Genome-Wide Analysis of the Trehalose-6-Phosphate Synthase (TPS) Gene Family and Expression Profiling of ScTPS Genes in Sugarcane

Round 1

Reviewer 1 Report

The authors took into consideration my comments.

Some work should be done regarding text editing (e.g. L331: "The average amino acid identity between Class I
(ScTPS1) and Class II (ScTPS2-9) is much lower than that among Class II")

Author Response

Response to Reviewer 1 Comments

Point 1: Some work should be done regarding text editing (e.g. L331: "The average amino acid identity between Class I (ScTPS1) and Class II (ScTPS2-9) is much lower than that among Class II")

Response 1: Thanks for your suggestions and the sentence was revised to: “The average amino acid identity is much lower between Class I-Class II pairwise comparisons than that within Class II pairwise comparisons”.

Reviewer 2 Report

The authors have addressed or answered most of my questions. However, for statistical analysis, the study still need to improve. For example, 1) the appropriateness of ANOVA needs to be adjusted. Since the data have outliers such as we can see in Figure 3, did the authors really test that ANOVA is appropriate for this kind of data? Were the multiple comparisons adjusted?

2) The statistical method sections need to contain what the study used for the analysis. Such as, add the contents that the authors responsed to the reviewer's concerns about the appropriateness of methods. 

Author Response

Response to Reviewer 2 Comments

Point 1:The authors have addressed or answered most of my questions. However, for statistical analysis, the study still need to improve. For example, 1) the appropriateness of ANOVA needs to be adjusted. Since the data have outliers such as we can see in Figure 3, did the authors really test that ANOVA is appropriate for this kind of data? Were the multiple comparisons adjusted?

Response 1: Thank you for your professional guidance. We adopted the Kruskal Wallis H test for comparison between different groups. Furthermore, we also used the Bonferroni method for multiple comparison when there was statistical difference between groups. The results of statistical analysis were the same as previous analysis shown in Figure 3.

Point 2: The statistical method sections need to contain what the study used for the analysis. Such as, add the contents that the authors responsed to the reviewer's concerns about the appropriateness of methods.

Response 2: Thanks for your good suggestions. We have revised this section “Regarding the data distribution and the homogeneity of variance, Kruskal Wallis H test was used to analyze the difference of protein sequence identity in the pairwise comparisons of TPS gene members from A. thaliana, O. sativa and sugarcane at p < 0.05 (SPSS 10.0, Inc., Chicago, IL, USA). Bonferroni method was further used for multiple comparison when there was statistical difference between groups. In the experiment of reverse transcription polymerase chain reaction analysis, the difference between relative gene expression was analyzed using one-way ANOVA test, LSD-t method was used for further comparison between two groups at p < 0.05 (SPSS 10.0, Inc., Chicago, IL, USA). The data of each time points were independent by the chi-square test.”

Round 2

Reviewer 2 Report

The authors have addressed my questions. 

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.

Round 1

Reviewer 1 Report

The statistical analyses have some fundamental problems.

Since the data were collected at 0, 6, 12, 24, and 48 h after the stress treatments were imposed, a longitudinal model should be used for the analysis. However, the study used Student‘s t-test instead. Did the study check the data distribution and consider the dependency of each time points?

3. Figures 5 and 6 are confusing. The data are longitudinal, we do not know how the results come from which models. What N=18 and “Bars with different letters differ significantly at (P< 0.05)” mean are not clear.  The legends are not clear.

4. The statistical analysis section should be included more details about the analysis and adjustment for the methods used for the analysis.

Reviewer 2 Report

The manuscript “Genome-Wide Analysis of the Trehalose-6-Phosphate Synthase (TPS) Gene Family and Expression Profiling of ScTPS1 Gene in Sugarcane” reports data concerning the TPS gene family in sugarcane. The authors suggest an evolutionary characterization and functional classification of the TPS gene family in sugarcane. I have a few minor suggestions for clarification:

1/ In the material and methods (L75,78), and results (L147-148) sections, the authors should add the version of the reference genomes used for classification of the TPS genes.

2/ L79-81: What are the parameters used for gene selection after the Blast analysis (e.g. % identity or gene length coverage)?

3/L117: What is the composition of the hydroponic solution used to grow the sugarcane?

3/ L120: “Six samples were collected for each time point”. Could the authors specify if these samples are biological or technical replicates?

4/ Paragraph 3.5 of the results: Why analyzed only the expression of ScTPS1?

5/ In the discussion, the authors should also compare their results with the results published in Junior et al. 2013 “Expression Analysis of Two Genes Coding for Trehalose-6-Phosphate Synthase (TPS), in Sugarcane (Saccharum Spp.) under Water Stress.” American Journal of Plant Sciences 4 (12): 91–99. https://doi.org/10.4236/ajps.2013.412A3011.

Minor comment: For atemporal facts, the authors should use the present tense: e.g.. L169 “The promoter regions of the ScTPS gene family were rich in both ...” could be replaced by “… are rich in both …”

Reviewer 3 Report

Authors have done an extensive study on the trehalose-6-phosphate synthase gene family which plays an important role in shaping plant abiotic resistance. Using bioinformatics approaches the authors have characterized 9 TPS genes from sugar cane, performed phylogenetic analysis and expression profiling. While sugar cane is an important agricultural crop, identifying key markers conferring resistance against drought, high salinity, and extreme temperatures will immensely help in crop protection strategy building. This study provides a valuable resource for the plant breeding and crop protection communities and therefore within the scope of this journal. However, major concerns listed below need to be addressed before the manuscript is suitable for publication in MDPI Agronomy. I suggest that manuscript be invited for resubmission after the following concerns are addressed.

1. It’s hard to understand why authors have decided to check the expression profile of just ScTPS1 and no other 8 identified genes. Even if they are down-regulated, it’s absolutely essential to include the expression matrix of all 9 ScTPS gene.

2. Out of 9 characterized TPS genes, the ScTPS1 gene structure is comparatively longer than other identified ScTPS genes. Moreover, ScTPS1 lacks one motif which is present in all other ScTPS genes. The discussion lacks a correlation as to why this particular gene lacks motif 2 and if this particular difference is responsible for this gene’s overexpression as compared to other ScTPS genes.

3. In Figure 2, I can easily identify the grouping of monocots in 3 clusters. Still authors have just mentioned two groups/clade, one is monocot and another is dicot (dicot group is not a cluster it is more like an outgroup here). The divergence is due to species and it does not show the actual divergence of genes within in-group. I would like to see more descriptive phylogenetic analysis and more discussion on the three clusters in the monocot group (something is already done here http://www.plantphysiol.org/content/177/1/12 but can be updated using ScTPS genes from sugarcane).

4. Inline 204, authors have talked about strong selective pressure on class II type ScTPS genes with fewer introns than class I, ScTPS1 gene. Instead of a general belief, it would help the manuscript if authors include an analysis of selection pressures/tests of selection (using PAML or data monkey.org algorithms) on ScTPS genes.

5. Please submit all 9 ScTPS gene sequences (exon + introns) to NCBI and provide accession number(s) with the manuscript.

6. All figure legends should be more descriptive (for example, in Figure 2, what are the values on nodes?).