Identification, Evolution and Expression Analysis of GRF Family Reveals Their Involvement in Shoot Growth and Abiotic Stress Response in Moso Bamboo
Round 1
Reviewer 1 Report
It is good to add the percentage of genome coverage of the identified GRFs
how large was the upstream start codon region for promoter analysis?
It is worth adding qRT-PCR to validate the expression analysis
no comment
Author Response
It is good to add the percentage of genome coverage of the identified GRFs
how large was the upstream start codon region for promoter analysis?
Answer: We used the 2000bp sequence upstream of the start codon region for promoter analysis.
It is worth adding qRT-PCR to validate the expression analysis
Answer：Thank you for your suggestion, we added qRT-PCR to validate the transcriptome data according to your suggestion. (Figure 8B and Figure 10B)
Reviewer 2 Report
The work entitled “Identifification, Evolution and expression of GRF Family in Moso Bamboo” identified 24 PheGRF, and the expression pattern, co-expression network were analized.
The results and methodology has been described nicely and according to my opinion the manuscript must be accepted after minor changes in language in introduction and discussion part.
And there are some mini problem should be revised. For example:
1. The figures (2, 3) are not labeled A-B.
2. Line 95: PGRFs should be PheGRFs.
3. The font of species names should be italics.
Minor editing of English language required
Author Response
The work entitled “Identifification, Evolution and expression of GRF Family in Moso Bamboo” identified 24 PheGRF, and the expression pattern, co-expression network were analized.
The results and methodology has been described nicely and according to my opinion the manuscript must be accepted after minor changes in language in introduction and discussion part.
And there are some mini problem should be revised. For example:
1. The figures (2, 3) are not labeled A-B.
Answer: Thank you for your reminder. We redrawn figure 2 and 3.
2. Line 95: PGRFs should be PheGRFs.
Answer: We revised it.
3. The font of species names should be italics.
Answer: The font of all Latin plant names has been modified to italics.
Reviewer 3 Report
The manuscript entitled “Identifification, Evolution and expression of GRF Family in Moso Bamboo” was reviewed.
The manuscript delivers novel finding related to GRF gene family in moso bamboo. However, few important issues are pending, including technical issues and weak discussion. Therefore, I do not recommend the publication of the manuscript in “Forests” unless major comments and corrections are applied (please see below).
1. General:
- The Language is good, however, please correct for common grammar mistakes and incorrect sentence phrasing.
- Typing errors: “Identifification”, “pitaya”, “enduced”, “comparinbg”, … etc.
2. Abstracts:
- Lines 11-15: useless introduction sentences. You have more valuable data to present instead
- Needs more data from your findings.
3. Results:
- Almost ALL figures are of low quality, you barely can read any text.
- Figure 9: you need to add a scale bar!
- Lines 236-247: you need to run qPCR to verify any RNAseq data!!!
4. Discussion:
- Relatively weak. You have good and novel results! But you barely discussed them!!!
5. Methods:
- Major technical data related to cloning, generated sequences, probe sequences, and primer sequences related to both “in situ hybridization” and “yeast two-hybrid assays” are MISSING. You need please to show all these data!
6. Conclusions:
- It is vague, please add more concrete conclusion NOT repeating your results!
7. References:
- Cited articles are too old, only 19 out of 50 (38%) are published in the last five years. You need to cite more recent articles and remove some of the old or one-time cited articles!
Author Response
The manuscript delivers novel finding related to GRF gene family in moso bamboo. However, few important issues are pending, including technical issues and weak discussion. Therefore, I do not recommend the publication of the manuscript in “Forests” unless major comments and corrections are applied (please see below).
- General:
- The Language is good, however, please correct for common grammar mistakes and incorrect sentence phrasing.
- Typing errors: “Identifification”, “pitaya”, “enduced”, “comparinbg”, … etc.
Answer: Thank you for your reminding, and we revised them.
- Abstracts:
- Lines 11-15: useless introduction sentences. You have more valuable data to present instead
- Needs more data from your findings.
Answer: We have abbreviated the introduction sentence and included more new findings in our revised manuscript (Line 12-35).
- Results:
- Almost ALL figures are of low quality, you barely can read any text.
Answer: Thank you for your suggestion. All images in our paper have a resolution greater than 300 dpi, and some even reach 600 dpi. The poor resolution in our upload manuscript may be caused by submission system and image compression feature in the Word program. In the revised version of the manuscript, we have cancelled this software feature. Additionally, all authors of this paper have prior experience publishing in SCI journals, and creating a high-resolution and journal suitable image is not a challenging task for us. Please trust us.
- Figure 9: you need to add a scale bar!
Answer: We added the scale bar
- Lines 236-247: you need to run qPCR to verify any RNAseq data!!!
Answer: We performed additional qRT-PCR experiments to validate the transcriptome data (Figure 8B and Figure 10B).
- Discussion:
- Relatively weak. You have good and novel results! But you barely discussed them!!!
- Methods:
- Major technical data related to cloning, generated sequences, probe sequences, and primer sequences related to both “in situ hybridization” and “yeast two-hybrid assays” are MISSING. You need please to show all these data!
Answer: We provided probe sequences, primer sequences as well as detailed experimental methods in our revised manuscript (Section: 4.6. In situ hybridization and 4.7. Yeast two-hybrid assays).
- Conclusions:
- It is vague, please add more concrete conclusion NOT repeating your results!
Answer: We have summarized the results of our research again, and the new conclusions are as follows: Here, 24 GRFs were identified in moso bamboo genome. During the evolution of PheGRF genes, whole-genome duplication events have played a significant role. Expression analysis has indicated that PheGRFs are responsive to cold and drought stress and are involved in the growth and development of bamboo shoots. Coexpression network analysis and the yeast two-hybrid system have unveiled a complex regulatory network in which PheGRFs participate. These findings will offer valuable insights for future investigations into the functional studies of PheGRFs. (5. conclusion)
- References:
- Cited articles are too old, only 19 out of 50 (38%) are published in the last five years. You need to cite more recent articles and remove some of the old or one-time cited articles!
Answer：Thank you for your suggestion, we rewrite our disccusion, and We have removed some outdated literature and added new research literature
Reviewer 4 Report
Comments:
In the present investigation entitled “Identification, Evolution and expression of GRF Family in Moso Bamboo”, authors have identified 24 GFR gene family members in moso bamboo and further these family members were characterized with all sort of bioinformatic analysis. Though, authors have tried to make this report technically sound. However, some comments and queries have been observed which need be rectified to improve the manuscript for global readability and acceptance. The comments observed during review of the manuscript are as follows: -
Comment #1 – Author should re-write the title of the manuscript with description. For example, they can write the suggestive title as “Identification, Evolution and Expression analysis of GRF Family reveals their involvement in shoot growth and abiotic stress response in Moso Bamboo”
Comment #2 – Authors must ensure about resolution of the figures embedded in the manuscript. All figures are not visible due to poor resolution and small font size of the labels. So, figures need to be improved at least 300 or more dpi scale.
Comment #3 – It is not clear about sequence type at line number 321. Whether it is genomic sequences or peptide sequencers, authors must clarify and same should be incorporated into the text.
Comment #4 – For identification of GRF family in Bamboo, which BAST module has been utilised (line no. 322), whether BLASTn or BLASTp? Authors should clarify.
Comment #5 –There is confusion about GIGAdb. GIGAdb is also belongs to a bacterial database (E. coli). So, authors should specify which data they have utilised to explore the sequences. Please quote the reference of database used (Line no. 328).
Comment #6 – Authors should quote the reference of Bayesian Information Criterion at line no. 331.
Comment #7 – Though, the methodology utilized in this study is quite interesting but it seems to be incomplete and not elaborated and described appropriately with quoting the references of each tools and software used. Authors should check the lacuna in methodology and it must be rectified.
Comment #8 – The methodology section 4.6 – “In situ Hybridization” need to be elaborated more at Line no. 354.
Comment #9 – Authors must describe the methods about extraction of seed sequences from Arabidopsis and Oryza.
Comment #10 - Authors have not constructed phylogenetic tree from 24 gene family members alone. It will help to decipher the actual grouping of the gene family members according to their structures and sequence homology.
Comment #11 - Authors must have mentioned the meristem expression related motifs at line no. 177.
Comment #12 – The gene expression analysis of these GRF genes in Bamboo is based on available RNA-seq data in public domain. So, for conclusive interpretation about the functional role of these gene family members in Bamboo, their wet lab validation must be performed that authors have not done in the present study. Why? Whether, it is possible to analyse 2-3 up- and 2-3 down-regulated genes using real time PCR analysis at this moment?
Comment #13 - Authors have claimed the GRF gene family members as involved in shoot growth in bamboo but they have not validated the genes during shoot growth development in bamboo using any wet lab expression.
Comment #14 - Authors must re-check the whole text of manuscript for any typological errors and spelling errors.
Authors must re-check the whole text of manuscript for any typological errors and spelling errors
Author Response
In the present investigation entitled “Identification, Evolution and expression of GRF Family in Moso Bamboo”, authors have identified 24 GFR gene family members in moso bamboo and further these family members were characterized with all sort of bioinformatic analysis. Though, authors have tried to make this report technically sound. However, some comments and queries have been observed which need be rectified to improve the manuscript for global readability and acceptance. The comments observed during review of the manuscript are as follows: -
Comment #1 – Author should re-write the title of the manuscript with description. For example, they can write the suggestive title as “Identification, Evolution and Expression analysis of GRF Family reveals their involvement in shoot growth and abiotic stress response in Moso Bamboo”
Answer: We revised the title.
Comment #2 – Authors must ensure about resolution of the figures embedded in the manuscript. All figures are not visible due to poor resolution and small font size of the labels. So, figures need to be improved at least 300 or more dpi scale.
Answer: Thank you for your suggestion, the poor resolution in our upload manuscript may be caused by submission system. All images in our paper have a resolution greater than 300 dpi, and some even reach 600 dpi. Additionally, all authors of this paper have prior experience publishing in SCI journals, and creating a high-resolution and journal suitable image is not a challenging task for us. Please trust us.
Comment #3 – It is not clear about sequence type at line number 321. Whether it is genomic sequences or peptide sequencers, authors must clarify and same should be incorporated into the text.
Answer: The sequence types are peptide sequences, and we have incorporated them into the text as per your suggestion.
Comment #4 – For identification of GRF family in Bamboo, which BAST module has been utilised (line no. 322), whether BLASTn or BLASTp? Authors should clarify.
Answer: We used BLASTp and have provided it in revised manuscript.
Comment #5 –There is confusion about GIGAdb. GIGAdb is also belongs to a bacterial database (E. coli). So, authors should specify which data they have utilised to explore the sequences. Please quote the reference of database used (Line no. 328).
Answer, thank you for your suggestion. GIGAdb contains some of the publicly available genomic data, including that of moso bamboo. In our revised article, we have referenced the relevant literature and website for the moso bamboo genome sequencing. We quote the reference of database used that you mentioned in Line 403.
Comment #6 – Authors should quote the reference of Bayesian Information Criterion at line no. 331.
Answer: We quote the reference (reference 61).
Comment #7 – Though, the methodology utilized in this study is quite interesting but it seems to be incomplete and not elaborated and described appropriately with quoting the references of each tools and software used. Authors should check the lacuna in methodology and it must be rectified.
Answerï¼›We quoted the references of each tools and software according to your suggestion.
Comment #8 – The methodology section 4.6 – “In situ Hybridization” need to be elaborated more at Line no. 354.
Answer: We have provided a more detailed description of the experimental steps for in situ hybridization according to your suggestion (Section: 4.6. In situ hybridization).
Comment #9 – Authors must describe the methods about extraction of seed sequences from Arabidopsis and Oryza.
Answer: The annotation of gene functions in the Phytozome v12.1 database for model plants is highly comprehensive. Therefore, all mentioned GRF members in model plants were searched using the keyword "growth-regulating factor" (4.1. Identification of GRF genes in moso bamboo reference genome).
Comment #10 - Authors have not constructed phylogenetic tree from 24 gene family members alone. It will help to decipher the actual grouping of the gene family members according to their structures and sequence homology.
Answer: The phylogenetic tree constructed based on the 24 moso bamboo GRF members demonstrated consistent grouping results, further indicating the reliability of our grouping method (line 100-102).
Comment #11 - Authors must have mentioned the meristem expression related motifs at line no. 177.
Answer: Thank you for your guidance, we provided the meristem expression related motifs in our revised version (line 189-195).
Comment #12 – The gene expression analysis of these GRF genes in Bamboo is based on available RNA-seq data in public domain. So, for conclusive interpretation about the functional role of these gene family members in Bamboo, their wet lab validation must be performed that authors have not done in the present study. Why? Whether, it is possible to analyse 2-3 up- and 2-3 down-regulated genes using real time PCR analysis at this moment?
Answer: Thank you for your suggestion. We conducted qRT-PCR validation on five randomly selected genes. The qRT-PCR results revealed an average Pearson correlation coefficient of 0.77 between the qRT-PCR results and the expression levels obtained from the transcriptomic data. This indicates that the qRT-PCR results generally reflect the transcriptomic data, although differences may be due to variations in the genetic background of the experimental seedlings.
Comment #13 - Authors have claimed the GRF gene family members as involved in shoot growth in bamboo but they have not validated the genes during shoot growth development in bamboo using any wet lab expression.
Answer: Thank you for your suggestion. We conducted qRT-PCR according to your suggestion (Figure 8B).
Comment #14 - Authors must re-check the whole text of manuscript for any typological errors and spelling errors.
Answer: Thank you for your reminding, and we re-check the whole text of manuscript.
Reviewer 5 Report
The authors present a manuscript providing further characterization of the GRF protein family in Moso bamboo. By employing the most recent genome annotation, the authors were able to successfully detect gene duplications and assess the relationships between paralog genes. In general, the results are well presented, but can be improved to better connect the ideas brought in each section.
The network presented in item 2.3, for example, is not mentioned in the discussion. Additionally, Supplementary Figures S1 and S2 are not mentioned in the manuscript, and their relevance to the presented results is questionable. Finally, the Conclusions section appears misplaced, since it is shown after the Methods section. I suggest it be placed right after the Discussion section. Please revise the structure of the manuscript and modify if agreed.
Please revise the presented Figures. Almost all of them are difficult to interpret due to small text or small scale. Consider changing the fonts or enlarging the figure for a better reading experience.
In the attached file you will find minor comments I hope will help improve the manuscript.
Comments for author File: 
Comments.docx
The English is fine overall, but editing by a more fluent speaker/writer would greatly improve the understanding of the manuscript.
Author Response
The authors present a manuscript providing further characterization of the GRF protein family in Moso bamboo. By employing the most recent genome annotation, the authors were able to successfully detect gene duplications and assess the relationships between paralog genes. In general, the results are well presented, but can be improved to better connect the ideas brought in each section.
The network presented in item 2.3, for example, is not mentioned in the discussion. Additionally, Supplementary Figures S1 and S2 are not mentioned in the manuscript, and their relevance to the presented results is questionable.
Answer: We cited Supplementary Figures S1 and S2 in revised manuscript. Besides, we discussed the interaction network in our revised Discussion.
the Conclusions section
Finally, the Conclusions section appears misplaced, since it is shown after the Methods section. I suggest it be placed right after the Discussion section. Please revise the structure of the manuscript and modify if agreed.
Answer: Our conclusion in the article is positioned after the methods section, in accordance with the formatting guidelines specified by the publishing journal.
Please revise the presented Figures. Almost all of them are difficult to interpret due to small text or small scale. Consider changing the fonts or enlarging the figure for a better reading experience.
Answer: Thank you for your suggestion. All images in our paper have a resolution greater than 300 dpi, and some even reach 600 dpi. The poor resolution in our upload manuscript may be caused by submission system and image compression feature in the Word program. In the revised version of the manuscript, we have cancelled this software feature.
In the attached file you will find minor comments I hope will help improve the manuscript. forests-2571790-peer-review-v1 Minor Comments
Page 1
Line 1: Please correct to Identification
Answer: We revised it.
Line 15: Please clarifiy if the identification of the GRFs was made in this work or in previous work.
Answer: The identification of the GRFs was made in this work. We added ‘In this study’ in our revised manuscript.
Line 18: Please clarify if the authors meant a single PheGRF or all PhGRFs?
Answer: we meant few PheGRFS, we revised it.
Line 21: Please indicate whether it is a single GRF or all GFRs.
Answer: we meant most PheGRFS, we revised it.
Line 22: I suggest changing this phrase to "A yeast two-hybrid screening".
Answer: We revised it.
Page 2
Line 39: Please specify the period to which this amount refers to and provide a reference to the data.
Answer: We provided the reference.
Line 40-42: I believe this phrase would be better placed as the start of the next paragraph.
Answer: We revised it.
Line 51: size
Answer: We revised it.
Line 64: The mutant name must be italicized.
Answer: We revised it.
Line 64: in comparison to
Answer: We revised it.
Line 63: Reduced or induced?
Answer: Induced, we confirmed it.
Line 74: I suggest changing the phrase “in that study” to "previously".
Answer: We changed it.
Line 74: I suggest changing “accomplish these” to "fulfill this"
Answer: We changed it.
Line 75: I suggest changing “second” to "the second" or "the most recent"
Answer: We changed it.
Page 4
Line 122: Please mention what the last sequence "Vun..." is in the text. I assume it was used as an outgroup, but please clarify this information in the text.
Answer: The most primitive Vigna unguiculata GRF was used as an outgroup. We provide this information according to your suggestion.
Page 5: Line 148: Please provide a new Figure with clear text. It is not possible to read the text in this figure.
Page 6: Line 167: Please provide a Figure with better quality. It is difficult to read the text and properly see the plots.
Answer: Thank you for your suggestion. All images in our paper have a resolution greater than 300 dpi, and some even reach 600 dpi. The poor resolution in our upload manuscript may be caused by submission system and image compression feature in the Word program. In the revised version of the manuscript, we have cancelled this software feature.
Page 7
Line 183-192: This section needs to be more detailed, to explain the network better. It is not clear which data is novel and which data is already published. Did the authors construct the network based on previous work only? Or the data generated in this manuscript was used together with previous data to build the network? Were any of the miRNAs validated in bamboo?
Answer: Thank you for your suggestion. The regulatory relationship between miRNA, lncRNA, and PheGRF in this study was predicted based on previously published research on degradome data and full-length transcriptome data. Following your advice, we have cited the data sources in the paper. However, unfortunately, the mentioned miRNAs in the paper were obtained through degradome data, and there is currently no study validating them through cloning. If you consider the predictions based on degradome data to be unreliable without experimental support, we will remove the regulatory relationship between miRNA and PheGRF in the revised version of the manuscript.
Page 8
Line 213: The identification of the growth media is inverted in this figure (SD -Trp -Leu and SD -Trp -Leu -His -Ade +X-a-gal are switched).
Answer: Thank you for your reminding, we revised this mistake.
a Line 214: What does DDO and QDO mean?
Answer: DDO means Double Dropout Supplements and QDO means Dropout Supplement. We provided full name according to your suggestion (line 239).
Line 215: Were auto-activation assays performed? To check if the PheGRF9b alone could activate transcription of the reporter genes? This test is very important in Y2H, and even more important when dealing with a transcriptional activator.
Answer: We supplemented the transcription of the self-activation experiment results in the article.
Page 10
Line 242: Please verify this information. There is no 1h timepoint in the provided Figure.
Answer: It’s 2 h, we revised it.
Line 242: Please check this information. I believe PheGRF8a was switched with PheGRF9a in this case.
Answer: Thank you for your reminding, and we revised it.
Line 236-243: Were any statistical method applied to verify the significance of changes in expression in the different treatments?
Answer: We used differential gene expression criteria (FDR value ≤0.05 and ≥1 fold change) from transcriptomic data to determine whether a gene was upregulated or downregulated. We provide this information in our revised manuscript (line 271).
Line 245: In this figure, concrete values could be given in the scale (upper left corner) instead of “row max” and “row min”.
Answer: Previously, we utilized a relative color scheme to represent the expression levels of PheGRFs. In new version, we have opted to use log2-transformed FPKM values as a more accurate measure of gene expression.
Page 11
Line 276: Suggestion: remove these last words, they are unnecessary.
Answer: We removed these words.
Page 12
Line 317: Should PheGRF be PheGRFs?
Answer: We revised it.
Line 321: If possible, please provide a list containing the accession numbers of the GRFs from each species.
Answer: We provide a list containing the accession numbers of all the GRFs in Table S2.
Line 324: Please clarify this method. The Swiss-Model website is a structural prediction tool. How were the domains checked in this tool? Please describe the steps of this analysis thoroughly for reproducibility.
Answer: Thank you for your reminding. In the previous version, we made an inaccurate description of the detection of conserved domains. We actually used another online software, PROSITE, provided by the Swiss Institute of Bioinformatics, for the detection of conserved domains. We revised our description (line 406).
Line 328: Please indicate the software version and properly cite the authors of ClustalW.
Answer: We added the reference.
Line 329: Please provide the version of the software and properly cite the authors of IQ-TREE.
Answer: We added the reference.
Line 332: Please cite the authors of FigTree.
Answer: We added the reference.
Line 334: Please clarify what the GFF is. Maybe the reader is not familiar with this type of file. One suggestion is: "A GFF file containing the genomic annotations of PheGRFs was uploaded to the gene...".
Answer: Thank you for your suggestion, we revised it.
Line 334: Please provide a citation for this tool (GSDS2).
Answer: We provide a citation.
Line 335: Please provide a software version and proper citation (MEME).
Answer: We provided the software version and citation in revised manuscript.
Line 340: Please clarify what this gff3 file refers to. Where do they come from and why are they used here? What information do they contain?
Answer: We have obtained the GFF3 file from the moso bamboo annotation databases, and we have provided this information and reference as per your suggestion. Additionally, the GFF3 file contains the positional information of all genes, which is necessary for mapping the collinear genes onto the chromosomes (line 430-432).
Line 341: Please indicate the version and citation of this software (MCScan).
Answer: We provided citation according to your suggestion. However, after consulting the software's documentation and description, we found that there is no version number specified for this software.
Page 13
Line 343: Please indicate the version and citation of this software (Circos).
Answer: We added the reference and version.
Line 348: These two words have the same meaning. Please revise (software/program).
Answer: We deleted ‘program’.
Line 347: If pertinent, please provide the software version and citation (DnaSP).
Answer: We added the reference and version.
Line 355-357: Please provide at least minimal descriptions of the assessed tissues, if the probe was specific, how long were the probes? Did they encompass the entire mRNA or just part of it?
Answer: The internode, obtained from the middle section of the winter bamboo shoot with a height of approximately 10 cm were used for In situ hybridization.We provided the probe sequences and detailed method in revised manuscript (line 448).
Line 359: Please clarify if a new library was constructed or if the authors used the same library from reference 27. Line 367: Why weren't interactions confirmed by switching AD an BD between interacting proteins, as was done by the authors in reference 27?
Answer: The methods we employed for the cDNA library construction and screening were consistent with those described in our previously published study. To be honest, in our research, we identified five genes associated with growth and development. However, we also discovered several other functional genes. Out of these five genes, two genes including PH02Gene11097.t1 (GIF3) and PH02Gene01921.t3(WD40) underwent pairwise testing by switching AD and BD between interacting proteins. Unfortunately, due to the first author having already obtained a master's degree and leaving the university, there is currently no suitable student available to continue the follow-up experiments in the near future. We plan to find an appropriate student later on to conduct further investigations on the functionality of these genes, including pairwise testing by switching AD and BD between putatitve interacting proteins for the remaining three genes. We kindly ask for your guidance on whether we should only include the results of the pairwise testing for the two interacting gene pairs or include the results for all five interacting gene pairs, including those that did not undergo this testing.
In addition, in the revised paper, we have provided a more detailed description of the vector construction and library screening methods (Section: 4.7. Yeast two-hybrid assays).
Line 374: Please indicate the version and citation of this tool (Morpheus).
Answer: We conducted a literature search and found no specific reference for the online heatmap generating tool Morpheus. Additionally, there is no documentation of a version number on the Morpheus website. In the revised manuscript, we have included the website URL for reference.
Page 14
Line 376-386: The same information is given twice. Please revise this section carefully.
Answer: We have removed the duplicated content.
Line 391: Please provide the software version and the citation (LncTar).
Answer: We added the reference and version.
Line 401: Please provide a citation to this algorithm (Cleaveland).
Answer: We added the reference and version.
Line 401: Please provide a URL or citation to this tool (oligomap).
Answer: We provided a URL according to your suggestion.
Line 404: Please provide a URL or citation to this tool (mirU).
Answer: We added the reference.
Line 410-411: Please provide more details about how the network was constructed. This section only mentions the method used for visualization. Was the network constructed in Cytoscape? Using a plugin or was it calculated by external methods? This information is important to ensure reproducibility of the method.
Answer: The expression levels of lncRNAs that regulate PheGRF were retrieved from transcriptomic data of different tissues, along with the expression levels of PheGRF. The Pearson correlation coefficient between the lncRNA expression levels and PheGRF expression levels was calculated using Excel. Only the absolute values of Pearson correlation coefficients greater than 0.3 were retained for constructing the co-expression network. Coefficients greater than 0.3 are considered as positive regulation, while those less than -0.3 are considered as negative regulation. Since the regulatory relationship between miRNA and PheGRF target genes is speculated based on degradation data, and based on the mechanism of miRNA action on target genes, the regulatory relationship between miRNA and PheGRF is considered as negative regulation. The obtained regulatory relationship between lncRNAs and PheGRF, as well as the regulatory relationship between miRNA and PheGRF, were inputted into Cytoscape 3.7.0 software in the form of an Excel table to generate a co-expression network graph. Positive regulatory relationships are represented by arrows, while negative regulatory relationships are represented by T-arrows. Adjust the size of the circles based on the number of gene connections.
We provided these information in our revised manuscript (Section: 4.12. Regulation network construction).
In the cases where I suggest citations for the software tools, they can be replaced by URLs in case no citation is provided by the software.
Answer: Thank you for your suggestion. We provided citations or URLs for all software tools in our revised manuscript.
Round 2
Reviewer 3 Report
21.9.2021
The revised manuscript "Identification, Evolution and Expression analysis of GRF Family reveals their involvement in shoot growth and abiotic stress response in Moso Bamboo" was reviewed.
The revised manuscript has been tangibly improved. And I would like to thank the authors for incorporating all comments and suggestions. Therefore, I recommend the publication of the revised manuscript along the new high resolution figures in "Forests".
Reviewer 4 Report
Authors have revised the manuscript appropriately.
Reviewer 5 Report
Dear authors,
Thank you for the replies provided in response to my comments. I believe the authors addressed my points of concern carefully and successfully. The manuscript provides a great starting point to research involving GRFs in Moso bamboo. My only comment is regarding the formatting of the manuscript, since it has several typos or differently formatted fonts and some figures are hard to read, but I believe this is easily solved during the editing/proofreading of the manuscript by the journal. After this minor corrections I believe the manuscript will be ready to be published.
Minor editing of English is necessary to ensure proper grammar in the manuscript.
