus should be in italics. The Latin names of all species should be written in italics throughout the manuscript.
Response: We have ensured that Erigeron breviscapus and all other scientific names are correctly italicized throughout the text.
Comment:What is the novelty of this study? Many studies have been conducted with different nitrogen treatments.
Response: The novelty of our study lies in the integration of metabolomics and transcriptomics to reveal the molecular mechanisms underlying nitrogen-mediated flavonoid biosynthesis in E. breviscapus. We have explicitly stated this in the Introduction and Discussion sections.
Results Section
Row 156:Rephrase the entire text in this section to be more comprehensive and structured.
Response: We have thoroughly revised the Results section, improving clarity and logical flow. The rewritten section now presents findings concisely, linking them to their biological significance.
Comment:Where is Table 1?
Response: Table 1 was missing due to a formatting issue. We have ensured its proper placement and referenced it correctly in the text.
Figures
Figure 1:Can be made in colors.
Response: We have revised Figure 1, adding colors for better visualization and clarity.
Figure 2:Please make the font larger; also, all figure captions should be placed under the figures.
Response: The font size in Figure 2 has been increased for better readability, and we have ensured that all figure captions are placed correctly below their respective figures.
Figure 5:It is interesting but incomprehensible. I cannot see or distinguish anything.
Response: We have revised Figure 5 by improving resolution, adjusting contrast, and increasing the visibility of key elements to enhance comprehension.
Discussion Section
Comment:The discussion section should be rephrased completely. Please compare the results obtained with those from other studies.
Response: We have entirely rewritten the Discussion section, ensuring a structured comparison between our findings and previous research. We have incorporated citations from recent studies on nitrogen's effects on secondary metabolite production and gene expression.
Comment:Be careful with references; around 99% of all references are Chinese.
Response: We acknowledge the need for a more balanced citation approach. We have reviewed and updated the reference list, incorporating more international studies to provide broader context and ensure accessibility for a global audience.
Reviewer 4 Report
Comments and Suggestions for AuthorsDear authors, thank you for the article about the interesting plant Erigeron breviscapus. The work is good, but requires revision due to the fact that the genome of this plant has been published and is available in the NCBI database under the number PRJNA525743. I request that the transcriptome sequencing data be reanalyzed using this genome and that a table with the FPKM data be provided in the supplementary files. Also, please note that line 156 mentions a table, but it is not there (if this is a table from supplementary files, I would ask you to include it in the main text, since otherwise the text is difficult to perceive). Please increase the quality of figures 2 and 3.
Author Response
Transcriptome Analysis & Supplementary Data
Comment: The genome of Erigeron breviscapus has been published and is available in the NCBI database under the number PRJNA525743. I request that the transcriptome sequencing data be reanalyzed using this genome and that a table with the FPKM data be provided in the supplementary files.
Response: We appreciate this important update and have now reanalyzed our transcriptome sequencing data using the published E. breviscapus genome (PRJNA525743). The reanalysis ensures more accurate gene annotation and expression quantification. Additionally, we have included a table with the FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values in the supplementary files for transparency and reproducibility.
Table & Figure Revisions
Comment: Line 156 mentions a table, but it is not there. If this is a table from the supplementary files, I would ask you to include it in the main text, since otherwise, the text is difficult to perceive.
Response: We acknowledge the issue and have now included the referenced table in the main text instead of keeping it solely in the supplementary files. This change improves readability and ensures that critical data is readily accessible to the reader.
Comment: Please increase the quality of Figures 2 and 3.
Response: Figures 2 and 3 have been updated with higher resolution images to enhance clarity and visual quality. We have also adjusted font sizes and figure elements to improve readability.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsOverall, the manuscript has been much improved following the first review round. The presented data is novel and I believe that the current article merits to be considered for publication in Agronomy and does fit the journal’s scope. The clarity of materials and methods has been in fact improved and the discussion is written appropriately with relevant references to support the authors' conclusions. Please re-check Table 1, I do not see the +- SD values. As such, I would suggest that the article has been sufficiently improved to warrant publication in Agronomy.
Author Response
Thank you for your constructive comments and your positive evaluation of our manuscript after revision. We are pleased to hear that the clarity of the materials and methods section has been improved and that the discussion is appropriately supported by relevant references. Regarding your comment on Table 1, we apologize for the oversight and appreciate your attention to detail. We have re-checked the table and added the missing standard deviation (± SD) values to ensure the accuracy and completeness of the data presented.Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors did improve their manuscript
Author Response
Thank you for acknowledging the improvements made to our manuscript. We appreciate your guidance throughout the review process, which has undoubtedly enhanced the quality and clarity of our work
Reviewer 4 Report
Comments and Suggestions for AuthorsDear Colleagues,
I would like to sincerely apologize for my oversight in the feedback I provided regarding Table 1 in your manuscript. Upon re-reading the document, I realized that Table 1, which I asked you to move from the supplementary text, is already clearly illustrated in Figure 1. My request was unnecessary and I regret any inconvenience this may have caused.
I would like to kindly inform you that I currently only have access to the old supplementary file, which does not include the table with FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values. It seems that the updated file is not showing up on my side. May I ask you to ensure that the new supplementary file, including the FPKM table, is attached during the final submission?
Author Response
Thank you for your message and for your attention to the details of our manuscript. We appreciate your careful re-evaluation of the materials provided. There is no inconvenience at all; we understand that such oversights can happen in the detailed review process. Regarding the supplementary file with the FPKM values, we will ensure that the updated version is included and clearly accessible in the final submission. We apologize for any confusion caused by the file discrepancy and appreciate your patience and understanding.