Identification and Analysis of Phosphatidylethanolamine-Binding Protein Family Genes in the Hangzhou White Chrysanthemum (Chrysanthemum morifolium Ramat)

Round 1

Reviewer 1 Report

Comment 1:

Why the authors have not attempted to sequence non-coding regions of the analysed genes?

Comment 2:

Why the authors have not attempted to study the chromosomal location of the genes?

Comment 3:

Have the authors attempted a comparative study for gene duplication in identified genes?

Other comments:

Check for the alignment and style of the manuscript.

Line no 22: Check the spelling of hormone ‘MeSA’.

Line no 46-47: “Most researchers considered MFT-like an ancestor of 46 FT-like and TFL1-like subfamilies”- Add references.

Line no 47-49: Check the reference is relevant.

Line no 61: check for grammatical error!

Author Response

Dear reviewer:

Thank you very much for your patience and comments on our manuscript. We have revised our manuscript according to your comments: 

The manuscript was carefully checked and corrected by all co-authors. We highlight the changes we make in the manuscript by using the "Track Changes" function in Microsoft Word.

The detail as follows:

Comment 1: Why the authors have not attempted to sequence non-coding regions of the analysed genes?

Response: Thanks for your kind suggestion. This is a valid and important question, we are also curious about the result and actively pursuing the answer. We know that non-coding regions are involved in almost all physiological activities such as embryonic development, cell proliferation, differentiation, apoptosis, infection, and immune response. However, we are unaware of any studies that provide the answer. For the analysis of the non-coding region, a higher quality genome is needed, and the amplification of the non-coding region is more difficult than that of the coding region. There is no open Genome database of Hangzhou White Chrysanthemum for our reference at present, we are going to continue our explore in the next step.

Comment 2: Why the authors have not attempted to study the chromosomal location of the genes?

Response: Thanks for your kind suggestion. There is no open Genome database of Hangzhou White Chrysanthemum for our reference at present, we are going to continue our explore in the next step.

Comment 3: Have the authors attempted a comparative study for gene duplication in identified genes?

Response: Thanks for your kind suggestion. There is no open Genome database of Hangzhou White Chrysanthemum for our reference at present, we are going to continue our explore in the next step.

Comment 4: Check for the alignment and style of the manuscript.

Response: We regret the problems with typesetting, and the paper has been carefully revised. We have referred to the format of papers published recently and found that the typesetting of authors was wrong. Now it has been correctly written under the title. If we still have typesetting errors in the manuscript, please inform and allow us to revise it again. We appreciate your warm work earnestly and hope that the correction will meet with approval.

Comment 5: Check the spelling of hormone ‘MeSA’.

Response: Thank you very much for discovering this error. We apologize for this spelling mistake and have changed the misspelled “methyl salicylat” to the correct “methyl salicylate”. In addition, we have carefully checked the full manuscript to ensure that similar mistakes did not occur. The revised details can be found on Page 1, Line 22.

Comment 6: Line no 46-47: “Most researchers considered MFT-like an ancestor of FT-like and TFL1-like subfamilies”- Add references.

Response: Thanks for your kind suggestion. We have added references “Yang, Z.; Chen, L.; Kohnen, M.V.; Xiong, B.; Zhen, X.; Liao, J.; Oka, Y.; Zhu, Q.; Gu, L.; Linet, C.; et al. Identification and Characterization of the PEBP Family Genes in Moso Bamboo (Phyllostachys heterocycla). Sci Rep. 2019, 9, 14998.” The revised details can be found on Page 2, Lines 50-51. 

Comment 7: Line no 47-49: Check the reference is relevant.

Response: Thanks for your kind suggestion. Thank you for your care. We have revised the cited references “Hedman, H.; Kallman, T.; Lagercrantz, U. Early evolution of the MFT-like gene family in plants. Plant Mol Biol. 2009, 70, 359-369.” The revised details can be found on Page 2, Lines 51-53.

Comment 8: Line no 61: check for grammatical error!

Response: We apologize for the poor language of our manuscript. We have now worked on both language and grammar and have also involved native English speakers for language corrections. We really hope that the language level has been substantially improved. Turn from “Zea mays [7], Oryza sativa [9], and Triticum aestivum [25,26] and Oncidium Gower Ramsey [27] hashave 17 to 30 PEBP genes, respectively. This complexity in the PEBP family genes in monocotyledons suggests that the functions of this family are more complex in monocotyledons than in the dicotyledon A. thaliana.” to “Zea mays [7], Oryza sativa [9], and Triticum aestivum [25,26] and Oncidium Gower Ramsey [27] have 17 to 30 PEBP genes, respectively. This complexity of PEBP family genes in monocotyledons suggests that the functions of this family are more complex in monocotyledons than in dicotyledons.” The revised details can be found on Page 2, Lines 64-67.

We would like to thank the referee again for taking the time to review our manuscript.

Reviewer 2 Report

This article studied Identification and Analysis of Phosphatidylethanolamine Binding Protein Family Genes in Hangzhou White Chrysanthemum (Chrysanthemum morifolium Ramat). This study will help to facilitate to understand identification, analysis and phylogenetic relationship of the gene family. Before recommending this article for publication, there are some shortcomings for that should be resolve.

Overall, the study is well designed and presented in a good way, but mostly the literature is not cited. Grammatical and typos must be revised

 “Expression patterns in response to different temperature and hormone stresses were investigated using qRT-PCR. TFL-like gene expression, which delays reproductive growth, was upregulated under heat stress. FT-like gene expression was upregulated under low temperature” In these lines add quantitative results and differences among different treatments briefly.

Line 70-71 should be cited with relevant study.

https://doi.org/10.1007/s10725-021-00785-7,

Line 106 to 121 is the summary of the work. No need of the summary here just add aims and objectives of the study.

Also add how this study is novel from other studies.

Section 2.5 should be cited with recent study. The following study could be cited https://doi.org/10.1016/j.indcrop.2022.116090

Italicize species and genes names in the whole MS.

Results are well presented but revise grammatical and typo mistakes

Compare the obtained with more recent studies.

Add reasons of radiations in the phylogenetic tree in the discussion section.

Author Response

Dear reviewer:

Thank you very much for your patience and comments on our manuscript. We have revised our manuscript according to your comments: 

The manuscript was carefully checked and corrected by all co-authors. We highlight the changes we make in the manuscript by using the "Track Changes" function in Microsoft Word.

The detail as follows:

Comment 1: Overall, the study is well designed and presented in a good way, but mostly the literature is not cited. Grammatical and typos must be revised.

Response: Thanks for kind suggestions. Eight recently published research articles in the introduction and discussion on genome-wide identification in other model crops were cited. The revised details can be found on Page 3 and Page 9-10. Meanwhile, the manuscript was carefully checked and corrected by all co-authors. In addition, it was polished and modified by native English editors. We really hope that the language level has been substantially improved.

Comment 2: “Expression patterns in response to different temperature and hormone stresses were investigated using qRT-PCR. TFL-like gene expression, which delays reproductive growth, was upregulated under heat stress. FT-like gene expression was upregulated under low temperature” In these lines add quantitative results and differences among different treatments briefly.

Response: Thanks for your kind suggestion. I'm awfully sorry, we are not sure whether you want us to write the results here. Two sentence “TFL-like gene expression, which delays reproductive growth, was upregulated under heat stress. FT-like gene expression was upregulated under low temperature” in the manuscript. This is the difference between TFL-like and FT-like of temperature treatment. If our understanding is wrong, please inform and give us the opportunity to revise it again.

Comment 3: Line 70-71 should be cited with relevant study. https://doi.org/10.1007/s10725- 021-00785-7

Response: Thanks for your kind suggestion. We have added references “Muhammad, N.; Khurram, S.; Saddam, S.; Asim S.; Nasrullah; Muhammad Y.; Muhammad I.A. The Solanum melongena COP₁LIKE manipulates fruit ripening and flowering time in tomato (Solanum lycopersicum). Plant Growth Regul. 2022, 96, 369-382.”. The revised details can be found on Page 2, Lines 75-77.

Comment 4: Line 106 to 121 is the summary of the work. No need of the summary here just add aims and objectives of the study. Also add how this study is novel from other studies.

Response: Thanks for your valuable feedback. Since we did not express it clearly, we have modified all of the corresponding parts on Lines 106-121. These newly revised sentences are as follows:

“With recent advances in sequencing technologies, a rapid increase in sequenced plant genomes has been accessed in the past few years. Although, genome sequence databases have provided researchers with a wealth of encoded information [46-49]. However, the gene family identified in the plant species genome is still uncharacterized, particularly in their function and regulation. Especially for polyploid plants with no genome or complex, comparative genomics is a very good method for mining family genes or transcription factors [46,47,49-51]. In this study, we used the sequence of PEBP family genes in the genome data of Arabidopsis and thirteen other species (Citrus clementina, C. sinensis, C. sativus, G. max, Medicago truncatula, O. sativa, Physcomitrella patens, P. trichocarpa, Prunus persica, Selaginella moellendorffii, Sorghum bicolor, Vitis vinifera, and Z. mays) to identify all potential PEBP family genes of Hangzhou White Chrysanthemum for the first time. Furthermore, we used bioinformatics methods to analyzed CmPEBP characteristics based on the results of gene structures, physical and biochemical properties, phylogenetic relationships and conserved motifs. Tissue-specific expression patterns and transcriptional responses to various abiotic stresses (low and high temperatures, and multi-hormone treatments) of identified CmPEBPs were analyzed to examine their possible involvement in flowering regulation. Our study can enable the investigation of detailed molecular and biological functions of PEBP members as well as provide a reference for the application of exogenous hormones to control flowering in Hangzhou White Chrysanthemum.” The revised details can be found on Page 3, Lines 112-141.

Comment 5: Section 2.5 should be cited with recent study. The following study could be cited https://doi.org/10.1016/j.indcrop.2022.116090

Response: Thanks for your kind suggestion. We have added references “Li, D.; Zaman, W.; Lu, J.; Niu, Q.; Zhang, X.; Ayaz, A.; Saqib, S.; Yang, B.; Zhang, J.; Zhao, H.; Lü, S. Natural lupeol level variation among castor accessions and the upregulation of lupeol synthesis in response to light. Ind Crop Prod. 2023, 192, 116090.” The revised details can be found on Page 5, Lines 236-237.

Comment 6: Italicize species and genes names in the whole MS.

Response: Thanks for your kind suggestion. We have paid special attention to the italics of species and genes in the revised manuscription, and the changes are highlighted by the "Track Changes" function in Microsoft Word. We appreciate for reviewers’ warm wok earnestly, and hope that the correction will meet with approval.

Comment 7: Results are well presented but revise grammatical and typo mistakes.

Response: Thank you for your recognition of our result. We have carefully modified the grammatical and spelling errors in the manuscription, and we hope that our manuscription can make some progress.

Comment 8: Compare the obtained with more recent studies.

Response: Thanks for your valuable feedback, we have obtained with more recent studies. The revised discussion is shown below:

“Due to the complexity of polyploid and the lack of genome of Hangzhou White Chrysanthemum, knowledge on CmPEBP family genes have remained relatively backward, which was similar to wheat [46-48,50]. In the present study, nine full-length CmPEBP coding genes were identified by homology studies using bioinformatics methods, and the results were consistent with the results of other species [3,4,7,9,18,19], all of which contain three subfamilies: MFT-like, TFL-like, and FT-like. This result agrees with that of other plants, indicating that the PEBP genes are conserved among species [3,4,18,19]. DPDxP and GxHR, which were found in the FT-like and TFL-like subfamilies, are considered two highly conserved characteristic short motifs of PEBPs [31,32]. Except for non-flowering plants (P. patens and S. moellendorffii), these sequences can be classified into three subfamilies, including A, B, and C.” The revised details can be found on Page 10, Lines 501-514.

Comment 9: Add reasons of radiations in the phylogenetic tree in the discussion section.

Response: Thanks for your kind suggestion. First of all, we are very sorry that our evolutionary tree has made some mistakes. When we analyzed the picture, we found that the FT-like of monocotyledonous plants may have more functions or research value, but it has no data support in this paper. We have modified the picture and related expressions. Meanwhile, we have added three sentences “FT-like also changed in two directions, one was to reduce the number of redundant members (dicotyledons), and the other was to increase the number of members (monocotyledons). FT-like genes were more conservative in dicotyledons and more diverse in monocotyledons and could be divided into three subgroups [3,20]. From the evolutionary tree, it can be clearly found that monocotyledons have more than 10 FT proteins at a higher level of classification and all member types of group A, whereas dicotyledons usually have only 1–2 FT proteins and only one of the member types of group A. FT-like plays an important role not only in the transformation and development of flowers but also in the regulation of tuber formation, seed germination, bud germination, branching, and stomatal opening [43,44]. More research may be verified in monocotyledonous plants.” The revised details can be found on Page 10, Lines 539-549.

We would like to thank the referee again for taking the time to review our manuscript.

Reviewer 3 Report

To,

The Editor-in-chief,

Agriculture, MDPI,

Manuscript ID: agriculture-2180827

Subject: Submission of comments on the manuscript in “Agriculture"

Dear Editor-in-chief Agriculture, MDPI,

Thank you very much for the invitation to consider a potential reviewer for the manuscript (ID: agriculture-2180827). My comments responses are furnished below as per each reviewer’s comments. 

In the reviewed manuscript, the authors identified nine CmPEBP genes that contained the PF01161 domain belonging to PEBPs were identified in Hangzhou White Chrysanthemum. Phylogenetic analysis classified CmPEBP genes into three subfamilies: MFT-like, TFL-like, and FT-like. Genes in these subfamilies displayed differential expression in the studied tissues. Each CmPEBP showed tissue-specific expression patterns, although the expression level varied greatly. Expression patterns in response to different temperature and hormone stresses were investigated using qRT-PCR. TFL-like gene expression, which delays reproductive growth, was upregulated under heat stress. FT-like gene expression was upregulated under low temperature. CmFT1 expression could be inhibited by GA (gibberellin), 6-BA (benzylaminopurine), ET (ethylene), and MeSA (methyl salicylat) but activated by IAA (indole-3-aceticacid), ABA (abscisic acid), and SA (salicylic acid) in the dark, whereas CmFT2 and CmFT3 expression levels were upregulated by ET, MeJA (methyl jasmonate), and ABA but downregulated by 6-BA, SA, and MeSA. GA, IAA, SA, and MeSA inhibited CmTFL gene expression under light and dark treatments. Further research on CmPEBP genes in Hangzhou White Chrysanthemum can better determine their roles in flowering and floral organ development, especially in response to prolonged spraying of exogenous hormones. In general, the manuscript represents a very big piece of information in a logical presentation. Therefore, it might be conditionally accepted subject to major revision. Authors need to address the following issues before it can be accepted for publication.

 

1. Most of the abstracts contain confusing and uninformative sentences. Please give more precise objectives here (such as in the Abstract). The abstract should highlight the most important results of the parameters and characteristics assayed.
2. General note: the figures in this section are quite low resolution and difficult to make out. Higher-resolution versions will be needed for publication. Further, text in the figures are not readable, for example, in Figures 1, 2, 3, 5, 6 and Figure S4 to S12.
3. Material methods most the citation is the webpage, some website is not working, hence, better to cite the original research paper.
4. The qRT-PCR methodology provided is also very vague and confusing. Please provide more details like the calibrator used in the study. I assume the authors have used the control as the calibrator. If so, the authors should not include the control within the bar graph as it represents the fold change between the treated vs control and a fold change of “1” for the ‘control’ doesn’t make any sense.  Also, would be good to provide details on what reagents (details of probes used, if any, if SYBR was used then details for that, etc.) and real-time PCR machines were used in the current study.
5. The discussion should be interpreted with the results as well as discussed in relation to the present literature and authors must cite recently published research articles in the introduction and discussion on genome-wide identification in other model crops, for instance, "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8880425/

https://www.frontiersin.org/articles/10.3389/fpls.2021.663118/full

https://pubmed.ncbi.nlm.nih.gov/33673010

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395832

https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-020-02576-0

https://www.frontiersin.org/articles/10.3389/fpls.2021.748146/ful

6.     The conclusion section is very poorly written. It should be extensively improved. 

 

 

 

Author Response

Dear reviewer:

Thank you very much for your patience and comments on our manuscript. We have revised our manuscript according to your comments: 

The manuscript was carefully checked and corrected by all co-authors. We highlight the changes we make in the manuscript by using the "Track Changes" function in Microsoft Word.

The detail as follows:

Comment 1: Most of the abstracts contain confusing and uninformative sentences. Please give more precise objectives here (such as in the Abstract). The abstract should highlight the most important results of the parameters and characteristics assayed.

Response: Thanks for your valuable feedback, we have revised the abstracts according to your comment. The revised Abstract is shown below:

Abstract: Hangzhou White Chrysanthemum (Chrysanthemum morifolium Ramat) is one of the “Zhejiang eight flavors” in traditional Chinese medicine. Phosphatidylethanolamine- binding protein (PEBP) plays an important role in flowering and floral organ development. Even so, the biological role of PEBP in Hangzhou White Chrysanthemum has not been studied, which attracted us. Here, nine CmPEBP genes that contained the PF01161 domain were identified in Hangzhou White Chrysanthemum for the first time, and their biological role in the flowering of Hangzhou White Chrysanthemum had been preliminarily studied. Phylogenetic analysis classified CmPEBP genes into three subfamilies: MFT-like, TFL-like, and FT-like. Differential expression analysis was performed under different tissue and stress using qRT-PCR. It showed that each CmPEBP displayed tissue-specific expression patterns. Expression patterns in response to different temperatures and hormone stresses were investigated. They were finally demonstrated to be differentially expressed. TFL-like gene expression, which delays reproductive growth, was upregulated under heat stress. FT-like gene expression was upregulated under low temperature. CmFT1 expression could be inhibited by GA (gibberellin), 6-BA (benzylaminopurine), ET (ethylene), and MeSA (methyl salicylat) but activated by IAA (indole-3-aceticacid), ABA (abscisic acid), and SA (salicylic acid) in the dark, whereas CmFT2 and CmFT3 expression levels were upregulated by ET, MeJA (methyl jasmonate), and ABA but downregulated by 6-BA, SA, and MeSA. GA, IAA, SA, and MeSA inhibited CmTFL gene expression under light and dark treatments. Further research on CmPEBP genes in Hangzhou White Chrysanthemum can better determine their roles in flowering and floral organ development, especially in response to prolonged spraying of exogenous hormones.

Comment 2: General note: the figures in this section are quite low resolution and difficult to make out. Higher-resolution versions will be needed for publication. Further, text in the figures are not readable, for example, in Figures 1, 2, 3, 5, 6 and Figure S4 to S12.

Response: Thanks for your kind suggestion. Thank you for your concern. I'm not sure whether you can't see clearly is the reason for the online display of the webpage. The word format of the manuscript I downloaded is clear. At the same time, all figures in the submission system were also provided with separate file pictures in the format of 300 dpi of tif.

Comment 3: Material methods most the citation is the webpage, some website is not working, hence, better to cite the original research paper.

Response: Thanks for your kind suggestion. Your consideration is very correct. We have cited with related research after all methods and websites. Methods and reference studies are as follows:

1. SMART

Letunic, I.; Doerks, T.; Bork, P. SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res. 2012, 40, 302-305

2. Pfam 31.0

Finn, R.D.; Coggill, P.; Eberhardt, R.Y.; Eddy, S.R.; Mistry, J.; Mitchell, A.L.; Potter, S.C.; Punta, M.; Qureshi, M.; et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016, 44, 279-28

3. NCBI

Johnson, M.; Zaretskaya, I.; Raytselis, Y.; Merezhuk, Y.; McGinnis, S.; Madden, T.L. NCBI BLAST: a better web interface. Nucleic Acids Res. 2008, 36, 5-9.

4. ProtParam tool

Wilkins, M.R.; Gasteiger, E.; Bairoch, A.; Sanchez, J.C.; Williams, K.L.; Appel, R.D.; Hochstrasser, D.F. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol. 1999, 112, 531-552.

5. WoLF PSORT

Horton, P.; Park, K.-J.; Obayashi, T.; Fujita, N.; Harada, H.; Adams-Collier, C.J.; Nakai, K. WoLF PSORT: protein localization predictor. Nucleic Acids Res. 2007, 35, W585-W587.

6. TargetP

Emanuelsson, O.; Brunak, S.; von Heijne, G.; Nielsen, H. Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc. 2007, 2, 953-971.

7. MEME analysis

Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.E.; Nobleet, W.S. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202-W208.

8. InterPro database

Hunter, S.; Apweiler, R.; Attwood, T.K.; Bairoch, A.; Bateman, A.; Binns, D.; Bork, P.; Das, U.; Daugherty, L.; Duquenne, L.; et al. InterPro: the integrative protein signature database. Nucleic Acids Res. 2009, 37, D211-D215.

The revised details can be found in Page 5, Lines 206-225.

Comment 4: The qRT-PCR methodology provided is also very vague and confusing. Please provide more details like the calibrator used in the study. I assume the authors have used the control as the calibrator. If so, the authors should not include the control within the bar graph as it represents the fold change between the treated vs control and a fold change of “1” for the ‘control’ doesn’t make any sense. Also, would be good to provide details on what reagents (details of probes used, if any, if SYBR was used then details for that, etc.) and real-time PCR machines were used in the current study.

Response: Thanks for your kind suggestion. We have added details of SYBR and real-time PCR machines “The StepOnePlus™ Real time PCR (Thermo Fisher Scientific, America) was used for the quantitative analysis. ROX Reference Dye was mixed with TB SYBR Premix Ex Taq II (Tli RNaseH Plus, TAKARA) in advance to normalize signal and ensure data integrity.” The revised details can be found on Page 5, Lines 227-231.

Meanwhile, we have added details of control “Controls were set up at each time point, and for all the above assays, plants of approximately equal size were selected for all treatments.” The revised details can be found on Page 4, Lines 171-172.

Comment 5: The discussion should be interpreted with the results as well as discussed in relation to the present literature and authors must cite recently published research articles in the introduction and discussion on genome-wide identification in other model crops, for instance, "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8880425/

https://www.frontiersin.org/articles/10.3389/fpls.2021.663118/full

https://pubmed.ncbi.nlm.nih.gov/33673010

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395832

https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-020-02576-0

https://www.frontiersin.org/articles/10.3389/fpls.2021.748146/full

Response: Thanks for your valuable feedback. The above six recently published research articles in the introduction and discussion on genome-wide identification in other model crops were cited.

These newly revised sentences in the introduction are as follows:

“With recent advances in sequencing technologies, a rapid increase in sequenced plant genomes has been accessed in the past few years. Although, genome sequence databases have provided researchers with a wealth of encoded information [46-49]. However, the gene family identified in the plant species genome is still uncharacterized, particularly in their function and regulation. Especially for polyploid plants with no genome or complex, comparative genomics is a very good method for mining family genes or transcription factors [46,47,49-51]. In this study, we used the sequence of PEBP family genes in the genome data of Arabidopsis and thirteen other species (Citrus clementina, C. sinensis, C. sativus, G. max, Medicago truncatula, O. sativa, Physcomitrella patens, P. trichocarpa, Prunus persica, Selaginella moellendorffii, Sorghum bicolor, Vitis vinifera, and Z. mays) to identify all potential PEBP family genes of Hangzhou White Chrysanthemum for the first time. Furthermore, we used bioinformatics methods to analyzed CmPEBP characteristics based on the results of gene structures, physical and biochemical properties, phylogenetic relationships and conserved motifs. Tissue-specific expression patterns and transcriptional responses to various abiotic stresses (low and high temperatures, and multi-hormone treatments) of identified CmPEBPs were analyzed to examine their possible involvement in flowering regulation. Our study can enable the investigation of detailed molecular and biological functions of PEBP members as well as provide a reference for the application of exogenous hormones to control flowering in Hangzhou White Chrysanthemum.” The revised details can be found on Page 3, Lines 112-141.

The revised discussion is shown below:

“Due to complex polyploid and the lack of genome and of Hangzhou White Chrysanthemum, knowledge on CmPEBP family genes have remained relatively backward, which was similar to wheat [46-48,50]. In the present study, nine full-length CmPEBP coding genes were identified by homology studies using bioinformatics methods, and the results were consistent with the results of other species [3,4,7,9,18,19], all of which contain three subfamilies: MFT-like, TFL-like, and FT-like. This result agrees with that for other plants, indicating that the PEBP genes are conserved among species [3,4,18,19]. DPDxP and GxHR, which were found in the FT-like and TFL-like subfamilies, are considered two highly conserved characteristic short motifs of PEBPs [31,32]. Except for non-flowering plants (P. patens and S. moellendorffii), these sequences can be classified into three subfamilies, including A, B, and C.” The revised details can be found on Page 10, Lines 501-514.

Comment 6: The conclusion section is very poorly written. It should be extensively improved.

Response: Thanks for your kind suggestion.

Thanks for your valuable feedback, we have revised the conclusion according to your comment. The revised conclusion is shown below:

“Nine CmPEBP genes, including three CmFTs, four CmTFLs, and two CmMFTs, were identified by comparative genomics analysis in Hangzhou White Chrysanthemum. Further, gene structures, physical and biochemical properties, phylogenetic relationships and conserved motifs analysis of the CmPEBP family was conducted.They contained the PBP (PF01161) domain belonging to PEBPs and were phylogenetically clustered into three subfamilies MFT-like, TFL-like, and FT-like. Each CmPEBP showed tissue-specific expression patterns, although the level of expression varied greatly. TFL-like genes, which delay reproductive growth, were upregulated under heat stress. Instead, FT-like gene expression was upregulated under low temperature. Based on the study of expression patterns, we identified candidate genes that might play important roles in response to exogenous multi-hormone stress. CmFT1 expression could be inhibited by GA, 6-BA, ET, and MeSA but activated by IAA, ABA, and SA treatments in the dark, whereas the expression levels of CmFT2 and CmFT3 were upregulated after ET, MeJA, and ABA treatments but downregulated by 6-BA, SA, and MeSA treatments. GA, IAA, SA, and MeSA treatments inhibited CmTFL gene expression under light and dark treatments. Our results provide a solid foundation for further understanding of the role PEBP plays in flowering and floral organ development and will provide the basis for future research on functional characterization of the CmPEBP family in response to exogenous hormones stresses.”

The revised details can be found on Page 12, Lines 636-660.

We would like to thank the referee again for taking the time to review our manuscript.

Reviewer 4 Report

The manuscript is well written in good English and, in general, methodically correct. However, the 100 uM concentration of active substances for spraying is slightly excessive. Some plant hormones, particularly IAA, may have more toxic effects at such a high concentration, affecting the obtained gene expression results. In any case, such concentrations are very far from physiological.
Furthermore, the authors should indicate which solvent they used to dissolve these substances. Absorption through leaf/green tissues is often problematic, and the choice of solvent is critical, particularly since there has to be a balance between active substance solubility, permeability and solvent toxicity to plant tissues.
The presentation and discussion of the result are generally acceptable. Nevertheless, the manuscript only marginally fits the journal's scope. Perhaps the authors should consider transferring it to another, more molecular biology-oriented journal from MDPI.

 

Author Response

Dear reviewer:

Thank you very much for your patience and comments on our manuscript. We have revised our manuscript according to your comments: 

The manuscript was carefully checked and corrected by all co-authors. We highlight the changes we make in the manuscript by using the "Track Changes" function in Microsoft Word.

The detail as follows:

Comment 1: The manuscript is well written in good English and, in general, methodically correct. However, the 100 uM concentration of active substances for spraying is slightly excessive. Some plant hormones, particularly IAA, may have more toxic effects at such a high concentration, affecting the obtained gene expression results. In any case, such concentrations are very far from physiological.

Response: Thanks for your kind suggestion. We also considered this problem in the pre-experiment. With regard to the selection of hormone concentration, we have carried out many pre-experimental treatments, and found that under the single treatment of 100 uM concentration, the plant did not show phenotypic and physiologically visible damage. Of course, the result is not necessarily correct. We will design the experiment more rigorously in the follow-up study. Thank you for your guidance.

The content of hormones in different plants varies greatly, even in the same species due to differences in cultivated species or specific growth periods. The following three papers are from Chen Fadi's team of Nanjing Agricultural University. In their three studies, the content of IAA in chrysanthemum reached 923.99 ng/g, 809 ng/g and 300-500 ng/g, respectively.

Therefore, the hormone content in this study may be within the safe range.

1. Zhang, S.; Chen, S.; Chen, F.; Teng, N.; Fang, W.; Guan, Z. Anatomical structure and gravitropic response of the creeping shoots of ground-cover chrysanthemum ‘Yuhuajinhua’. Plant Growth Regul. 2008, 56, 141.
2. Jiang, B.; Miao, H.; Chen, S.; Zhang, S.; Chen, F.; Fang, W. The Lateral Suppressor-Like Gene, DgLsL, Alternated the Axillary Branching in Transgenic Chrysanthemum (Chrysanthemum × morifolium) by Modulating IAA and GA Content. Plant Mol Biol Rep. 2010, 28, 144-151.
3. Han, S.; Chen, S.; Jiang, J.; Fang, W.; Cuan, Z.; Chen F. Hormone Levels and Gene Expression Analysis of Chrysanthemum Cultivar ‘Puma Sunny’ Under Low Light Intensity. Scientia Agricultura Sinica. 2015, 48, 324-333.

Comment 2: Furthermore, the authors should indicate which solvent they used to dissolve these substances. Absorption through leaf/green tissues is often problematic, and the choice of solvent is critical, particularly since there has to be a balance between active substance solubility, permeability and solvent toxicity to plant tissues.

Response: Thanks for your kind suggestion. We added more details about the solvent “For fat-soluble hormones (IAA, GA, etc.), use an appropriate amount of 95% ethanol to dissolve, and then dilute with distilled water to reach the working solution concentration (the final concentration of ethanol is 1%). For water-soluble hormones (ABA, etc.), 1% ethanol distilled water is directly used as the solvent for preparation. The control (CK) uses 1% ethanol as the working solution.” The revised details can be found on Page 4, line 163-167.

 

Comment 3: The presentation and discussion of the result are generally acceptable. Nevertheless, the manuscript only marginally fits the journal's scope. Perhaps the authors should consider transferring it to another, more molecular biology-oriented journal from MDPI.

Response: Thanks for your kind suggestion. Your opinion is very good for us. Hangzhou White Chrysanthemum (Chrysanthemum morifolium Ramat), belongs to typical crops, is one of the famous “Zhejiang eight flavors” in traditional Chinese medicine. Although this study is partly based on molecular biology, it is also expected to reveal the relationship between hormones and flowering. The team feels that this article published in agricultural journals may be more meaningful.

Besides we have found some papers based on molecular biology accepted by the journal recently, some relevant papers are showed below:

Goyal, N.; Bhuria, M.; Verma, D.; Garewal, N.; Singh, K. Genome-Wide Identification of BTB Domain-Containing Gene Family in Grapevine (Vitis vinifera L.). Agriculture 2023, 13, 252. https://doi.org/10.3390/agriculture13020252.

Li, Q.; Li, X.; Sun, Y.; Tian, X.; Zhu, S.; Wang, Y.; Gao, H.; Shi, C.; Zhu, X. Transcriptome−Based Identification and Characterization of Genes Associated with Resistance to Beta−Cypermethrin in Rhopalosiphum padi (Hemiptera: Aphididae). Agriculture 2023, 13, 235. https://doi.org/10.3390/agriculture13020235.

Yang, Q.; Liu, J.; Yang, Y.; Lu, Y. Transcriptome Analysis of Propylaea quatuordecimpunctata L. (Coleoptera: Coccinellidae) under High Temperature Stress. Agriculture 2022, 12, 1088. https://doi.org/10.3390/agriculture12081088.

Eom, S.H.; Hyun, T.K. Comprehensive Analysis of the Histone Deacetylase Gene Family in Chinese Cabbage (Brassica rapa): From Evolution and Expression Pattern to Functional Analysis of BraHDA3. Agriculture 2021, 11, 244. https://doi.org/10.3390/agriculture11030244.

We would like to thank the referee again for taking the time to review our manuscript.

Round 2

Reviewer 3 Report

Dear Editor,

Thank you for providing the opportunity to review the revised manuscript. The manuscript is improved considerably after revision according to the reviewer's comment. Now this study is a suitable contribution to Agriculture. I recommend the manuscript for publication.

Thank you

 

With best regards