Genome-Wide Identification, Evolution, and Expression Analysis of the TCP Gene Family in Rose (Rosa chinensis Jacq.)

Round 1

Reviewer 1 Report

This paper comprises basic bioinformatics analysis to provide a description of the TCP transcription factor family in Rose. While there is some useful information presented, the paper lacks proper detail relating to both the methodology and data presentation/ analysis (lack of proper descrption, labels etc.), and has a strong tendancy towards overconclusion based on the limited data available. The English is generally in need of considerable improvement. The Figures are not well-explained in the text nor in the legends. The clarity and precision of language needs major improvement, and a clearer rationale often needs to be provided and the reasoning behind conclusions made more robust. The paper does not really constitute a significant advance in the field and the data would be better integrated within a manuscript that addresses at least some of the functions of the gene using more empirical experiments, rather than overreliance on inferring functions based on informatics approaches. The drought stress experiment seems to be an afterthought and does not integrate well into the overall narrative.

Author Response

Dear Editor,

We would like to submit our revised manuscript entitled “Genome-Wide Identification, Evolution, and Expression Analysis of the TCP Gene Family in Rose (Rosa chinensis Jacq.)” for publication in Horticulturae. The manuscript was previously sent for appreciation under the ID: horticulturae-1892485.

The manuscript underwent considerable revision following the valuable suggestions provided by the reviewers and the editorial office (please see the Track-Change version). The criticisms and questions raised by the reviewers were addressed point-by-point as follows.

    In addition, we have added Yufei Chang as co-authors (Yi Hou^#, Chunguo Fan^#, Jingrui Sun^#, Yufei Chang, Jun Lu, Jingjing Sun, Changquan Wang and Jinyi Liu*) due to her significant contribution in revision period. All authors agree and confirmed it. We also changed the Acknowledgements for funding information. Sorry for your inconvenience.

 

Sincerely yours

 

Jinyi Liu

Dr./Associate Professor

College of Horticulture

Nanjing Agricultural University, P.R. China, 210095

No. 1 of Weigang, Nanjing City, Jiangsu Province, P.R., China,

Email: [email protected]

Mobile phone: +86-13851796756

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Title: Genome-Wide Identification, Characterization, and Expression Analysis of the TCP Gene Family in Rose (Rosa chinensis Jacq.)

 

In this work the authors identify the TPC plant-specific transcription factor gene family in Rosa chinensis. The topic is interesting, and several studies was performed for identify these genes. The authors also suggest the role of TPC genes in biological processes against drought stress. However, the manuscript must be improved in the materials and methods and results sections. Several experiments in the work are hardly comprehensible because the flow of the work in the materials and methods section is not clear and several information are missing. It is also not clear what type of material was used for the investigation. There is often no correspondence between the two sections. There are many errors that make it difficult to understand the methods used and invalidate the results shown.

Often there are phrases in the results that should be included in the other sections, materials and methods or discussion. Some figures are not adequately cited and described. In the text the Supplementary Materials is never mentioned in the text

Dear editor:

This is the modified version of the paper titled “Genome-Wide Identification, Characterization, and Expression Analysis of the TCP Gene Family in Rose (Rosa chinensis Jacq.)”. We wish to thank you for the time and efforts you have spent reviewing our paper. Following your comments, we have tried to fix all the problems you mentioned. What we changed was highlighted in yellow in the text, and the following are point-to-point responses to editor and reviewers.

• See below for details:
• The manuscript does not even have line numbering. This makes revision difficult

 

 

In the manuscript there are errors relating to the correct indication of the names of the species: I remember that excluding the abstract, the names must be complete (e.g. Rosa chinensis) only the first time, then the following times only the initial of the genus must be written followed by specie name (e.g. R.chinensis). Names must always be italicized. Correct throughout the manuscript.

• Abstract: ‘TCP gene family’ add the TPC long name
• TCP gene family have been performed for many species, such as Arabidopsis.
• What does arabidopsis mean? Either put the species name or put Arabidopsis spp ..
• Arabidopsis alone is a genus.

 

-Thank you for your suggestions. We have corrected the specie name throughout the manuscript.

-The TCP long name is TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR LAND 2. We have added the long name of it in manuscript.

-Here, the “arabidopsis” means “Arabidopsis thaliana”. We have corrected according to your comment.

 

• ‘The whole genome of Rosa chinensis has been sequenced.’

Add reference

 

-Thanks for your reminder, we have replaced the sentence with “Along with the release of R. chinensis cv. ‘Old blush ’ genome, one of the important progenitors of modern roses, roses have been proposed to be an ideal model species for studying the molecular basis of woody plants”, and added reference of it. 

 

• ‘All rose genome sequence and the gff3 files were obtained from GDR (Genome Da[1]tabase for Rosaceae, https://www.rosaceae.org). And the arabidopsis TCP sequences were obtained from phytozome（https://phytozome-next.jgi.doe.gov/）.

What does 'rose' mean? Correctly indicate which species you selected genes from. In all materials and methods make it clear which species you used for each test you did. There is no clarity on this.

Specify also what it refers to gff3 files

-Thanks for the comments,the 'rosa' in the manuscript represents the Rosa Chinensis, and we made a mistake in writing, which has now been corrected.

 In addition, the genomic data of roses come from GDR, and the gff3 file is annotation file of the genome, which are included in the genome file and downloaded from the GDR website. Therefore, the gff3 file does not need to be specified separately, so we removed the related representation.

• Change ‘(Genome Da[1]tabase for Rosaceae,’: with (Genome Da[1]tabase for Rosaceae family,

‘based on the arabidopsis TCP sequences..’

You can't write such a sentence in an article. Correctly indicate the species or species you have used

 

-Thanks for the comments, the long name of GDR is ‘Genome Database for Rosaceae’,

We have corrected ‘based on the arabidopsis TCP sequences’ to ‘with the sequences of AtTCP proteins as queries’

• ‘A phylogenetic tree was constructed using all protein sequences of TCP domain us[1]ing 0in rose and arabidopsis.’

Specify better (see previous suggestions)  in addition write MEGA11.0 software

 

-Thanks for the comments. We have specified the species in manuscript, which are R. chinensis and A. thaliana. We have also added the web address of the MEGA11.0 to the manuscript

• ‘Dentification of TCP structural domains in Arabidopsis and Luna using NCBI's CD-Search’

Luna? Specify. Then specify also NCBI GenBank the first time.

 

-Thanks for the comments, As mentioned in the manuscript, we used MEME suite to identify motifs in TCP protein, not NCBI, so we deleted this sentence.

• Malus domestica genome sequence and gff3 files we

Spec-ify what it refers to gff3 files

 

-Thanks for the comments. As mentioned above, the gff3 file is annotation file of the genome, which are included in the genome file and downloaded from the GDR website. Therefore, the gff3 file does not need to be specified separately, so we removed the related representation.

 

• Change: ‘Malus domestica genome sequence and gff3 files were obtained from GDR and Oryza sativa genome sequence and gff3 files were obtained from Ensembl Genomes  https://plants.ensembl.org/Oryza_sativa/Info/Index) to investigate the syntenic relationship among the TCP genes of rose and other related species. MCScanX was used to detect gene synteny and collinearity with the default parameters.
• With: The syntenic and collinearity relationship among the TCP genes of rose and other related species was investigated using MCScanXwith the default parameters. Malus domestica genome sequence and gff3 files were obtained from GDR and Oryza sativa genome sequence and gff3 files were obtained from Ensembl Genomes  https://plants.ensembl.org/Oryza_sativa/Info/Index).

 

Thanks for your suggestions. We have revised both of the above passages in the original text to more clearly state where our data came from and how we used them.

 

 

 

• Not clear: The transcript levels of the various RcTCPs were analyzed using the following tran[1]scriptome data sets retrieved: ROSAseq web interface database[13] (http://iant.tou[1]louse.inra.fr/R.chinensis), Rosa chinensis Raw sequence reads (accession number PRJNA663119)，Phosphorylation of RhPIP2;1, an aquaporin from rose (accession number PRJNA486271)，Rosa chinensis cultivar Old Blush (accession number PRJNA351281)[14], Rosa chinensis Transcriptome or gene expression (accession number PRJNA398090, PRJNA325324)[15, 16], Transcriptome of the floral transition in Rosa chinensis ‘Old Blush’

(accession number PRJCA000258)[17]. The expression levels were illustrated based on the

log2 transformed FPKM values visualized by TBtools

Excluded for the FPMK value of FMK, it is not clear how you used these data.

 

-Thanks for your suggestions. We found a clutter in this passage, which has now been corrected. In addition, we downloaded the published transcriptome data from public database platforms, processed them with Kallisto to obtain their FPKM values, and finally visualized them with tbtools.We have added the above to the manuscript

 

 

-  PlantCARE

.(add link)

 

Thanks for your suggestions. We have added the link here.

 

• ‘Plant Material and Treatments’

Title not clear add which type of treatment tested in the title

-RNA Isolation and qRT-PCR Analysis

Add qRT-PCR complete name the first time

-Thanks for the comments. Both of your proposed issues have been corrected in the manuscript. We have added ‘PEG’ before ‘Treatment’. The complete name of qRT-PCR is Quantitative Real-time PCR.

 

• ‘Total RNA was extracted using the BioTeke Quick RNA isolation Kit (Cat. #: RP3301, BioTeke Corporation, Beijing, China) and 1 μg of high quality total RNA was reverse transcribed using the PrimeScriptTM RT reagent Kit (Cat. #: RR047A, TaKaRa, Dalian, China) according to the manufacturer’s instructions. For qRT-PCR, RcGADPH was used as the reference gene, as described previously[18]. Three biological replicates and three technical replicates were used for each experiment. The relative expression levels of each gene were calculated in accordance with the 2- ∆∆CT method.’

There are few indications on the protocol (e.g. PCR reaction, the primers you used and how you selected them).

If some data are in the supplemental material, you must indicate them in the text

Add references for DDCt.

Thanks for the comments. All missing data have been supplemented in the manuscript.

 

• ‘while no possible TCP genes were found on chromosomes 3 and 6.’

Modify with

while no putative TCP genes were found on chromosomes 3 and 6.

 

Thanks for your suggestions. We have revised the manuscript as you said.

 

• No tandem replication events were found by retrieval. These results suggest that fragment replication plays an important role in the amplification of rose TCP gene family.
• Include in the Discussion section

 

Thanks for the comments. We have removed this sentence from the Results section.

 

• A phylogenetic tree was constructed for 130 protein sequences of rose, arabidopsis,

plum, apple, and Fragaria vesca by MEGA11.0 in order to explore the evolutionary and

phylogenetic relationship between the TCP protein of roses and other known TCP pro[1]teins.

 

You did not write this in the Materials and Methods section.

 

Thanks for your suggestions. In Materials and Methods, we have completed this part.

 

-  The TCP gene family can be divided into two subfamilies, class I, TCP-P or PCF,

and class II, TCP-C. In addition, Class II is subdivided into two subclasses: the CyC/TB1

subclass and the CIN subclass. Each plant has all subfamilies with a different number of

TCP genes, as shown in the figure.

-  Which figure? Figure3A? Figure 3B? Indicates correctly.

 

Thanks for the comments. In the new paragraph, we have clearly indicated the corresponding pictures.The phylogenetic tree of TCP genes from R. chinensis and A. thaliana were represented for classification of R. chinensis (Figure 2A). Interestingly, for each subclass, the copy number of TCP genes varied significantly (Figure 2B).

 

-  Each plant has all subfamilies

It is better to write species not plants

 

Thanks for the comments. We have showed all the species and subfamilies in Figure 2B.

 

-  Perhaps this is due to the differentiation of TCP family genes

before the separation of their ancestral species.

I suggest removing this sentence or phrasing it better.

 

Thanks for the comments. We have removed this sentence.

 

• method（Figure 3）

It’s Figure 3A? correct

 

Thanks for the comments. We have changed Figure 3 to Figure 2A.

 

All arabidopsis TCP belong to the same branch as previously reported,

which confirms the reliability of the phylogenetic tree.

???

Inappropriate comment, you must know before using them to make the phylogenetic tree the correctness of the sequences you have selected! Delete

 

Thanks for the comments. We have removed this sentence.

 

• ‘In rose, the TCP[1]C class has the largest number of members with nine, accounting for about 56% of the total number of RcTCP proteins, while the TCP-P class has seven members, accounting for about 44%. In class II, CyC/TB1 subclass is the smallest, with only 4 members; The CIN subclass has 5 members, accounting for about 31% of the total. Based on these findings, TCP genes might have evolved species-specifically’
• Do you show it in the picture? Which?

 

Thanks for the comments. We have a new description of the results of the phylogenetic tree in the manuscript.

 

• Figure 3. The phylogenetic tree of RcTCPs. (A) The phylogenetic tree of TCP gene family genes fromrose and arabidopsis. (B)Number of each subclass of TCP gene in each plant.This legend it’s not clear. What do you mean rose and arabidopsis? Some tips: in figure 3A there are stars and triangles, indicate what they refer to. You write about roses and Arabidopsis. Then the panel B in the figure shown that you have analyzed species belonging to the Rosaceae and Arabidopsis thaliana. I suggest therefore to write species (instead of plants) belonging to the Rosaceae family and Arabidopsis thaliana.

 

Thanks for the comments. We added what the triangle and star refer to in the text, and modified plant to species.

 

• ‘Conservative Motifs,’
• Which motifs? You reported only 1,2,3,

 

Thanks for the comments. These motifs have been added to the graph by us, see Figure 3 for details. All six motifs are described in the legends of figure 3.

 

• Figure 4
• Adjust the legend. A?

Thanks for the comments. Figure 4 has been adjusted.

 

- ‘To better understand the function of RcTCP gene in rose, transcript levels of RcTCP gene in different tissues were downloaded and analyzed, and heatmap was visualized the expression pattern of RcTCP genes. As shown in Figure 7, highly expressed genes in different tissues mainly exist in PCF and CIN subclass. Some RcTCP genes have similar ex[1]pression profiles in different tissues….

 

You write about gene expression in different treatments, but all this is not written in the corresponding paragraph on materials and methods section.

 

Thanks for the comments. Different processing methods we have modified in the figure, adding them to Figure 6.

 

• ‘Figure 7. The gene expression pattern of rose TCP genes in different tissues and developmental stages. (A) Phylogenetic tree of 23 arabidopsis TCP and 16 rose TCP genes;’

Where arabidopsis?

 

Thanks for the comments. We have corrected this error in the article and removed ‘arabidopsis’

 

• ‘To investigate the expression pattern of TCP genes in the growth and developmentof rose organs, transcriptome data from the database were used to analyze the expression profile of TCP genes during the differentiation of rose organs to full opening.’
• Thanks for the comments.This part is shown in Figure 6D and has been annotated in the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Title: Genome-Wide Identification, Characterization, and Expression Analysis of the TCP Gene Family in Rose (Rosa chinensis Jacq.)

 

In this work the authors identify the TPC plant-specific transcription factor gene family in Rosa chinensis. The topic is interesting, and several studies was performed for identify these genes. The authors also suggest the role of TPC genes in biological processes against drought stress. However, the manuscript must be improved in the materials and methods and results sections. Several experiments in the work are hardly comprehensible because the flow of the work in the materials and methods section is not clear and several information are missing. It is also not clear what type of material was used for the investigation. There is often no correspondence between the two sections. There are many errors that make it difficult to understand the methods used and invalidate the results shown.

Often there are phrases in the results that should be included in the other sections, materials and methods or discussion. Some figures are not adequately cited and described. In the text the Supplementary Materials is never mentioned in the text

 

. See below for details:

The manuscript does not even have line numbering. This makes revision difficult

 

In the manuscript there are errors relating to the correct indication of the names of the species: I remember that excluding the abstract, the names must be complete (e.g. Rosa chinensis) only the first time, then the following times only the initial of the genus must be written followed by specie name (e.g. R.chinensis). Names must always be italicized. Correct throughout the manuscript

 

-          Abstract: ‘TCP gene family’ add the TPC long name

 

-          TCP gene family have been performed for many species, such as arabidopsis

What does arabidopsis mean? Either put the species name or put Arabidopsis spp ..

Arabidopsis alone is a genus

 

-          ‘The whole genome of Rosa chinensis has been sequenced.’

Add reference

 

-          ‘All rose genome sequence and the gff3 files were obtained from GDR (Genome Da[1]tabase for Rosaceae, https://www.rosaceae.org). And the arabidopsis TCP sequences were obtained from phytozome（https://phytozome-next.jgi.doe.gov/）.’

What does 'rose' mean? Correctly indicate which species you selected genes from. In all materials and methods make it clear which species you used for each test you did. There is no clarity on this.

Specify also what it refers to gff3 files

 

-          Change ‘(Genome Da[1]tabase for Rosaceae,’: with (Genome Da[1]tabase for Rosaceae family,

‘based on the arabidopsis TCP sequences..’

You can't write such a sentence in an article. Correctly indicate the species or species you have used

 

-          ‘A phylogenetic tree was constructed using all protein sequences of TCP domain us[1]ing MEGA11.0 in rose and arabidopsis.’

Specify better (see previous suggestions)  in addition write MEGA11.0 software

 

-          ‘Dentification of TCP structural domains in Arabidopsis and Luna using NCBI's CD-Search’

Luna? Specify. Then specify also NCBI GenBank the first time

 

-          Malus domestica genome sequence and gff3 files we

Spec-ify what it refers to gff3 files

 

-          Change: ‘Malus domestica genome sequence and gff3 files were obtained from GDR and Oryza sativa genome sequence and gff3 files were obtained from Ensembl Genomes  https://plants.ensembl.org/Oryza_sativa/Info/Index) to investigate the syntenic relationship among the TCP genes of rose and other related species. MCScanX was used to detect gene synteny and collinearity with the default parameters

With: The syntenic and collinearity relationship among the TCP genes of rose and other related species was investigated using MCScanX with the default parameters. Malus domestica genome sequence and gff3 files were obtained from GDR and Oryza sativa genome sequence and gff3 files were obtained from Ensembl Genomes  https://plants.ensembl.org/Oryza_sativa/Info/Index).

 

-          Not clear: The transcript levels of the various RcTCPs were analyzed using the following tran[1]scriptome data sets retrieved: ROSAseq web interface database[13] (http://iant.tou[1]louse.inra.fr/R.chinensis), Rosa chinensis Raw sequence reads (accession number PRJNA663119)，Phosphorylation of RhPIP2;1, an aquaporin from rose (accession number PRJNA486271)，Rosa chinensis cultivar Old Blush (accession number PRJNA351281)[14], Rosa chinensis Transcriptome or gene expression (accession number PRJNA398090, PRJNA325324)[15, 16], Transcriptome of the floral transition in Rosa chinensis ‘Old Blush’

(accession number PRJCA000258)[17]. The expression levels were illustrated based on the

log2 transformed FPKM values visualized by TBtools

Excluded for the FPMK value of FMK, it is not clear how you used these data

 

-          PlantCARE

.(add link)

 

-          ‘Plant Material and Treatments’

Title not clear add which type of treatment tested in the title

-RNA Isolation and qRT-PCR Analysis

Add qRT-PCR complete name the first time

 

-          ‘Total RNA was extracted using the BioTeke Quick RNA isolation Kit (Cat. #: RP3301, BioTeke Corporation, Beijing, China) and 1 μg of high quality total RNA was reverse tran￾scribed using the PrimeScriptTM RT reagent Kit (Cat. #: RR047A, TaKaRa, Dalian, China) according to the manufacturer’s instructions. For qRT-PCR, RcGADPH was used as the reference gene, as described previously[18]. Three biological replicates and three technical replicates were used for each experiment. The relative expression levels of each gene were calculated in accordance with the 2- ∆∆CT method.’

There are few indications on the protocol (e.g. PCR reaction, the primers you used and how you selected them).

If some data are in the supplemental material, you must indicate them in the text

Add references for DDCt

-          ‘while no possible TCP genes were found on chromosomes 3 and 6.’

Modify with

while no putative TCP genes were found on chromosomes 3 and 6.

 

-          No tandem replication events were found by retrieval. These results suggest that fragment replication plays an important role in the amplification of rose TCP gene family.

Include in the Discussion section

 

-          A phylogenetic tree was constructed for 130 protein sequences of rose, arabidopsis,

plum, apple, and Fragaria vesca by MEGA11.0 in order to explore the evolutionary and

phylogenetic relationship between the TCP protein of roses and other known TCP pro[1]teins.

 

You did not write this in the Materials and Methods section

 

-          The TCP gene family can be divided into two subfamilies, class I, TCP-P or PCF,

and class II, TCP-C. In addition, Class II is subdivided into two subclasses: the CyC/TB1

subclass and the CIN subclass. Each plant has all subfamilies with a different number of

TCP genes, as shown in the figure.

 

-          Which figure? Figure3A? Figure 3B? Indicates correctly

 

-          Each plant has all subfamilies

It is better to write species not plants

 

-          Perhaps this is due to the differentiation of TCP family genes

before the separation of their ancestral species.

I suggest removing this sentence or phrasing it better.

 

-          method（Figure 3）

It’s Figure 3A? correct

 

-          All arabidopsis TCP belong to the same branch as previously reported,

which confirms the reliability of the phylogenetic tree.

???

Inappropriate comment, you must know before using them to make the phylogenetic tree the correctness of the sequences you have selected! Delete

 

-          ‘In rose, the TCP[1]C class has the largest number of members with nine, accounting for about 56% of the

total number of RcTCP proteins, while the TCP-P class has seven members, accounting for

about 44%. In class II, CyC/TB1 subclass is the smallest, with only 4 members; The CIN

subclass has 5 members, accounting for about 31% of the total. Based on these findings,

TCP genes might have evolved species-specifically’

Do you show it in the picture? Which?

 

-          Figure 3. The phylogenetic tree of RcTCPs. (A) The phylogenetic tree of TCP gene family genes from

rose and arabidopsis. (B)Number of each subclass of TCP gene in each plant.

This legend it’s not clear. What do you mean rose and arabidopsis? Some tips: in figure 3A there are stars and triangles, indicate what they refer to. You write about roses and Arabidopsis. Then the panel B in the figure shown that you have analyzed species belonging to the Rosaceae and Arabidopsis thaliana. I suggest therefore to write species (instead of plants) belonging to the Rosaceae family and Arabidopsis thaliana..

 

-          ‘Conservative Motifs,’

Which motifs? You reported only 1,2,3,

 

-          Figure 4

Adjust the legend. A?

 

-          Co-linearity analysis?

 

-          ‘To better understand the function of RcTCP gene in rose, transcript levels of RcTCP gene in different tissues were downloaded and analyzed, and heatmap was visualized the expression pattern of RcTCP genes. As shown in Figure 7, highly expressed genes in different tissues mainly exist in PCF and CIN subclass. Some RcTCP genes have similar ex[1]pression profiles in different tissues….

 

You write about gene expression in different treatments, but all this is not written in the corresponding paragraph on materials and methods section.

 

-          ‘Figure 7. The gene expression pattern of rose TCP genes in different tissues and developmental stages. (A) Phylogenetic tree of 23 arabidopsis TCP and 16 rose TCP genes;’

Where arabidopsis?

 

-          ‘To investigate the expression pattern of TCP genes in the growth and development

of rose organs, transcriptome data from the database were used to analyze the expression

profile of TCP genes during the differentiation of rose organs to full opening.’

Author Response

Dear Editor,

We would like to submit our revised manuscript entitled “Genome-Wide Identification, Evolution, and Expression Analysis of the TCP Gene Family in Rose (Rosa chinensis Jacq.)” for publication in Horticulturae. The manuscript was previously sent for appreciation under the ID: horticulturae-1892485.

The manuscript underwent considerable revision following the valuable suggestions provided by the reviewers and the editorial office (please see the Track-Change version). The criticisms and questions raised by the reviewers were addressed point-by-point as follows.

    In addition, we have added Yufei Chang as co-authors (Yi Hou^#, Chunguo Fan^#, Jingrui Sun^#, Yufei Chang, Jun Lu, Jingjing Sun, Changquan Wang and Jinyi Liu*) due to her significant contribution in revision period. All authors agree and confirmed it. We also changed the Acknowledgements for funding information. Sorry for your inconvenience.

 

Sincerely yours

 

Jinyi Liu

Dr./Associate Professor

College of Horticulture

Nanjing Agricultural University, P.R. China, 210095

No. 1 of Weigang, Nanjing City, Jiangsu Province, P.R., China,

Email: [email protected]

Mobile phone: +86-13851796756

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Title: Genome-Wide Identification, Characterization, and Expression Analysis of the TCP Gene Family in Rose (Rosa chinensis Jacq.)

 

In this work the authors identify the TPC plant-specific transcription factor gene family in Rosa chinensis. The topic is interesting, and several studies was performed for identify these genes. The authors also suggest the role of TPC genes in biological processes against drought stress. However, the manuscript must be improved in the materials and methods and results sections. Several experiments in the work are hardly comprehensible because the flow of the work in the materials and methods section is not clear and several information are missing. It is also not clear what type of material was used for the investigation. There is often no correspondence between the two sections. There are many errors that make it difficult to understand the methods used and invalidate the results shown.

Often there are phrases in the results that should be included in the other sections, materials and methods or discussion. Some figures are not adequately cited and described. In the text the Supplementary Materials is never mentioned in the text

Dear editor:

This is the modified version of the paper titled “Genome-Wide Identification, Characterization, and Expression Analysis of the TCP Gene Family in Rose (Rosa chinensis Jacq.)”. We wish to thank you for the time and efforts you have spent reviewing our paper. Following your comments, we have tried to fix all the problems you mentioned. What we changed was highlighted in yellow in the text, and the following are point-to-point responses to editor and reviewers.

• See below for details:
• The manuscript does not even have line numbering. This makes revision difficult

 

 

In the manuscript there are errors relating to the correct indication of the names of the species: I remember that excluding the abstract, the names must be complete (e.g. Rosa chinensis) only the first time, then the following times only the initial of the genus must be written followed by specie name (e.g. R.chinensis). Names must always be italicized. Correct throughout the manuscript.

• Abstract: ‘TCP gene family’ add the TPC long name
• TCP gene family have been performed for many species, such as Arabidopsis.
• What does arabidopsis mean? Either put the species name or put Arabidopsis spp ..
• Arabidopsis alone is a genus.

 

-Thank you for your suggestions. We have corrected the specie name throughout the manuscript.

-The TCP long name is TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR LAND 2. We have added the long name of it in manuscript.

-Here, the “arabidopsis” means “Arabidopsis thaliana”. We have corrected according to your comment.

 

• ‘The whole genome of Rosa chinensis has been sequenced.’

Add reference

 

-Thanks for your reminder, we have replaced the sentence with “Along with the release of R. chinensis cv. ‘Old blush ’ genome, one of the important progenitors of modern roses, roses have been proposed to be an ideal model species for studying the molecular basis of woody plants”, and added reference of it. 

 

• ‘All rose genome sequence and the gff3 files were obtained from GDR (Genome Da[1]tabase for Rosaceae, https://www.rosaceae.org). And the arabidopsis TCP sequences were obtained from phytozome（https://phytozome-next.jgi.doe.gov/）.

What does 'rose' mean? Correctly indicate which species you selected genes from. In all materials and methods make it clear which species you used for each test you did. There is no clarity on this.

Specify also what it refers to gff3 files

-Thanks for the comments,the 'rosa' in the manuscript represents the Rosa Chinensis, and we made a mistake in writing, which has now been corrected.

 In addition, the genomic data of roses come from GDR, and the gff3 file is annotation file of the genome, which are included in the genome file and downloaded from the GDR website. Therefore, the gff3 file does not need to be specified separately, so we removed the related representation.

• Change ‘(Genome Da[1]tabase for Rosaceae,’: with (Genome Da[1]tabase for Rosaceae family,

‘based on the arabidopsis TCP sequences..’

You can't write such a sentence in an article. Correctly indicate the species or species you have used

 

-Thanks for the comments, the long name of GDR is ‘Genome Database for Rosaceae’,

We have corrected ‘based on the arabidopsis TCP sequences’ to ‘with the sequences of AtTCP proteins as queries’

• ‘A phylogenetic tree was constructed using all protein sequences of TCP domain us[1]ing 0in rose and arabidopsis.’

Specify better (see previous suggestions)  in addition write MEGA11.0 software

 

-Thanks for the comments. We have specified the species in manuscript, which are R. chinensis and A. thaliana. We have also added the web address of the MEGA11.0 to the manuscript

• ‘Dentification of TCP structural domains in Arabidopsis and Luna using NCBI's CD-Search’

Luna? Specify. Then specify also NCBI GenBank the first time.

 

-Thanks for the comments, As mentioned in the manuscript, we used MEME suite to identify motifs in TCP protein, not NCBI, so we deleted this sentence.

• Malus domestica genome sequence and gff3 files we

Spec-ify what it refers to gff3 files

 

-Thanks for the comments. As mentioned above, the gff3 file is annotation file of the genome, which are included in the genome file and downloaded from the GDR website. Therefore, the gff3 file does not need to be specified separately, so we removed the related representation.

 

• Change: ‘Malus domestica genome sequence and gff3 files were obtained from GDR and Oryza sativa genome sequence and gff3 files were obtained from Ensembl Genomes  https://plants.ensembl.org/Oryza_sativa/Info/Index) to investigate the syntenic relationship among the TCP genes of rose and other related species. MCScanX was used to detect gene synteny and collinearity with the default parameters.
• With: The syntenic and collinearity relationship among the TCP genes of rose and other related species was investigated using MCScanXwith the default parameters. Malus domestica genome sequence and gff3 files were obtained from GDR and Oryza sativa genome sequence and gff3 files were obtained from Ensembl Genomes  https://plants.ensembl.org/Oryza_sativa/Info/Index).

 

Thanks for your suggestions. We have revised both of the above passages in the original text to more clearly state where our data came from and how we used them.

 

 

 

• Not clear: The transcript levels of the various RcTCPs were analyzed using the following tran[1]scriptome data sets retrieved: ROSAseq web interface database[13] (http://iant.tou[1]louse.inra.fr/R.chinensis), Rosa chinensis Raw sequence reads (accession number PRJNA663119)，Phosphorylation of RhPIP2;1, an aquaporin from rose (accession number PRJNA486271)，Rosa chinensis cultivar Old Blush (accession number PRJNA351281)[14], Rosa chinensis Transcriptome or gene expression (accession number PRJNA398090, PRJNA325324)[15, 16], Transcriptome of the floral transition in Rosa chinensis ‘Old Blush’

(accession number PRJCA000258)[17]. The expression levels were illustrated based on the

log2 transformed FPKM values visualized by TBtools

Excluded for the FPMK value of FMK, it is not clear how you used these data.

 

-Thanks for your suggestions. We found a clutter in this passage, which has now been corrected. In addition, we downloaded the published transcriptome data from public database platforms, processed them with Kallisto to obtain their FPKM values, and finally visualized them with tbtools.We have added the above to the manuscript

 

 

-  PlantCARE

.(add link)

 

Thanks for your suggestions. We have added the link here.

 

• ‘Plant Material and Treatments’

Title not clear add which type of treatment tested in the title

-RNA Isolation and qRT-PCR Analysis

Add qRT-PCR complete name the first time

-Thanks for the comments. Both of your proposed issues have been corrected in the manuscript. We have added ‘PEG’ before ‘Treatment’. The complete name of qRT-PCR is Quantitative Real-time PCR.

 

• ‘Total RNA was extracted using the BioTeke Quick RNA isolation Kit (Cat. #: RP3301, BioTeke Corporation, Beijing, China) and 1 μg of high quality total RNA was reverse transcribed using the PrimeScriptTM RT reagent Kit (Cat. #: RR047A, TaKaRa, Dalian, China) according to the manufacturer’s instructions. For qRT-PCR, RcGADPH was used as the reference gene, as described previously[18]. Three biological replicates and three technical replicates were used for each experiment. The relative expression levels of each gene were calculated in accordance with the 2- ∆∆CT method.’

There are few indications on the protocol (e.g. PCR reaction, the primers you used and how you selected them).

If some data are in the supplemental material, you must indicate them in the text

Add references for DDCt.

Thanks for the comments. All missing data have been supplemented in the manuscript.

 

• ‘while no possible TCP genes were found on chromosomes 3 and 6.’

Modify with

while no putative TCP genes were found on chromosomes 3 and 6.

 

Thanks for your suggestions. We have revised the manuscript as you said.

 

• No tandem replication events were found by retrieval. These results suggest that fragment replication plays an important role in the amplification of rose TCP gene family.
• Include in the Discussion section

 

Thanks for the comments. We have removed this sentence from the Results section.

 

• A phylogenetic tree was constructed for 130 protein sequences of rose, arabidopsis,

plum, apple, and Fragaria vesca by MEGA11.0 in order to explore the evolutionary and

phylogenetic relationship between the TCP protein of roses and other known TCP pro[1]teins.

 

You did not write this in the Materials and Methods section.

 

Thanks for your suggestions. In Materials and Methods, we have completed this part.

 

-  The TCP gene family can be divided into two subfamilies, class I, TCP-P or PCF,

and class II, TCP-C. In addition, Class II is subdivided into two subclasses: the CyC/TB1

subclass and the CIN subclass. Each plant has all subfamilies with a different number of

TCP genes, as shown in the figure.

-  Which figure? Figure3A? Figure 3B? Indicates correctly.

 

Thanks for the comments. In the new paragraph, we have clearly indicated the corresponding pictures.The phylogenetic tree of TCP genes from R. chinensis and A. thaliana were represented for classification of R. chinensis (Figure 2A). Interestingly, for each subclass, the copy number of TCP genes varied significantly (Figure 2B).

 

-  Each plant has all subfamilies

It is better to write species not plants

 

Thanks for the comments. We have showed all the species and subfamilies in Figure 2B.

 

-  Perhaps this is due to the differentiation of TCP family genes

before the separation of their ancestral species.

I suggest removing this sentence or phrasing it better.

 

Thanks for the comments. We have removed this sentence.

 

• method（Figure 3）

It’s Figure 3A? correct

 

Thanks for the comments. We have changed Figure 3 to Figure 2A.

 

All arabidopsis TCP belong to the same branch as previously reported,

which confirms the reliability of the phylogenetic tree.

???

Inappropriate comment, you must know before using them to make the phylogenetic tree the correctness of the sequences you have selected! Delete

 

Thanks for the comments. We have removed this sentence.

 

• ‘In rose, the TCP[1]C class has the largest number of members with nine, accounting for about 56% of the total number of RcTCP proteins, while the TCP-P class has seven members, accounting for about 44%. In class II, CyC/TB1 subclass is the smallest, with only 4 members; The CIN subclass has 5 members, accounting for about 31% of the total. Based on these findings, TCP genes might have evolved species-specifically’
• Do you show it in the picture? Which?

 

Thanks for the comments. We have a new description of the results of the phylogenetic tree in the manuscript.

 

• Figure 3. The phylogenetic tree of RcTCPs. (A) The phylogenetic tree of TCP gene family genes fromrose and arabidopsis. (B)Number of each subclass of TCP gene in each plant.This legend it’s not clear. What do you mean rose and arabidopsis? Some tips: in figure 3A there are stars and triangles, indicate what they refer to. You write about roses and Arabidopsis. Then the panel B in the figure shown that you have analyzed species belonging to the Rosaceae and Arabidopsis thaliana. I suggest therefore to write species (instead of plants) belonging to the Rosaceae family and Arabidopsis thaliana.

 

Thanks for the comments. We added what the triangle and star refer to in the text, and modified plant to species.

 

• ‘Conservative Motifs,’
• Which motifs? You reported only 1,2,3,

 

Thanks for the comments. These motifs have been added to the graph by us, see Figure 3 for details. All six motifs are described in the legends of figure 3.

 

• Figure 4
• Adjust the legend. A?

Thanks for the comments. Figure 4 has been adjusted.

 

- ‘To better understand the function of RcTCP gene in rose, transcript levels of RcTCP gene in different tissues were downloaded and analyzed, and heatmap was visualized the expression pattern of RcTCP genes. As shown in Figure 7, highly expressed genes in different tissues mainly exist in PCF and CIN subclass. Some RcTCP genes have similar ex[1]pression profiles in different tissues….

 

You write about gene expression in different treatments, but all this is not written in the corresponding paragraph on materials and methods section.

 

Thanks for the comments. Different processing methods we have modified in the figure, adding them to Figure 6.

 

• ‘Figure 7. The gene expression pattern of rose TCP genes in different tissues and developmental stages. (A) Phylogenetic tree of 23 arabidopsis TCP and 16 rose TCP genes;’

Where arabidopsis?

 

Thanks for the comments. We have corrected this error in the article and removed ‘arabidopsis’

 

• ‘To investigate the expression pattern of TCP genes in the growth and developmentof rose organs, transcriptome data from the database were used to analyze the expression profile of TCP genes during the differentiation of rose organs to full opening.’
• Thanks for the comments.This part is shown in Figure 6D and has been annotated in the manuscript.

Round 2

Reviewer 2 Report

The authors have  integrate in the manoscript the corrections previous suggested.

Author Response

Dear Reviewer,

We would like to submit our revised manuscript entitled “Genome-Wide Identification, Evolution, and Expression Analysis of the TCP Gene Family in Rose (Rosa chinensis Jacq.)” for publication in Horticulturae. The manuscript was previously sent for appreciation under the ID: horticulturae-1892485.

The manuscript underwent revision following the valuable suggestions provided by the reviewers and the editorial office (please see the Track-Change version). The criticisms and questions raised by the reviewers were addressed point-by-point as follows.

  -We have corrected some spelling and grammar mistakes in the manuscript.

  -We have deleted the sentence in 2.3 - "the tertiary structure of TCP protein in R. chinensis was predicted and visualized using the Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2)." Because the relevant results are not mentioned in Part 3.

  -We have changed the format of the references and checked that all references were relevant to the manuscript.

  -We have changed all qRT-PCR in the manuscript to RT-qPTR.  

Sincerely yours

Author Response File: Author Response.docx