Expression Profile of Selected Genes Involved in Na⁺ Homeostasis and In Silico miRNA Identification in Medicago sativa and Medicago arborea under Salinity Stress

Round 1

Reviewer 1 Report

The objectives of this research manuscript are clearly defined. These results built on a previous report of the same group (Tani et al. 2018) and further analyse the gene expression of selected salt-tolerance marker genes.

The written English is quite good and the manuscript is easy to understand; however, there are few minor language-related issues that should be addressed (for example, line 40 “which is caused due to low water availability”).

The research fits very well with the scope of the Journal.

I can only propose minor suggestions and recommendations.

Medium points:

1) Fig 1: it’ll be good to include the name of the analysed gene in each panel. Please, also add in at least one panel what the black (leaves) and grey (roots) bars stand for. It’ll also be good to add more information about the treatment in the legends: how long was the 50-100mM NaCl and the 100mM NaCl treatments? How many plants were analysed?

2) It is important to clearly describe previous results showing that M. sativa is more tolerant than M. alborea to salt stresses in the Introduction and at the end of the paragraph 2.1 Gene expression levels.

3) Figure 2: does the psRNAΤarget tool provide a statistical or significant value for each miRNA-gene pair? I understand that more than 13 consecutive bases have been shown to be sufficient; it’ll be good to add a statistical value to further support the proposed hypothesis.

4) The importance of the miRNAs in abiotic stress tolerance is clearly introduced, however a better connection with the study of the expression of the salt-tolerance marker genes. For example, are any of the analysed genes regulated by miRNA in plants?

Minor suggestions:

5) Line 88, 109, 114, 118, 140 and 141: change “overexpresses” to expression higher than xxx.

6) Line 106: remove “t”

7) The section about the miRNA should be presented as an independent paragraph 2.2.

8) It is good that Authors clearly stated that further experimental validation will be required to confirm the putative role of miR167b-AKT1 and miRNA156-AVP modules in salt tolerance. I appreciate the proper interpretation of the analysis.

Author Response

Thank you very much for your fruitful comments. Please find attached the responses to your comments.

Thank you for helping in improving our manuscript.

Sincerely

The corresponding authors

Author Response File: Author Response.docx

Reviewer 2 Report

NO

Author Response

Thank you very much for your encouraging comments.

Sincerely

The corresponding authors

Reviewer 3 Report

The overall data of this study is relatively small. 

First, it should try to show whether there are any differences in growth state and physiological phenotype of the two different plants (M. sativa and M. arborea) after salt treatment, then it will be more meaningful to display gene expression changes by salt using qPCR. 

The resolution of Figure 1 is not enough. And the labels in the figure are not clear enough. For example, the leaves and roots should be marked in the figure, and the six genes’ name should also be marked in the figure but not only in the legend.

It is not explained why the relative expression of some genes in M. sativa leaves is lower than 0 in figure 1.

"…gradual step acclimatization every 10 days…" in the text is not clear how 50 mM salt change into 100 mM.

The stage of the plant used for salt treatment is also obscure.

Figure 2 is purely bioinformatics analysis without verification. If it is not easy to verify in M. sativa and M. arborea, it should try to discuss whether similar regulatory mechanism exist in other model plants, such as Arabidopsis thaliana.

Author Response

Thank you very much for your fruitful comments. Please find attached our responces to all the comments.

Sincerely

The corresponding authors.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The article is well written and I recommend it for publication.

Author Response

Thank you very much for your valuable comments. We improved the quality of the figure according to your suggestions.

Sincerely

The corresponding editor