Combined Analysis of Transcriptomes and Metabolomes Reveals That MeJA-Mediated Flavonoid Biosynthesis Is Crucial for Pigment Deposition in Naturally Colored Green Cotton Fibers
, Asigul Ismayil 1, Xiang Jin 3, Fei Wang 1, Haifeng Liu 2,* and Hongbin Li 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript contains research results on the molecular mechanism of pigment deposition in green cotton fibres, which may apply to the breeding of naturally coloured cotton using genetic engineering techniques.
I would like to ask the Authors to supplement the content of the manuscript with their opinion on the practical applicability of the research results obtained. Can they do this immediately?
Comments
Results
Figures are clear and needed.
Materials and Methods
In which year was the research carried out?
Please include a brief description of the variety studied.
In the manuscript, I found no information about the producer of the statistical software used for data analysis.
References
A large number of publications – 62. Those published before 2015 I propose to remove and limit to the most recent.
Author Response
Reviewer1
The manuscript contains research results on the molecular mechanism of pigment deposition in green cotton fibres, which may apply to the breeding of naturally coloured cotton using genetic engineering techniques.
Comment 1: I would like to ask the Authors to supplement the content of the manuscript with their opinion on the practical applicability of the research results obtained. Can they do this immediately?
Response: Thank you for your comment. Our discovery provides a molecular basis for enhancing the pigment stability and strength of natural colored cotton through genetic engineering. These strategies not only meet the needs of sustainable development in the textile industry but also accelerate their practical applicability.
This part has already been added to the conclusion section in line 472-476.
Comment 2: Figures are clear and needed.
Response: Thank you very much.
Comment 3: Materials and Methods
In which year was the research carried out?
Response: Thank you for your question. The experiment was carried out in 2023-2025. For more detailed information, please refer to the Materials and Methods section of the manuscript. We have added this information to the Materials and Methods section in line 398-400.
Comment 4: Please include a brief description of the variety studied.
Response:Thank you for your suggestion. In the Plant Materials section, we have added detailed information about the colored cotton materials in line 395-398.
Comment 5: In the manuscript, I found no information about the producer of the statistical software used for data analysis.
Response: Thank you for your question. We have included the statistical software and information in the Materials and Methods section in line 466-477.
Comment 6: References
A large number of publications – 62. Those published before 2015 I propose to remove and limit to the most recent.
Response: Thank you for the advice.We made appropriate modifications to the citations of the references.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis manuscript investigates the molecular mechanisms underlying pigment deposition in naturally colored green cotton fibres (GCFS), focusing on flavonoid biosynthesis. Through a combination of transcriptomic and metabolomic analyses, the authors identify methyl jasmonate (MeJA) as a key signalling molecule influencing the upregulation of flavonoid pathway genes, especially chalcone synthase (CHS), in GCFS compared to white cotton fibres (WCFS). The study provides new insights into plant secondary metabolism and offers potential strategies for improving GCF breeding and colouration.
Comment 1: The background literature review is generally adequate, but could benefit from a more focused discussion on the known roles of MeJA in fibre development and colour regulation. The authors do not sufficiently address previous studies on transcriptional regulation of pigmentation in cotton.
Comment 2: Please specify the RNA integrity threshold used for sequencing library preparation.
Comment 3: Can the authors clarify whether the observed association between MeJA and CHS expression is correlative or causative?
Comment 4: Were any other plant hormones or signalling molecules examined in the analysis that might interact with MeJA or influence flavonoid biosynthesis in green cotton fibres?
Comment 5: Have the authors considered whether their findings could be applied to enhance pigment stability or intensity in commercial GCF cultivars through exogenous MeJA application?
Comment 6: Why were certain known genes or pathways involved in fiber coloration (e.g., proanthocyanidin or tannin pathways) not explored in more detail?
Author Response
Reviewer2
Comments and Suggestions for Authors
This manuscript investigates the molecular mechanisms underlying pigment deposition in naturally colored green cotton fibres (GCFS), focusing on flavonoid biosynthesis. Through a combination of transcriptomic and metabolomic analyses, the authors identify methyl jasmonate (MeJA) as a key signalling molecule influencing the upregulation of flavonoid pathway genes, especially chalcone synthase (CHS), in GCFS compared to white cotton fibres (WCFS). The study provides new insights into plant secondary metabolism and offers potential strategies for improving GCF breeding and colouration.
Comment 1: The background literature review is generally adequate, but could benefit from a more focused discussion on the known roles of MeJA in fibre development and colour regulation. The authors do not sufficiently address previous studies on transcriptional regulation of pigmentation in cotton.
Response: Thank you for the advice. We have incorporated the known regulatory effects of MeJA on color development and its research progress regarding cotton fiber development in the Introduction section in line 77-83.
Comment 2: Please specify the RNA integrity threshold used for sequencing library preparation.
Response: Thank you for the advice. We have added this information to the Materials and Methods section in line 411.
Comment 3: Can the authors clarify whether the observed association between MeJA and CHS expression is correlative or causative?
Response: Thank you for your question. Based on previous studies and our experimental results, both exogenous application of MeJA and endogenous jasmonic acid synthesis can regulate the expression of CHS, thereby influencing cotton fiber development (see Lines 69, 128, 164, 357 and 378 for details).
Comment 4: Were any other plant hormones or signalling molecules examined in the analysis that might interact with MeJA or influence flavonoid biosynthesis in green cotton fibres?
Response:Thank you for your question. We have indeed identified other hormones, such as salicylic acid (SA) and brassinosteroids (BR), that are closely associated with both MeJA and flavonoid biosynthesis in green cotton fibers. These findings constitute an ongoing focus of our research and, consequently, have not been extensively elaborated in the current manuscript.
Comment 5: Have the authors considered whether their findings could be applied to enhance pigment stability or intensity in commercial GCF cultivars through exogenous MeJA application?
Response: Thank you. We sincerely appreciate this valuable suggestion and will systematically implement your proposed approach in our future investigations.
Comment 6: Why were certain known genes or pathways involved in fiber coloration (e.g., proanthocyanidin or tannin pathways) not explored in more detail?
Response:Thank you for the comment. We conducted an integrated transcriptomic and metabolomic analysis in this study, with the metabolomic component specifically focused on targeted flavonoid profiling, which represents the core focus of our current work. While we did not perform targeted analysis of proanthocyanidins in this investigation, we sincerely appreciate your suggestion and will certainly consider incorporating targeted proanthocyanidin metabolomics combined with transcriptomic analysis in our future research. Thank you again for this valuable recommendation.
Author Response File: Author Response.pdf
