Physiological and Transcriptomic Analyses Reveal the Mechanisms Underlying Methyl Jasmonate-Induced Mannitol Stress Resistance in Banana

Comments 1: The authors used four set of samples as, MeJA treated plants for 0h and 8h, and with 2days mannitol treatment for both. Thus, the samples are 0h-MeJA, 8h-MeJA, 0hMeJa+48hMa, 8hMeJa+48hMa.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have changed the names of group methods and applied them to the figure 1. And please see manuscript for details.

Line 92 – line 97: We changed the description of processing method.

Comments 2: The authors did not clearly mention which samples were taken for RNA-seq analysis.

Response 2: The description of the sample used for transcriptome sequencing had given in both the part of results and materials & methods. However, we made adjustments to avoid duplication.

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Comments 3: The figure legends does not carry sufficient information, (for eg. The authors did not even mention and explain about A0, B0, A8 and B8, in the figure legend of Fig. 2).

Response 3: Thank you for helping us to notice that the legend was not comprehensive. We have added the legend for figures.

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Comments 4: The results and materials & methods and overlaps one another.

Response 4: Agree, and we rewrite the method we treat plant material and made adjustment to other parts.

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Comments 5: The promoter was mentioned as reporter in materials and methods.

Response 5: Agree. Thank you very much. We have fixed this mistake.

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Comments 6: The discussion volumes less.

Response 6: Agree. We contributed to the discourse regarding the involvement of MeJA in enhancing plant resilience and elucidated the reaction of bananas towards adverse conditions.

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Comments 7: The references are not included in the text body of the article

Response 7: Agree. We have updated the references for the full article.

Comments 8: The supportive documents are missing.

Response 8: Thank you for your comments. We have included a supplementary document at the conclusion of the manuscript.

Comments 9: They should have compared this data with 0hMeJa vs 0hMeJa+48hMa, to pool out genes that are specifically regulated with Mannitol treatment. The this data should be compared with DEGs pooled out from 0hMeJa+48hMa vs 8hMeJa+48hMav, to get the genes that are MeJA responsive as well as with role in drought resistance.

Response 9:

Response to Comments on the Quality of English Language

Point 1: The article carries grammatical and syntax errors and requires thorough English correction.

Response 1: We have conducted a comprehensive English revision of this article. The revised sections in the article have been highlighted but are currently hidden. You will be able to view all the revisions when reviewing it.

5. Additional clarifications

All revisions made to the article have been appropriately highlighted, while specific modifications have been concealed to ensure protection against any amendments that could affect the overall lines number of the manuscript.

Comments 1: In the title, mention to mannitol stress in our title.

Response 1: Many thanks for your suggestions, we had added the ‘mannitol stress’ in the title.

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Comments 2: The figures need to be improved. For Figure 9, add statistical analysis. The resolution of figures must be improved.

Response 2: Agree. Thank you for your suggestion. We have made adjustments to ensure that the figures are clearly visible, including resizing the words and enhancing the clarity of the figures.

Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 9

Comments 3: Improve the keywords. under osmotic stress, cannot be use under as a keyword.

Response 3: Agree. We modified "Under osmotic stress" to "Mannitol-induced stress".

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Comments 4: The introduction section must be improved by adding more information on the sensitivity of banana to stress.

Response 4: We appreciate your advice. We have incorporated additional information regarding the sensitivity of bananas to stress in the introduction.

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Comments 5: Add more information on the biochemical change of the aim of the work must be rewritten.

Response 5: Thanks for your suggestion we have added a description of the aim for biochemical measurement.

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Comments 6: The references of introduction should be revised. In the discussion section, the references should be edited major errors has been found. The references should be updated throughout the manuscript.

Response 6: Agree. We have updated the references for the full article.

Comments 7: The conclusion should be rewritten.

Response 7: Thanks for your suggestion, we have revised the conclusion part.

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Comments 8: Why did the authors not include a control without addition of mannitol? What is the number of plants used per replica? how many replicates per treatment?

Response 8: We are currently unaware of the part which the control without additional mannitol. However, we have rewritten the treatment method of plant material mentioned in the article. We added the number of biological replicates in the experiment and the number of plants used in each replicate.

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Additional clarifications

All revisions made to the article have been appropriately highlighted, while specific modifications have been concealed to ensure protection against any amendments that could affect the overall lines number of the manuscript.

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

Comments 1:

Line 168-169, Is 13,265 DEGs too much? As I learned about 36,000 genes expressed in bananas, 1/3 of genes are differentially expressed in roots. Do the authors have any explanations for that? I do suggest authors set a cutoff of the expression level (e.g., FPKM>=1) before performing further analysis.

Line 164, what do authors mean by setting |log2 fold-change (FC)| > 0? Why did the authors set the parameter logFC without filtering any candidate genes?

Response 1: Response to Reviewer 3, thank you so much for your valuable comments. We have observed an inadvertent error in setting the fold-change (FC) threshold for screening differentially expressed genes (DEGs), prompting us to revise the criteria to | log2 fold-change (FC)| > 1. Consequently, a reduction in the number of DEGs has been noted. Specifically, our revised analysis reveals that there are now 7,966 differentially expressed genes in the root, contrasting with the previously reported number of 13,256. These updated findings have been incorporated into both the part of results 2.3 and Figure 2 of this study.

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Comments 2:  Libe 193, the same for the GO analysis with all the DEGs, are 4,140 GO terms too much? Besides, are there any different enriched terms for upregulated and downregulated genes?

Response 2: I agree. Thanks for your inquiries and suggestions regarding the GO enrichment analysis results section. Based on your comments and the most recent findings from DEGs screening, we have revised and updated both the GO enrichment results and Fig. 3 accordingly. The enriched terms of upregulated and downregulated genes are included there.

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Comments 3:  Line 147, if 87% is the average percentage, please indicate it’s an average percentage.

Response 3: Thank you for your inquiry, which has brought to our attention that the provided description lacks clarity. It should be noted that the mentioned 87% represents the minimum percentage rather than an average. However, we have revised the data to reflect an average percentage and ensured its explicit clarification.

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Comments 4:  Fig 2B. “Differential Expressed Genes” -> “Differentially Expressed Genes”. Please label the genes without any differences (blue dots) in the figure.

Response 4: Thanks for your comments. We changed the DEGs filters and updated the results in Fig. 2B, C. And we label the genes without any differences (gray dots).

Figure 2

Comments 5:  Check the manuscripts including the formats (e.g., number format in the main text, fig.2, and Supp. Tables is not consistent), references, etc.

Response 3: Thank you very much for your comments. We have reunified the text format and size in the manuscripts.

Response to Comments on the Quality of English Language

Point 1: Need minor improvement.

Response 1: We further improved the language and established a standardized writing format.

Additional clarifications

Based on the newly acquired differentially expressed genes (DEGs), we conducted a re-analysis of the KEGG pathways, resulting in slight modifications as depicted in Figure 4. In banana leaves, the pathways "photosynthesis-antenna proteins" (mus00196), "photosynthesis" (mus00195), and "linoleic acid metabolism" (mus00591) still exhibit the highest enrichment index. Similarly, in banana roots, the pathways with the highest enrichment index remain unchanged: "linoleic acid metabolism" (mus00591), "selenocompound metabolism" (mus00450), and "biotin metabolism" (mus00780). These pathways are still worth discussing and do not affect subsequent results.

Figure 4

Point 1: language edition should be done.

Response 1: We appreciate your comments. We further improved the language and established a standardized writing format.