Expression of Genes Related to Plant Hormone Signal Transduction in Jerusalem Artichoke (Helianthus tuberosus L.) Seedlings under Salt Stress

Round 1

Reviewer 1 Report

 In this manuscript, the author studied the expression of genes related to plant hormone signal transduction in Jerusalem artichoke (Helianthus tuberosus L.) seedlings under salt stress. Authors obtained high-quality transcriptome from leaves and roots of Jerusalem artichoke exposed to salinity (300 mM NaCl) for 0 h, 6 h, 12 h, 24 h, and 48 h, with 150,129 unigenes and 9023 DEGs (Differentially Expressed Genes). The RNA-seq data were clustered into time-dependent groups (nine clusters each in leaves and roots); gene functions were distributed evenly among the group's convergence. KEGG enrichment analysis showed the genes related to plant hormone signal transduction were enriched in almost all treatment comparisons. Under salt stress, genes belonging to PYL (abscisic acid receptor PYR / PYL family), PP2C (Type 2C protein phosphatases), GH3 (Gretchen Hagen3), ETR (ethylene receptor), EIN2/3 (ethylene-insensitive protein 2/3), JAZ (Genes such as jasmonate ZIM-domain gene) and MYC2 (Transcription factor MYC2) had extremely similar expression patterns. The results of qPCR of 12 randomly selected genes confirmed the accuracy of RNA-seq. Conclusions: Under the impact of high salinity (300mM) environment, Jerusalem artichoke in the seedling stage was difficult to survive for a long time, and the phenotype was severe in the short term. Based on the expression of genes on the time scale, the authors found that the distribution of gene functions in time is relatively even. Upregulation of the phytohormone signal transduction had a crucial role in the response of Jerusalem artichoke seedlings to salt stress, the genes of abscisic acid, auxin, ethylene, and jasmonic acid had the most obvious change pattern.

The manuscript has good data however for the betterment of the manuscript I have a few suggestions for the authors:

1. Why is the supplementary figure being in the main text and that too in material and method?
2. Paper is not in clear agronomy format and alignment also not good. Figures are too small to read. Enlarge the figures or divide them to see clearly.
3. Consider changing the annotation title in table 1 with another title.
4. In table 3 it is not a functional abbreviation it is a gene name. Change it.
5. Figure 11 is incomplete. All are connected with each other. Make a comprehensive inclusive figure. Before plant hormones, there must be some signal that triggers these hormones. Also, there should be a final function of these hormones. If it is not well characterized in this plant then consider finding it in other plants with question marks and dotted lines. Your study is not conclusive so you should not put a clear line instead use a dotted line and in the title say hypothetical prediction based on the finding of this study.
6. Do in-silico protein-protein analysis using a free web server and use it as figure 10. and explain it in the text.
7. The introduction is short. The author should include recent expression analysis studies such as a. Genome-Wide Identification, and Characterization of PIN-FORMED(PIN) Gene Family Reveals Role in Developmental and Various Stress Conditions in Triticum aestivum. b. Genome-wide identification and expression pattern analysis of the KCS gene family in barley. C. Genome-wide identification and characterization of abiotic stress-responsive lncRNAs in Capsicum annuum. d. Genome-Wide Identification and Characterization of the Brassinazole-resistant (BZR) Gene Family and Its Expression in the Various Developmental Stage and Stress Conditions in Wheat (Triticum aestivum). e. Genome-wide identification and functional characterization of natural antisense transcripts in Salvia miltiorrhiza. f. Genome-wide identification and expression analysis of the AT-hook Motif Nuclear Localized gene family in soybean. g. Strigolactone signaling genes showing differential expression patterns in Arabidopsis max mutants.

Author Response

Thank you for your valuable suggestions, we have made a revision of the paper.

1. Why is the supplementary figure being in the main text and that too in material and method?

A: This is really strange, we have moved the information about the figure to the results.

2. Paper is not in clear agronomy format and alignment also not good. Figures are too small to read. Enlarge the figures or divide them to see clearly.

A: This does cause difficulties for readers, we tried to split the image and enlarge the font.

3. Consider changing the annotation title in table 1 with another title.

A: This was a problem indeed, which made people unable to understand the meaning of annotation. Here, we have made improvements. We used the protein sequence of sunflower as a reference (Sunflower is the plant known to be the closest to Jerusalem artichoke) to give these unigene functional explanations, and add these 12 to the qPCR quantitative result graph in Figure 10. The specific information of unigenes predicted CDS was used as a supplement to in-silico protein-protein analysis.

4. In table 3 it is not a functional abbreviation it is a gene name. Change it.

A: As you said, we modified the expression now.

5. Figure 11 is incomplete. All are connected with each other. Make a comprehensive inclusive figure. Before plant hormones, there must be some signal that triggers these hormones. Also, there should be a final function of these hormones. If it is not well characterized in this plant then consider finding it in other plants with question marks and dotted lines. Your study is not conclusive so you should not put a clear line instead use a dotted line and in the title say hypothetical prediction based on the finding of this study.

A: As you said, we have optimized the description of Figure 11 again to make it more complete.

6. Do in-silicoprotein-protein analysis using a free web server and use it as figure 10. and explain it in the text.

A: In order to allow readers to have a clearer understanding of the specific functions of these 12 unigenes, we compared them with sunflower data and visualized them in Figure 10.

7. The introduction is short. The author should include recent expression analysis studies such as a. Genome-Wide Identification, and Characterization of PIN-FORMED(PIN) Gene Family Reveals Role in Developmental and Various Stress Conditions in Triticum aestivum. b. Genome-wide identification and expression pattern analysis of the KCS gene family in barley. C. Genome-wide identification and characterization of abiotic stress-responsive lncRNAs in Capsicum annuum. d. Genome-Wide Identification and Characterization of the Brassinazole-resistant (BZR) Gene Family and Its Expression in the Various Developmental Stage and Stress Conditions in Wheat (Triticum aestivum). e. Genome-wide identification and functional characterization of natural antisense transcripts in Salvia miltiorrhiza. f. Genome-wide identification and expression analysis of the AT-hook Motif Nuclear Localized gene family in soybean. g. Strigolactone signaling genes showing differential expression patterns in Arabidopsis max mutants.

A:You have provided very good suggestions. In the introduction section of the article, we have added the results of these studies.

Reviewer 2 Report

The manuscript by Zhou et al. presents transcriptomic studies related to hormonal signalling in Jerusalem artichoke leaves and roots under salt stress. Although the topic is interesting, some issues should be improved/explained before the publication.

General. The other results could be included in the manuscript, e.g. from biochemical analyzes or physiological measurements that would support conclusions based only on transcriptomic results. Please consider it.

Highlights do not reflect the most important conclusions. This section should be definitely rewritten or removed.

Abstract (especially “Background” part) should be also improved towards a better introduction to work.

Materials and methods

I cannot find the plant breeding details. Was there 1 biological repetition (1 independent experiment)? In what period (season) was the experiment conducted? Did other factors, e.g. temperature,  not affect the greenhouse experiment?

I also cannot find the details of RNA samples preparation. Was the material pooled?

The descriptions of the figures are illegible (Figure S1, 2, 9, 10). It is difficult to analyze.

Conclusions need to be improved. Currently, this section repeats the results.

 

Author Response

Thank you very much for your comments, your comments are very helpful to our research.

1. The first is the content of biochemical analysis or physiological measurement. Biochemical analysis is helpful, but our article mainly focuses on changes in the time gradient of the transcriptome. The addition of physical and chemical information seems to deviate slightly from our research. We will use our physical and chemical information in the next article. The focus of the next article is the joint analysis between omics. This information will be very critical.
2. The highlight and summary section did have the problem and we corrected it, the highlight has been deleted.
3. The detailed information of plant breeding is mentioned in the “Plant Material Preparation and Salt Treatment”section of the material method. When preparing the samples, three biological replicates were prepared. RNA-seq mixed the three samples into one and performs RNA extraction. Tubers were collected in February, plant breeding experiments were conducted in March (supplemented in materials and methods), and the entire experiment was carried out in an incubator environment (supplemented instructions in materials and methods), to minimize The influence of other factors on the experimental results.
4. The steps and results of RNA extraction are described and corrected in “cDNA Library Establishment and Illumina RNA sequencing”
5. Related charts have been rearranged and made to make them more readable.
6. Consistent with the highlights and abstracts, the conclusions have also been revised.

Round 2

Reviewer 1 Report

Excellent. Manuscript quality improved a lot. It can be processed in its current format but there are still aligning issue and again supplementary information in the main text.

Author Response

Thank you very much for your comments, and your comments are very helpful to our research.

Reviewer 2 Report

Dear Authors,

I am satisfied with the revision of the manuscript. I have no further comments. However, before publication, I recommend checking carefully the entire manuscript again, there are still minor mistakes. Examples: NanoDrop (capital letter), Helianthus tuberosus, page 22, line 511 and in the References (should be in italic), references should be formatted as required by Agronomy.

Author Response

Thank you for your suggestion. We have checked carefully the entire manuscript again and improved it.