Genome-Wide Identification of ERF Transcription Factor Family and Functional Analysis of the Drought Stress-Responsive Genes in Melilotus albus

Round 1

Reviewer 1 Report

In this manuscript, the authors take on an important challenge to identify the members of ERF Transcription Factor Family and their potential role in drought response in Melilotus albus. Although I think this might be an important contribution, I have a number of comments concerning certain statements, experiments and conclusions which, in my opinion, should be addressed in order to consider the manuscript for publication:

-       Check the English throughout the text. There are multiple style and grammar mistakes.

-       Line 19. Clarify the statement.

-       Line 22. How does the comparison to yeast system can prove that these genes are drought-responsive? I think this rather suggests and definitely doesn’t prove that they “are indeed responsive” to drought stress. Why the yeast system was chosen?

-       Line 25. What is “response theory” in this case? Perhaps a different formulation should be used?

-       Line 38-68. The description of AP2/ERF family and following subdivisions is very cryptic. A proper description should be added in the introduction part.

-       Line 77-80. Why did the authors choose yeast and not more simple plant models for verification of MaERF? Please provide the justification.

-       Figure 1. Why some of MaERFs are indicated on the chromosome maps and others are left in red triangles? Elaborate the legend accordingly.

-       Line 178. Why these particular time points of drought treatment were chosen? Please justify. Are they associated with certain physiological changes in M.albus?

-       Line 180. How did the authors decide that these are the drought-induced MaERFs? Based on the heatmap presented in Figure 6, multiple MaERFs are strongly induced after 3hours – what does it mean and why they are not selected? At the same time, there seems to be a significant difference between root and shoot, this aspect is largely ignored, but it’s very important. I think the authors should work on elaboration of the data presented in Figure 6, perhaps pointing out different subgroups with a similar behaviour in different parts of M.albus and at different time points would be very beneficial. It is not clear to me at all how the authors select drought-responsive candidates and what is reasoning behind it.

-       Line 194. What part of M.albus was taken for qRT-PCR analysis? This is important as they have different responses in root or shoot. This aspect is ignored here for some reason. 

-       Line 195-201. Also I have doubts how these candidate MaERFs were initially selected, I also don’t understand why a more detailed analysis of the behaviour of selected MaERFs was performed here and not really discussed for the data in Figure 6?

-       Line 229. I have hard time to correlate the contribution of MaERFs to drought tolerance with transgenic yeast assays. A proper justification should be provided of why they were not tested in other plant systems, such as A.thaliana.

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript is relevant to the research topic and provides an informative dataset for the future research regarding this topic specially in legumes.

I would make some few comments to the authors that might further the manuscript:

 

#comment-1:

Using the sequences of identified proteins to construct the protein-protein interaction (PPI) network would give further information to reveal the interaction and relationship between different proteins. Such protein-protein interaction (PPI) networks of the upregulated DAPs are important to get better knowledge about proteins mainly involved in the interaction network.

 

# comment-2

Figure 6. I would recommend to use a heatmap of two-dimensional visualization to understand the similarity among root and shoot levels too.

 

Minors

1-      Lines 36-37 please correct the sentence

2-      Figure 1, Explain the color scale bar in the legend.

3-      Table 1, Explain pI, GRAVY in the footnote.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Also, the authors significantly improved the manuscript and addressed many comments, I still have several important comments which I think should be addressed:

 

-       Point 3: Line 22. How does the comparison to yeast system can prove that these genes are drought-responsive? I think this rather suggests and definitely doesn’t prove that they “are indeed responsive” to drought stress. Why the yeast system was chosen? 

 

-       Response 3: Thanks for your suggestion. Firstly, we are sorry for the inaccurate description, and we have revised “In transgenic yeast, overexpression MaERF008, MaERF037, MaERF054 and MaERF058 increased tolerance to drought, further confirming that these genes are indeed responsive to drought stress in M. albus” as “Furthermore, four up-regulated ERFs (MaERF008, MaERF037, MaERF054 and MaERF058) under drought stress were overexpressed in yeast and indicated their biological functions to confer the tolerance to drought.” as shown in line 22-25. Secondly, both plants and yeast belong to eukaryotes, and yeast has been widely used as a model research system to study the gene functions of eukaryote. Some previous studies have used yeast to verify the function of transcription factors under multiple abiotic stresses, so we chose the yeast to study these gene functions preliminary. The examples are as follows. [1] Li X, Zhang D, Li H, et al. EsDREB2B, a novel truncated DREB2-type transcription factor in the desert legume Eremosparton songoricum, enhances tolerance to multiple abiotic stresses in yeast and transgenic tobacco[J]. BMC Plant Biology, 2014, 14(1): 1-16. [2] Li H, Zhang D, Li X, et al. Novel DREB A-5 subgroup transcription factors from desert moss (Syntrichia caninervis) confers multiple abiotic stress tolerance to yeast[J]. Journal of Plant Physiology, 2016, 194: 45-53.

 

-       This is my opinion, but I still don’t understand how the studies in yeast can help us to understand the drought responses in M.albus at the whole plant, organ level and considering the specific and complex ERF-dependent signaling module.

 

-       Point 8: Line 178. Why these particular time points of drought treatment were chosen? Please justify. Are they associated with certain physiological changes in M.albus?-

 

-       Response 8: That’s a great question. Our previous unpublished results showed that in the drought-treated M. albus seedlings, the content of H2O2 were peaked up-to 24 h and then decreased, the content of malondialdehyde (MDA), proline, and soluble sugar were increased continuously up-to 48 h, while the soluble protein content remained basically unchanged for 24 h and then decreased significantly (Figure 1 in this response file). In addition, considering that transcription factors are usually able to rapidly respond to adversity stress in plants, so we finally chose 3 h and 24 h as the drought treatment time points in our study.

 

-       How do all these time points affect the growth of M.albus? are there any anatomical changes? How do the authors correlate the changes they see in malondialdehyde, proline, and soluble sugar with drought responses? Elaborate in the text and provide appropriate references.

 

-       Point 9: Line 180. How did the authors decide that these are the drought-induced MaERFs? Based on the heatmap presented in Figure 6, multiple MaERFs are strongly induced after 3hours – what does it mean and why they are not selected? At the same time, there seems to be a significant difference between root and shoot, this aspect is largely ignored, but it’s very important. I think the authors should work on elaboration of the data presented in Figure 6, perhaps pointing out different subgroups with a similar behaviour in different parts of M.albus and at different time points would be very beneficial. It is not clear to me at all how the authors select drought-responsive candidates and what is reasoning behind it. 

 

-       Response 9: The main purpose of this part is to gain a comprehensive understanding of the expression patterns of all genes in response to drought stress, which would provide a reference for the subsequent selection and verification of drought-responsive functional genes. Just as pointed out by the reviewer that there are multiple MaERFs induced after 3 h drought treatment in M. albus, especially in the root tissue, so we chose these genes mainly focused on their expression levels (all up-regulated more than 10-fold times). It is a great suggestion to select genes based on their expression profiles 

-       in different subgroups, and we will take this approach in our future researches. Thanks again for your valuable suggestion.

 

-       I appreciate the extensive analysis the authors have performed; it is very informative and important. At the same time, I still don’t see a good explanation why only these particular MaERFs candidates were selected?

 

Point 10: Line 194. What part of M.albus was taken for qRT-PCR analysis? This is important as they have different responses in root or shoot. This aspect is ignored here for some reason. 

 

Response 10: We are sorry for this mistake. The tissue used for qRT-PCR analysis is root, and this information has been added as shown in line 420.

 

-       Why only the roots were taken considering different behavior of different MaERFs in different parts of the plant? I think the analysis in shoot should be performed as well. 

 

 

 

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf