Genome-Wide Analysis of NAC Gene Family in Betula pendula

Round 1

Reviewer 1 Report

Thanks for your quick resubmission. However, I believe some more points should be concerned for the publication.

Since this will be the first report for NAC family of Betula pendula, the member identification should be carefully done. The total number of 96 members should be verified because most of the other species have more members (e.g., Populus trichocarpa (170), Arabidopsis thaliana (113), Oryza sativa (141), Prunus persica (115)). Xylem sampling should be described in more detail. For example, which part of the tree was used and how old are they? For tissue-specific expression of NAC members, other tissues (e.g., phloem, cambium, bark) together with xylem should be examined since xylem tissues were collected by using the sequential tangential cryosectioning. It is intriguing that the xylem-specific BpNACs do not have VND orthologous genes (VND1~VND7), which are crucial in xylem development. This fact further emphasizes the careful identification of BpNACs.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Though the manuscript has been improved in some parts, I suggest the Authors to read the articles below to shape up the manuscript nicely. 

Moreover, in the methodology part: the sequencing place must be added. 

The phylogenetic tree with color coding of the clades should be added to the result in the main text due to it is the crux of this study, table 2 must be moved to supplementary instead. 

Please find more minor comments on the pdf attached. 

 

Su et al. 2013. Genome-wide analysis and identification of stress-responsive genes of the NAM–ATAF1,2–CUC2 transcription factor family in apple. Plant Physiology and Biochemistry 71: 11-21.

Diao et al. 2018. Genome-Wide Analyses of the NAC Transcription Factor Gene Family in Pepper (Capsicum annuum L.):

Chromosome Location, Phylogeny, Structure, Expression Patterns, Cis-Elements in the Promoter, and Interaction Network. Int. J. Mol. Sci. 2018, 19, 1028.

Lv et al. 2016. Global Expressions Landscape of NAC Transcription Factor Family and Their Responses to Abiotic Stresses in Citrullus lanatus. Scientific Reports | 6:30574.

Sun et al. 2018. Comprehensive analysis of NAC transcription factors uncovers their roles during fiber development and stress response in cotton. BMC Plant Biology (2018) 18:150

Liu et al. 2018. Comprehensive analysis of NAC transcription factors and their expression during fruit spine development in cucumber (Cucumis sativus L.). Horticulture Research (2018) 5:31

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

The Manuscript “Genome-wide Analysis of NAC Gene Family in Betula pendula” (forests-571798) by Chen and co-authors reports a combined  bioinformatic and transcriptomic analysis of the NAC transcription factor family in Betula pendula. The study is mostly well executed and could be highly interesting with a better presentation of, and more relevant reflections on, the results in relation both to model organisms and other woody species. The authors report that NAC gene family in the B. pendula comprises 96 members. That is a relatively low number compared other woody species such as Eucalyptus and Malus (examples of sequenced Rosid woody species)and in Eucalyptus specific expansions are seen in clades associated with stress responsiveness (Hussey et al 2014, New phytol.) It is also known that the phylogenetic relationships of the NAC TF gene family clustering is reflected in the genes expression patterns (Jensen et al 2010 Biochemical Journal, eg Jensens cluster VI-3 is strongly upregulated by cold temperatures) Therefore I wonder how expansion and contractions distribute over the different clades reported in in B. pendula (compare Jin et al 2017 Front. Plant Sci.), and how phylogeny interact with expression patterns in the current study? -This is not touched upon with any greater depth in the discussion.

 

Additional comments

L46 and 76 the format of Arabidopsis (thaliana) is inconsistent

 

L53 give the latin name Eriobotrya japonica for loquat-

 

L63 Is NAC domains implicated in only development and in abiotic stress? I would argue that there are NAC TFs that are linchpins  in biotic stress responses too.

 

L114 What is the TAIR abiotic stress database? I cannot find any such database  at arabidopsis.org

 

L200-201 more introns compared to what?

 

L228 “Gene family arose from multiple ways including tandem duplication, duplicative transposition and whole genome duplication (WGD), followed by mutation and divergence..” this sentence makes little sense and must be thoroughly revised. I would also recommend to transfer this reasoning on gene family evolution to the introduction, perhaps together with a small section on NAC gene family evolution?

 

L250-251 I do not think that a study published a decade ago is recent. Also in this context I would argue that both Jensen et al 2010 Biochemical Journal and Jin et al 2017 Front. Plant Sci. present relevant material to compare to, see comments above

 

L277-278 “The results indicated that BpNACs may be implicated in various aspects of plant development.” This sentence is extremely generic. Approximately 50% of your  identified NAC genes contain low temperature regulatory elements or “stress-responsive elements” (what ever that is, please be more stringent) that means that a large fraction of the promoters do not contain the specified elements and thus should not respond to these cues. Do they instead contain other recognized motifs? This final statement seems to imply this.

 

Fig 1 Please write out Trimmed Mean of M-values  in the figure text. Also an arrow or similar indicating which rows that  represent  the root and xylem  data would be helpful as the legend is  quite small  in the figure. And similarly use a symbol to mark the BpNACs with specific expression patterns.

 

Fig S6 lacks clade numbers which makes it very difficult to use for the reader

Table S5. Please mark which BpNAC/timepoint values which are significantly different from the other timepoints (compare L313) Also what are the values you are showing? Count data or Fold change data?

 

Figure 2 The authors write. “For the convenience of drawing, the total expression level of each gene was normalized to 1” so basically we are looking at fractions of total counts  associated with this gene in the RNAseq experiment? I think it would have been interesting to use the count data instead to also get the information if the genes were highly expressed or not (compare table S5).

 

L329-337 and Figure 3 what statistical test was used to determine up-regulation in response to low temperature stress one-way ANOVA? I cannot see any indications of differences between treatments in Figure 3. Proper statistics must be made.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

Thanks for your quick resubmission again. I think this revised manuscript was improved substantially for the publication in the journal Forests. Thanks again for your efforts. Good Luck.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This manuscript titled “Genome-wide Analysis of BpNAC Gene Family in Betula Pendula” describes an identification of NAC family in B. pendula with expressional analysis using four different tissues and cold temperature stress. Although highly descriptive, the results provided in this manuscript might be helpful for scientists interested in NAC family especially of woody species. Following points should be answered or revised for publication in Forests.

 

1. Authors first identified a total of 110 BpNACs but reduced to 96 after by BLAST search. How about the remaining 14 BpNACs? Are those not BpNACs only because of having no homologous genes in other plant species? More logical explanation should be provided.

2. Figs 1, 2, 3, 4, 5 should be supplemental since no significant biological information are delivered.

3. In the tissue specific gene expression analysis, how did you prepare the four different tissues, especially xylem tissue? Detailed methods should be provided in the materials and method section.

4. It will be great if there is a more detailed comparative analysis with Arabidopsis and Populus trichocarpa in terms of biological function of each NAC member. As you know, NAC family has been extensively studied.

Reviewer 2 Report

In this manuscript, the Authors have identified and characterised NAC genes in silver birch. The manuscript presents significant information that is of interest to the scientific community of functional genomics researchers. Though it is well presented and thorough, there are several minor grammatical errors and inconsistency in using verb forms throughout the manuscript. Past passive form should be followed through the manuscript.

In the conclusion part, the method must be removed and the crux of the findings, the impact and future directions should be explained. 

All suggestions are enclosed in the manuscript PDF attached. Please make all necessary changes to the manuscript as suggested in the enclosed revised version.

Comments for author File: Comments.pdf

Reviewer 3 Report

Brief summary

The first objective of the paper was to identify and characterise the NAC transcription factors gene family in the Birch (Betula pendula) genome. In addition, this study aimed to provide some information on the Birch NACs (BpNACs) regarding their potential physiological role and molecular mechanism associated. Using bioinformatic and phylogenetic analyses, the authors identified 96 BpNACs divided in seven subfamilies. BpNACs gene structure, chromosome location and BpNACs conserved protein motif were identified. Using available Betula pendula expression data, BpNACs tissue expression profile was investigated and the results showed that BpNACs expression was higher in xylem and roots compare to other tissues. Finally, RNA-seq analysis was used to determine the expression profiles of BpNACs when exposed to low-temperature. 19 BpNACs were differentially expressed under low-temperature and associated gene co-expression networks identified.

 

Broad comments:

 

In the overall the manuscript is original with a content that has potential interest for the reader. The manuscript is correctly written with some figures that can be improved for better readability. However, this study needs to be improved by adding some data in the results (More details below) and more discussions regarding previous studies on NACs. Additionally, I have some concern with the accuracy of the references in the manuscript and the lack of enough information in the methods to fully understand and reproduce the results.

 

Main comments:

1) The authors wanted to provide insight into the evolution of BpNAC genes but didn’t include in their phylogenetic analysis any other plant NAC gene families that are already well characterised. Including at the minimum Arabidopsis NAC family would have been expected and potentially other forest trees NAC family (E.g Eucalyptus or Populus). This would add and support some results/conclusions related to potential role of BpNAC in xylem biology, lignin biosynthesis and in cold birch resistance. Authors hypothesize that xylem-specific BpNACs may be involved in lignin biosynthesis. There is at least four Arabidopsis NACs shown to be involved in lignin biosynthesis (SND1, NST1, VND6, VND7), therefore a comparative phylogenetic analysis with AtNAC and BpNAC would add some interesting results/discussions on potential conserved/divergent function of NACs in Birch. The fact that Birch is a cold resistant tree is something interesting and I agree that potentially NAC genes might be part of this resistance. Again here, the authors should use the literature on NAC and cold stress to compare their results with known characterised plant NAC involved in cold stress tolerance (e.g. MaNAC1, Apple MdNAC029 , Arabidopsis LOV1,….). In summary, I recommend the authors to include at the minimum Arabidopsis NAC genes in their phylogenetic analysis and compare BpNACs with functionally characterised NACs involved in xylem biology, lignin biosynthesis and in cold.

 

2) The authors found 110 putative NACs in the reference genome of Betula pendula. From 110, only 96 were found in NCBI database. The authors decide to consider only this 96. But I don’t understand why the remaining 14 were not considered. This is all the point to use a reference genome as you expect to cover all the NAC genes that are probably not present in Non-redundant database such as NCBI. Moreover, what is the gene locus ID in table 1? Does that correspond to annotated genes from the Betula genome? If yes, how many genes annotated in the birch genome correspond to NAC genes?

 

3) The Material and methods section are missing some details and important information. I suggest the authors to review the Methods and add necessary details bearing in minds that other labs need to be able to repeat these results. For example on the section 2.4. What is the experimental design of this experiment (how many leaf per samples, how many independent experiment was done,….)? what are the culture conditions if any (lights, soil)? What is the age of the plants? How the cold treatment was done? What are the controls? log2FC, FC compare to what?

 

4) To be more confident on the cold experiment. I would add a usual control for RNAseq experiment such as validation of few BpNAC genes by qRT-PCR analysis.

 

5) The introduction and discussion section need to include more information on the role of NACs in in xylem biology, lignin biosynthesis and in abiotic stress resistance.

 

Specific comments

 

-          Line 33: Reference 10 seems not correct, this reference is not about transcription factor biology

 

-          Line 37: Reference 11 doesn’t seem to show that “In plant genomes, about 10% of the products of coding genes are TFs” and reference 13 is not on the role of TF in plants.

Ø  As there are few problems in the references, the authors need to double-check all the references and make sure they are accurate.

 

-          Line 64: to confirm the presence of the NAC domain?

-          Line 84: didn’t find the expression data in this database, could you add more details and/or adjust the link.

-          Line 106: explain how you retrieved the NAC from NCBI?

-          Line 116: what are the molecular weights 6.26 and 39.54 kDa? Line 115 are the range of BpNACs molecular weight.

-          Line 129, Figure1: Add the legend in the x axis (axis legends is missing as well in Fig.6 and Fig. 7). What are the blue boxes? Specify in the figure legend

-          Line 234, Figure7: what is the control of this experiment? What means CK and 4T in the figure?

-          Line 251, Figure8: what are the data used as input for the co-expression network. A Go enrichment histogram would be good for each network presented.

Reviewer 4 Report

I have read the MS “Genome-wide analysis of BpNAC gene family in Betula pendula” (forests-512809) by Chen and co-workers and I appreciate the large amount of work that has been invested in the bioinformatics analysis and generation of figures. However, in its present form the manuscript presents several major weaknesses.

It is not clear from the authors results presentation, or text, how the conserved amino acid motifs identified by MEME relates to previously identified conserved motifs (eg Ooka et al 2003, Shen et al 2009  references nr 22 and 54 in the paper or Jensen et al 2010 Biochemical Journal) or how these conserved domains relate to NAC domain classifications. I would also suggest that when the authors are dealing with only seven motifs the sequences should be integrated into Figure 2.

Figure 5; on what basis did the authors divide the sequences into seven clades; structure similarity bootstrap values? I would suggest that the authors report the significant bootstrap values.

The authors need to present more information on their RNA seq work eg the authors sequenced 11 libraries for the tissue panel of Birch how were these libraries distributed over the four tissues? What stage of tissues were sampled to generate these tissue libraries etc . The same questions apply to the low-temperature experiment; How was it set up? How many biological and technical replicates were used etc etc? How many raw reads were obtained per sequence library, how may were retained after quality filtering? What settings and reference files were used in the bioinformatics pipeline? -there are many ways to run RSEM, edgeR and WGCNA depending on the settings and models used.  Given that at least three of the genes that are active in the xylem appear to be highly similar how did the authors handle ambiguously-mapping reads in their RSEM-edgeR pipeline?

The figures (except Figures 4, 5 and 7) are illegible, gene names cannot be read and domain structure et cannot be seen, please think about different formatting to improve legibility.  Figure 3 and 4 are redundant.

References nr 30 and 49 are duplicated