Genome-Wide Identification and Evolutionary Analysis of AOMT Gene Family in Pomegranate (Punica granatum)

Round 1

Reviewer 1 Report

In general, the manuscript is interesting. However, the content of this manuscript does not fit well with the direction and scope of Agronomy, judging from the quality and contents of the data included in the manuscript. This manuscript may be a better fit for more specified journals related to genome analysis. In order to submit the manuscript to Agronomy, new data and discussion related to subject areas of Agronomy must be added.

 

Author Response

Dear reviewer:

Many thanks for the insightful comments and suggestions of the referees. I have made corresponding revision according to your advice. Words in red are the changes I have made in my manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

In their article "Genome-wide identification and evolutionary analysis of AOMT gene family in pomegranate (Punica granatum)", Zhang and colleagues describe a gene family study of Anthocyanin O-methyltransferases in Punica, where these genes are important for the production of the characteristic secondary metabolite profile of pomegranate fruits and flowers.
Overall, the paper seems solid.
That said, the gene transfer aspect (Figure 4) should be completely removed. It is the major weakness of this manuscript.

In the Introduction:
The authors highlight the role of the anthocyanins play in nutrition and
Part of the important stress relevant and nutritionally beneficial secondary metabolites of Punica is in its oil (see and cite doi: 10.1186/s12906-016-1237-3 "Chemical analysis of Punica granatum fruit peel and its in vitro and in vivo biological properties" in BMC Complementary Medicine and Therapies and doi: 10.1016/j.lwt.2020.109519 "Microencapsulation of pomegranate (Punica granatum L.) seed oil by complex coacervation: Development of a potential functional ingredient for food application" in LWT - Food Science and Technology)
Indeed, lipid droplets that make up the seed oils are an ancient stress response mechanism -- and these droplets can accumulate secondary metabolites. The authors should hence highlight this and cite doi: 10.1016/j.tplants.2020.07.017 "Ties between Stress and Lipid Droplets Pre-date Seeds" in Trends in Plant Science

Phenylpropanoid metabolisms and anthocyanin production is not limited to Punica. Indeed, the molecular mechanisms that regulate this process are found across land plants (read and cite doi: 10.1111/nph.15002 "Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants" in New Phytologist) and probably even in the algal relatives of land plants (read and cite doi: 10.1093/pcp/pcx037 "How Embryophytic is the Biosynthesis of Phenylpropanoids and their Derivatives in Streptophyte Algae?" in Plant & Cell Physiology.

M&M and results on RNAseq:
The RNAseq analyses are not describe in sufficient detail. Even though the data were obtained from published sources, sufficient information must be included within the manuscript that the authors present here.
How was the pre-processing done? Were RNAseq data quality checked using FASTQC? Was trimming performed? How exactly were the reads mapped? How many reads mapped onto the genome (i.e. mapping rate). Were those reads paired end & stranded; if so, how many reads mapped concordantly?
Please also describe more explanation of where these reads were exactly stemming from (i.e. how were the tissues separated).

Results in general:
Figure 1: While Class I is a monophyletic clade (which is good!), Class II is paraphyletic and includes an assortment of several, sometimes quite small clades. Class II should be drawn smaller as only including GSVIVT01010468001 up to XM_022870224.1

Figure 4: It is highly unlikely that gene transfer occurred between for example Arabidopsis and apple -- eukaryote to eukaryote LGT is extremely rare (read and cite doi: 10.1002/bies.201700115 ) and just because the tool Jane v4.0 makes these predictions does not mean that this makes much biological sense. This would be an exceptional claim that requires exceptional evidence. All putative transfers can more likely be explained by ancient duplications follow by several losses as well as weakness of the trees.
In fact, Jane 4 is a tool designed for host-parasite co-evolution. It does not do any sophisticated tests for loss vs. Gene transfer (indeed, such tests are impossible, as there are no proper empirical data on gene transfer between eukaryotes in nautre)
The gene transfer aspect should be completely removed. It is in my opinion the major weakness of this manuscript.

 

Author Response

Dear reviewer:

Many thanks for the insightful comments and suggestions of the referees. I have made corresponding revision according to your advice. Words in red are the changes I have made in my manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

The research presented in the manuscript “Genome-wide identification and evolutionary analysis of AOMT gene family in pomegranate (Punica granatum)” is interesting and worth publishing.

Comments:

The main problem concerns the description of the materials and methods. The manuscript mainly consists of the analysis of information already known and stored in databases. However some experimental (laboratory) work was also carried out, aiming at candidate genes sequencing and studying of differential expression of AOMT genes in plant tissues.

The experimental part is described very briefly, data on some stages of the work (e.g. PCR) are missing. There is no detailed information on the methods used to analyze gene expression (RNA-seq?). Judging by the part "Materials and methods" (Sample collection, Expression analyses of PgAOMT gene family members) such studies have been carried out.

Author Response

Dear reviewer:

Many thanks for the insightful comments and suggestions of the referees. I have made corresponding revision according to your advice. Words in red are the changes I have made in my manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have been well revised to reflect the opinions of reviewers. In particular, content and references were added in connection with agricultural characteristics. 

Author Response

Dear reviewers

Thank you for your positive reply.

Reviewer 2 Report

The authors have successfully addressed all my comments.

However, one important issue remains that should be fixed before this is ready for publication. 

In Figure 1, there are still issues with how the classes are labeled — the current labelling is not supported by the topology of the phylogeny.

Class II should be the clade supported by 0.93, that is the purple, blue and orange branches.

Class I should be the clade supported by 0.96, that is from Pg026019.1 down to the "Tandem".

The sequences XM_022878853.1 as well as Pg006183.1 do not fall into any of these two classes. Therefore, they should either be labeled as class III or not labeled at all. The latter is probably the more elegant solution -- not all sequences have to be assigned to a class! Please also amend the nomenclature throughout the manuscript accordingly.

Regarding the statements on the algal relatives of land plants, the following reference should be cited, too: Evo-physio: on stress responses and the earliest land plants, Journal of Experimental Botany, Volume 71, Issue 11, 11 June 2020, Pages 3254–3269, https://doi.org/10.1093/jxb/eraa007

Author Response

Dear reviewer:

We have modified the figure 1, amend the nomenclature throughout the manuscript accordingly and cited new reference based on your suggestion in the manuscript.