Genome-Wide Identification and Expression Analysis of the CmHAK Gene Family in Melon (Cucumis melo L.)

Round 1

Reviewer 1 Report

Please provide a brief description of data RNA seq analysis to show gene expression Pattern. 

Line 36. Review the sentence, it´s confusing. ... as a typical potassium loving crop, potassium plays an important role in its growth and 37 stress processes....

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript from Fu et al. presents an in silico study of the melon hak gene family. The authors have identified 14 putative genes encoding potassium transporters in the complete NCBI genome sequence. The genes were numbered 1 to 14. How the numbering was done is unspecified but it looks to have been at random. Unless the genes were already given 1 to 14 numbers in the genome database, changes are requested. Because this a pioneering work, the gene numbering could follow some logics. The genes could have been ranked according to their position in the genome or better, according to their sequence homology, indicating their phylogenetic relatedness. Since the proteins are grouped in different clusters (fig. 6), it is suggested that their numbering should follow their grouping. For example, HAK6, 7, 9 and 8 which are in cluster IA should be numbered 1 to 4; 5 and 12 (IIA) should be 5 and 6; 3, 13, 4 and 11 (IIB) should be 7 to 10; 1 and 2 (IIIA) should be 11 and 12; 10 and 14 (IIIB) should be 13 and 14. 

 

This work must give access to gene and protein sequences. This is why, in table 1, the instability index, aliphatic index and gravy should be replaced by the NCBI gene ID, chromosome number and the precise position of each gene on their chromosome (nucleotide numbers of start and end of coding sequences in the chromosome sequence), and number of introns. The columns "number of transcripts", "signal peptides" and "location" should be deleted because they are the same for all members, which is stated in the text.

 

A phylogenetic tree should replace the cladogram of figure 2 to show a more accurate relatedness of the various melon sequences. 

 

The order of the protein sequences shown in figure 4 does not follow their relatedness. The sequences should be ordered as they are in the phylogenetic tree. Similar ordering should be used in figure 2 and 3.

 

Because of the status of Arabidopsis as a model dicot species, it would be informative to identify putative Cucumis-Arabidopsis orthologous pairs. This is difficult to find in figure 6 because there are too many entries in the phylogenetic tree. The presence of the rice sequences makes the branches longer and makes it more difficult to see the grouping of the various dicot sequences. This tree could be shown in a supplementary figure and replaced by a simpler tree showing only the Arabidopsis and melon sequences. In the caption of figure 6, it has to be stated that the tree is based upon amino acid sequence comparison. Also in the caption, add Fragaria and annuum.

 

Line 254: the ref. is not correct

Line 294 and elsewhere: "conserved" instead of "conservative"

Line 328-329: it is not the structures of the genes that are likely different in the two varieties but the regulations of their expression. 

 

The discussion recalls some results but those are not discussed in relation with published data. For example, how do protein and gene structures compare with those of Arabidopsis? Does the cold-regulated expression of hak genes in melon relate to the expression of the orthologous genes in Arabidopsis? 

 

The conclusion is a mere repetition of the discussion, which is partly itself a repetition of the results. That makes a lot of redundancy. The whole text under the conclusion headline should be deleted and replaced by the last paragraph of the discussion. 

 

The supplementary figure 1 is of no use and could be removed.

Although the text is understandable, the syntax could be improved. The whole text would benefit from editing. For example, in the abstract only:

- line 16: delete "gene family"

- line 18: start a new sentence after "technology" 

- line 21: start a new sentence after "7 chromosomes": Each member contains 3-11 introns and 4-12 exons and the family can be divided... 

- line 22: start a new sentence after "phylogeny" 

- line 23: "varies between 610 and 878"

- line 24: start a new sentence after "resistances"

- line 28: correct "family"

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript is well-written and addresses an important subject in horticulture but shows signs of English as a second language. The reviewer provided some examples of grammar deficiencies and poor language construction below. Many words are inappropriately capitalized in the manuscript and should be addressed by the authors. For example, “Melon” should not be capitalized unless it leads a sentence.

Detailed suggestions for improving the manuscript are provided below:

Abstract

Line 22) “…phylogeny, and…” change to “…phylogeny and…”.

Line 23) “…varies…” change to “…varied…”.

Line 23) “…stress, most…” change to “…stress most…”.

Line 24) “…resistances, the…” change to “…resistances. The…”. (new sentence)

Line 28) “…famil、y…” change to “…family,…”.

Line 29) “…and cultivation…” the authors should be more specific about the findings of the study relative to aspects of cultivation that maximize yield or fruit quality in stress-tolerant melons.

Introduction

Line 33) “…crop,…” change to “…crop…”. The authors should review the entire manuscript for inappropriate uses of commas. The reviewer will cease to highlight such issues from this point to the end of the manuscript.

Line 36) “…spread…” do the authors intend the word “widespread”? This is not clear.

Lines 36-38) “Melon, as a typical potassium loving crop, potassium plays an important role in its growth and stress processes…” suggest change to “Potassium plays an important role in the growth and stress responses of melon…”.

Line 38) “. Potassium…” change to “and…”. (no new sentence).

Line 42) “…plant, potassium…” change to “plant. Potassium…” (new sentence).

Line 49) “Plant absorbs and transports…” change to “Plants absorb and transport…”.

Line 49 “…Ion…” should be lower case.

Line 54) “…transporter, responsible for potassium ion absorption,…” change to “…transporter and is responsible for potassium ion absorption.”.

Line 54) “…if found in soil yeast, it is called…” change to “If found in soil yeast it is called the…” (new sentence).

Line 55) “KT/HAK/KUP gene…” change to “The KT/HAK/KUP gene…”. This grammatical error recurs and change should be made throughout the manuscript.

Lines 59-60) “…function; subfamily I…” suggest change to “…function. Subfamily I…”. (new sentence).

Line 61) “…roots; Subfamily II…” suggest change to “…roots. Subfamily II…”. (new sentence).

Line 62) “…regulation; there…” change to “…regulation. There…”. (new sentence).

Line 69) “…parts, the AtHAK5 gene…” change to “…parts. The AtHAK5 gene…”. (new sentence).

Lines 70-78) The text here consists of one long run-on sentence punctuated with semicolons and commas. This text should be broken up into shorter sentences to be clearer.

Line 80) “…some HAK genes have been cloned and Functional verification…” change to “…some HAK genes have been cloned and the functions of gene products verified…”.

Materials and Methods

Lines 92-92) “…which were taken as the construction of a hidden Markov model with HMMER 3.0…” this statement is unclear and should be rewritten without jargon for better clarity.

Lines 99-100) “Use the aligned sequences as candidate HAK family sequences and combine them into candidate HAK protein sequences.” This sentence is not grammatically correct and is not clear; must be rewritten to be correct and clear.

Line 101) “…annotated…” annotated how and using which criteria?

Line 110) “…using an easy-to-use web tool…” readers don’t care if the tool is easy but it must be accessible to other researchers and widely accepted as the preferred tool for this application. When I clicked on the provided link no information about this tool appeared. All links should be verified before they are included in the manuscript. This statement should be rewritten to reflect these needs.

Line 124) “…TF binding sites…” TF = transcription factor; define all acronyms if not previously defined in the body of the manuscript.

Line 135) “…is used…” change to “…was used…”.

Results

Lines 145-146) “To identify potential CmHAK proteins in Melon genome, a total of 14 CmHAK genes were identified.” Change to “A total of 14 CmHAK genes was identified.”.

Line 186) “…only has…” change to “…has only…”.

Lines 197-198) “…in Figure 5, a total of 12 cis-effects were identified in the promoter region…” This phrase is formatted with a smaller font size than the adjoining text.

Line 198) “Elements can be…” the reviewer presumes that the authors are referring to sequences within the 200 bp upstream region that are acting as modulators of expression. If so, the authors should improve the clarity of this statement.

Line 203) “…). And…” change to “…and…” (no new sentence).

Lines 217-218) “However, 14 CmHAK proteins in Cucumis melo belong to group I, II and III.” Change to “The 14 CmHAK proteins in Cucumis melo were assigned to groups I, II, and III.”

Lines 222-223) “The results of the phylogenetic analysis certified that most of CmHAK proteins tended to be clustered together.” The reviewer suggests that this sentence be rewritten to “The results of the phylogenetic analysis showed that most of the CmHAK proteins were similar in sequence and origin.”

Line 235) “But no tandem replication gene pairs were found…” change to “No tandem replicated gene pairs were found…”.

Line 236) “It indicates that some…” change to “This indicates that some…”.

Lines 239-240) “Calculate the non-synonymous substitution value (Ka), synonymous substitution value (Ks), and Ka/Ks ratio of three identified replication gene pairs.” This sentence makes no sense and must be rewritten to be clearer and grammatically correct.

Lines 246-247) “mainly perFormed…” suggest change to “…experienced…”.

Line 255) “…explored, and the…” change to “…explored. The…”. (new sentence).

Line 266) “Especially…” the reviewer suggests that this word be deleted and the rest of the sentence left as-is.

Discussion

Line 278) “…which makes…” suggest change to “…making…”.

Line 278) “…three major elements…” change to “…three major chemical elements…”.

Lines 294-295) “…conservative, which may be the reason why HAKs of Melon are highly conservative.” Change to “…conserved, which may be the reason why HAKs of melon or highly conserved.”.

Lines 316-317) “There is a significant difference in transcriptome data for the PhaHAK2 gene in reed between salt-tolerant materials and controls…” The reviewer is confused by this sentence and suggests it be rewritten to be clearer.

Lines 319-320) “Under the treatment of growth hormone, the K+/H+ cotransport activity of OsHAK5 in rice was significantly improved…” it was not clear to the reviewer why this statement was included. How does it relate to the new study in melons?

The manuscript is well-written and addresses an important subject in horticulture but shows signs of English as a second language. The reviewer provided some examples of grammar deficiencies and poor language construction below. Many words are inappropriately capitalized in the manuscript and should be addressed by the authors. For example, “Melon” should not be capitalized unless it leads a sentence.

Detailed suggestions for improving the manuscript are provided below:

Abstract

Line 22) “…phylogeny, and…” change to “…phylogeny and…”.

Line 23) “…varies…” change to “…varied…”.

Line 23) “…stress, most…” change to “…stress most…”.

Line 24) “…resistances, the…” change to “…resistances. The…”. (new sentence)

Line 28) “…famil、y…” change to “…family,…”.

Line 29) “…and cultivation…” the authors should be more specific about the findings of the study relative to aspects of cultivation that maximize yield or fruit quality in stress-tolerant melons.

Introduction

Line 33) “…crop,…” change to “…crop…”. The authors should review the entire manuscript for inappropriate uses of commas. The reviewer will cease to highlight such issues from this point to the end of the manuscript.

Line 36) “…spread…” do the authors intend the word “widespread”? This is not clear.

Lines 36-38) “Melon, as a typical potassium loving crop, potassium plays an important role in its growth and stress processes…” suggest change to “Potassium plays an important role in the growth and stress responses of melon…”.

Line 38) “. Potassium…” change to “and…”. (no new sentence).

Line 42) “…plant, potassium…” change to “plant. Potassium…” (new sentence).

Line 49) “Plant absorbs and transports…” change to “Plants absorb and transport…”.

Line 49 “…Ion…” should be lower case.

Line 54) “…transporter, responsible for potassium ion absorption,…” change to “…transporter and is responsible for potassium ion absorption.”.

Line 54) “…if found in soil yeast, it is called…” change to “If found in soil yeast it is called the…” (new sentence).

Line 55) “KT/HAK/KUP gene…” change to “The KT/HAK/KUP gene…”. This grammatical error recurs and change should be made throughout the manuscript.

Lines 59-60) “…function; subfamily I…” suggest change to “…function. Subfamily I…”. (new sentence).

Line 61) “…roots; Subfamily II…” suggest change to “…roots. Subfamily II…”. (new sentence).

Line 62) “…regulation; there…” change to “…regulation. There…”. (new sentence).

Line 69) “…parts, the AtHAK5 gene…” change to “…parts. The AtHAK5 gene…”. (new sentence).

Lines 70-78) The text here consists of one long run-on sentence punctuated with semicolons and commas. This text should be broken up into shorter sentences to be clearer.

Line 80) “…some HAK genes have been cloned and Functional verification…” change to “…some HAK genes have been cloned and the functions of gene products verified…”.

Materials and Methods

Lines 92-92) “…which were taken as the construction of a hidden Markov model with HMMER 3.0…” this statement is unclear and should be rewritten without jargon for better clarity.

Lines 99-100) “Use the aligned sequences as candidate HAK family sequences and combine them into candidate HAK protein sequences.” This sentence is not grammatically correct and is not clear; must be rewritten to be correct and clear.

Line 101) “…annotated…” annotated how and using which criteria?

Line 110) “…using an easy-to-use web tool…” readers don’t care if the tool is easy but it must be accessible to other researchers and widely accepted as the preferred tool for this application. When I clicked on the provided link no information about this tool appeared. All links should be verified before they are included in the manuscript. This statement should be rewritten to reflect these needs.

Line 124) “…TF binding sites…” TF = transcription factor; define all acronyms if not previously defined in the body of the manuscript.

Line 135) “…is used…” change to “…was used…”.

Results

Lines 145-146) “To identify potential CmHAK proteins in Melon genome, a total of 14 CmHAK genes were identified.” Change to “A total of 14 CmHAK genes was identified.”.

Line 186) “…only has…” change to “…has only…”.

Lines 197-198) “…in Figure 5, a total of 12 cis-effects were identified in the promoter region…” This phrase is formatted with a smaller font size than the adjoining text.

Line 198) “Elements can be…” the reviewer presumes that the authors are referring to sequences within the 200 bp upstream region that are acting as modulators of expression. If so, the authors should improve the clarity of this statement.

Line 203) “…). And…” change to “…and…” (no new sentence).

Lines 217-218) “However, 14 CmHAK proteins in Cucumis melo belong to group I, II and III.” Change to “The 14 CmHAK proteins in Cucumis melo were assigned to groups I, II, and III.”

Lines 222-223) “The results of the phylogenetic analysis certified that most of CmHAK proteins tended to be clustered together.” The reviewer suggests that this sentence be rewritten to “The results of the phylogenetic analysis showed that most of the CmHAK proteins were similar in sequence and origin.”

Line 235) “But no tandem replication gene pairs were found…” change to “No tandem replicated gene pairs were found…”.

Line 236) “It indicates that some…” change to “This indicates that some…”.

Lines 239-240) “Calculate the non-synonymous substitution value (Ka), synonymous substitution value (Ks), and Ka/Ks ratio of three identified replication gene pairs.” This sentence makes no sense and must be rewritten to be clearer and grammatically correct.

Lines 246-247) “mainly perFormed…” suggest change to “…experienced…”.

Line 255) “…explored, and the…” change to “…explored. The…”. (new sentence).

Line 266) “Especially…” the reviewer suggests that this word be deleted and the rest of the sentence left as-is.

Discussion

Line 278) “…which makes…” suggest change to “…making…”.

Line 278) “…three major elements…” change to “…three major chemical elements…”.

Lines 294-295) “…conservative, which may be the reason why HAKs of Melon are highly conservative.” Change to “…conserved, which may be the reason why HAKs of melon or highly conserved.”.

Lines 316-317) “There is a significant difference in transcriptome data for the PhaHAK2 gene in reed between salt-tolerant materials and controls…” The reviewer is confused by this sentence and suggests it be rewritten to be clearer.

Lines 319-320) “Under the treatment of growth hormone, the K+/H+ cotransport activity of OsHAK5 in rice was significantly improved…” it was not clear to the reviewer why this statement was included. How does it relate to the new study in melons?

The manuscript shows signs of English as a second language, but is mostly acceptable. The reviewer provided some examples of grammar deficiencies and poor language construction.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The major issues of the first draft have been addressed. 

 

Two changes are requested:

 

The genomic sequences in supplementary table 1 are of little use. They should be replaced by the cDNA sequences. 

 

As already requested in the review of the first version, in the caption of Supplementary figure 3, Fragaria and annuum should be added before vesca and after Capsicum, respectively. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf