Transcriptomic and Metabolomic Analyses of Seedlings of Two Grape Cultivars with Distinct Tolerance Responses to Flooding and Post-Flooding Stress Conditions

Round 1

Reviewer 1 Report

The article is interesting with a large amount of complementary data.I think there is a serious problem with the figures. The letters that refer to the figures are poorly arranged and not very descriptive.There are enzyme numbers, the images are not aligned. This demerit the job.

I suggest a major revision of the figures.

Author Response

Response to Reviewer 1 Comments:

Point 1: I suggest a major revision of the figures.

Response 1: We revised all the figures in this manuscript, please check the new figures in the revised version.

Reviewer 2 Report

The authors worked with two cultivars (“SO4” and “Kyoho”) and analyzed the effect of flooding and post-flooding stress by transcriptome and LC-MS/MS methods. The plant is grapevine that a crop with economic importance and it may be affected by these conditions. They evaluate the stress effect on roots.

Abstract – it needs to be improved

1)      They mention that SO4” and “Kyoho “ have difference tolerance. It is known which one is more tolerant?

2)      It mentions it is the first time that endoplasmic reticulum-related E3 ubiquitin ligase gene. Ok, but what is the role? What is the hypothesis? Why is giving emphasis to this. We need more information about.

3)      Why it is not mentioned any data from LC-MS/MS method in the abstract

Results

4)      Make a table with all the libraries and all the conditions used. It will be easier to understand the figure 1. Who is KF7 or KCK0? It is in the text, but if we have a table make simple to follow. And add this information in the figure legend too.

5)      Explain better what PCA analysis allowed to observe.

6)      Explain better why the comparation between different libraries. What is the hypothesis and what expect and what got it – figure 2

7)      Improve figure 3 presentation, it is difficult to analyze.

8)      Improve figure 4c and explain better in the text.

9)      Improve figure 6b it is impossible to be analyze even if we increase the zoom or find a different way to present. They have important data presented in this figure.

10)   Figure 8 is really good. It is good this type of regulation. Great

11)   The authors bring important data how these two cultivars respond to these conditions. But how these data can be used in plant breeding or crop management? They can add this information in the last paragraph.

12)   They can add a model how grapevine respond to these conditions.

Methods

13)   They can add a graph with all the steps from methods

Sup material – all the tables are important.

Comments for author File: Comments.pdf

The authors need to check english

Author Response

Response to Reviewer 2 Comments

Point 1: They mention that SO4” and “Kyoho “ have difference tolerance. It is known which one is more tolerant?

Response 1: We changed “two grape cultivars (“SO4” and “Kyoho”) differing in flood tolerance” to ““SO4” (tolerant to flooding) and “Kyoho” (sensitive to flooding)” in line 22-23.

Point 2: It mentions it is the first time that endoplasmic reticulum-related E3 ubiquitin ligase gene. Ok, but what is the role? What is the hypothesis? Why is giving emphasis to this. We need more information about.

Response 2: In order to meet the word number limitation (200 words), we deleted “For the first time, we report that endoplasmic reticulum-related E3 ubiquitin ligase genes could be involved in post-flooding responses, and March6 in SO4 seedlings in recovery expressed 2.33 times higher than the control”.

Point 3: Why it is not mentioned any data from LC-MS/MS method in the abstract

Response 3: We added “the abundant of many metabolites in phenylpropanoids and polyketides superclass, organic acids and derivatives superclass, and organic oxygen compounds superclass changed in different patterns between Kyoho and SO4 under flooding and post-flooding” in line 24-26.

Point 4: Make a table with all the libraries and all the conditions used. It will be easier to understand the figure 1. Who is KF7 or KCK0? It is in the text, but if we have a table make simple to follow. And add this information in the figure legend too.

Response 4: We added “The treatment of sample groups in this study were listed in Table 1” in line 109, and we added Table 1 in line 117.

Point 5: Explain better what PCA analysis allowed to observe.

Response 5: We changed “The principal component analysis (PCA) of gene expression data revealed high reproducibility between the biological replicates, with the six groups clearly separated, except for SO4 recovered for 3 days after flooding (SF7R) and control seedlings of SO4 (SCK0) (Figure 1A). This suggested that flooded SO4 might already have fully recovered within 3 days after drainage.” to “The principal component analysis (PCA) of gene expression data revealed high reproducibility between the biological replicates (Figure 1A). Principal component 1 (PC1), which accounted for 56.47% of the variance, separated the three treatments. Principal component 2 (PC2), which accounted for 16.49% of total variance, separated the two cultivars. All sample groups but SCK0 and SF7R were apparently separated, this suggested that sample groups had different characteristics at transcriptional level, and flooded SO4 might already have fully recovered within 3 days after drainage.” In line 123 to 128.

Point 6: Explain better why the comparation between different libraries. What is the hypothesis and what expect and what got it – figure 2

Response 6: We added “The enriched pathways analysis between treatment and control of the same cultivar could reveal which pathway the transcriptional responses were focused on under a certain treatment.  A number of enriched pathways were usually different between cultivars, and those different enriched pathways might explain how Kyoho and SO4 respond differently to the flooding and post-flooding” in line 175-180.

Point 7: Improve figure 3 presentation, it is difficult to analyze.

Response: We improved the figure 3, please check it in line 240-248.

Point 8: Improve figure 4c and explain better in the text.

Response 8: We improved the figure 4c, please check it in line  ; we changed “The top-five superclasses containing the most DEMs were lipids and lipid-like molecules, organic oxygen compounds, phenylpropanoids and polyketides, organic acids and derivatives, and organoheterocyclic compounds.” to “The seven superclasses containing the more than 10 DEMs were lipids and lipid-like molecules superclass, phenylpropanoids and polyketides superclass, organic oxygen compounds superclass, organic acids and derivatives superclass, organoheterocyclic compounds superclass, Benzenoids and Nucleosides, nucleotides and analogues superclass.” in line 278-281 ; we added “lipids and lipid-like molecules superclass and” in line 282; we changed “many more” to “a lot more DEMs” in line  ; we added “, as well as Benzenoids” in line 284-285; we changed “The DEMs in the above three superclasses might play crucial roles in shaping the different responsive traits of the two grapevine cultivars to flooding and post-flooding recovery.” to “In organoheterocyclic compounds superclass and Nucleosides, nucleotides and analogues superclass, the DEM numbers were bigger under flooding than post-flooding situation.” in line 286-288.

Point 9: Improve figure 6b it is impossible to be analyze even if we increase the zoom or find a different way to present. They have important data presented in this figure.

Response 9: We improved the figure 6b, please check it in line 348.

Point 10: Figure 8 is really good. It is good this type of regulation. Great

Response 10: Thanks.

Point 11: The authors bring important data how these two cultivars respond to these conditions. But how these data can be used in plant breeding or crop management? They can add this information in the last paragraph.

Response 11: We added “, and the DEGs in SO4, like TF genes in ERFs, WRKYs, and bHLHs family, could be applied for further study on the breeding of flood-tolerant rootstock cultivars” in line 822-823.

Point 12: They can add a model how grapevine respond to these conditions.

Response 12: We added “and the mechanism of flooding response and post-flooding response in SO4, the flood-tolerant cultivar, were briefly illustrated in Figure 12” in line 82-823, and we added Figure 12.

Point 13: They can add a graph with all the steps from methods

Response 13: We added Figure 11, please check line 695.

Point 14: all the tables are important

Response 14:  We added the titles of Supplementary Tables.

Reviewer 3 Report

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 3 Comments:

Point 1: Regarding the introduction, I am pleased with how well you have described why you decided to pursue this type of study and also why you chose the species.

Response 1: Thanks.

Point 2: Line 115: In section 2.2, when one begins to discuss the results, it is difficult to understand who is what. That is, it would be better at the beginning of the results to include the full name of the cultivar and the experimental condition and not just the acronym. I would insert for each SO4 recovered for 3 days after (SFR7) and so on. So that it is clearer at the beginning how to read the results and the graphs, which are numerous and complex

Response 2: We added Table 1 to describe the cultivar and the experimental condition in line 117.

Point 3: For figures and tables: the quality of the images needs to be improved as some of them look grainy. For example in figure 2, the first two overlap and the references cannot be read clearly. I would try arranging the figures so that one is next to the other and/or group them 2 by 2 (cultivar by cultivar) and not stack them on top of each other.

Response 3: We improved all Figures, please check the new ones.

Point 4: For the discussions, instead, because the analyses were different and so were the results, I would divide the discussions into sub-sections. Because by not dividing them, it is difficult to understand the conclusion drawn from the study.

Response 4: We divided the discussions into 6 sub-sections, please check the title of sub-sections in line 526, line 536, line 578, line 604, line 625 and line 658.

Point 5: But the thing that needs to be more systematized is the materials and methods. We have chosen to do a huge work on transcriptomics and metabolomics, so we need to better describe the methods of extraction, especially for metabolomics. I found the description for proteins, but sugars, amino acids? Plus a section on materials and methods regarding hormones? How were they detected?

Response 5: We were not sure the exact DEMs before this study, so we applied a non-targeted LC-MS analysis in this research by using one extracting method which could obtain the biggest number of metabolites, the targeted extraction methods will be used based the result of metabolomics in this study. We added Figure 11 in line 695 to describe the research steps.

Reviewer 4 Report

The manuscript by Peng et al. considers changes in transcriptome and metabolome of grape under flooding stress condition. The manuscript seems to be interesting. However, I have some comments.

(1) Is flooding an important problem for grape? Please, note regions with this problem. How often floods are observed in these regions?

(2) Please, check correctness of colorbars in Figure 3b (right), Figure 5a (right).

(3) Please, add scheme that briefly summarize results of the work.

Author Response

Response to Reviewer 4 Comments

Point 1: Is flooding an important problem for grape? Please, note regions with this problem. How often floods are observed in these regions?

Response 1: Flooding is an important problem for common crops and trees, including grape. It is hard to conclude the frequency of flooding in all those regions, because it is more likely a global situation. We described this global event in the first paragraph of introduction section, please check line 38-45. We added “in both Northern and Southern Hemispheres” in line 79, and we added “like other common crops” in line 80.

Point 2: Please, check correctness of colorbars in Figure 3b (right), Figure 5a (right).

Response 2: we revised Figure 3b and Figure 5a in line 243 and line 312, respectively.

Point 3: Please, add scheme that briefly summarize results of the work.

Response 3: we added Figure 12 in line 824, and we added “and the mechanism of flooding response and post-flooding response in SO4, the flood-tolerant cultivar, were briefly illustrated in Figure 12” in line 797-799.

Round 2

Reviewer 1 Report

The manuscript has been improved. I think it is ready to be published