Identification and Expression Profile Analysis of WOX Family Genes in the Formation of Eucalyptus Adventitious Root

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

I would like to thank you for giving such opportunity for reviewing the MS. The MS covered majority of the in silico analysis, and their characterization through gene expression analysis. Still, it needs more improvisation in terms of presentation.

 

1.      The title of the MS was not clear, and the abstract also need to re write

2.      The introduction needs to reorganize and clearly explain the hypothesis and importance of WOX family

3.      Authors needs to provide the information of shoot induction and root induction medium

4.      Authors can predict WOX SSRs which strengthen the MS.

5.      It is interesting if authors perform the in-silico promoter analysis and discuss their functional roles.

6.      Authors need to perform conserved motif and gene structure (exon-intron) prediction. It attracts more audience if authors can discuss the conserved domains with motif results (perform MEME), calculate evolutionary relationship ratios (ka/ks), and compare the exon-introns with evolutionary existence of genes in different crops.

7.      It attracts more audience if authors draw the molecular mechanism of Wox family based on their findings which provide better conclusion.

 

 

 

Author Response

Dear Reviewer1,

 

On behalf of all the contributing authors, I would like to express our sincere appreciations of the reviewer’s constructive comments concerning our article (Manuscript No.: forests-2821523). These comments are all valuable and helpful for improving our article. According to the reviewers’ comments, we have made extensive modifications to our manuscript and supplemented extra data to make our results convincing. In this revised version, changes to our manuscript within the document were all highlighted by using revisions mode. Point-by-point responses to the reviewers are listed below this letter.

 

Note: original text of email received from the Journal is shown below in bold, black text of Arial font.

Our action and response to each question/suggestion is shown in blue text of Arial font.

 

 

1. The title of the MS was not clear, and the abstract also need to re write.

Response: We all think this is an excellent suggestion. The title of this manuscript has been changed to “Identification and expression profile analysis of WOX family genes in the formation of eucalyptus adventitious root”.

Meanwhile, the abstract has been revised.

 

2. The introduction needs to reorganize and clearly explain the hypothesis and importance of WOX family.

Response: We are very sorry for our negligence of the hypothesis and importance of WOX family, we have been revised this part (Line 61-77).

3. Authors needs to provide the information of shoot induction and root induction medium.

Response: Thank the reviewer for the insightful advice. We are in agreement with the Reviewer’ suggestion, and the information of shoot induction and root induction medium has been added in the manuscript.

4. Authors can predict WOX SSRs which strengthen the MS.

Response: Thank you for your nice comments on our article. It is very unfortunate that we did not screen out effective SSRs in the WOX genes.

5. It is interesting if authors perform the in-silico promoter analysis and discuss their functional roles.

Response: We fully agree with your opinion. We attempted to perform the in-silico promoter analysis on E. grandis and C. citridora, and obtained some results. However, due to the lack of relevant information on E. urophylla × E. grandis and E. pellita, we were unable to analyze these two species. Therefore, after discussion, we decided unanimously not to add this part of the analysis.

6. Authors need to perform conserved motif and gene structure (exon-intron) prediction. It attracts more audience if authors can discuss the conserved domains with motif results (perform MEME), calculate evolutionary relationship ratios (ka/ks), and compare the exon-introns with evolutionary existence of genes in different crops.

Response: Considering the Reviewer’s suggestion, we have been identifed the WOX protein motifs of Eucalyptus using the online MEME program.

As for the evolutionary relationship ratios (ka/ks), both E. urophylla × E. pellita and E. pellita only have the transcriptome data, so the ratio cannot be calculated.

7. It attracts more audience if authors draw the molecular mechanism of Wox family based on their findings which provide better conclusion.

Response: Thank you for your nice comments on our article. According to your suggestions, we have added some discussion on the molecular mechanism of the WOX family on the basis of the research results.

Thank you again for your positive comments and valuable suggestions to improve the quality of our manuscript. If there are any other modifications we could make, we would like very much to modify them and we really appreciate your help. We hope that our manuscript could be considered for publication in your journal. Thank you very much for your help.

Finally, as the Chinese New Year is approaching, I wish you a happy New Year, a successful work and a happy family.

 

All authors of the manuscript (forests-2821523)

Reviewer 2 Report

Comments and Suggestions for Authors

The presented manuscript is devoted to the study of WUSCHEL-related homeobox (WOX) gene family in Eucalyptus: bioinformatic and molecular-genetic analyses (peculiarities of spatiotemporal WOX-genes expression) were carried out. Despite relevance of the topic and some interesting results obtained by the authors there are drawbacks and inaccuracies.

Introduction

 Reference 6 (Li, R.; Ge, H.; Dai, Y.; Yuan, L.; Liu, X.; Sun, Q.; Wang, X. Genomewide analysis of homeobox gene family in apple (Malus domestica Borkh.) and their response to abiotic stress. J. Genet. 2019 ,98,13.) is not appropriate and could be exchanged with the more recent publication - Zhang, ZA., Liu, MY., Ren, SN. et al. Identification of WUSCHEL-related homeobox gene and truncated small peptides in transformation efficiency improvement in Eucalyptus. BMC Plant Biol 23, 604 (2023). https://doi.org/10.1186/s12870-023-04617-w

 Materials and Methods

The section 2.2. is missing

Line 104: "the protein and amino acid sequences of the WOX family of  E. pellita and E. urophylla × E. grandis were obtained from unpublished data". Why the protein sequences were not submitted?

How many protein sequences of the WOX family were used in the study? In the Table S2 - 44 sequences, Table S3 and line 188 (physicochemical properties) - 31sequences, Line 214 (phylogenetic analysis) - 62 sequences.

Line 150: In “2.5 RNA extraction and cDNA synthesis” : the amount of RNA should be defined: how many mg or µg were taken for cDNA synthesis. Not the volume (7 μL).

Line 155: RT-qPCR should be instead of qRT-PCR

Line 158: The reference for checking the primer specificity given by the authors is http://plants.ensembl.org/hordeum_vul-158 gare/info/index and it is referred to the Hordeum vulgare (barley). Why the authors used this plant instead of Eucalyptus grandis (the genome sequence is presented in the Ensembl Plants mentioned by the authors).

The another option that could be used for analyzing primer specificity is Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome)

Line 161: 2x TransStart Top Green qPCR SuperMix should be instead of TransStart Top Green qPCR SuperMix

Lines 162-163: the concentration/amount of Forward and Reverse primers should be written instead of volume

Line 165: reference of 2^-ΔΔCt method should be provided (Livak, Kenneth J., and Thomas D. Schmittgen. "Analysis of relative gene expression data using real-time quantitative PCR and the 2^{− ΔΔCT} method." methods 25.4 (2001): 402-408.)

Results

Line 202: “the 13 conserved sites previously reported.” Provide reference.

Line 208: Figure 1 (written twice)

Lines 214-215: “Three distinct groups were identified among the 62 WOX protein sequences, which corresponded to the modern, intermediate, and ancestral evolution of plant WOX proteins.” How the authors differentiated groups (modern, intermediate, and ancestral), according to what principle?

Lines 234, 235: the terms “more conserved”, “more homologous” should be specified (for example, define %)

Line 239: instead of “higher degree of homology” should be used “similarity”.

Lines 243-244: the statement “Based on this finding, we hypothesized that the amino acid sequences of WOX proteins were more conserved between Eucalyptus hybrids and their parent species” could be not the hypothesis: it is predictable and expected that genomes and proteins of species belonging to the same genus are more similar than between different genus (such as Eucalyptus and Arabidopsis, for example).

The data of expression patterns of the EupWOX genes (sections 3.4. and 3.5) are hard to understand. It is not clear what the numbers in Tables S5 and S6 (supplementary): Ct or relative fold (2^-ΔΔCt). And what was the control sample (2^-ΔΔCt = 1)?

Lines 265-267: the statement "This might be because EupWOX11 plays an active role in the rooting of the line 30–66 to some extent" should be confirmed and explained.

Line 284: 16 - is not appropriate reference.

Lines 294-295: "the rooting results were consistent with the expression of the WOX genes." Should be explained (at least one example) and confirmed.

 Since data on genes expression analysis and their representation in Figures are not understandable completely and clear presented, the authors' selection of WOX1, WOX5, WOX11 and WOX13 genes as the markers and conclusion about the role of  these genes in adventitious roots development and growth are doubtful and сontroversial. Data on genes expression should be presented in the appropriate and correct way, re-analysed and edited to explain authors' conclusions in more details and conformation.

The authors obtained a big massive of data but their analysis and conclusions should be re-done.

Author Response

Dear Reviewer2,

 

On behalf of all the contributing authors, I would like to express our sincere appreciations of the reviewer’s constructive comments concerning our article (Manuscript No.: forests-2821523). These comments are all valuable and helpful for improving our article. According to the reviewers’ comments, we have made extensive modifications to our manuscript and supplemented extra data to make our results convincing. In this revised version, changes to our manuscript within the document were all highlighted by using revisions mode. Point-by-point responses to the reviewers are listed below this letter.

 

Note: original text of email received from the Journal is shown below in bold, black text of Arial font.

Our action and response to each question/suggestion is shown in blue text of Arial font.

 

 

Materials and Methods

1. The section 2.2. is missing

Response: We were really sorry for your careless mistakes. We have corrected it. Thank you for your reminding.

2. Line 104: "the protein and amino acid sequences of the WOX family of E. pellitaand E. urophylla× E. grandis were obtained from unpublished data". Why the protein sequences were not submitted?

Response: The WOX family of E. pellita and E. urophylla × E. grandis were obtained from the genome of E. pellita and E. urophylla × E. grandis, respectively. These two genomes were provided by our research team, but they have not been published or submitted to the public database yet.

3. How many protein sequences of the WOX family were used in the study? In the Table S2 - 44 sequences, Table S3 and line 188 (physicochemical properties) - 31sequences, Line 214 (phylogenetic analysis) - 62 sequences.

Response: Thanks for your careful checks. The protein sequence in table S2 is the sequence for constructing the phylogenetic tree, including E. grandis, Corymbia citriodora, Populus tomentosa, P. trichocarpa and Arabidopsis thaliana. The protein sequences of E. pellita and E. urophylla × E. grandis come from unpublished data. Therefore, 62 protein sequences were used in the phylogenetic tree with Line 214 of conserved domain sequences. The protein sequence in Table S3 is the physical and chemical characteristics of the members of the WOX gene family in eucalyptus, including E. pellita, E. grandis, C. citriodora, E. urophylla ×E. grandis.

4. Line 150: In “2.5 RNA extraction and cDNA synthesis”: the amount of RNA should be defined: how many mg or µg were taken for cDNA synthesis. Not the volume (7 μL).

Response: According to your suggestion, we have corrected these mistakes based on your suggestions. The sentence has been changed to “Reverse transcription was performed using 7 µL of total RNA (1µL of total RNA:100mg) and the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Tokyo).”

5. Line 155: RT-qPCR should be instead of qRT-PCR

Response: Thanks for your careful checks. We have replaced RT-qPCR with qRT-PCR.

6. 6. Line 158: The reference for checking the primer specificity given by the authors is http://plants.ensembl.org/hordeum_vul-158 gare/info/index and it is referred to the Hordeum vulgare (barley). Why the authors used this plant instead of Eucalyptus grandis (the genome sequence is presented in the Ensembl Plants mentioned by the authors). The another option that could be used for analyzing primer specificity is Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome)

Response: We are really sorry for our careless mistakes. EnsemblPlants is a websit could be used for analyzing primer specificity.

7. Line 161: 2x TransStart Top Green qPCR SuperMix with TransStart Top Green qPCR SuperMix.

Response: Thank you for your reminding. We have corrected these contents to “2x TransStart Top Green qPCR SuperMix”.

8. Lines 162-163: the concentration/amount of Forward and Reverse primers should be written instead of volume.

Response: Thanks. We have corrected these mistakes based on your suggestions.

9. Line 165: reference of 2^-ΔΔCtmethod should be provided (Livak, Kenneth J., and Thomas D. Schmittgen). "Analysis of relative gene expression data using real-time quantitative PCR and the 2^{− ΔΔCT}method." methods 25.4 (2001): 402-408.)

Response: Thank you again for your positive comments and valuable suggestions. The reference has been added.

 

Results

1. Line 202: “the 13 conserved sites previously reported.” Provide reference.

Response: According to your nice suggestions, we have been added the reference into the manuscript.

2. Line 208: Figure 1 (written twice)

Response: We are sorry for our carelessness. We have been revised it.

3. Lines 214-215: “Three distinct groups were identified among the 62 WOX protein sequences, which corresponded to the modern, intermediate, and ancestral evolution of plant WOX proteins.” How the authors differentiated groups (modern, intermediate, and ancestral), according to what principle?

Response: Thanks for your suggestions. The three clades (ancient, intermediate, and modern) was based on previous reports in Arabidopsis, Eucalyptus and other species (Nardmann & Werr, 2013; Lin et al., 2013; Feng et al., 2021; Zhang et al., 2023).

4. Lines 234, 235: the terms “more conserved”, “more homologous” should be specified (for example, define %)

Response: It is really a giant mistake to the whole quality of our article. We feel sorry for our carelessness. We have deleted them and we also feel great thanks for your point out.

5. Line 239: instead of “higher degree of homology” should be used “similarity”.

Response: According to your suggestion, we have corrected the “higher degree of homology” into “similarity”.

6. Lines 243-244: the statement “Based on this finding, we hypothesized that the amino acid sequences of WOX proteins were more conserved between Eucalyptushybrids and their parent species” could be not the hypothesis: it is predictable and expected that genomes and proteins of species belonging to the same genus are more similar than between different genus (such as Eucalyptus and Arabidopsis, for example).

Response: Thanks for your suggestions. We have corrected it to “Based on this finding, we predicted that genomes and proteins of species belonging to the same genus between Eucalyptus hybrids and their parent species are more similar than between different genus the amino acid sequences of WOX proteins and were more conserved.”

7. The data of expression patterns of the EupWOX genes (sections 3.4. and 3.5) are hard to understand. It is not clear what the numbers in Tables S5 and S6 (supplementary): Ct or relative fold (2^-ΔΔCt). And what was the control sample (2^-ΔΔCt= 1)?

Response: Thanks for your suggestions. The numbers in Tables S5 and S6 (supplementary) are the RT-qPCR values, and presented the results significance analysis. The control sample (2^-ΔΔCt = 1) is “0d” and added their values into the tables.

8. Lines 265-267: the statement "This might be because EupWOX11 plays an active role in the rooting of the line 30–66 to some extent" should be confirmed and explained.

Response: According to the reviewer’s comments, we have added some explanations in the corresponding positions. (Line 305-310)

9. Line 284: 16 - is not appropriate reference.

Response: Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have got the reference 15 in the right place. (Line 287)

10. Lines 294-295: "the rooting results were consistent with the expression of the WOX genes." Should be explained (at least one example) and confirmed.

Response: Thanks for your nice suggestions. We have changed the sentence to “Combined with the rooting after 24 d (Table 1), the expression of four EupWOX (EupWOX1, 5, 11 and 13) were significantly different among lines, and significant differences were observed in the rooting rate, the average number of roots, and the average root length of each asexual line.”

11. Since data on genes expression analysis and their representation in Figures are not understandable completely and clear presented, the authors' selection of WOX1, WOX5, WOX11 and WOX13 genes as the markers and conclusion about the role of  these genes in adventitious roots development and growth are doubtful and сontroversial. Data on genes expression should be presented in the appropriate and correct way, re-analysed and edited to explain authors' conclusions in more details and conformation.

The authors obtained a big massive of data but their analysis and conclusions should be re-done.

Response: Thank you for your nice comments on our article. According to your suggestions, we have re-analysed the gene expression data, and presented them in another appropriate way. Please see the details in Line 263-389.

 

Thank you again for your positive comments and valuable suggestions to improve the quality of our manuscript. If there are any other modifications we could make, we would like very much to modify them and we really appreciate your help. We hope that our manuscript could be considered for publication in your journal. Thank you very much for your help.

Finally, as the Chinese New Year is approaching, I wish you a happy New Year, a successful work and a happy family.

 

All authors of the manuscript (forests-2821523)

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made significant corrections and edited their manuscript. But there are still some corrections should be done.

 According to the Instructions for Authors (https://www.mdpi.com/journal/forests/instructions):

Accession numbers of RNA, DNA and protein sequences used in the manuscript should be provided in the Materials and Methods section.

Lines 106-107: "the protein and amino acid sequences of the WOX family of E. pellita and E. urophylla × E. grandis were obtained from unpublished data."

Thus, as it was written in the first review, the authors should provide the Accession numbers. Especially as the authors mentioned "These two genomes were provided by our research team, but they have not been published or submitted to the public database yet." It won't take much time to submit them and get the accession numbers.

Line 155: "Reverse transcription was performed using 7 µL of total RNA (1µL of total RNA:100mg)".   It can not be 700 mg for cDNA synthesis, probably, it is µg, not mg.

Line 164: it still contains the reference to barley database: (http://plants.ensembl.org/hordeum_vulgare/info/index).

So, again: Why the authors used this plant instead of Eucalyptus grandis (the genome sequence is presented in the Ensembl Plants mentioned by the authors).

Lines: 229-230 (repeating question) “Three distinct groups were identified among the 62 WOX protein sequences, which corresponded to the modern, intermediate, and ancestral evolution of plant WOX proteins.” How the authors differentiated groups (modern, intermediate, and ancestral), according to what principle?

Please, provide the information as "on the basis of domain sequences" or "phylogenetic tree topology" etc.

Lines 249-250: NO CORRECTIONS WERE MADE although authors replied:

Lines 234, 235: the terms “more conserved”, “more homologous” should be specified (for example, define %)

Response: It is really a giant mistake to the whole quality of our article. We feel sorry for our carelessness. We have deleted them and we also feel great thanks for your point out.

 Repeating questions:

The data of expression patterns of the EupWOX genes (sections 3.4. and 3.5) are hard to understand. It is not clear what the numbers in Tables S5 and S6 (supplementary): Ct or relative fold (2^-ΔΔCt). And what was the control sample (2^-ΔΔCt = 1)?

Table S5 is titled “The EupWOX gene expression in different tissues.” According to the authors’ answer:

The numbers in Tables S5 and S6 (supplementary) are the RT-qPCR values, and presented the results significance analysis. The control sample (2^-ΔΔCt = 1) is “0d” and added their values into the tables.

How the significance can be “2429.88”?

It is better to give the information about the Ct values or the ratio (2^-ΔΔCt)

 Figure 6: please, provide the description of the colour scale: what the numbers are meaning.

 

 

Author Response

Dear Reviewer2,

On behalf of all the contributing authors, I would like to express our sincere appreciations of the reviewer’s constructive comments concerning our article (Manuscript No.: forests-2821523). These comments are all valuable and helpful for improving our article. According to the reviewers’ comments, we have made extensive modifications to our manuscript and supplemented extra data to make our results convincing. In this revised version, changes to our manuscript within the document were all highlighted by using revisions mode. Point-by-point responses to the reviewers are listed below this letter.

 

Note: original text of email received from the Journal is shown below in bold, black text of Arial font.

Our action and response to each question/suggestion is shown in blue text of Arial font.

 

 

 According to the Instructions for Authors (https://www.mdpi.com/journal/forests/instructions):

1. Accession numbers of RNA, DNA and protein sequences used in the manuscript should be provided in the Materials and Methods section.

Lines 106-107: "the protein and amino acid sequences of the WOX family of E. pellita and E. urophylla × E. grandis were obtained from unpublished data."

Thus, as it was written in the first review, the authors should provide the Accession numbers. Especially as the authors mentioned "These two genomes were provided by our research team, but they have not been published or submitted to the public database yet." It won't take much time to submit them and get the accession numbers.

Response: We all think this is an excellent suggestion. Therefore, we are already processing and uploading the WOX sequences of the relevant E. pellita and E. urophylla × E. grandis to the NCBI database, but since it will take about a week to obtain the serial number, we will contact the journal in time to add the serial information to the article once we have obtained the serial number.

2. Line 155: "Reverse transcription was performed using 7 µL of total RNA (1µL of total RNA:100mg)".   It can not be 700 mg for cDNA synthesis, probably, it is µg, not mg.

Response: According to your suggestion, we have corrected these mistakes based on your suggestions. The sentence has been changed to “Reverse transcription was performed using 7 µL of total RNA (1µL of total RNA: 100 ng) and the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Tokyo).”

3. Line 164:it still contains the reference to barley database: (http://plants.ensembl.org/hordeum_vulgare/info/index).

So, again: Why the authors used this plant instead of Eucalyptus grandis (the genome sequence is presented in the Ensembl Plants mentioned by the authors).

Response: We are really sorry for our careless mistakes. The sentence has been changed to “The specificity of primers to their target genes was evaluated on the website Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi?LINK_LOC=BlastHome).

4. Lines: 229-230 (repeating question)“Three distinct groups were identified among the 62 WOX protein sequences, which corresponded to the modern, intermediate, and ancestral evolution of plant WOX proteins.” How the authors differentiated groups (modern, intermediate, and ancestral), according to what principle?

Please, provide the information as "on the basis of domain sequences" or "phylogenetic tree topology" etc.

Response: Thank you for your reminding. We have added “On the basis of domain sequences, and refer to the grouping of WOX family members of E.grandis.” to the section on building evolutionary trees.

5. Lines 249-250:NO CORRECTIONS WERE MADEalthough authors replied:

Lines 234, 235: the terms “more conserved”, “more homologous” should be specified (for example, define %)

Response: We feel sorry for our carelessness. We have added the alignment of several WOX protein sequences and wrote out the similarity between the sequences in article.

6. The data of expression patterns of the EupWOXgenes (sections 3.4. and 3.5) are hard to understand. It is not clear what the numbers in Tables S5 and S6 (supplementary): Ct or relative fold (2^-ΔΔCt). And what was the control sample (2^-ΔΔCt = 1)?

Response: The numbers in Table S5 is the calculated value of the relative expression of EupWOXs from different tissues with rooting 24d of lines 30-66 and 46-99(2^-ΔΔCt); Table S6 is the calculated value of the relative expression of EupWOXs in 10 lines with different rooting times (2^-ΔΔCt); 0d of rooting seedlings was used as the control sample (2^-ΔΔCt = 1).

7. How the significance can be “2429.88”? It is better to give the information about the Ct values or the ratio (2^-ΔΔCt).

Response: Thank you again for your positive comments and valuable suggestions. The “2429.88” is the calculated value of the relative expression.

8. Figure 6: please, provide the description of the colour scale: what the numbers are meaning.

Response: Thanks for your suggestions. The heatmap in Figure 6 was obtained after normalization of the columns, where the relative expression of each gene obtained at different times through the 10 asexual lines was normalized to obtain an array. Changes in expression trends of each gene at different times in different samples. We have added the appropriate additions to the text.

 

Thank you again for your positive comments and valuable suggestions to improve the quality of our manuscript. If there are any other modifications we could make, we would like very much to modify them and we really appreciate your help. We hope that our manuscript could be considered for publication in your journal. Thank you very much for your help.

 

All authors of the manuscript (forests-2821523)