Genome-Wide Analyses of Tea Plant Stress-Associated Proteins (SAPs) Reveal the Role of CsSAP12 in Increased Drought Tolerance in Transgenic Tomatoes

Round 1

Reviewer 1 Report

In this study,  the author's identified SAP genes in the tea genome by bioinformatics tools.  They studied the structure and domains and drew phylogenetic trees for CsSAPs proteins. They also investigated the expression patterns of CsSAPs in different tissues and plants exposed to drought stress. In their study authors,  also confirmed the function of CsSAP12 by overexpressing this gene in tomato plants. They found that transgenic tomatoes have an increased tolerance to drought stress compared with the wild type.

This MS contains good data. However, I found the presentation of this data is weak. Therefore, MS can be improved. There are some mistakes (such as cold stress treatments mentioned in the abstract not available in methods and results). Details of the experiments should be provided and some sections should be rewritten. For more suggestions please check the attached file. 

Comments for author File: Comments.pdf

Author Response

Point 1: In this study, the author's identified SAP genes in the tea genome by bioinformatics tools. They studied the structure and domains and drew phylogenetic trees for CsSAPs proteins. They also investigated the expression patterns of CsSAPs in different tissues and plants exposed to drought stress. In their study authors, also confirmed the function of CsSAP12 by overexpressing this gene in tomato plants. They found that transgenic tomatoes have an increased tolerance to drought stress compared with the wild type.

This MS contains good data. However, I found the presentation of this data is weak. Therefore, MS can be improved. There are some mistakes (such as cold stress treatments mentioned in the abstract not available in methods and results). Details of the experiments should be provided and some sections should be rewritten. For more suggestions please check the attached file. 

Response 1: The whole manuscript has been carefully checked and modified in the revised version, and the details of the experiments were added and modified in Lines 88-94, Lines 173-178 and Lines 180-186. Detailed modifications were marked in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Article entitled “Genome-Wide Analyses…………… in Transgenic Tomatoes” described about identification of Plant stress-associated proteins encoding genes in tea plant genome, and overexpression of CsSAP12 gene in tomato for enhanced drought stress tolerance.

Study is good, but followings are expected for a publication of transgenic studies: -

1. Molecular confirmation, copy numbers, GUS assay of Transgenics
2. Morphological study (growth parameters) of transgenic under stress compared to WT
3. Seed germination assay of transgenics
4. ROS generation and localization study in Transgenics under stress
5. Osmolyte analysis in Transgenics under stress
6. Drought stress and plant recovery analysis of Transgenics
7. Possible mechanism of CsSAP12 in drought tolerance

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

1. Website addresses should be provided for TPIA (Tea Plant Information Archive) database (line 93), the tea plant genome database (lines 80 and 103), and the Plant CARE database (line 116).
2. Line 80 – delete the parentheses containing the binomial name of tea plant. If the tea plant genome database is publicly available, the website should be provided in the parentheses.
3. English writing needs to be checked. Lines 142 – 143, “The transgenic seeds were screened out with kanamycin monosulfate were after harvested.” Is an example.
4. Line 147 – what is the meaning of “trained” plants?
5. Table 1 – Information in this table indicated that the tea plant genome database is composed of DNA sequences in scaffolds only. These sequences are not located to the tea chromosomes yet. The heading “chromosome location” would be inappropriate.
6. Line 213 - Actinidia chinensis and Vitis vinifera, and other species names in the text, should be in italic font.
7. Carefully check for the missing commas and spaces, e.g. lines 325, 327 etc.
8. Supplementary Figure S1 – The AN1 domain was not marked.
9. Supplementary Table 1 – Annealing temperatures and other PCR conditions were not included.

Author Response

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Author Response File: Author Response.docx

Reviewer 4 Report

The authors performed detailed analysis of SAP proteins. They also used qPCR for expression analysis and developed transgenic lines. I have the following comments. 

1. The manuscript needs a lot of improvement in writing up.
2. Why the authors did not do classification in four groups instead of 2 groups based on domain presence. 
3. The author used NJ method, they should use the ML approach. 
4. The introduction should provide details about the studies which reported SAPs in other species and performed the genome wide analysis. 
5. The discussion should also expand based on my above comment.  
6. The description of the figures should be improved as the numbering in the figure such 1,2,3  not clear in figure 2. 
7. The phylogenetic tree is confusing. I think the grouping can be shown by alternate way or using subgrouping approach. However, the cladogram is an obstacle to me to provide clear advice on it. Authors need to provide actual tree instead of ignoring the branch length (may be supplementary). 

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

In possible mechanism, you are stating role of oxygen free radical BUT ROS generation and osmolyte analysis will be detected in transgenic tomatoes and it will be another story in future.  

These two experiment can be performed within a week.  BUT you are stating that no seeds nor seedlings of CsSAP12 transgenic tomatoes are available to perform the assay at present? How is it possible ?

Surprisingly, You do not have material to re-validate your experiments?

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

line 82 - isolated from tea 

Author Response

 Dear reviewer,

  ‘form’ was  was modified to be ‘from’ in the revised version (Line 83).

  Additionally, we have used MDPI English Editing service for English editing in the revised version.

Best regard,

Dan-Dan Liu

Reviewer 4 Report

The manuscript can be accepted. However, the suggestion of one of the reviewers needs to be addressed. Authors can improve methodology section and discussion regarding transgenic work. 

I think the content fulfils publication in the journal. 

Regards

Author Response

Point 1: The manuscript can be accepted. However, the suggestion of one of the reviewers needs to be addressed. Authors can improve methodology section and discussion regarding transgenic work. 

Response 1: Sorry for the misunderstanding caused by the inappropriate words in the original responses. When the question was raised, WT and transgenic tomatoes were small with only two-cotyledons that were not suitable for performing the experiments. After submitting the revised manuscript, we immediately prepared the experiments of ROS determination and osmotic analysis using the seedlings with two true leaves. Additionally, we added the related methods, results and discussion in Lines 193-202, 417-440 and 533-539, respectively.

     Additionally, we have used MDPI English Editing service for English editing in the revised version.

Round 3

Reviewer 2 Report

Accepeted