Identification, Expression, and Functional Study of Seven NAC Transcription Factor Genes Involved in Stress Response in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn.)

Round 1

Reviewer 1 Report

The study is important for understanding the genes that imprint plant resistance to stressors, in the context of the opportunity to create new plant genotypes so that they can be grown even in harsh environmental conditions that occur under climatic changes.
The research methods are current and the wording is correct.

Minor corrections:

rows 34, 87 and other: ”Arabidopsis” should be written in italics.

rows 225 and 226: species names should be written in italics.

Author Response

Thanks for your suggestion. We have revised all the Latin names of species to be italic throughout the manuscript, including Arabidopsis, Oryza sativa, Fagopyrum tataricum, Cajanus cajan, and so on.

 

Reviewer 2 Report

Overall the authors of the manuscript have studied the function and expression level of 7 NAC transcription factors in Tartary buckwheat. They introduce the general function of NAC transcription factors in other species in the introduction section. The materials and Methods section provided enough information about the technical setup of experiments. In silico analysis of 7 NAC transcription was presented in the first part of the results. Authors also qualified the expression levels of NAC transcription factors under biotic and abiotic stresses. At the end of the study, they have shown overexpression lines of NAC70 confer drought resistance in the model plant Arabidopsis.  Please consider this few suggestions :

1) Figure 2 has low quality and is not readable

2) Line 250: what figure are the authors referring to? 

3) Drought treatment on OE line plants in pots has not been described. 

Author Response

 

 

Reviewer 3 Report

General

Overall, the authors have made a good set of experiments to observe the expression of the NAC TF genes in tartary buckwheat. However, it will be much better if some explanations are added to improve the strength of this paper and attractiveness for the reader. There are also gaps between the results and discussions that require to be connected in order to strengthen the theory. There are some missing details that the authors have to revise.

Specific

Title – Please add the author of the botanical name (e.g., Oryza sativa L.)

Introduction

Line 39-42 – Please be consistent on adding the author of the botanical names (e.g., you’ve added one for rice, Oryza sativa L., longan, Dimocarpus longan Lour., but none for cotton and for the star of this research, the tartary buckwheat)

Line 47-55 – You mentioned that NAC TF genes play essential role for plant growth, development, stress, and senescence. I think it will be important to also add 3-7 sentences to elaborate shortly on how the transcription factor genes are involved. Are there pathways of genes regulated by NAC TF? Perhaps you want to check on Nurruzaman et al. (2015; https://doi.org/10.3389/fpls.2015.00676) for it.

Line 56-89 – What are the relationships between NAC TF genes and other stress response pathway? You mentioned how the processes are mediated by JA and ABA. You might want to explain how, i.e., for salt/drought tolerance, is it related to glycine betaine or other signaling pathways? How about cold? Check Nurruzaman et al. (2013; https://doi.org/10.3389/fmicb.2013.00248) and Nakashima et al. (2011; https://doi.org/10.1016/j.bbagrm.2011.10.005). You used cold treatment for your experiment, but the introductory information is missing.

Additionally at the end of the Introduction, add short explanations on how significant of tartary buckwheat for the field of study. Describe the novelty, you might want to compare it with the other crop plants.

Materials and Methods

Line 152 – qRT-PCR is Real Time Quantitative (for the q) Reverse Transcription (RT) PCR. If you refer it as Quantitative Real Time, then qPCR (to quantify DNA, not cDNA from RNA). However, from your manuscript, I believe that you want to check the expression and involved cDNA synthesis, not DNA, hence qRT-PCR. Please fix it.

Discussions

Line 371-400 – The authors describe how each FtNAC gene plays important roles of its own. Are the explanations here based on previous study on the same plant, or Arabidopsis? If so, please mention it on the text, for example “Based on the study on Arabidopsis…” to avoid misinterpretation. Also, please recheck if the specific FtNAC genes also work the same on the other species. I think there are plenty of references for this.

FtNAC11 and 28 is for detoxification and abiotic response, 12 is for nitrate response, senescence and defense (again herbivory or stress?), 65 is for senescence, 26 is for proteotoxic stress and protein homeostasis. 11, 28, 12, 65, 26 have similar function… of which one? Please rephrase! FtNAC70 is for embryo and flower pattern formation, pathogen-induced cell death and salt stress resistance and 78 is suggested for development process. Could you correlate these facts to your result (Fig. 4)? Based on your perspective and the references, why then FtNAC11 is abundant in flower, 12 in root and stem, 26 in stem, 28 in flower, 65 in leaf (flower and root as well, next in order of expression), 70 in seed, and 78 in seed? What is the significance of this distribution and the possible impact in stress tolerance responses?

Line 409-417 – The authors explain that some of the FtNAC genes are induced, and some are inhibited by the stress, etc. Are these explanations based on Figure 5? If correct, you need to mention the figure here. Add more discussion based on the CAREs results on how you come to the statements to bridge the results to the discussion part. Because, without this, it feels like you make an assumption despite you already state it based on the results.

Please also connect the outcome of the transformation into Arabidopsis (as Arabidopsis-OE) versus the wild type in the discussion. Use it to fortify your explanation on how the genes work under stress environment in the Arabidopsis.

Author Response

Dear reviewer,

Many thanks for you because you spent lots of times to give us many good suggestions for our manuscript to improve its quality. The point-to-point response are as follows.

 

Title – Please add the author of the botanical name (e.g., Oryza sativa L.)

Response: Revised.

 

Introduction

Line 39-42 – Please be consistent on adding the author of the botanical names (e.g., you’ve added one for rice, Oryza sativa L., longan, Dimocarpus longan Lour., but none for cotton and for the star of this research, the tartary buckwheat)

Response: Thanks for your suggestion. We have all the botanical names for cotton and Tartary buckwheat (Line 41-43).

 

Line 47-55 – You mentioned that NAC TF genes play essential role for plant growth, development, stress, and senescence. I think it will be important to also add 3-7 sentences to elaborate shortly on how the transcription factor genes are involved. Are there pathways of genes regulated by NAC TF? Perhaps you want to check on Nurruzaman et al. (2015; https://doi.org/10.3389/fpls.2015.00676) for it.

Response: Thanks for your suggestion. We have added (Line 51-60).

 

Line 56-89 – What are the relationships between NAC TF genes and other stress response pathway? You mentioned how the processes are mediated by JA and ABA. You might want to explain how, i.e., for salt/drought tolerance, is it related to glycine betaine or other signaling pathways? How about cold? Check Nurruzaman et al. (2013; https://doi.org/10.3389/fmicb.2013.00248) and Nakashima et al. (2011; https://doi.org/10.1016/j.bbagrm.2011.10.005). You used cold treatment for your experiment, but the introductory information is missing.

Response: We have added (Line 73-78).

 

Additionally at the end of the Introduction, add short explanations on how significant of tartary buckwheat for the field of study. Describe the novelty, you might want to compare it with the other crop plants.

Response: We have added (Line 86-88).

 

Materials and Methods

Line 152 – qRT-PCR is Real Time Quantitative (for the q) Reverse Transcription (RT) PCR. If you refer it as Quantitative Real Time, then qPCR (to quantify DNA, not cDNA from RNA). However, from your manuscript, I believe that you want to check the expression and involved cDNA synthesis, not DNA, hence qRT-PCR. Please fix it.

Response: Thanks a lot. We have revised it (Line 167).

 

Discussions

Line 371-400 – The authors describe how each FtNAC gene plays important roles of its own. Are the explanations here based on previous study on the same plant, or Arabidopsis? If so, please mention it on the text, for example “Based on the study on Arabidopsis…” to avoid misinterpretation. Also, please recheck if the specific FtNAC genes also work the same on the other species. I think there are plenty of references for this.

Response: Revised (Line 401-403).

 

FtNAC11 and 28 is for detoxification and abiotic response, 12 is for nitrate response, senescence and defense (again herbivory or stress?), 65 is for senescence, 26 is for proteotoxic stress and protein homeostasis. 11, 28, 12, 65, 26 have similar function… of which one? Please rephrase!

Response: Revised (Line 403-413).

 

FtNAC70 is for embryo and flower pattern formation, pathogen-induced cell death and salt stress resistance and 78 is suggested for development process. Could you correlate these facts to your result (Fig. 4)? Based on your perspective and the references, why then FtNAC11 is abundant in flower, 12 in root and stem, 26 in stem, 28 in flower, 65 in leaf (flower and root as well, next in order of expression), 70 in seed, and 78 in seed? What is the significance of this distribution and the possible impact in stress tolerance responses?

Response: We have revised this paragraph based on your comments and the result of Figure 4 (Line 414-418). Six of the FtNAC genes were discussed in this paragraph except for FtNAC70.

FtNAC70 is discussed in detail in the Lines of 460-467, because of it was transformed into Arabidopsis.

 

Line 409-417 – The authors explain that some of the FtNAC genes are induced, and some are inhibited by the stress, etc. Are these explanations based on Figure 5? If correct, you need to mention the figure here. Add more discussion based on the CAREs results on how you come to the statements to bridge the results to the discussion part. Because, without this, it feels like you make an assumption despite you already state it based on the results.

Response: Revised (Line 430-445).

 

Please also connect the outcome of the transformation into Arabidopsis (as Arabidopsis-OE) versus the wild type in the discussion. Use it to fortify your explanation on how the genes work under stress environment in the Arabidopsis.

Response: Revised (Line 467-471).

 

Thanks in advance for your time and suggestion.

 

Author Response File: Author Response.docx

Reviewer 4 Report

Reviewer comments
Manuscript agronomy-1631169 - Identification, Expression, and Functionally Study of Seven NAC Transcription Factor Genes Involved in Stress Response in Tartary Buckwheat (Fagopyrum tataricum)

The authors dentified seven Tartary buckwheat FtNAC genes, namely FtNAC11, FtNAC12, FtNAC26, FtNAC28, FtNAC65, FtNAC70, and FtNAC78. Multiple sequence alignments revealed all of them contained the conserved NAM domain, which consisted of A, B, C, D, and E subdomain. Seven NAC proteins were classified into six subgroups based on phylogenetic analyses. The authors provided valuable information about the molecular mechanism of abiotic stress regulated by NAC TFs in Tartary buckwheat.

The statistical methods are correct.
The English of the text is well written and well readable.
The uniqueness of the text is more than 90% by AntiPlagiarism.NET.
The text contains some misspellings and typos.

There are following comments:

Line 15 - functional >> functionally
Line 16 - resistance >> resistant
Line 19 - subdomain >> subdomains
Line 21 - abiotic response and hormone response >> abiotic responses and hormone responses
line 18 - revealed >> revealed that
Line 49 - ang >> and
Line 78 - functional >> functionally
Line 85 - understand >> understanding
Line 80 - limit >> limited
Line 73 -  that are high >> that has a high
Line 60 - JA mediate >> JA-mediated
Line 66 - JA mediate >> JA-mediated
Line 67 - process >> processes
Line 86 - Why the authors was transformed FtNAC70 into Arabidopsis? Why the authors did not study the expression of FtNAC70 in buckwheat?
Line 120 - monoclone >> monoclones
Line 172 - was >> were
Line 182 -  treatment >>  treatments
Line 194 - Previously >> Previously,
Line 210 - reveals >>  revealed
Line 233 - most closed >> most closer
Line 234 - most closed >> most closer
Line 235 - most closed >> most closer
Line 236 - most closed >> most closer
Line 237 - most closed >> most closer
Line 250 - abiotic response or hormone response >> abiotic responses or hormone responses
Line 241 - Figure 2. - There is no link to the Figure 2 in the text.
Line 291 -  Among >> Within
Line 334 - meaned >> meant
Line 300 -  stress >>  stresses
Line 306 - but FtNAC65 >> but not FtNAC65
Line 359 - To data >> To date
Line 349 - the >> The
Line 362 - functional >> functionally
Line 365 - tolerant >> tolerance
Line 390 - maintaining >> maintenance
Line 389 - plant >> plants
Line 380 - most closed >> most closer
Line 427 - indirectly manner >> indirect manner

Please improve the manuscript according to the above comments.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Many thanks for your careful correcting for the mistakes and other suggestions for our manuscript. The point-to-point response are as follows.

 

Line 15 - functional >> functionally

Response: Revised.

Line 16 - resistance >> resistant

Response: Revised.

Line 19 - subdomain >> subdomains

Response: Revised.

Line 21 - abiotic response and hormone response >> abiotic responses and hormone responses

Response: Revised.

line 18 - revealed >> revealed that

Response: Revised.

Line 49 - ang >> and

Response: Revised.

Line 78 - functional >> functionally

Response: Revised.

Line 85 - understand >> understanding

Response: Revised.

Line 80 - limit >> limited

Response: Revised.

Line 73 -  that are high >> that has a high

Response: Revised.

Line 60 - JA mediate >> JA-mediated

Response: Revised.

Line 66 - JA mediate >> JA-mediated

Response: Revised.

Line 67 - process >> processes

Response: Revised.

 

Line 86 - Why the authors was transformed FtNAC70 into Arabidopsis? Why the authors did not study the expression of FtNAC70 in buckwheat?

Response: The molecular study of buckwheat is backward. There is not an effective method for the genetic transformation of buckwheat. So we transformed FtNAC70 into Arabidopsis to study its function.

 

Line 120 - monoclone >> monoclones

Response: Revised. (Line 135)

Line 172 - was >> were

Response: Revised. (Line 187)

Line 182 -  treatment >>  treatments

Response: Revised. (Line 197)

Line 194 - Previously >> Previously,

Response: Revised. (Line 209)

Line 210 - reveals >>  revealed

Response: Revised. (Line 225)

 

Line 233 - most closed >> most closer

Line 234 - most closed >> most closer

Line 235 - most closed >> most closer

Line 236 - most closed >> most closer

Line 237 - most closed >> most closer

Response: All “most closed” were revised to “most closer” based on your suggestion. (Line 248-252)

 

Line 250 - abiotic response or hormone response >> abiotic responses or hormone responses

Response: Revised. (Line 265-266)

 

Line 241 - Figure 2. - There is no link to the Figure 2 in the text.

Response: We have added figure 2 in the text (Line 238, 239, and 247).

 

Line 291 -  Among >> Within

Response: Revised. (Line 306)

Line 334 - meaned >> meant

Response: Revised. (Line 350)

Line 300 -  stress >>  stresses

Response: Revised. (Line 315)

Line 306 - but FtNAC65 >> but not FtNAC65

Response: Revised. (Line 321)

Line 359 - To data >> To date

Response: Revised. (Line 377)

Line 349 - the >> The

Response: Revised. (Line 367)

Line 362 - functional >> functionally

Response: Revised. (Line 380)

Line 365 - tolerant >> tolerance

Response: Revised. (Line 383)

Line 390 - maintaining >> maintenance

Response: Revised. (Line 412)

Line 389 - plant >> plants

Response: Revised. (Line 412)

Line 380 - most closed >> most closer

Response: Revised. (Line 398)

Line 427 - indirectly manner >> indirect manner

Response: Revised. (Line 455)

Please improve the manuscript according to the above comments.

Response: Thanks for your careful revision. We have revised all the incorrect words or description throughout the manuscript based on your suggestion as well as the others.

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The authors have done a good measure on revising the article, and in my opinion, this is close to acceptance. The outline regarding how the manuscript explains the idea from introduction, e.g., the significance of this NAC genes for stress control in the tartary buckwheat, a plant chose due to its importance for food. Only minor things require improvements.

Line 167, title for 2.4 - I think you have to choose, between "Real Time Reverse Transcription PCR" or simply "Quantitative Reverse Transcription PCR". Both "Real Time" and "Quantitative" can be synonymously referred as "q", so adding both terms are simply redundant. Pick one.

Last paragraph (Line 456 - 475) - I suggest to the authors to try add a future prospect of this research in your manuscript. Let's say, just add max 3 sentences. Now that we know how the NAC genes in action after you screen them from the plant itself and transformed to Arabidopsis, what next? Adding this, will provoke your reader to continue your work, citing this paper as a foundation of work, while you can also do your work in the future based on this research as well. Then, add 1 sentence at the end of the conclusion section (Line 485).