Genome-Wide Analysis and Expression of MYC Family Genes in Tomato and the Functional Identification of slmyc1 in Response to Salt and Drought Stress
Round 1
Reviewer 1 Report
The manuscript is well planned, well organized and well written.
Some typo mistakes in scientific name ( line 203, 175, 246 etc) need to be corrected.
Please download the attachment and revise according the report.
Comments for author File: 
Comments.pdf
Author Response
Dear editor,
Based on the comment and request, we have made extensive modification on the original manuscript. Here, we resubmit the revised manuscript, for your approval. Every question from the referee was also answered below. Thank you and all the reviewers for the kind advice.
Sincerely yours,
Huini Xu
Dear reviewers:
I am very grateful to your comments for the manuscript. According with your advice, we amended the relevant part in the manuscript. Some of your questions were answered below.
1] Some typo-mistakes in scientific name (line 203, 175, etc), word “conservative” line 216 need to be corrected.
Response: We have changed these mistakes.
2] In Fig. 2, please add description of short terms like Nt, Zm, Ta etc in fig legend.
Response: We have added this part.
3] Fig.1, Please show SLMYC gene distribution on chromosomes by synteny relationship.
Response: We have redrawn the figure and the synteny relationship was added.
4] Put a reference for the work on “low nitrogen stress”-line 315.
Response: The RNA-seq data was not published yet. So we added data not shown.
5] Please describe ROS content measurements in Materials and Methods section.
Response: The method was added.
6] Finally, the manuscript needs English language edit.
Response: The language of the manuscript was edited by a language editing company.
Sincerely yours,
Huini Xu
Author Response File: Author Response.pdf
Reviewer 2 Report
The study, "Genome-wide analysis and expression of MYC family genes in tomato and the functional identification of slmyc1 in response 3 to salt and drought stress," discussed the role of MYC family genes in tomato concerning stress conditions. Although the study covers most aspects, a few shortcomings need to be addressed for a proper understanding of the work.
1. in Figures 6, 7, and 8, authors provided P values to present the significant differences between the expression pattern of genes under induced stress conditions. Are these values calculated using all pairwise comparisons? The asterisk signs are present on one column and sometimes on more. So it is difficult to distinguish the meaning and comprehend the results. So it will be appropriate to clarify this by using proper statistical analysis or removing these asterisk signs.
2. The authors have discussed Expression patterns of SlMYC under MeJA, NaCl, and mannitol stress. However, they have failed to provide any evidence of stress effects by data or figures. It will be appropriate to provide documentary evidence of the stress effect of tomato plants (figures showing the impact of stress conditions) or phenotypic data demonstrating the effects.
3. Discussion needs improvement. Focus on discussing your results instead of repeating the results in the discussion section.
Author Response
Dear reviewer 2:
I am very grateful to your comments for the manuscript. According with your advice, we amended the relevant part in the manuscript. Some of your questions were answered below.
The study, "Genome-wide analysis and expression of MYC family genes in tomato and the functional identification of slmyc1 in response to salt and drought stress," discussed the role of MYC family genes in tomato concerning stress conditions. Although the study covers most aspects, a few shortcomings need to be addressed for a proper understanding of the work.
- in Figures 6, 7, and 8, authors provided P values to present the significant differences between the expression pattern of genes under induced stress conditions. Are these values calculated using all pairwise comparisons? The asterisk signs are present on one column and sometimes on more. So it is difficult to distinguish the meaning and comprehend the results. So it will be appropriate to clarify this by using proper statistical analysis or removing these asterisk signs.
Response: The values calculated was not used all pairwise comparisons. One-way analysis of variance (ANOVA) of the t test was done to the data. The data after MeJA, NaCl and mannitol stress for 3h, 6h, 12h was compared with the data of 0 h treatment with t test, respectively. p-values < 0.05 are summarized with one asterisk, and < 0.01 are summarized with two asterisks. Otherwise, there is no asterisk, which means no significant after stress treatment, compared with the 0 h treatment.
- The authors have discussed Expression patterns of SlMYC under MeJA, NaCl, and mannitol stress. However, they have failed to provide any evidence of stress effects by data or figures. It will be appropriate to provide documentary evidence of the stress effect of tomato plants (figures showing the impact of stress conditions) or phenotypic data demonstrating the effects.
Response: Thank you for your good advice. After 150 mM NaCl, 100 µM MeJA, or 100 mM mannitol treatment for 0, 3 h, 6 h, 12 h, there is no phenotypic changes. We just analyze the gene expression.
- Discussion needs improvement. Focus on discussing your results instead of repeating the results in the discussion section.
Response: Thank you for your good advice. The discussion part was revised.
Sincerely yours,
Huini Xu
Reviewer 3 Report
Review for agronomy-2184928-peer-review-v1
Feng et al., in their manuscript “Genome-wide analysis and expression of MYC family genes in tomato and the functional identification of slmyc1 in response to salt and drought stress,” identified 14 SlMYC genes based on the analysis of the tomato genomic data. They further carried out the phylogenetic, expression, and functional domain prediction to characterize these genes. They showed that the loss-of-function of SlMYC1 (Slmyc1) reduced resistance to NaCl and mannitol stress. The work done systematically for the MYC in Solanum lycopersicum is crucial for understanding this gene family evolution and its roles in stress responses. The new findings in this work pave the way for future detailed analyses.
Here are my comments:
There are errors in the numbering system in the Material and Methods section. Please revise.
Results:
Page 5, 3.3, figure 2: Please provide the bootstrap supports for each branch on the phylogeny.
Page 5, 3.4, figure 3: Please indicate whether the tree (A) is rooted or unrooted.
Page 7, 3.5, figure 4: Please indicate whether the tree (A) is rooted or unrooted.
Page 9, 3.8, figure 9: Please indicate what CK stands for in Figures 9C, D, and E.
Conclusion:
Page 12, lines 402-403: “Our results provide a novel idea for future research on crop breeding.” Be specific about the novel idea you referred to here or remove this sentence.
Author Response
Dear reviewer 3:
I am very grateful to your comments for the manuscript. According with your advice, we amended the relevant part in the manuscript. Some of your questions were answered below.
Here are my comments:
There are errors in the numbering system in the Material and Methods section. Please revise.
Response: Sorry about the mistakes we made. We have revised it.
Results:
Page 5, 3.3, figure 2: Please provide the bootstrap supports for each branch on the phylogeny.
Response: Thank you for your good advice. The bootstrap was added on the phylogeny.
Page 5, 3.4, figure 3: Please indicate whether the tree (A) is rooted or unrooted.
Response: We have added “The tree is rooted” in Figure legends.
Page 7, 3.5, figure 4: Please indicate whether the tree (A) is rooted or unrooted.
Response: We have added “The tree is rooted” in Figure legends.
Page 9, 3.8, figure 9: Please indicate what CK stands for in Figures 9C, D, and E.
Response: We have added as below: The tomato nutrient solution treatment was employed as a control (CK).
Conclusion:
Page 12, lines 402-403: “Our results provide a novel idea for future research on crop breeding.” Be specific about the novel idea you referred to here or remove this sentence.
Response: We have deleted this sentence.
Sincerely yours,
Huini Xu
Reviewer 4 Report
In this paper the authors perform a genome-wide characterization of MYC transcription factors in S. lycopersicum and describe a loss of function mutant obtained with Crispr/Cas9.
The work presented is sound and provides a nice overview of the MYC family in tomato underlining the relationship with abiotic stress of the members of the family followed by a direct demonstration with a mutant harboring a (putative) non functional SlMYC1.
I have a couple of questions regarding the approach used for assessing the activity of SlMYC1. I can understand you used leaf tissues for qRT-PCR given the mean overall higher expression level but I wonder why you did not use root tissue, given that you provide mannitol and NaCl.
The root phenotype is very nice, but what puzzles me is why this strong phenotype occurs in the tissue where SlMYC1 is expressed the least (Fig. 5). I would like you also to discuss why similar phenotype occurs despite a different response of leaves with the 2 stress agents. Could that be due to a concurrent decrease in the expression levels of other MYCs? In the paper you do not provide elements to assess the specificity (ie absence of off-targets) in you CrispR experiment: was the sequences you select specific and not similar to other MYCs?
Moreover, given such a phenotype, a qRT-PCR in root tissues may also be useful to provide other elements to support your claims.
Other than that, I have only a few other minor items:
- line 91: provide a link or a reference for TBtools
- line 94: you mean blastN?
- paragraph 1.2: please provide the method you used for transcript quantification
- line 143: please explain what the primers amplify
- line 168: I think there is a typo ("2000")?
- line 316: please provide a reference for that claim (or "data not shown" if non public)
- a paper describing another putative role for MYC1 and 2 was recently published (Swinnen et al, 2022): it could be worth including it in introduction and possibly discussed with respect to your findings (if nomenclature matches)
- Table 1: is the numbering of MYCs the same as found in previous literature or did you renumber it? This is useful for comparisons
- Figure 2: please add info useful for the retrival of MYC proteins used for the dendrogram
- Figure 4B: the colors are not very easy to see, could you change the tones?
- Figure 9B: please add gene structure (start, stop, exons) to make easier to spot where the deletion occurred
Author Response
Dear reviewer 4:
I am very grateful to your comments for the manuscript. According with your advice, we amended the relevant part in the manuscript. Some of your questions were answered below.
In this paper the authors perform a genome-wide characterization of MYC transcription factors in S. lycopersicum and describe a loss of function mutant obtained with Crispr/Cas9.
The work presented is sound and provides a nice overview of the MYC family in tomato underlining the relationship with abiotic stress of the members of the family followed by a direct demonstration with a mutant harboring a (putative) non functional SlMYC1.
I have a couple of questions regarding the approach used for assessing the activity of SlMYC1. I can understand you used leaf tissues for qRT-PCR given the mean overall higher expression level but I wonder why you did not use root tissue, given that you provide mannitol and NaCl.
The root phenotype is very nice, but what puzzles me is why this strong phenotype occurs in the tissue where SlMYC1 is expressed the least (Fig. 5). I would like you also to discuss why similar phenotype occurs despite a different response of leaves with the 2 stress agents. Could that be due to a concurrent decrease in the expression levels of other MYCs? In the paper you do not provide elements to assess the specificity (ie absence of off-targets) in you CrispR experiment: was the sequences you select specific and not similar to other MYCs?
Moreover, given such a phenotype, a qRT-PCR in root tissues may also be useful to provide other elements to support your claims.
Response: Thank you for yours good advice. According to the expression of SlMYC in different tissues, most SlMYC genes expression is higher than root. So we analyze the expression of SlMYC in the leaves under MeJA, NaCl and mannitol stress.
Other than that, I have only a few other minor items:
- line 91: provide a link or a reference for Tbtools
Response: Thank you for yours good advice. The reference was added.
- line 94: you mean blastN?
Response: Thank you for yours good advice. Pfam website search bHLH-MYC_ N domain (PF14215) and HLH domain (PF00010) needs further screening to delete redundant genes. So we need blastN.
- paragraph 1.2: please provide the method you used for transcript quantification
Response: We added the sentence below in the manuscript. “The relative expression of specific genes was quantified using the 2−ΔΔCt method”.
- line 143: please explain what the primers amplify
Response: The primers was used to amplify the PCR product including target 1 and target 2 in the genomic DNA. Then the PCCR product was sequenced to know the gene editing sequence. We added the sentence as below: A pair of primers were designed to amplify PCR product including target 1 and target 2 in the genomic DNA.
- line 168: I think there is a typo ("2000")?
Response: Sorry about the mistakes we made. “2000” was deleted.
- line 316: please provide a reference for that claim (or "data not shown" if non public)
Response: Thank you for yours good advice. The RNA-seq data was not published. We added "data not shown" in the manuscript.
- a paper describing another putative role for MYC1 and 2 was recently published (Swinnen et al, 2022): it could be worth including it in introduction and possibly discussed with respect to your findings (if nomenclature matches)
Response: Thank you for yours good advice. The reference was added in the introduction.
- Table 1: is the numbering of MYCs the same as found in previous literature or did you renumber it? This is useful for comparisons
Response: Thank you for yours good advice. TaMYC, NtMYC and AtMYC are the same as the published names. The names of SlMYC1, SlMYC2, SlMYC2-like-1, SlMYC2-like-2 and SlMYC3-like are the same on NCBI. The remaining SlMYC is renumber.
- Figure 2: please add info useful for the retrival of MYC proteins used for the dendrogram.
Response: Thank you for yours good advice. We have supplemented relevant references.
- Figure 4B: the colors are not very easy to see, could you change the tones?
Response: Thank you for yours good advice. We have changed the tones.
- Figure 9B: please add gene structure (start, stop, exons) to make easier to spot where the deletion occurred
Response: Thank you for yours good advice. We redraw the diagram. But SlMYC1 has no exon.
Sincerely yours,
Huini Xu
