Genome-Wide Identification of NAC Genes Associated with Bast Fiber Growth in Ramie (Boehmeria nivea L.)
, Yanzhou Wang 2 and Touming Liu 1,*
Round 1
Reviewer 1 Report
The paper by Zeng et al. describes the identification of NAC genes in ramie. The authors also present phylogenetic analyses, expression analyzes of the studied genes and the results of overexpression experiments.
In general, I find the work clear, legible and informative.
However, I would like to raise a few points that I think should be changed or improved:
- the description of expression analysis in the methods is so general that I am not able to understand how these tests were actually performed. On what basis were the expression levels of individual genes estimated, and were the mRNA levels analyzed? what method?
- in the description of experiments with overexpression: in which A. thaliana line was overexpression performed? Was it a WT line? or maybe some mutant with a silenced gene/genes were used? What was the possible native expression level of the tested gene? How was the expression level of the introduced construct measured?
- why was subcellular localization done on tobacco?
Besides, I got the impression that the discussion is quite general and focuses in large part on reporting the results described in other papers.
The authors mention that only 3 of the 10 identified genes show an increase in expression in fiber under growing, but they cannot explain this phenomenon. Finally, they claim that in the plant varieties they study, all 10 identified genes are involved in fiber growing, but they provide no evidence of this. They claim that these genes are important at different stages of development, other than those studied in the paper. However, they do not show any results for this either.
Minor points: I belive gene names are capitalized and italic, e.g. NAC genes.
I believe that the work can be accepted after eliminating the above shortcomings.
Author Response
The paper by Zeng et al. describes the identification of NAC genes in ramie. The authors also present phylogenetic analyses, expression analyzes of the studied genes and the results of overexpression experiments.
In general, I find the work clear, legible and informative.
However, I would like to raise a few points that I think should be changed or improved:
Response: Thank you for taking the time to conduct a thorough review of our submitted manuscript, and give us valuable comments.
- the description of expression analysis in the methods is so general that I am not able to understand how these tests were actually performed. On what basis were the expression levels of individual genes estimated, and were the mRNA levels analyzed? what method?
Response: Thanks for your suggestion. We have added some detailed information in the revision.
- in the description of experiments with overexpression: in which A. thaliana line was overexpression performed? Was it a WT line? or maybe some mutant with a silenced gene/genes were used?
Response: It is wild Arabidopsis. We have indicated in revision.
What was the possible native expression level of the tested gene?
Response: Thank you for comment. The native expression level has been showed in Figure 2.
How was the expression level of the introduced construct measured?
Response: Thank you for comment. In our constructs, expression of ramie NAC genes were driven by overexpression promoter CaMV 35S, and they should be continuous expression at a high level in transgenic plants. The observation of new phenotype also validated the high expression of exogenous gene. Actually, few previous studies focused the expression level of genes that were driven by CaMV 35S.
- why was subcellular localization done on tobacco?
Response: Thank you for comment. Generally, the subcellular localization was done either in leaves of tobacco or plant protoplast. Because experiment in leaves of tobacco is easier in operation, most of studies for performing subcellular localization were carried out in leaves of tobacco. In this study, we also used this universal method.
Besides, I got the impression that the discussion is quite general and focuses in large part on reporting the results described in other papers.
Response: Thank you for comment. In the section of “Discussion”, we cited the previous findings as an evidence, further to explain our results and to support our perspective.
The authors mention that only 3 of the 10 identified genes show an increase in expression in fiber under growing, but they cannot explain this phenomenon. Finally, they claim that in the plant varieties they study, all 10 identified genes are involved in fiber growing, but they provide no evidence of this.
Response: Thank you for comment. There were seven NAC genes that showed either down-regulation or slight up-regulation in fiber under growing. To explain this phenomenon, we performed overexpression analysis for three NAC genes. Additionally, two NAC genes, Bnt08G012573 and Bnt03G004997, have been validated with a role in promoting fiber growth. Therefore, five of these seven genes have distinct evidences to support their involvement in fiber growth.
They claim that these genes are important at different stages of development, other than those studied in the paper. However, they do not show any results for this either.
Response: Your suggestion is good. In this study, only expression evidence could support our perspective. We have corrected the presentation in the “Discussion.”
Minor points: I belive gene names are capitalized and italic, e.g. NAC genes.
Response: Thanks for your suggestion. We have corrected it thoroughly.
I believe that the work can be accepted after eliminating the above shortcomings.
Response: Thanks for your recommendation. Also, we thank once again for your great comments and suggestions, which significantly help in improving the quality of our manuscript.
Reviewer 2 Report
This article is part of a larger project and continues previous research, application of genomic and transriptomic to plant physiology and investigation of function of certain gene.
The article successfully combined methods of sequencing, transgenic mutants and microscopy to study role of genes that were associated with fiber growth and regulation the secondary wall-thickening in fiber crop ramie (Boehmeria nivea L.)
Minor revision:
Figure 1: Please, clear what means yellow and pink colours (Arabidopsis and ramie genes, as I can suppose).
Line 140: "The result revealed five NAC genes without any expression in 140 the investigated tissues of two varieties."
What genes do authors mean?
Line 141: "Additionally, three of 9 genes only expressed in the leaves and/or roots of ZSZ1, but not in any investigated tissues of tenacissima."
What genes do authors mean?
Line162: "Furthermore, expression level of these 10 NAC genes were compared between the barks from the top and middle section of stems where fiber growth is under different stages [29], and revealed that the transcript abundance of eight genes had more than two-fold difference (Figure 2d), indicating a potential roles of these nine NAC regulator in fiber growth of ramie."
Eight or nine? An explanation is given below, in lines 225-230, but here these statements looks inconsistent.
Line 183: "To validate the role of these NAC genes in fiber growth, three of these five genes, Bnt01G000429, Bnt03G004081, and Bnt14G020028"
Why were these genes chosen? I mean, for example, Bnt03G004081 - its expression fold MPS/TPS 0,487, on the treshold of no differential expression. Other genes have expression fold 0,042 and 0.299. Why not to take BNT10G015609 with 7,17?
In general, the manucript is well-suited for Agronomy journal and can be accepted for publication.
Author Response
This article is part of a larger project and continues previous research, application of genomic and transriptomic to plant physiology and investigation of function of certain gene. The article successfully combined methods of sequencing, transgenic mutants and microscopy to study role of genes that were associated with fiber growth and regulation the secondary wall-thickening in fiber crop ramie (Boehmeria nivea L.)
Response: Thank you for taking the time to conduct a thorough review of our submitted manuscript, and give us valuable comments.
Minor revision:
Figure 1: Please, clear what means yellow and pink colours (Arabidopsis and ramie genes, as I can suppose).
Response: Thanks for your suggestion, and we have corrected it.
Line 140: "The result revealed five NAC genes without any expression in 140 the investigated tissues of two varieties."
What genes do authors mean?
Response: It is Bnt04G006656, Bnt05G007366, Bnt05G008828, Bnt13G018465, and BntUnG020221. We have indicated these gene ID in the revision.
Line 141: "Additionally, three of 9 genes only expressed in the leaves and/or roots of ZSZ1, but not in any investigated tissues of tenacissima."
What genes do authors mean?
Response: We have indicated these gene ID in the revision.
Line162: "Furthermore, expression level of these 10 NAC genes were compared between the barks from the top and middle section of stems where fiber growth is under different stages [29], and revealed that the transcript abundance of eight genes had more than two-fold difference (Figure 2d), indicating a potential roles of these nine NAC regulator in fiber growth of ramie."
Eight or nine? An explanation is given below, in lines 225-230, but here these statements looks inconsistent.
Response: I am sorry for our unclear explanation. Of these then NAC genes, nine displayed relatively high expression in stems of ZSZ1. Additionally, eight of these ten NAC genes showed differential expression between the barks where fiber growth is under different stages. We have corrected the sentence.
Line 183: "To validate the role of these NAC genes in fiber growth, three of these five genes, Bnt01G000429, Bnt03G004081, and Bnt14G020028"
Why were these genes chosen? I mean, for example, Bnt03G004081 - its expression fold MPS/TPS 0,487, on the treshold of no differential expression. Other genes have expression fold 0,042 and 0.299. Why not to take BNT10G015609 with 7,17?
Response: Thanks for your comment. Of these eight differentially expressed NAC genes, five displayed down regulation in expression. As the description of the second of paraphrase in “Discussion”, it is puzzling for the down-regulation in the expression of these five genes during fiber growing stage. Of these five genes, one had been performed for functional validation in previous study, and therefore, the remaining four were performed for overexpression. However, one gene failed to obtain positive transgenic seeds. Therefore, in manuscript, overexpression results of three NAC genes were showed finally.
In general, the manucript is well-suited for Agronomy journal and can be accepted for publication.
Response: Thanks for your recommendation and for your great comments and suggestions, which significantly help in improving the quality of our manuscript.
Reviewer 3 Report
Based on genomic and trancriptomic data from A. thaliana and Boehmeria nivea the Authors have selected three NAC transcription factors, which overexpressed stimulate growth or division of fiber cells in xylem of A. thaliana, as well as cell wall thickening of xylem cells. Hopefully this preselection of the three transcription factors in a model plant will be helpful for Boehmeria nivea breeders.
Before publishing some editorial problems should be solved however:
The following sentences should be reformulated to make the content more understandable for the readers:
Two sentences in lines: 53-56: “low ability of transcriptome”, “far less than in other plants”
Line 63: “removing proteins from alternative splicing”
91 “three….genes was amplified”
119 “transcribed by alternative splicing way”?
121 “into as 11 group”
127 “extremely large conservation with…” ?
130 “ramie members”
131 “orthologs mediated Arabidopsis….”
160 “differentially expressed pattern”?
242 “they are not redundancy”
Before publishing some editorial problems should be solved:
The following sentences should be reformulated to make the content more understandable for the readers:
Two sentences in lines: 53-56: “low ability of transcriptome”, “far less than in other plants”
Line 63: “removing proteins from alternative splicing”
91 “three….genes was amplified”
119 “transcribed by alternative splicing way”?
121 “into as 11 group”
127 “extremely large conservation with…” ?
130 “ramie members”
131 “orthologs mediated Arabidopsis….”
160 “differentially expressed pattern”?
242 “they are not redundancy”
Author Response
Comments and Suggestions for Authors
Based on genomic and trancriptomic data from A. thaliana and Boehmeria nivea the Authors have selected three NAC transcription factors, which overexpressed stimulate growth or division of fiber cells in xylem of A. thaliana, as well as cell wall thickening of xylem cells. Hopefully this preselection of the three transcription factors in a model plant will be helpful for Boehmeria nivea breeders.
Before publishing some editorial problems should be solved however:
Response: Thank you for taking the time to conduct a thorough review of our submitted manuscript, and give us valuable comments.
The following sentences should be reformulated to make the content more understandable for the readers:
Two sentences in lines: 53-56: “low ability of transcriptome”, “far less than in other plants”
Response: Yes, we have corrected it.
Line 63: “removing proteins from alternative splicing”
Response: Yes, we have corrected it.
91 “three….genes was amplified”
Response: Yes, we have corrected it.
119 “transcribed by alternative splicing way”?
Response: Yes, we have corrected it.
121 “into as 11 group”
Response: Yes, we have corrected it.
127 “extremely large conservation with…” ?
Response: Yes, we have corrected it.
130 “ramie members”
Response: Yes, we have corrected it.
131 “orthologs mediated Arabidopsis….”
Response: Yes, we have corrected it.
160 “differentially expressed pattern”?
Response: Yes, we have corrected it.
242 “they are not redundancy”
Response: Yes, we have corrected it.
