Systematic Investigations of the ZF-HD Gene Family in Tobacco Reveal Their Multiple Roles in Abiotic Stresses

Round 1

Reviewer 1 Report

The ms reports the structural and functional characterization of the ZFHD gene family (32 members) in Nicotiana tabacum. The ms is based on numerous results obtained from bioinformatics analyses and lab experiments.

One phylogenetic analysis was conducted from the protein sequences of the 32 Nt ZFHD members (including tomato and Arabidopsis protein sequences). NtZFHD genes are distributed in the phylogenetic groups defined from previous study (Wang et al. 2014, Khatun et al. 2017). Structural (intron/exon distribution, conserved motifs) and functional (RT qPCR, VIGS) analyses were conducted and interpreted in this phylogenetic context.

Even if the manuscript shows a lot of work and represents the first characterization of this family in tobacco, (i) it suffers from numerous innacurracies which minimizes the significance of the results and (ii) perhaps lacks originality in the approach and conclusions (i.e. involvement of some orthologous proteins ATHD1 and NtHD21 in the tolerance to abiotic stress already identified in Arabidopsis).

1. Phylogenetic analysis and its interpretation should be reinforced (as it is the basis of the results interpretation).

• NJ method is not today considered as the best reconstruction method. The authors should test other method (ML method using MEGA for example).
• Bootstrap tests were conducted by values are not indicated in the figure. It seems difficult to build robust groups without this information.
• The authors could have brought originality to this analysis by taking into account the polyploid nature of N. tabacum: they could include protein sequences from the two ancestors N. tomentosiformis and sylvestris (some of them are available). These data could increase the robustness of the phylogenetic analysis and bring interesting data for evolutionary interpretation.
• The addition of sequences from ancestral plant lineages like green algae (before gene family expansion) could be also interesting to root the tree (a few sequences (1?) in Klebsormidium nitens)
• Generally, the use of appropriate terms to qualify “duplicated forms” such as orthologous or paralogous should be privileged and could facilitate the understanding.
• Lanes 171-172 : what does the term “numbers” mean? : duplicated form? “members”  ?
• Lane 172 : group VI perhaps includes two different lineages (it is very heterogenous)? A better phylogenetic reconstruction (including monocots to identify orthologous groups and see above) could help to conclude.
• Lane 176 : what are these WD events ? (see De Smet et al. 2012 (https://doi.org/10.1073/pnas.1300127110)

 

2. As expected, the synteny analysis confirms a better synteny with closer related species (dicot Vs monocots), reflecting organismal phylogeny, but is hard to follow :

• What does the terms “synchronized” mean?
• Link between Figure 3B and, Supplementary Table S5 should be better explained

 

3. Promoter analysis of NtZF-HD family genes

- The clustering obtained could be compared with phylogenetic groups

- What is the significance of the heat map (legend of fig 4)?

 

4. RT PCR analysis needs statistics to conclude

 

5. Several conclusions (ideas) needs to be better argued (in results or in conclusion) :

e.g. lane 207 “The specific distribution of conserved motifs may contribute to reflect the various functions of NtZF-HD genes in tobacco.”

 lane 243 “These results provide a valuable reference for functional studies of NtZF-HD genes in various stress responses.”

Lane 362 “Overall, this study provides useful information for the further functional characterization of NtZF-HD genes in tobacco.”

 

Minor points :

Lane 42 : Arabidopsis is not a model of C4 plants at my knowledge

Lane 219 : Colinearity instead of collinearity, S. lycopersicum instead of S. lycopesicum in fig3A

References list should be homogenized according to the instructions

Author Response

Dear Reviewer:

Thanks for the comments on our manuscript entitled “Systematic Investigations of the ZF-HD Gene Family in Tobacco Reveal Their Multiple Roles in Abiotic Stresses”. Those comments are valuable and helpful for revising and improving our work. We have studied your comments carefully and tried our best to revise our manuscript according to the instructions. Attached please find the revised version, which we would like to submit for your kind consideration.

Best regards.

Response to Reviewer 1 Comments:

Point 1: Even if the manuscript shows a lot of work and represents the first characterization of this family in tobacco, (i) it suffers from numerous innacurracies which minimizes the significance of the results and (ii) perhaps lacks originality in the approach and conclusions (i.e. involvement of some orthologous proteins ATHD1 and NtHD21 in the tolerance to abiotic stress already identified in Arabidopsis).

Response 1: Based on your kind comments, we have rewritten some of the results and discussion to explore the significance of the results. In line: 170-189, 199-204, 255-237, 253-255, 322-326, 334-359.

Based on：NtZF-HD21 was clustered into group I with AtZF-HD1 and may share similar function. The explorations of this gene in biological function could provide clues to verify our hypothesis, and more important, to prove the homologs inferred from our analysis maybe reliable.

Point 2: Phylogenetic analysis and its interpretation should be reinforced (as it is the basis of the results interpretation).

NJ method is not today considered as the best reconstruction method. The authors should test other method (ML method using MEGA for example).

Bootstrap tests were conducted by values are not indicated in the figure. It seems difficult to build robust groups without this information.

Response 2: Because of the low conservation, the NJ method is commonly adopted in reported studies of ZF-HD family in plants. According to your valuable comments, we tried to construct the phylogenetic tree with ML method, but the topological structure is chaos. Therefore, we choose NJ method to construct the phylogenetic tree.

The support values were added on the tree.

Point 3: The authors could have brought originality to this analysis by taking into account the polyploid nature of N. tabacum: they could include protein sequences from the two ancestors N. tomentosiformis and sylvestris (some of them are available). These data could increase the robustness of the phylogenetic analysis and bring interesting data for evolutionary interpretation. The addition of sequences from ancestral plant lineages like green algae (before gene family expansion) could be also interesting to root the tree (a few sequences (1?) in Klebsormidium nitens).

Response 3: According to your kind comments, the N. tomentosiformis and sylvestris ZF-HD proteins were added in the phylogenetic tree, in Figure 1.

We carefully checked the literature and found that the ZF-HD transcription factors only existed in higher plants. (Wang, Wet al. 2016 [http://dx.doi.org/10.1007/s00438-015-1136-1])

Point 4: Generally, the use of appropriate terms to qualify “duplicated forms” such as orthologous or paralogous should be privileged and could facilitate the understanding.

Lanes 171-172 : what does the term “numbers” mean? : duplicated form? “numbers”   ?

The Lane 176 : what are these WD events ? (see De Smet et al. 2012 (https://doi.org/10.1073/pnas.1300127110)

Response 4: Lanes 171-172 : corrected. In line: 177-178.

The Lane 176: This section was rewritten. In line: 179-189.

Point 5: Lane 172 : group VI perhaps includes two different lineages (it is very heterogenous)? A better phylogenetic reconstruction (including monocots to identify orthologous groups and see above) could help to conclude.

Response 5: The ZF-HD proteins of rice were added in phylogenetic tree , in Figure 1.

Point 6: As expected, the synteny analysis confirms a better synteny with closer related species (dicot Vs monocots), reflecting organismal phylogeny, but is hard to follow :

What does the terms “synchronized” mean?

Link between Figure 3B and, Supplementary Table S5 should be better explained.

Response 6: This section were rewritten. In line: 225-230.

Point 7: Promoter analysis of NtZF-HD family genes

-The clustering obtained could be compared with phylogenetic groups.

Response 7: Promoter analysis of NtZF-HD family genes was compared with phylogenetic groups in “4. Discussion”. In line: 347-345.

Point 8: What is the significance of the heat map (legend of fig 4)?

Response 8: The legend information of Figure 4 was changed. In line: 256-257.

Point 9: RT PCR analysis needs statistics to conclude

Response 9: One of reviewers suggested to plot a heatmap of NtZF-HD genes expression patterns in different tissues. We divided Figure 5 into two parts: Supplementary Figure S2 and Figure 4. And the Figure 4 was added the significant statistical differences of RT-PCR analysis. In Figure 4.

Point 10: Several conclusions (ideas) needs to be better argued (in results or in conclusion):

e.g. lane 207 “The specific distribution of conserved motifs may contribute to reflect the various functions of NtZF-HD genes in tobacco.”

lane 243 “These results provide a valuable reference for functional studies of NtZF-HD genes in various stress responses.”

Lane 362 “Overall, this study provides useful information for the further functional characterization of NtZF-HD genes in tobacco.”

Response 10: 

lane 207, corrected. In line: 219-220.

lane 243, this section was rewritten. In line: 253-255.

Lane 362, corrected. In line: 368-369.

Point 11: Minor points :

Lane 42 : Arabidopsis is not a model of C4 plants at my knowledge

Lane 219 : Colinearity instead of collinearity, S. lycopersicum instead of S. lycopesicum in fig3A.

Response 11: Thank you very much for pointing out our mistakes.

Lane 42 : We changed the sentence to “Previous researches revealed that ZF-HD genes participate in many essential biological processes in plants, for example Arabidopsis”. In line: 44-45.

Lane 219 : corrected. In Figure 2A.

Point 12: References list should be homogenized according to the instructions

Response 12: Corrected. In line: 384, 391, 395, 405 412-413, 405, 420, 423, 427, 439, 444, 455, 458, 460, 476, 486.

 

Furthermore, we have already checked the language of this article carefully and corrected the typographical errors. Then, the text has undergone English language editing by MDPI officially and the revised sections are highlighted with red in the latest manuscript. MDPI English language editor certify that this article has been edited to a level suitable for reporting research in a scholarly journal.

Author Response File: Author Response.doc

Reviewer 2 Report

Overall, the study is well conducted and presented coherently. I have few minor concerns like:

Line 117: Please enlist all abiotic stresses instead of just using the word “various” and indicating only salt and drought stresses. Methods should be detailed.

Line 128: Please add, how did you make cDNA? Mention the method/kit used before jumping to RT-qPCR.

Line 129-130: You have mentioned using three biological replicates, did you use any technical replicates? If yes, please mention that too.

Line 131: Please provide reference having details of the 2−∆∆CT data analysis method you have mentioned.

Line 136 and 145-146: Which strain of Agrobacterium (competent cells) was used?

Primers for the house keeping gene (ribosomal protein gene L25) are missing from the Supplementary Table S2.

Is there any specific reason to choose NtZF-HD21 for subcellular localization and VIGS analysis? In methods you have mentioned subcellular localization studies of only NtZF-HD21. In results, you have mentioned the prediction of subcellular localization of the whole family. Please mention the software you have used for the prediction of subcellar localization also under subheading 2.7 in methods. Also results section 3.7 you should mention the predicted subcellular localization results instead of section 3.1.

Line 170 , 171 and 178 : A typo “numbers” should be replaced with “members”.

Line 172-175: Please rephrase the sentence, it is confusing.

Line 186-187: You have written, “coding sequences of the NtZF-HD genes were interrupted by introns”, whereas line 188-189 is saying that 15 gene are intronless. Similarly, line 185-186 is contradicting to what you are saying in lines 190-193. Looking at the figures, I think your generalized sentences (lines 185-187 are not appropriate). Same thing in discussion line 314-315, please clarify what do you mean? Are you talking about the gene families in all understudy plants or just tobacco?  

Line 214-215: There is a typo “synthetic” should be “syntenic”

Lines 217-219: Please look at the sentence, it grammatically incorrect.

Lines 293-294: Please look at the sentence, I think “plant resistant” should be replaced with something like “plant defense” Or you should rephrase the whole sentence to clarify what you mean.

Line 297 –  You have mentioned “NtZF-HD21 was randomly selected” , just wondering if you have found stress responsive motifs in the genes, why didn’t you choose according to the presence of a certain stress responsive motif containing gene and the respective stress type in your studies?

Lines 342-343: Please rephrase the sentence. I think you should say “These findings suggest that” instead of  “These suggested that”.

 Lines 349-350: Please rephrase the sentence. I think you should say “These results” instead of  “This result”.

Line 352: The word “various” shouldn’t be used when you have only presented the results for drought and salinity.

Line 360: Biotic stress is not being tested here, remove the word “biotic”.

 

 

Author Response

Dear Reviewer:

Thanks for the comments on our manuscript entitled “Systematic Investigations of the ZF-HD Gene Family in Tobacco Reveal Their Multiple Roles in Abiotic Stresses”. Those comments are valuable and helpful for revising and improving our work. We have studied your comments carefully and tried our best to revise our manuscript according to the instructions. Attached please find the revised version, which we would like to submit for your kind consideration.

Best regards.

Response to Reviewer 2 Comments:

Point 1: Overall, the study is well conducted and presented coherently. I have few minor concerns like:

Line 117: Please enlist all abiotic stresses instead of just using the word “various” and indicating only salt and drought stresses. Methods should be detailed.

Response 1: Corrected in line: 122 and 126-127.

 

Point 2: Line 128: Please add, how did you make cDNA? Mention the method/kit used before jumping to RT-qPCR.

Response 2: Added in line: 130-132.

 

Point 3: Line 129-130: You have mentioned using three biological replicates, did you use any technical replicates? If yes, please mention that too.

Response 3: Added in line: In line 137-138.

 

Point 4: Line 131: Please provide reference having details of the 2^−∆∆CT data analysis method you have mentioned.

Response 4: We added the reference, Livak, K.J. et al. (2001 (https://doi.org/10.1006/meth.2001.1262).

 

Point 5: Line 136 and 145-146: Which strain of Agrobacterium (competent cells) was used?

Response5: We added the strain of Agrobacterium, “GV3101”. In line: 147 and 156.

 

Point 6: Primers for the house keeping gene (ribosomal protein gene L25) are missing from the Supplementary Table S2.

Response 6: L25 primer sequences were added in the Supplementary Table S2. In Table S2.

 

Point 7: Is there any specific reason to choose NtZF-HD21 for subcellular localization and VIGS analysis? In methods you have mentioned subcellular localization studies of only NtZF-HD21. In results, you have mentioned the prediction of subcellular localization of the whole family. Please mention the software you have used for the prediction of subcellar localization also under subheading 2.7 in methods. Also results section 3.7 you should mention the predicted subcellular localization results instead of section 3.1.

Response 7: Based on：NtZF-HD21 was clustered into group I with AtZF-HD1 and may share similar function. The explorations of this gene in biological function could provide clues to verify our hypothesis, and more important, to prove the homologs inferred from our analysis maybe reliable.

The software for prediction of subcellar localization were added. In line 140-141.

We added the predicted subcellular localization results at results section 3.7 instead of section 3.1. In line 283-285.

 

Point 8: Line 170 , 171 and 178 : A typo “numbers” should be replaced with “members”.

Response 8: Corrected in line: 177, 178 and 191.  

 

Point 9: Line 172-175: Please rephrase the sentence, it is confusing.

Response 9: Line 172-175: Corrected in line: 176-177.

 

Point 10: Line 186-187: You have written, “coding sequences of the NtZF-HD genes were interrupted by introns”, whereas line 188-189 is saying that 15 gene are intronless. Similarly, line 185-186 is contradicting to what you are saying in lines 190-193. Looking at the figures, I think your generalized sentences (lines 185-187 are not appropriate). Same thing in discussion line 314-315, please clarify what do you mean? Are you talking about the gene families in all understudy plants or just tobacco?  

Response 10:  We changed the describe in line 198-203.

 

Point 11: Line 214-215: There is a typo “synthetic” should be “syntenic”

Response 11: Corrected in line: 224.

 

Point 12: Lines 217-219: Please look at the sentence, it grammatically incorrect.

Response 12: Corrected in line: 237-240.

 

Point 13: Lines 293-294: Please look at the sentence, I think “plant resistant” should be replaced with something like “plant defense” Or you should rephrase the whole sentence to clarify what you mean.

Response 13: Corrected in line: 305.

 

Point 14: Line 297–You have mentioned “NtZF-HD21 was randomly selected” , just wondering if you have found stress responsive motifs in the genes, why didn’t you choose according to the presence of a certain stress responsive motif containing gene and the respective stress type in your studies?

Response 14: We selected NtZF-HD21 after careful consideration. NtZF-HD21 was clustered to into group I with AtZF-HD1. The AtZF-HD1-overexpressing transgenic plants improved Arabidopsis tolerance to drought stress. NtZF-HD21 with tress-responsive elements (TC-rich repeats) was up-regulated significantly under dehydration treatment implied that NtZF-HD21 may be involved in the response to tobacco drought stress. Therefore, we selected NtZF-HD21 to verify its function by subcellular localization and VIGS analysis. Therefore, we deleted “randomly”. In line: 309.

 

Point 15: Lines 342-343: Please rephrase the sentence. I think you should say “These findings suggest that” instead of  “These suggested that”. ]

Response 15: Corrected in line: 350.

 

Point 16: Lines 349-350: Please rephrase the sentence. I think you should say “These results” instead of  “This result”.

Response 16: This section was revised in line: 356-358.

 

Point 17: Line 352: The word “various” shouldn’t be used when you have only presented the results for drought and salinity. 

Response 17: We changed the “various” to “drought and salinity”.

 

Point 18:

Line 360: Biotic stress is not being tested here, remove the word “biotic”.

Response 18: Corrected in line: 364.

 

Furthermore, we have already checked the language of this article carefully and corrected the typographical errors. Then, the text has undergone English language editing by MDPI officially and the revised sections are highlighted with red in the latest manuscript. MDPI English language editor certify that this article has been edited to a level suitable for reporting research in a scholarly journal. 

Author Response File: Author Response.doc

Reviewer 3 Report

The investigation of ZF-HD gene family is very interesting since the encoded transcription factors are involved in the control of development and stress response.  The authors used complex bioinformatic and molecular biological approach to elucidate the importance and role of these genes. However, the discussion is not well-written. Its several part is a repetition of the results. It does not explain the mechanism by which these transcription factors explicate their effects. The possible target genes are not mentioned. The conclusions section is a summary of the results, therefore it should be rewritten.

Other remarks:

1. 2: The letters are too small.
2. 4: What is the meaning of the colours?
3. 5: There are too many small graphs. They should be replaced heatmaps.
4. 6: The legends of C and D should be changed.
5. 293-295: This part belongs to the introduction.
6. The typing and grammatical errors should be corrected.

Author Response

Dear Reviewer:

Thanks for the comments on our manuscript entitled “Systematic Investigations of the ZF-HD Gene Family in Tobacco Reveal Their Multiple Roles in Abiotic Stresses”. Those comments are valuable and helpful for revising and improving our work. We have studied your comments carefully and tried our best to revise our manuscript according to the instructions. Attached please find the revised version, which we would like to submit for your kind consideration.

Best regards.

Response to Reviewer 3 Comments:

Point 1: The investigation of ZF-HD gene family is very interesting since the encoded transcription factors are involved in the control of development and stress response.  The authors used complex bioinformatic and molecular biological approach to elucidate the importance and role of these genes. However, the discussion is not well-written. Its several part is a repetition of the results. It does not explain the mechanism by which these transcription factors explicate their effects. The possible target genes are not mentioned. The conclusions section is a summary of the results, therefore it should be rewritten.

Response 1: According to your valuable comments, the discussion has been rewritten. In line: 321-324, 327-331, 334-358.

Point 2: 2: The letters are too small.

Response 2: Based on your kind comments, we modified the letters in the Figure 2. The letters size is twice the original. And we put Figure 2 in the “Supplementary Materials”. Revised in Figure S2.

Point 3: 4: What is the meaning of the colours?

Response 3: We changed the legend information of Figure 4. In line: 256-257.

Point 4: 5: There are too many small graphs. They should be replaced heatmaps.

Response 4: According to your valuable comments, we divided Figure 5 into two parts: Supplementary Figure S2 and new Figure 4. Figure S2 was “The expression pattern of the selected NtZF-HD genes in different tissues”which was a heatmap. And the new Figure 4 was “The expression patterns of selected NtZF-HD genes under (A) drought and (B) NaCl.” which was composed of some small graphs. We did not make new Figure 4 into a heatmap because it needs to add the significant statistical differences. Revised in Figure S2 and Figure 4.

Point 5: 6: The legends of C and D should be changed.

Response 5: Corrected in line: 301-303.

Point 6: 293-295: This part belongs to the introduction.

Response 6: Based on your kind comments, the sentence in line 293-295 was deleted. And in the last paragraph of the introduction. In line: 71-73.

Point 7: The typing and grammatical errors should be corrected.

Response 7: We have already checked the language of this article carefully and corrected the typographical errors. Then, the text has undergone English language editing by MDPI officially and the revised sections are highlighted with red in the latest manuscript. MDPI English language editor certify that this article has been edited to a level suitable for reporting research in a scholarly journal. 

Author Response File: Author Response.doc

Round 2

Reviewer 3 Report

The authors considered all my suggestion by the revision of their manuscript. However, minor errors still remained in the text. I found those ones listed below, but you should check the whole manuscript again.

 

Remarks:

1. 44: in Arabidopsis (I always typed the corrected form).
2. 82, 96, 167, 184: in sylvestris
3. 122: for 0, 3
4. 131: was the transcript of
5. 132: repeats of all reactions were performed
6. 140-142: The sentence should be checked.
7. 223: may existed
8. 224: has maintained
9. 294: The sentence must be rewritten.
10. l 356: genes suggesting

Author Response

Dear Reviewer:

 Thanks for the additional suggestions for our manuscript, which were so valuable and helpful for promoting our work. Best wishes with you and happy Chinese new year !

Best regards.

Response to Reviewer 3 Comments:

Point 1: 44: in Arabidopsis (I always typed the corrected form).

Response 1: Corrected.

Point 2: 82, 96, 167, 184: in sylvestris.

Response 2: Corrected.

Point 3: 122: for 0, 3

Response 3: Corrected.

Point 4: 131: was the transcript of

Response 4: Corrected.

Point 5: 132: repeats of all reactions were performed

Response 5: Corrected.

Point 6: 140-142: The sentence should be checked.

Response 6: Checked and corrected.

Point 7: 223: may existed

Response 7: Corrected.

Point 8: 224: has maintained

Response 8: Corrected.

Point 9: 294: The sentence must be rewritten.

Response 9: Corrected.

Point 10: 356: genes suggesting

Response 10: Corrected.

Author Response File: Author Response.doc