Comparative Analyses of Superoxide Dismutase (SOD) Gene Family and Expression Profiling under Multiple Abiotic Stresses in Water Lilies
Round 1
Reviewer 1 Report
Article review
1. EVALUATION OF THE PAPER MANUSCRIPT
Title of the Manuscript:
"Comparative analyses of Superoxide dismutase (SOD) gene
family and expression profiling under multiple abiotic stresses
in water lilies"
Manuscript number: horticulturae-2441373
The article horticulturae-2441373 has comprehensively identified and characterized the SOD family in four water lilies species based on various bioinformatics approaches.
Then, expression patterns of the SOD genes in various organs/tissues under different treatments were explored by using qRT-PCR. Article horticulturae-2441373 is slightly suited to Horticulture as per aims and scope. However, to improve the quality of the article, some points should be noted:
- Please, provide some sentences to introduce the water lily in the third paragraph in the Introduction part.
- It would be very significant if the authors provide the conserved domain of SOD proteins in the Pfam server (PF????) in the method.
- Authors should clarify the criteria of the protein features (physic-chemical properties) in the method.
- Authors state that 2 Kb sequences upstream to the transcription start site were retrieved for the prediction of cis-regulatory elements. How did the transcription start site get determined? Furthermore, the PlantCARE is out of date because the authors had to manually search for many well-characterized stress-responsive cis-acting elements. The Results and Discussion section related to this part should concentrate on the stress- and hormone-responsive cis-regulatory elements.
- If possible, please provide the references for methods 2.6, 2.7, 2.8, and 2.9.
- If possible, please carry out a GFP assay to determine the subcellular localization of several interesting SOD proteins in four water lily species. The reviewer is looking forward to seeing this part in the revised manuscript.
- Gene duplication is a very important part to get insight into the evolution of the SOD gene family in water lily species. Please, carry out this in silico part.
- The author should explain the unpublished data in section 3.6.
- Please, define specifically-expressed, highly-expressed, and expressed genes in the Method related to the re-analysis of transcriptome data.
- The list of references should be exactly followed the journal style.
Several minor points should be checked:
- Please check the tense in the Introduction part. The authors should carefully check the correct verb tense in this part. Some mistakes were recently highlighted in the pdf file.
- Gene's name should be italicized. Please, check the whole text.
- Provide the scientific name of higher plant species at the first occurrence. Please, check the whole text, like pea, wheat, water lily, etc.
- Please, rephrase some sentences in the Introduction to avoid the similarity between them and the Abstract.
- Check the font sizes in the whole text.
- Please check for grammatical and spelling mistakes as many in the pdf file.
Comments for author File: 
Comments.pdf
Author Response
Dear Editor
We are pleased to re-submit our revised manuscript, “Comparative analyses of Superoxide dismutase (SOD) gene family and expression profiling under multiple abiotic stresses in water lilies’’ (Manuscript ID: horticulturae-2441373) to be considered for publication in “Horticulturae”.
Thank you very much for making a critical assessment of the original version of our manuscript. We are glad to learn that the editor as well as the reviewer(s) considered our findings meaningful. However, the reviewer(s) provided some valuable comments and suggestions for the potential improvement of the manuscript. Based on the comments, you encouraged us to re-submit a revised version, which takes into account all of the points raised by the reviewer(s). We have now completed a thorough revision as per the recommendations of the reviewer(s). Revised portions are marked in red in the paper. Our response to reviewers’ comments has been listed below point-by-point.
Answers to Reviewer 1:
The article horticulturae-2441373 has comprehensively identified and characterized the SOD family in four water lilies species based on various bioinformatics approaches.
Then, expression patterns of the SOD genes in various organs/tissues under different treatments were explored by using qRT-PCR. Article horticulturae-2441373 is slightly suited to Horticulture as per aims and scope. However, to improve the quality of the article, some points should be noted:
Thank you very much for your comments! We deeply appreciate your kind words and valuable suggestions, which have improved our manuscript.
Point 1: Please, provide some sentences to introduce the water lily in the third paragraph in the Introduction part.
Response 1: The section has been modified as suggested. (In third paragraph in introduction part)
Point 2: It would be very significant if the authors provide the conserved domain of SOD proteins in the Pfam server (PF????) in the method.
Response 2: The conserved domain and Pfam (Pfamv34.0-19178pSSMs) numbers has been provided as suggested. (Added in method section 2.1)
Point 3: Authors should clarify the criteria of the protein features (physic-chemical properties) in the method.
Response 3: Thank you very much for your suggestions. We have modified the text in the materials and methods section 2.2.
Point 4: Authors state that 2 Kb sequences upstream to the transcription start site were retrieved for the prediction of cis-regulatory elements. How did the transcription start site get determined? Furthermore, the PlantCARE is out of date because the authors had to manually search for many well-characterized stress-responsive cis-acting elements. The Results and Discussion section related to this part should concentrate on the stress- and hormone-responsive cis-regulatory elements.
Response 4: Thank you very much for pointing out the mistake. We have modified the text in the manuscript. We retrieved the 2000bp sequence upstream of the CDS sequence, not the transcription start site. Thanks for understanding.
Point 5: If possible, please provide the references for methods 2.6, 2.7, 2.8, and 2.9.
Response 5: Thank you so much. We have added the references as suggested.
Point 6: If possible, please carry out a GFP assay to determine the subcellular localization of several interesting SOD proteins in four water lily species. The reviewer is looking forward to seeing this part in the revised manuscript.
Response 6: Thank you so much for your suggestions. We are establishing the protocols for GFP expression in water Lily species for subcellular localization and other methods to validate the role of these genes using latest biotechnological tools. In our lab, we are trying our best to explore the genome of these species especially those gene families which have a role in tolerance. We are hopeful; we will establish these protocols in our lab and will reveal in future publications. Thanks for understanding.
Point 7: Gene duplication is a very important part to get insight into the evolution of the SOD gene family in water lily species. Please, carry out this in silico part.
Response 7: Thanks for the suggestion. Our lab is trying to get the insightful data from the un-published genome of Water lily species. Due to some restrictions, we cannot get the gene-duplication data from the sequenced data. Because the genome sequence data off all species of water lilies are not published yet. We are very hopeful that, soon, the sequenced genomic data will be available for the public, and then we will do in-silico gene duplication analysis in other genome-wide publications. Thank you very much for understanding.
Point 8: The author should explain the unpublished data in section 3.6.
Response 8: Thank you for suggestion. We have explained the data in section 3.6.
Point 9: Please, define specifically-expressed, highly-expressed, and expressed genes in the Method related to the re-analysis of transcriptome data.
Response 9: Thanks for your suggestion, the section has been modified as suggested.
Point 10: The list of references should be exactly followed the journal style.
Response 10: Thank you very much. All references are modified as per journal format.
Point 11: Several minor points should be checked
Response 11: Thank, we have checked it throughout the manuscript.
Point 12: Please check the tense in the Introduction part. The authors should carefully check the correct verb tense in this part. Some mistakes were recently highlighted in the pdf file.
Response 12: Thank you very much for pointing out the mistake. We have checked it in introduction part as well as in throughout the manuscript.
Point 13: Gene's name should be italicized. Please, check the whole text.
Response 13: Thank you very much. We have italicized all gene’s name in whole manuscript.
Point 14: Provide the scientific name of higher plant species at the first occurrence. Please, check the whole text, like pea, wheat, water lily, etc.
Response 14: We have checked it throughout the manuscript, and added the scientific names of all higher plant.
Point 15: Please, rephrase some sentences in the Introduction to avoid the similarity between them and the Abstract.
Response 15: Thank you very much for indicating the mistake. We have rephrased all sentences which were similar between introduction and abstract.
Point 16: Check the font sizes in the whole text.
Response 16: We have checked and made the font size according to journal format.
Point 17: Please check for grammatical and spelling mistakes as many in the pdf file.
Response 17: Thank you so much for highlighting the mistakes. We have Checked carefully all the text and corrected the grammatical mistakes as well as spelling mistake.
Author Response File: Author Response.pdf
Reviewer 2 Report
Abstract and 2.10, 3.8, Figure 8, discussion,: "qRT-PCR", It's RT-qPCR, the quantitative part is the PCR not the Real Time, even when this term is quite misused in the literature.
Why separate the 4 species in section 2.1? N. colorata, N. thermarun, N. mexicana (unpublish) from N. minuta (unpublish) ? Why not "N. colorata, N. thermarun, N. mexicana, and N. minuta (unpublish)?? "
Sp names in lowercase: N. Mexicana --> N. mexicana
Did you sequence the 4 genomes? how can we have access to the genomic data?
2.8. When you talk about RIN>9 to construct database, What database are you refering?
3.1 Can you explain what are reduntant sequences you remove for STable2?
pag 5 "previuos research has shown that..." this is discussion not results.
Why did you use only N. colorata for RT-qPCR? justify this point
Figure 2. "Both phylogenetic tree and sequence motif pattern support the clssification of SOD genes in to two groups". This looks like a discussion and should be descriptive for images. E.g "Classification of... groupping ....Depiction of ..."
Figure 2a. Why did you use NJ method instead a ML method for tree reconstruction?? in Methods you say "both NJ or ML.." but I did not see ML along the Results section. Please, clarifty this point.
Figure 2. Please, describe Amt in text (A. trichopoda; at the top of branches), this is not set before.
Figure 3a is not an easy to compare. It would be more useful to understand if you group the sequences by phylogeny, as in Figure 2a.. then we would discover patterns by group
Figura 4. N. thermarun have 10 sequences but only 9 are shown.
Figure 4 is not informative as it's presented. We see different protein structures, but we cannot compare between orthologs, groups, a reference sequences? Maybe A. thaliana sequences? You can overlap specific structures with a reference. E.g. the MnSOD with a AtSOD-Fe.. if this is not possible, perhaps grouping sequences by subfamily? SOD-Fe. SOD-Cu/Zn, SOD-Mn... this will make it easier to compare differences and similarities.
Section 3.6 Expression Examination of .... You don't mention anything about RNA-seq methodology in section 2.8. Even when is from unpublished data, you should describe the methodological steps... how many replicates, the software used to obtain the count matrix, the normalization method, etc. This part seem from other research history and just used the data to explore your genes.
Why you use pollen and ovules? You do not justify why you chose these tissues and their relevance.
section 3.7 What is the relevance to get the interaction among 7 SOD genes from N. colorata? Do these proteins interact with other families of proteins? I know you can extend the interaction network to see other protein related groups from other metabolic functions. You can use a more annotaed Reference sequences to see other interactors... other srtess related proteins, signalling cascade, etc.
I think the Figure 8 is a valuable contribution for this gene family.
Author Response
Dear Editor
We are pleased to re-submit our revised manuscript, “Comparative analyses of Superoxide dismutase (SOD) gene family and expression profiling under multiple abiotic stresses in water lilies’’ (Manuscript ID: horticulturae-2441373) to be considered for publication in “Horticulturae”.
Thank you very much for making a critical assessment of the original version of our manuscript. We are glad to learn that the editor as well as the reviewer(s) considered our findings meaningful. However, the reviewer(s) provided some valuable comments and suggestions for the potential improvement of the manuscript. Based on the comments, you encouraged us to re-submit a revised version, which takes into account all of the points raised by the reviewer(s). We have now completed a thorough revision as per the recommendations of the reviewer(s). Revised portions are marked in red in the paper. Our response to reviewers’ comments has been listed below point-by-point.
Answers to Reviewer 2:
Point 1: Abstract and 2.10, 3.8, Figure 8, discussion,: "qRT-PCR", It's RT-qPCR, the quantitative part is the PCR not the Real Time, even when this term is quite misused in the literature.
Response 1: Thank you so much for indicating the mistake, we have corrected and wrote RT-qPCR in whole manuscript.
Point 2: Why separate the 4 species in section 2.1? N. colorata, N. thermarun, N. mexicana (unpublish) from N. minuta (unpublish) ? Why not "N. colorata, N. thermarun, N. mexicana, and N. minuta (unpublish)?? "
Response 2: Because the genomes of N. colorata, and N. thermarun already published and available online. While we sequenced the genomes of N. mexicana and N. minuta but not yet published. Soon we will publish the both genome. That’s why we mentioned both species separately to viewers understand clearly. Thank you
Point 3: Sp names in lowercase: N. Mexicana --> N. mexicana
Response 3: Thank you so much for highlighting, we have changed in materials and methods section 2.1.
Point 4: Did you sequence the 4 genomes? how can we have access to the genomic data?
Response 4: The genomes of two species already published. N.colorata (available online at Phytozome and NCBI). N.thermarum genome also published and available at NCBI database. We sequenced the genomes of N.mexicana and N.minuta and soon we will publish and it will be available online. Thanks
Point 5: 2.8. When you talk about RIN>9 to construct database, What database are you refering?
Response 5: Thanks. We mistakenly added the RIN values. We have deleted the text from the manuscript. Thanks again for pointing out the mistake.
Point 6: 3.1 Can you explain what are redundant sequences you remove for STable2?
Response 6: Thank you for highlighting the mistake. AT3G10920.1 and AT5G23310.1 were the redundant sequences and removed manually.
Point 7: pag 5 "previous research has shown that..." this is discussion not results.
Response 7: Thank you so much for point out the mistake. We have checked and removed from result section and explained in discussion part.
Point 8: Why did you use only N. colorata for RT-qPCR? justify this point
Response 8: Basically our main focus on this specie. Our future studies on the specie will focus on gene engineering and comprehensive analysis, integrating genomics, transcriptomics, proteomics, and metabolomics.
Point 9: Figure 2. "Both phylogenetic tree and sequence motif pattern support the classification of SOD genes in to two groups". This looks like a discussion and should be descriptive for images. E.g "Classification of... groupping ....Depiction of ..."
Response 9: Thank you very much. We have removed from figure and explained in discussion part as well as given the title to Figure 2 as suggested.
Point 10: Figure 2a. Why did you use NJ method instead a ML method for tree reconstruction?? in Methods you say "both NJ or ML.." but I did not see ML along the Results section. Please, clarify this point.
Response 10: Thank you for indicating the mistake. We only used the NJ method; in writing mistake we also mentioned the ML method. I have removed the ML word from paragraph in method section.
Point 11: Figure 2. Please, describe Amt in text (A. trichopoda; at the top of branches), this is not set before.
Response 11: We have added the full name of Amborella trichopoda with Amt.
Point 12: Figure 3a is not an easy to compare. It would be more useful to understand if you group the sequences by phylogeny, as in Figure 2a.. then we would discover patterns by group.
Response 12: Thanks for the suggestions. We modified the figure 3a as shown in manuscript
Point 13: Figure 4. N. thermarum have 10 sequences but only 9 are shown.
Response 13: Thank you so much for pointing out the mistake. We have added the structure of NtFSD5 gene which was missed by mistake.
Point 14: Figure 4 is not informative as it's presented. We see different protein structures, but we cannot compare between orthologs, groups, a reference sequences? Maybe A. thaliana sequences? You can overlap specific structures with a reference. E.g. the MnSOD with a AtSOD-Fe.. if this is not possible, perhaps grouping sequences by subfamily? SOD-Fe. SOD-Cu/Zn, SOD-Mn... this will make it easier to compare differences and similarities.
Response 14: We divided all protein structures according to their sub-families (SOD-Cu/Zn, SOD-Fe and SOD-Mn. We mentioned every sub-family of each specie and compared with each other (Figure 4).
Point 15: Section 3.6 Expression Examination of .... You don't mention anything about RNA-seq methodology in section 2.8. Even when is from unpublished data, you should describe the methodological steps... how many replicates, the software used to obtain the count matrix, the normalization method, etc. This part seem from other research history and just used the data to explore your genes.
Response 15: Respected reviewer thanks for your suggestion, the expression data was obtained from our own RNA sequencing raw data which is unpublished yet and needs some modifications, as our group already published the genome of N, colorata in Nature (https://doi.org/10.1038/s41586-019-1852-5), (Phytozome info: N.colorata v1.2 (doe.gov)), that’s why we used our own data in the current study which belongs to us and not taken from any other source. Thanks for understanding.
Point 16: Why you use pollen and ovules? You do not justify why you chose these tissues and their relevance.
Response 16: Pollen and ovules are reproductive tissues that are highly vulnerable to environmental stresses, including high light intensity, heat, cold, drought, and oxidative damage. To protect these tissues from such stresses, they possess antioxidant defence systems. We choose pollen and ovules, because they contain different types of antioxidant enzymes like (SOD), (CAT), (POD), (POX) etc and play important roles against abiotic stresses. Thanks for understanding.
Point 17: Section 3.7 what is the relevance to get the interaction among 7 SOD genes from N. colorata? Do these proteins interact with other families of proteins? I know you can extend the interaction network to see other protein related groups from other metabolic functions. You can use a more annotated Reference sequences to see other interactors... other stress related proteins, signalling cascade, etc.
Response 17: Thank you very much for the suggestion. We have already did in silico protein interaction but we didn’t find any interaction of this seven SOD genes with other species including N.minuta, N.thermarum and N.mexicana. Even in out of nine SOD genes from N.colorata we can only find protein interaction of seven family members. Thanks for understanding.
Point 18: I think the Figure 8 is a valuable contribution for this gene family.
Response 18: Thank you so much, for your appreciation.
Author Response File: Author Response.pdf
