Genome-Wide Identification, Evolution and Expression Profile Analysis of NAC Transcription Factor in Simmondsia chinensis

Round 1

Reviewer 1 Report

The manuscript extends the work of Sturtevant et al. (Ref. 25) who sequenced the genome.  The current work utilizes the gene models from Ref. 25 and bioinformatically identifies NAC genes based on sequence similarity to the Arabidopsis NAC genes using BlastP. These sequences were further searched for the NAM sequence using the presence of the NAM domain using HMM's from Pfam.  The authors used whole seed development expression from Ref. 25 to observe differential expression using fold change of the seed stages.  The authors state that they generated additional RNA-seq data for additional seed tissues using dissected seeds.  They do not explain their method of doing this in the Methods section nor do they give accession numbers for the data. The authors present diagrams of promoter regions of the genes with colored glyphs of TF binding domains that lack recognizable names for the for the colors.  The authors presented pictures of the exonic structures and motifs that are coded by color but no table listing the individual motifs that were included with each color.  This makes interpretation and re-use of the data impossible.   These and other issues are itemized below:

Why don’t you specify that the RNA-seq reads were in GSE130603 in the text? Why make us dig for it.  Less words.

Is there a supplemental table that lists the members of the groups in Fig. 1?

Is there a supplemental table that lists all of the “motifs” referenced in Fig. 2? The figure legend just uses Motif1 to Motif20 so it would be hard to impossible to use that data in other contexts. What does groups I - VIII represent?

Is there a supplemental table that lists Cis-acting elements in the promoter region of each NAC gene in Fig. 3? You should use the proper “name” for each cis element.

You should add text to Fig. 5 that explains the color scheme of the chromosomes.

In Fig. 6 you have groupings indicated by Roman numerals.  You need to explain the groups in the legend. You refer to some of them in lines 215-216 but you don’t explain what the groups represent as a whole. You did not specify in the text or legend whether these data represent the mean of the log2 expression of these genes or the expression value of a single observation.  The variance between biological and technical replicates can be unfortunately large so it it impossible to draw conclusions about differential expression between the seed tissues until this issue is resolved.

In Fig. 7, you must explain what the groups I - VIII mean in the text and in the legend. You did not specify in the text or legend whether these data represent the mean of the log2 expression of these genes or the expression value of a single observation.  The variance between biological and technical replicates can be unfortunately large so it it impossible to draw conclusions about differential expression between the seed stages and tissues (Fig. 6 and Fig.7) until this issue is resolved.

Figure 8 is very pretty, but you failed to state what tissue and developmental stage was used to generate the data in the legend. In the text you state that NAC genes in cotyledon and embryonic axis was used, but you did not specify the developmental stage of that tissue. Since the NAC genes are transcription factors, developmental stage is probably very important.  I think you should have done multiple stages of embryonic development and compared the FC  of each gene between each stage to identify stage specific expression patterns. Also you could use that data to identify general “growth” transcriptional factors from stage/tissue specific NAC genes. You state that all relevant data is in the text or supplemental files. 

Where is the new RNA-seq data for “manually dissected tissues” you refer to on lines 114-115. It is not in reference 25. If you did derive new RNA-seq data where is that data and how did you process it prior to loading into the R-package pheatmap? Did you normalize your counts for differences in expression level (RPMK, FPMK, TM)?  You must include a table in the supplemental section that links your naming system to the S. Chinensis gene model name.

P-values or Q-values for significance should be chosen at the beginning of the experiment and values that exceed those are “significant”. There is no significant*, more significant**, more highly significant***.  

Does the Supplemental contain the RNA-seq pipeline? Is it published in a public repository? If not it must be an item in the supplemental files and is should detail each program used and the options chosen.

The gene structure display server appears to do an ab-initio discovery of intron/exon boundaries.  Is there any RNA validation of the predicted exons?

No attempt to identify Arabdopsis orthologs and name Sc genes accordingly
 

Line 71 you state that you downloaded the genomic data from the BIG data center, but you do not supply the BIG project number or biosample numbers

Line 72 you refer to the TF identification process and cite reference 25.  You should briefly state what the procedure was in the text.

Line 77-78 you refer to reference 27 for your technique of using the Pham HHM of the NAM Domain.  You should state briefly what that technique was along with citing Ref. 27

Establishing your own name for each NAC gene in S. chinensis does not serve the community well.  In figure 1., you establish the phylogenetic relationships of S. Chinensis NAC genes with its homolog in Arabidopsis thaliana.  You should use that data to name the NAC genes in S. Chinensis.  You should explain your naming system in the methods section. Most plant species transfer the “name” of the At homolog to their genes, thus the Sc homolog of AtADH1 becomes ScADH1 in jojoba.  You may have to add a numeral or letter to the homolog name due to differences in genome duplication events. Without a naming convention and attention to precedence, your ScNAC57 becomes somebody else’s ScNAC21 and chaos in sues.

Where is the data that supports your conclusion in lines 323-325? What does “increasing gradually” mean?

The conclusion in lines 338-339 may be true, but it is not supported by your data. Had you also included non-seed tissues in your RNA expression experiments, you might be able to support the statement.  But you did not and that sentence should be removed.

While lines 339 - 345 are interesting, what it has to do with NAC (NaC-type???) genes is not stated.  Even if you intended to write “NAC”, you are making inferences between grasses (Poaceae) and eudicots that may not be supported.  None of the corn genes mentioned seem to involve TAG synthesis.  If they were associated with the biosynthesis of corn oil, then it may be a valid comparison.

There was no supplementary files available to me so I cannot verify the veracity of the “Data Availability Statement”, lines 367-368

 

 

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Reviewer 2 Report

This paper deals with the comprehensive investigation of the NAC transcription factor genes in Jojoba. The authors are interested in the NAC genes involved in seed development. Due to the high value of this plant in respect to commercial products, I think this study field is important for plant science. I have a minor comment.

I am glad if the authors present more detailed explanation regarding the method and result of Figure 8. What is the different point with Figure 6?

line 97, "Mircorsynteny" is this correct?

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Round 2

Reviewer 1 Report

See the attached file.

Comments for author File: Comments.pdf

There were many grammatical errors that the EDITOR should have identified before sending this manuscript out.

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