Genome-Wide Identification, Characterization and Expression Profile of F-Box Protein Family Genes Shed Light on Lateral Branch Development in Cultivated Peanut (Arachis hypogaea L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The main aim of the manuscript “Genome–Wide Identification, Characterization and Expression Profile of F–box Protein Family Genes Shed Light on Lateral Branch Development in cultivated peanut (Arachis hypogaea L.)” was to provide gene resources that enhance the understanding of the molecular mechanisms in which F-box associated genes contribute to peanut branching development. In this work the authors performed several routine analyses with gene data that are available in an online database, however, I believe that the major contribution is to obtain the expression pattern of F-box genes in peanut.

Abstract. The authors highlight more the methodology and bioinformatics analyses of data extracted from the peanutbase website (http://peanutbase.org/), but do not highlight the most important results which are the gene expression analyses of the F-box proteins. The authors should highlight their gene expression results more than the other analyses.

Method. RNA extracted and qRT-PCR analysis is not detailed. The authors should show this item in detail. For example, how many individuals were used? How much material was used from each tissue for RNA extraction? among others.

I have a doubt, are the primers you designed for qRT-PCR specific of the gene? did you evaluate if a certain primer pair amplifies other F-box genes?

Results. Much of the results are devoted to analyzing and characterizing the F-box protein genes, which is easy to do because data from the same peanutbase website is used. However, when the authors show the gene expression patterns (Figure 5 and 6), the most important in my opinion, the plots look very small and are not understandable. In addition, the authors do not describe whether the gene was induced, repressed or is constitutive. In the column diagram in Figure 6, the x-axis is not labeled.

Author Response

Dear reviewer，

      Thank you for your comments. Acoording to your comments, all the questions and comments in the postil were answered and revised in the MS. A few main Q&A were as follows:

Question 1：Abstract. The authors highlight more the methodology and bioinformatics analyses of data extracted from the peanutbase website (http://peanutbase.org/), but do not highlight the most important results which are the gene expression analyses of the F-box proteins. The authors should highlight their gene expression results more than the other analyses.

Answer: The point was taken. We have added main results of the gene expression analyses in Abstract and highlighted in red (Lines 26-35) of the revised MS.

Question 2: Method. RNA extracted and qRT-PCR analysis is not detailed. The authors should show this item in detail. For example, how many individuals were used? How much material was used from each tissue for RNA extraction? among others.

Answer: The point was taken. We have described the methods of RNA extracted and qRT-PCR analysis in M&M and highlighted in red (Lines 179-187) of the revised MS.

Question 3: I have a doubt, are the primers you designed for qRT-PCR specific of the gene? did you evaluate if a certain primer pair amplifies other F-box genes?

Answer: The points were taken. We have added the estimate values of each primer specificity and uniqueness and the corresponding gene sequence number in Table S1.

Question 4: Results. Much of the results are devoted to analyzing and characterizing the F-box protein genes, which is easy to do because data from the same peanutbase website is used. However, when the authors show the gene expression patterns (Figure 5 and 6), the most important in my opinion, the plots look very small and are not understandable. In addition, the authors do not describe whether the gene was induced, repressed or is constitutive. In the column diagram in Figure 6, the x-axis is not labeled.

Answer: Thank you for your comments. We previously focused on the research of peanut lateral branch development via transcriptome and found F-box proteins were greatly enriched in the differentially expressed genes. Thus, we analyzed the F-box protein family. In this manuscript, the main results were the expression of F-box genes relating to the development of lateral branch that is a plant developmental process. So our expression results included the tissue expression pattern and time-course expression during lateral branch development. The response to stresses that were more common in the gene family analysis is not related to our focus, so there is no much inducible, repressed or constitutive expression results.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

the manuscript “Genome–Wide Identification, Characterization and Expression Profile of F–box Protein Family Genes Shed Light on Lateral Branch Development in cultivated peanut (Arachis hypogaea L.)” reports interesting information relating to the molecular characterization of the F-box gene family in Arachis hypogaea L. which was accompanied by a gene expression study which led to the identification of some genes of the family specifically active in the development of the lateral branches.

In my opinion, the manuscript provides a lot of information and can be accepted for publication after minor revisions to the text and figures.

Line 52 Please, remove the comma before "tomato"

Line 239 Change 3.6 to 3.5

Line 255 Change 3.5 to 3.6

Line 291 Insert "tissues" after "22 different"

Line 411 Group II is missing

Lines 413-414 Group IV was included twice

It is advisable to enlarge and improve the quality of Figure 3 B which is difficult to read.

In Figure 5 it is recommended inserting a legend to explain the acronyms of the plant tissues.

Author Response

Dear reviewer,

      Thank you for your comments. Acoording to your comments, all the questions and comments in the postil were answered and revised in the MS. A few main Q&A were as follows:

Question 1: Line 52 Please, remove the comma before "tomato".

Answer: The point was taken. We had been remove the comma before "tomato" and highlighted in red (Line 55) of the revised MS.

Question 2: Line 239 Change 3.6 to 3.5.

Answer: The point was taken. We had been changed 3.6 to 3.5 and highlighted in red (Line 256) of the revised MS.

Question 3: Line 255 Change 3.5 to 3.6.

Answer: The point was taken. We have changed 3.5 to 3.6 and highlighted in red (Line 272) of the revised MS.

Question 4: Line 291 Insert "tissues" after "22 different".

Answer: The point was taken. highlighted in red (Line 310) of the revised MS.

Question 5: Line 411 Group II is missing.

Answer: Thank you for your comment. We have not found DEGs in Group II and have corrected describe in the lines 438-439 of the revised MS, and highlighted in red.

Question 6: Lines 413-414 Group IV was included twice.

Answer: The point was taken. We have corrected it and highlighted in red (Line 452) of the revised MS..

Question 7: It is advisable to enlarge and improve the quality of Figure 3 B which is difficult to read.

Answer: The point was taken. We have replaced a high resolution Figure 3 in Results.

Question 8:In Figure 5 it is recommended inserting a legend to explain the acronyms of the plant tissues.

Answer: The point was taken. We have replaced the acronyms to complete name of the plant tissues in Figure 5.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript of Li et al. aims "to investigate the expression patterns of F-box protein genes in various tissues of cultivated peanuts, with a focus on their patterns during lateral branch development". The proposal is interesting, and the authors have all the results and data to carry out a detailed analysis as proposed. However, the results were presented in a superficial, descriptive way, focusing on numbers: numbers of motifs, exons, cis-elements, etc., There is little discussion or hypotheses to correlate the expression of F-box genes during lateral branch development and in the 2 varieties with different branching habits (erect x prostrate).

Additional points to be considered:

Lines 26-2: The authors did not present or discuss domestication in this manuscript…

Lines 39-40: “The Skp-Cullin-Fbox (SCF) protein, a subset…” The SCF is a complex and not a protein. Please correct.

Lines 40-41: “… subset of ligase E3, is characterized by the involvement of SKP-1 and SKP-1 like proteins in substrate recognition.” To my knowledge, the protein responsible for the substrate recognition is the F-box and NOT the SKP-1.

Lines 45-49: Please replace the pattern with DOMAIN or MOTIF in the sentence “F-box proteins possess a specific structural pattern consisting of approximately 40-50 amino acids.”

Lines 56-57: “By constructing a pps mutant, …” What is a pps mutant? Please clarify.

Lines 84-85: Please, rewrite for clarity. “Among these genes, Araip.E64SW could be negatively regulated the lateral branch development, …” Additionally, please identify to which F-box gene Araip.E64SW corresponds. I could not find this information in the manuscript or the Supplementary Material.

On items “2.6 Expression analysis in lateral branch development of peanut” and “2.7 RNA extracted and qRT–PCR analysis”, please inform the number of biological samples used. Were the same biological samples used in both experiments?

Lines 179-181: All the 95 AhFBX genes have the same typical F-box domain (ILSRLPTKHLARTSCVSKRWR)? No variation of this sequence was found or allowed during the search in the Arachis hypogaea genome?

Lines 186-187: “There were Arahy.10 contained 2 or fewer members. Specially, AhFBX95 was positioned on scaffold_50:16019-23949.” I do not understand the meaning of these sentences. Please clarify.

Lines 210-213: “The FBA, FBD, FBK, FBT, FBP, FBL, and FBO subfamilies were each defined by their respective structural domains: FBA, FBD, Kelch repeat, Tub, PP2, LRR (leucine–rich repeat), and FBO_C respectively.” Please define the structural domains.

Please improve figure legends, detailing and informing what is shown, acronyms, etc. For example, the legend of Figure 2B.

Line 226 and Figure 3: It is important to introduce and explain the 10 conserved motifs found. Please clarify.

Figure 3 is very small, and very little can be understood from what is written there. What is the meaning of the trees in A and B? Is there some correlation between them?

Line 260: Please introduce Aradu.04/Arahy.04, Aradu.09/Arahy.09, Araip.04/Arahy.14 and Araip.09/Arahy.19 and where the data was obtained.

Line 268: “… with the exception of AhFBX36/AhFBX83, indicated…” Please identify the corresponding points in Figure 4C.

Lines 277-279: “To explore the expression patterns of AhFBX genes in different tissues, we analyzed the expression profiles of 22 tissues (Table S8, Figure 5). Of these genes, 24 up-regulated expression genes and 47 down-regulated expression genes were found in all tissues.” Please introduce the 22 tissues studied.  What does it mean to be up-regulated and down-regulated here? Up-regulated and down-regulated in relation to what?

Line 282: “… and negative expression levels in all tissues…” What does it mean by negative expression level?

Line 283: “… 25 genes showed tissue-specific expression patterns in all tissues.” How can a gene have a tissue-specific expression in ALL tissues?

Line 286: “Interestingly, a part of homoeologous genes from same group…” Please clarify. Show which the homeologous genes are and their expressions.

Please, improve the legend of Figure 5, present the acronyms and what is being shown.

Line 295: How DEGs were established? What were the comparisons made? Differentially expressed in relation to what?

The first paragraph of the Discussion refers to the total number of genes for F-box proteins in the genome of peanut and other species. The number of genes reported in Arachis is much smaller than in other species. Some hypotheses were presented to explain this fact. However, at no point were the following discussed: how the search was carried out, the quality of the genome sequencing, etc.

Lines 349-351: Please, provide the complete name for “SAMs”, “ETH”, “PAL”.

Line 356: “ Overexpressed AtTLP9 mutants in Arabidopsis emonstrated ABA sensitivity…” I do not understand overexpressed and mutant… Can you please clarify what you are referring to?

Lines 365-370: Where is the discussion about the architecture of the plant? Here are 2 general, speculative sentences with no connection to the experiments or what is shown in the article

Lines 373-374: I'm afraid I have to disagree that the collinearity and the estimation of Ka/Ks ratios confirm the allotetraploid origin of the peanut genome.

Lines 442-444: “Among them, AhFBX8, AhFBX31, AhFBX45, AhFBX46, AhFBX55, AhFBX57, AhFBX90 and AhFBX91 showed specific expression in lateral branch development.” I didn't see where the authors showed this data. Please make it clear.

 Several places need correction, as some listed here:

Line 38 “… participates in varous of physiological…” Please correct.

Line 44: “… play criitical roles…” Please correct.

Line 55: “… growth and development had been clarified.” Please replace it with HAVE BEEN.

Line 93: “This process highllights the …” Please correct.

Line 97: “… patterns of AhFBXs aross different peanut… “ Please correct.

Line 356: “… Arabidopsis emonstrated…” Please correct.

Comments on the Quality of English Language

The manuscript could benefit from a good English revision.

Author Response

Dear reviewer,

     Thank you for your comments. Acoording to your comments, all the questions and comments in the postil were answered and revised in the MS. A few main Q&A were as follows:

Question 1: Lines 26-2: The authors did not present or discuss domestication in this manuscript.

Answer: The point was taken. We have discussed evolutionary of peanut F-box proteins in Abstract and Discussion of revised MS and highlighted in red (Lines 23-25 and 398-414).

Question 2: Lines 39-40: “The Skp-Cullin-Fbox (SCF) protein, a subset…” The SCF is a complex and not a protein. Please correct.

Answer: The point was taken. We have corrected and highlighted in red (Line 43).

Question 3: Lines 40-41: “… subset of ligase E3, is characterized by the involvement of SKP-1 and SKP-1 like proteins in substrate recognition.” To my knowledge, the protein responsible for the substrate recognition is the F-box and NOT the SKP-1.

Answer: The point was taken. We have corrected and highlighted in red (Lines 44-45).

Question 4:Lines 45-49: Please replace the pattern with DOMAIN or MOTIF in the sentence “F-box proteins possess a specific structural pattern consisting of approximately 40-50 amino acids.”

Answer: The point was taken. We have revised in MS and highlighted in red (Lines 48-49).

Question 5: Lines 56-57: “By constructing a pps mutant, …” What is a pps mutant? Please clarify.

Answer: The point was taken. We have clarified in MS and highlighted in red (Line 60).

Question 6: Lines 84-85: Please, rewrite for clarity. “Among these genes, Araip.E64SW could be negatively regulated the lateral branch development, …” Additionally, please identify to which F-box gene Araip.E64SW corresponds. I could not find this information in the manuscript or the Supplementary Material.

Answer: Thank you for your comments. We have rewrited “Among these genes, Araip.E64SW could be negatively regulated the lateral branch development, …” to “Of these genes, Araip.E64SW could be involved in lateral branch development, potentially leading to a spreading or prostrate appearance” in MS and highlighted in red (Lines 88-89). Additionally, we have found that Araip.E64SW corresponded to AhFBX58, with a similarity of 77.4% (E-value = 5e-18) based on the Basic Local Alignment Search Tool analysis. So we think that Araip.E64SW and AhFBX58 are not same gene.

Question 7: On items “2.6 Expression analysis in lateral branch development of peanut” and “2.7 RNA extracted and qRT–PCR analysis”, please inform the number of biological samples used. Were the same biological samples used in both experiments?

Answer: The point was taken. We have added and described in M&M and highlighted in red (Lines 186-188). 

Question 8: Lines 179-181: All the 95 AhFBX genes have the same typical F-box domain (ILSRLPTKHLARTSCVSKRWR)? No variation of this sequence was found or allowed during the search in the Arachis hypogaea genome ?

Answer: Thank you for your comments. In order to ensure the accuracy of the identified F-box in cultivated peanut, we used the HMM profile of FBX (accession: PF00646, E-value ≤ 1e-10) to search the peanut local protein database, and incomplete, variant and putative F-box domain protein sequences were excluded.

Question 9: Lines 186-187: “There were Arahy.10 contained 2 or fewer members. Specially, AhFBX95 was positioned on scaffold_50:16019-23949” I do not understand the meaning of these sentences. Please clarify.

Answer: The point was taken. We have modified this sentence in MS and highlighted in red (Lines 201-202).

Question 10: Lines 210-213: “The FBA, FBD, FBK, FBT, FBP, FBL, and FBO subfamilies were each defined by their respective structural domains: FBA, FBD, Kelch repeat, Tub, PP2, LRR (leucine–rich repeat), and FBO_C respectively.” Please define the structural domains.Please improve figure legends, detailing and informing what is shown, acronyms, etc. For example, the legend of Figure 2B.

Answer: The point was taken.We have clarified and defined these subfamilies in revised MS and highlighted in red (Lines 224-229).

Question 11: Line 226 and Figure 3: It is important to introduce and explain the 10 conserved motifs found. Please clarify.

Answer: The point was taken.We have explained it and highlighted in red (line 244-246).

Question 12: Figure 3 is very small, and very little can be understood from what is written there. What is the meaning of the trees in A and B? Is there some correlation between them?

Answer: The points was taken.We have changed it in Figure 3. The tree in Fig.3A is divided according to the motif, and the gene names of Fig.3B and Fig.3A also correspond one by one.

Question 13: Line 260: Please introduce Aradu.04/Arahy.04, Aradu.09/Arahy.09, Araip.04/Arahy.14 and Araip.09/Arahy.19 and where the data was obtained.

Answer: The point was taken. We have added it in M&M and Results of the revised MS and highlighted in red (lines 142-143 and 275-279).

Question 14: Lines 277-279: “To explore the expression patterns of AhFBX genes in different tissues, we analyzed the expression profiles of 22 tissues (Table S8, Figure 5). Of these genes, 24 up-regulated expression genes and 47 down-regulated expression genes were found in all tissues.” Please introduce the 22 tissues studied. What does it mean to be up-regulated and down-regulated here? Up-regulated and down-regulated in relation to what?

Answer: The points were taken. We have corrected this content in 3.7 0f revised MS and highlighted in red (lines 296-303).

Question 15: Line 282:“… and negative expression levels in all tissues…” What does it mean by negative expression level?

Answer: The point was taken. We have corrected this content in 3.7 0f revised MS and highlighted in red (lines 296-303).

Question 16: Line 283: “… 25 genes showed tissue-specific expression patterns in all tissues.” How can a gene have a tissue-specific expression in ALL tissues?

Answer: The point was taken. We have corrected this content in 3.7 0f revised MS and highlighted in red (lines 296-303).

Question 17: Line 286: “Interestingly, a part of homoeologous genes from same group…” Please clarify. Show which the homeologous genes are and their expressions.

Answer: The point was taken. We have corrected this content in 3.7 0f revised MS and highlighted in red (lines 304-306).

Question 18: Please, improve the legend of Figure 5, present the acronyms and what is being shown.

Answer: The point was taken. We have replaced the acronyms to complete name of the plant tissues in Figure 5.

Question 19: Line 295: How DEGs were established? What were the comparisons made? Differentially expressed in relation to what?

Answer: The points were taken. We have added it in M&M and Results of revised MS and highlighted in red (lines 171-177 and 313-316).

Question 20: The first paragraph of the Discussion refers to the total number of genes for F-box proteins in the genome of peanut and other species. The number of genes reported in Arachis is much smaller than in other species. Some hypotheses were presented to explain this fact. However, at no point were the following discussed: how the search was carried out, the quality of the genome sequencing, etc.

Answer: The point was taken. We have added the relevant content and highlighted in red (lines 354-356).

Question 21:Lines 349-351: Please, provide the complete name for “SAMs”, “ETH”, “PAL”.

Answer: The point was taken. We have added the complete name for “SAMs”, “ETH”, “PAL” and highlighted in red (lines 377-380).

Question 22:Line 356: “Overexpressed AtTLP9 mutants in Arabidopsis emonstrated ABA sensitivity…” I do not understand overexpressed and mutant… Can you please clarify what you are referring to?

Answer: The points was taken, we have corrected this sentence and highlighted in red (lines 385-387).

Question 23: Lines 365-370: Where is the discussion about the architecture of the plant? Here are 2 general, speculative sentences with no connection to the experiments or what is shown in the article.

Answer: The point was taken. We have corrected this sentence and highlighted in red (lines 395-397).

Question 24: Lines 373-374: I'm afraid I have to disagree that the collinearity and the estimation of Ka/Ks ratios confirm the allotetraploid origin of the peanut genome.

Answer: The point was taken. We have corrected this sentence and highlighted in red (lines 412-414).

Question 25:Lines 442-444: “Among them, AhFBX8, AhFBX31, AhFBX45, AhFBX46, AhFBX55, AhFBX57, AhFBX90 and AhFBX91 showed specific expression in lateral branch development.” I didn't see where the authors showed this data. Please make it clear.

Answer: The point was taken. we have added it in the Figure 6A.

3.Several places need correction, as some listed here:

Line 38 “… participates in varous of physiological…” Please correct.

Line 44: “… play criitical roles…” Please correct.

Line 55: “… growth and development had been clarified.” Please replace it with HAVE BEEN.

Line 93: “This process highllights the …” Please correct.

Line 97: “… patterns of AhFBXs aross different peanut… “ Please correct.

Line 356: “… Arabidopsis emonstrated…” Please correct.

Comments on the Quality of English Language

The manuscript could benefit from a good English revision.

Our response: Thanks for your comments. We have corrected the spelling mistakes and modified grammar in revised MS.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

no comments