Genome-Wide Analysis of the Trehalose-6-Phosphate Synthase Gene Family in Rose (Rosa chinensis) and Differential Expression under Heat Stress

Round 1

Reviewer 1 Report

Dear Editor

I carefully reviewed the manuscript entitled “Characterization of the Trehalose-6-phosphate Synthase Family and Analysis of Its Differential Expression in Rose (Rose chinensis) under Heat Stress”, by Wei et al., which submitted to Horticulture. The manuscript topic is adequate for the aims and scope of the journal. However, I feel that it is suitable for publication after minor revision. The authors should address the following comments and revise the manuscript accordingly:

1-There are many papers about the abiotic stress effects on plants. The authors have not cited sufficient references to get findings or conclusions. 

2- In section 2.1: When the crop was planted, and harvested? What was the plant sowing rate? There was no information about harvest time of plants, crop nutrition, and plant protection! In which stage they were harvested. Please explain it carefully in the mentioned section.

3-How much substrate was used in every pot? Please add it in the text.

4-I have not find any sentence about statistical analysis methods used in this paper. The Statistical analyses were performed by what? Please describe it carefully in the paper.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled “Characterization of the Trehalose-6-phosphate Synthase Family and Analysis of Its Differential Expression in Rose (Rose chinensis) under Heat Stress” it is considered well organized for the journal format, however there is in need of some small changes as followed:

 

I recommend to extend the aim of the research, at the end of the Introduction section.

The cutting and the seedling are not the same thing, please reconsider which one is the correct:

            -cutting: a part of a mature plant, which was cut off and rooted

            -seedlings: a young plant, especially one raised from seed and not from a cutting.

L 90–91: “2-year-old healthy cutting seedlings of the same size”–same size which means…....cm?

L 89–100: the R. chinensis were irrigated during the 7 days incubator culture. If yes, then with what kind of water?

L 437–439: suggest to use for all plants the binominal nomenclature.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Title:  Characterization of the Trehalose-6-phosphate Synthase Family and Analysis of Its Differential Expression in Rose (Rose chinensis) under Heat Stress

Authors: Wei et al.

General comments: The manuscript deals with the analysis multiple members of Trehalose-6-phosphate Synthase (RsTPS) Family in R. chinensis. The analysis includes in silico analysis of gene, transcript and protein and expression analysis in different tissues in response to heat-stress. A lot of information is presented for the first time; however, the study has not addressed some important aspects. For example, whether the conditions used for heat-stress (high-temperature stress) in the study for 72 h, will give only heat stress to the plant and not osmotic stress? How it was ensured that there will not be water loss despite high temperature? The involvement of genome duplication events and its impact on the multiple RcTPS genes (if any) should have been investigated by synteny analysis. This would have given a different perspective to look into the diversity of the RcTPS members in R. chinensis. An important concern is whether the trehalose is getting increased due to heat stress or is it conferring the tolerance to the plant under heat stress. Although, the second point is mentioned few times, the evidence is not presented. Whether the R. chinensis is heat-stress tolerant or not is not clarified clearly. Heat-stress and High-temperature stress has been used interchangeably at several places, which should be avoided. Also abiotic stress responses are complex and multilayered. Hence, projecting only a single candidate (in this case trehalose) with limited analysis may not be promising.  Overall, the information w.r.t. RcTPS may be novel however, some of the aspects mentioned above could have been addressed. Several concerns are indicated in the PDF file of the manuscript, while some important section-specific comments are detailed below.

Section specific comments

Title:                    The title of the manuscript is very long and may be modified to make is more focussed ad short. The scientific name of the plant species analysed should be ‘Rosa chinensis’ and not Rose chinensis, kindly check.

Suggestion: ‘Genome-wide Analysis of Trehalose-6-Phosphate Synthase Gene family in Rose (Rosa chinensis) and Differential Expression under Heat-Stress'.

Abstract:             Abstract looks over simplified and needs to be improved in terms of defining an appropriate rationale behind the study. Certain statements may need to be re-assessed for the information content and may be rewritten if needed. Overall outcome of the study is also not getting highlighted properly, other than outcome of different analyses.

 Introduction:     The length of the introduction section is appropriate and written in a fine manner. However, at several places minor changes have been suggested (see PDF file for details) to improve the description further. Some are briefly mentioned here

Line 28:               impact of heat stress may be elaborated a little bit more.

Line 35:               check and rectify usage of ‘Heat-stress’ vs ‘High-temperature stress’.

Lines 47-57:        the description is modified to reduce unnecessary details without losing the content.

Line 58-69:         the content is again reduced a little bit, and some terms and phrases need to be clarified.

Line 73-75:         the optimum temperature for Rose should be indicated and what temperature constitute heat-stress for this species. Is any information known about the thermotolerance of Rose plants, as it is indicated that the stress is detrimental to these plants, including its ornamental value?

Lines 76-86:        this section should be able to convey why trehalose is indicated to be important for Rose plants, under heat stress. And then a more appropriated scientific rationale for the basis of this should be indicated. 

Line 85:               idea of this study providing ‘theoretical help’ is not very clear.       

Material and methods: This section provided sufficient details of methodologies used, however

Lines 88-95:        how it was ensured that the Heat-Stress conditions used in the growth chambers (for 3-days) will not cause water deficiency or osmotic stress to the plants?

Lines 92-94:        what were the humidity conditions used in the growth chamber/ green house, during initial growth phase?

Line 95:               what was the basis of choosing 40degree as the heat-stress temperature? Is it based on some previous study?

Lines 103-104:   Something is missing in the statement, need to be re-written for better clarity.

Lines 109-110:   The software used in the MSOffice package may be indicated. For example, if only Excel was used.

Line 113:             Since the RcTPS members are present in multiple copies, does some of these originated due to duplications? Is this evident in previous studies on other plant species? Was this aspect addressed in this study or not and why?

Lines 155-156:   First paragraph of material and methods indicates usage of Pfam tool? But the tool is not mentioned in this section on domain analysis, why?

Lines 157-159:   Analysis of ‘chromosomal locations’ should also have included the 'Synteny analysis' for duplication events?

Lines 161-171:   Kindly see whether ‘qRT-PCR’ or ‘RT-qPCR’ is appropriate term for the analysis.

 Results:               Results section needs to be improved significantly at several places. In particular, the content/description should be written in a manner to avoid unnecessary lengthy details and content which is also evident in the figure/tables. Also, the experimental details that are already in the materials and methods section should not be again detailed, unless really important. ‘Heat-stress’ and sometimes ‘high-temperature stress’ terms are used to convey same meaning. Instead, one term should be used in a consistent manner.

Lines 194-195:   The RcTPS members show more than 100 aa length variations. Does this length difference cause any problem in alignment? The alignment file is not available, it may be included as supplementary file. Alignment errors, if any, may also affect the phylogenetic inferences.

Lines 220-228:   The involvement of duplication events and its effect on diversity should have been addressed.

Lines 242-243:   Statements discussing the implication of previous studies should be ideally part of discussion, unless very important. Kindly check at other places in the results section.

Lines 243-246:   The projections related to role of RcTPS by modulation of ABA signalling may need additional experimental evidence, or support from studies in other systems.

Lines 299:           Does the motifs properly aligned in the MSA file?

Line 309:             The proteins have only two conserved domains and similar motifs. What type of functional diversity is indicated here?

Lines 311-340:   In general, the description in this section is given in too much detail, and may be minimized by removing the repetitive information content w.r.t. the Figure 5.

Line 324:              The bootstrap value support, though mentioned, is not evident in the dendrogram.

Lines 338-339:   Is there any possibility that the relationships between TPS members (closeness/divergence) of different species may change if more species are included in the analysis?

Lines 348-361:   This analysis related to the ‘positive selection’ may be more elaborated in materials and methods section, and the results section should only mention the results.

Line 383:             Majority of the TPS genes are down-regulated in the three tissue. What does this indicate w.r.t. importance of trehalose synthesis in heat stress?

Discussion:         Discussion section needs to be shortened by keeping previous literature and description relevant to the focus of the present work. The section headings may not be needed as they are appears similar to the Results section headings. The overall outcome tries to project the elevation of trehalose is important for conferring heat-stress tolerance in Rose. However, the results show that despite continuous heat-stress, the trehalose levels actually reduce after 24h or so. Whether, the increase in trehalose make it a long term protecting molecule to heat stress is not very clear. The optimum conditions and heat-stress detrimental to the Rose plants should have been discussed appropriately. Several statements of discussion are very similar to the results section, which may be avoided.

Lines 400-401:   How much fold was the increase? Does this much increase make it a long term protecting molecule to heat stress?

Lines 404-406:   Here, it is suggested that the roots are synthesizing trehalose and it goes to stem and leaves. In fact, this contention is unlikely, as the data show that leaves have much higher levels of trehalose compared to roots and stem and there is not significant increase for upto 24 h, and after that it decreases. Also stem have comparable levels w.r.t. roots. Some evidence should have been presented in the supports of these projections.

Lines 433-434:   TPS enzyme is a key signalling molecule or trehalose? Check the statement to see if it need to be rewritten.

Lines 454-455:   The other members have been validated for TPS activity like in A. thaliana or not validated yet?

Lines 467-470:   These genes were not continuously up-regulated (as in Fig 6), but at certain time points. The same was true for may other TPS genes. Why RcTPS7a and 7b termed important?

Lines 487-488:   The duplication events affecting TPS genes, if any, needs to addressed in an appropriate manner, particularly in the R. chinensis.

Conclusions:      This section also needs to be improved a little bit and should include some of the important information from the study and previous studies in an integrated manner. For example, does the R. chinensis survive heat-stress? How heat-stress tolerant is this species? Is the trehalose increase due to heat stress? and whether the trehalose content makes the plant heat tolerant, and by how much?

 Figures: There are few minor concerns related to the Figures. In general, the figure legends/captions should have been a little bit made more informative.  

Figure 1: Different graphs in the figure may be labelled as A, B and C.

Figure 2: Indicate what the scale on LHS indicates, in the caption.

Figure 3A: The blue colour coded region at 5' and 3' end does not represent the UTR completely. in fact, it indicates the UTR as well as intronic regions upstream and downstream to the coding region. Kindly verify it and do changes in the description or figure labels, if needed.

Figure 3B: The length of the upstream promoter region may be represented from -2000 bp to 1 bp.

Figure 5: Instead of a circular/radial tree, it is better to include a NJ tree, with a root (out group) to show the direction of evolution.

Figure 6: Indicate which panel (A,B, C) of the figures is for which tissue.

Line 514:              Supplementary data provided in the web link is not available. The link does not give any of the listed files. The availability of the files may be re-checked.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Manuscript ID: horticulturae-1688885-version2

Revised Title:  Genome-wide Analysis of Trehalose-6-Phosphate Synthase Gene family in Rose (Rosa chinensis) and Differential Expression under Heat-Stress

Authors: Wei et al.

General comments: The revised manuscript (horticulturae-1688885v2) has been considerably improved. The concerns raised have been appropriately addressed. Section wise comments have also been listed below: 

1)    The revised tittle now title ‘Genome-wide Analysis of Trehalose-6-Phosphate Synthase Gene family in Rose (Rosa chinensis) and Differential Expression under Heat-Stress' now looks appropriate, and the species name has been corrected

2)    The revised abstract is affine and has been updated as per the changes incorporated in the manuscript.

3)    The revised introduction has been improved by incorporating the information relevant to the content of the manuscript along with the citations, and removing the non-relevant description. Consistency in the usage of certain terms have been improved. Background information known about diversity in the thermotolerance of Rose plant species has been incorporated. 

4)    The Material and methods section has also been improved by adding experimental details to certain sections and basis of using stress treatment conditions, which are necessary for proper understanding of the experimental design used. It is suggested to give versions and company names of the all the software used. It is missing for GraphPad, kindly check for others also. Required information has been updated in certain sections e.g. Selective Pressure Analysis and Gene Chromosomal Location and Collinearity Analysis etc.), which was needed.

5)    Results section has also been revised in an appropriate manner, and as per considering the comments/ suggestions. It looks simplified, non-relevant and repetitive details have been minimized/removed, and additional results (such as duplication analysis) incorporated. Statements more appropriate for discussion has been moved from results. Better 

Suggestions: 
Section 3.2 title may be updated to ‘Identification and analysis of characteristics of TPS Family members in R. chinensis.

Font size of labels in Figure 2 may be increased and appropriate colour may be used for better visibility. Other figures may also be checked.

6)    The concerns in the discussion section has also been appropriately address and the revised version seems fine and focussed on the present work. 

7)    The figures and captions improved as suggested. Kindly note in Figure 3 B, the length of the upstream promoter region may be represented from -2000 bp towards 5’ end to 1 towards 3’ end, as often represented conventionally by taking +1 as the first site to be translated. Kindly refer to few published papers to place it correctly.

8)    In Supplementary data Table S1 the term ‘Sequence’ in the column headings is misleading. It is actually TPS family member designation used in the study. It may be rectified if applicable. In Figure S1 the algorithms used for phylogenetic analysis should be indicated. In Table S2 the 5’- and 3’ ends of oligonucleotide primers should be indicated in the column heading, and in Figure S2 the parameters used for the MSA should indicated in the figure caption.

Author Response

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Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.