Identification and Functional Analysis of Transcription Factor NF-Y Family during Flower Bud Dormancy in Prunus mume

Round 1

Reviewer 1 Report

Line 21: remove ‘However’.

Line 25: change ‘PmNF-Y gene’ to ‘’PmNF-Y genes’’

Line 43: correct: ‘…connect two α, the helix based on…’

Line 45: change ‘its’ to ‘’their’’

Line 49: change  ‘[5]; [6]’ to ‘’[5,6]’’ do the same for lines: 49, 63, 67 and so on.

Line 50: change ‘Among them,’ to ‘’The’’

Line 52-56: rewrite the sentences: ‘The transcription and protein values of CO and NF-Y transcription factors change with time, which enables the regulation of FT by NF-YA/B/C trimer and NF-YB/C-CO trimer change regularly, and dynamically modulates flowering process.… ‘. There are several of such strangely formulated sentences. You should definitely consider an English editing option.  

Something like: Irregular time-dependent differential expression rate of CO and NF-Y transcription factors and the protein availability is believed to regulate the FT by NF-YA/B/C trimer and NF-YB/C-CO trimer that are involved in flowering processes…

Line 56-58: Bring a reference where it has been validated.

Line 60: (Fig. 1A) is missing or it was intended to refer to Fig S (Suppl.) 1A?

Line 74-75: avoid using such general statements:  ‘The modulation of blossoming in woody plants differs from that of herbaceous species. This distinctive process of woody plants needs further study.’ Be more specific and focus on physiology of flowering to justify your ternscriptomic objectives.

76: Give the full name here: Prunus mume var.!!! and remove ‘However,’

Line 94: this is very important to give a clear description of the samples and their identity. Photos are welcome to be shown in supplementary materials. Better write the sentences to be more understandable. E.g. : ‘The branches with a length of 30-40cm and full branches of one year were collected …’. It doesn’t give me a clear idea about the branches. The one-year-old branches are 30-40 cm? Also you just mentioned in the introduction that there are several cultivars of late and early flowering. Which one did you choose and why? The definition of ‘groups’ is not clear here. Do you mean 4 treatments? How did you apply the GA₄;  ABA;  MeJA? Was it sprayed or the branches were place in a solution? What was the applied volume? Also in the introduction you should a valid foundation to justify the nature of your treatments and the selected growth regulators or stimulants! Clearly describe the phenology of the selected branches/samples, their age (approx.), etc.

Line 133: delete ‘However, ’do not over use it in your MS. Maybe the applied Bootstrap values can also be mentioned here.

Line 139: there is no information of RNA-Seq analysis! Provide more details about the sequencing and also I recommend to give the raw data (at least the statistically different fold changes) as a supplementary material or if the bio-project is deposited somewhere, give the access link of the database website. Also in your results you mentioned the leaves, roots, buds, stems, and fruits!!! How many RNA-Seq analyses have been done with their number of replicates and how may RNA samples were obtained. You should clarify these technical details as you just said you froze the branches! What about the dormancy stage (Para, Endo Eco…) sampling??? Is the 0, 1, 3 and 5 days of treatment considered as dormancy stages? You need to clarify these precisely. Also you should clarify how your RNA-Seq data was normalized! What was considered as control or base value? The non-treated buds? If yes in which stage? Or in every stages? We are talking about hundreds of RNA-Seq analyses if every treatment is analyzed 3 times?

Treatments (GA₄;  ABA;  MeJA and control)

Dormancy stage (Para, Endo Eco… and release)

Tissues (leaves, roots, buds, stems, and fruits)

3 replicates!

Line 146: what do you mean by ‘bioraector’?

 

Line 342: In which tissue? 

Author Response

RESPONSES TO THE TWO REVIEWERS COMMENTS ON THE Manuscript ID: horticulturae-2046844

 

Dear Editor,

We are resubmitting the revised version of our manuscript (Horticulturae-2046844) with point-by-point responses to the reviewers’ comments. We are grateful to you, and the reviewers for the opportunity to improve the scientific quality of our manuscript. Below are point-by-point responses to issues raised by the two reviewers. Corrections were effected in the manuscript with track changes in Microsoft word.

 

Reviewer 1

 

Comments and Suggestions for Authors

 

#Line 21: remove ‘However’.

 

#Response: We have removed the ‘’However” by considering your suggestion. Thank you.

 

Line 25: change ‘PmNF-Y gene’ to ‘’PmNF-Y genes’’

 

#Response: We have changed the “PmNF-Y gene” to “PmNF-Y genes” as you suggest. Thank you.

 

Line 43: correct: ‘…connect two α, the helix based on…’

#Response: Thank you for your remark. We have corrected “…connect two α, the helix based on…” to “NF-YA protein contains a core conserved region consisting of 53 conservative amino acids. The core region consists of two conserved α-helices and a conserved gap sequence connecting the two α-helices . As with NF-YB and NF-YC proteins, the primary role of their conserved domain is to bind to the CCAAT-box and interact with the other two subsets”  

 

Line 45: change ‘its’ to ‘’their’’

 

#Response: We have changed the “its” to “their” as you suggest. Thank you.

 

Line 49: change  ‘[5]; [6]’ to ‘’[5,6]’’ do the same for lines: 49, 63, 67 and so on.

#Response: Thank you for your remark. We have edited the reference bibliograpy and formatted up to your requirement.  

 

Line 50: change ‘Among them,’ to ‘’The’’

 

#Response: We have changed the “Among them” to “The” as you suggest. Thank you.

 

Line 52-56: rewrite the sentences: ‘The transcription and protein values of CO and NF-Y transcription factors change with time, which enables the regulation of FT by NF-YA/B/C trimer and NF-YB/C-CO trimer change regularly, and dynamically modulates flowering process.… ‘. There are several of such strangely formulated sentences. You should definitely consider an English editing option.

Something like: Irregular time-dependent differential expression rate of CO and NF-Y transcription factors and the protein availability is believed to regulate the FT by NF-YA/B/C trimer and NF-YB/C-CO trimer that are involved in flowering processes…

 

#Reponse: Thank you for your suggestion. Here, we rewrite the sentences: “The transcription and protein values of CO and NF-Y transcription factors change with time, which enables the regulation of FT by NF-YA/B/C trimer and NF-YB/C-CO trimer change regularly, and dynamically modulates flowering process.” by “Irregular time-dependent differential expression rate of CO and NF-Y transcription factors and the protein availability is believed to regulate the FT by NF-YA/B/C trimer and NF-YB/C-CO trimer that are involved in flowering processes” as you suggested.

 

Line 56-58: Bring a reference where it has been validated.

 

#Response: Thank you for your remark. We have added the reference in the manuscript. It’s [5,8]

(Kumimoto RW, Zhang Y, Siefers N, et al. NF–YC3, NF–YC4 and NF–YC9 are required for CONSTANS‐mediated, photoperiod‐dependent flowering in Arabidopsis thaliana [J]. The Plant Journal, 2010, 63(3): 379-91.

Siriwardana CL, Gnesutta N, Kumimoto RW, et al. Nuclear factor Y, subunit A (NF-YA) proteins positively regulate flowering and act through flowering locus T [J]. PLoS Genet, 2016, 12(12): e1006496).

 

Line 60: (Fig. 1A) is missing or it was intended to refer to Fig S (Suppl.) 1A?

 

#Response: Thank you for your remark. There is no (Fig. 1A) to refer. We have removed “(Fig 1A)”. Thank you.

 

Line 74-75: avoid using such general statements:  ‘The modulation of blossoming in woody plants differs from that of herbaceous species. This distinctive process of woody plants needs further study.’ Be more specific and focus on physiology of flowering to justify your ternscriptomic objectives.

 

#Reponse: Thank you for your suggestion. Here we rewrite the statement and be more focus on the physiology of flowering. The new sentences is: “So knowing more about the regulation of flowering, which is also connected with bud dormancy, would be more than welcome”

 

Line 76: Give the full name here: Prunus mume var.!!! and remove ‘However,’

 

#Response: Thank you for your remark. We have removed “However” and started the sentence by the full name of Japanese apricot “Prunus mume Sieb. et Zucc”.

 

Line 94: this is very important to give a clear description of the samples and their identity. Photos are welcome to be shown in supplementary materials. Better write the sentences to be more understandable. E.g. : ‘The branches with a length of 30-40cm and full branches of one year were collected …’. It doesn’t give me a clear idea about the branches. The one-year-old branches are 30-40 cm? Also you just mentioned in the introduction that there are several cultivars of late and early flowering. Which one did you choose and why? The definition of ‘groups’ is not clear here. Do you mean 4 treatments? How did you apply the GA₄;  ABA;  MeJA? Was it sprayed or the branches were place in a solution? What was the applied volume? Also in the introduction you should a valid foundation to justify the nature of your treatments and the selected growth regulators or stimulants! Clearly describe the phenology of the selected branches/samples, their age (approx.), etc.

 

#Response: Thank you for your suggestion. Here, we rewrite all the paragraph on “Plant materiel and treatment” by “The plant materials were collected from the national Field Genebank for Prunus mume of Nanjing Agricultural University. The materials were from a 5-year-old early flowering cultivar "Taoxingmei", with a strong growth. The sampling period was after the leaves of "Taoxingmei" had fallen. Annual branches of 30-40cm with full and substantial buds were collected. The branches were divided into four groups, replicated three times with five annual branches. The base of the branch was cut evenly and submerged at a height of 3cm into 200 μ mol/L GA4 solution; 200 μ mol/L ABA solution; 200μ Mol/L MeJA solution and distilled water respectively. After that they were immediately put in a light incubator for cultivation. The conditions of GA4, ABA, and MeJA treatment were: day and night temperature 21 ± 3 ℃, light/dark hours 12/12 h, light intensity 55 mmol m-2 s-1, and relative air humidity 70% respectively. The conditions of 4℃ treatment are: day and night temperature 4 ± 3 ℃, light/dark hours 12/12 h, light intensity 55 mmol m-2 s-1, and relative air humidity 70% respectively. After every 3 days 2 mm base was cut to expose the new stubble in the media. After 0, 1, 3 and 5 days of treatment, the buds were collected from each treatment and stored at -80℃ for future use.”

 

Line 133: delete ‘However, ’do not over use it in your MS. Maybe the applied Bootstrap values can also be mentioned here.

 

#Response: We have removed the "However" and the applied Bootstrap values have been added with your suggestion. By the way, we have mentioned in the manuscript the applied Bootstrap values. Thank you.

 

Line 139: there is no information of RNA-Seq analysis! Provide more details about the sequencing and also I recommend to give the raw data (at least the statistically different fold changes) as a supplementary material or if the bio-project is deposited somewhere, give the access link of the database website. Also in your results you mentioned the leaves, roots, buds, stems, and fruits!!! How many RNA-Seq analyses have been done with their number of replicates and how may RNA samples were obtained. You should clarify these technical details as you just said you froze the branches! What about the dormancy stage (Para, Endo Eco…) sampling??? Is the 0, 1, 3 and 5 days of treatment considered as dormancy stages? You need to clarify these precisely. Also you should clarify how your RNA-Seq data was normalized! What was considered as control or base value? The non-treated buds? If yes in which stage? Or in every stages? We are talking about hundreds of RNA-Seq analyses if every treatment is analyzed 3 times?

 

Treatments (GA4;  ABA;  MeJA and control)

 

Dormancy stage (Para, Endo Eco… and release)

 

Tissues (leaves, roots, buds, stems, and fruits)

3 replicates!

 

#Reponse: Thank you! We have taken your comment into consideration and rewritten the RNA-Seq analysis section with additional information. The Materials and Methods section has also been revised and supplemented with additional information.

 

 

Line 146: what do you mean by ‘bioraector’?

 

#Response: Thank you for your remark. It’s not ‘bioraector’ but bioreactor. We have corrected in the manuscript.

 

Line 342: In which tissue?

 

#Reponse: Thank you for your suggestion. The tissue is bud. We precise in the manuscript now it’s the flower bud.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Functional characterization and evolutionary changes of NF-Y transcription is essential to understand their role in many biological processes of plants. While for high standard author suggested to address the following issues

1.       Author focused functional characterization of PmNF-Y genes by qRT-PCR study, while missed elaborate discussion why PmNF-Y genes expression essential under GA4, ABA, MeJA, and low temperature treatments in discussion part. Author suggested to provide elaborate discussion.

2.       Author suggested to provide clear image in figure-2.

3.       In figure author suggested to avoid pink color with green which create visibility problem.

Author Response

RESPONSES TO THE TWO REVIEWERS COMMENTS ON THE Manuscript ID: horticulturae-2046844

 

Dear Editor,

We are resubmitting the revised version of our manuscript (Horticulturae-2046844) with point-by-point responses to the reviewers’ comments. We are grateful to you, and the reviewers for the opportunity to improve the scientific quality of our manuscript. Below are point-by-point responses to issues raised by the two reviewers. Corrections were effected in the manuscript with track changes in Microsoft word.

 

Reviewer 2

Comments and Suggestions for Authors

 

Functional characterization and evolutionary changes of NF-Y transcription is essential to understand their role in many biological processes of plants. While for high standard author suggested to address the following issues

 

#Reponse: Thank you for your valuable comment to improve the scientific quality of our manuscript. We thank you for your valuable comments to improve the scientific quality of our manuscript. We have revised and rewritten the introduction with emphasis on why NF-Y transcription is essential to be studied.

 

Author focused functional characterization of PmNF-Y genes by qRT-PCR study, while missed elaborate discussion why PmNF-Y genes expression essential under GA₄, ABA, MeJA, and low temperature treatments in discussion part. Author suggested to provide elaborate discussion.

 

#Reponse: Thank you for your suggestion. Here we rewrite have write why PmNF-Y genes expression essential under GA₄, ABA, MeJA, and low temperature treatments and add to the discussion

 

Author suggested to provide clear image in figure-2.

 

#Reponse: Thank you for your suggestion. We have provided a new figure 2.

 

In figure author suggested to avoid pink color with green which create visibility problem.

 

#Reponse: Thank you for your remark. We have changed the color in the figure as you suggested.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Please consider the below remarks one more time.

Line 146: I still recommend giving the raw data (at least the statistically different fold changes) as supplementary materials (just some excel files).

 Also in your results you mentioned the leaves, roots, buds, stems, and fruits!!! How many RNA-Seq analyses have been done with their number of replicates and how may RNA samples were obtained. You should clarify these technical details as you just said you froze the branches! What about the dormancy stage (Para, Endo Eco…) sampling??? Is the 0, 1, 3 and 5 days of treatment considered as dormancy stages? You need to clarify these precisely. Also you should clarify how your RNA-Seq data was normalized! What was considered as control or base value? The non-treated buds? If yes in which stage? Or in every stages? We are talking about hundreds of RNA-Seq analyses if every treatment is analyzed 3 times?

Treatments (GA4;  ABA;  MeJA and control)

Dormancy stage (Para, Endo Eco… and release)

Tissues (leaves, roots, buds, stems, and fruits)

3 replicates!

Line 161: what do you mean by ‘bioreactor?

 Line 375: In which tissue?

 

Author Response

RESPONSES TO THE REVIEWER’S COMMENTS ON THE Manuscript ID: horticulturae-2046844

 

Dear Editor,

We are resubmitting the revised version of our manuscript (Horticulturae-2046844) with point-by-point responses. We are grateful to you, and the reviewers for the opportunity to improve the scientific quality of our manuscript. Below are point-by-point responses to issues raised by the reviewer. Corrections were affected in the manuscript with track changes in Microsoft word.

 

Line 146: I still recommend giving the raw data (at least the statistically different fold changes) as supplementary materials (just some excel files).

 

#Response: Thank you very much for your recommandation. The statistically different fold changes have been added now to the supplementary materials.

 

Also, in your results you mentioned the leaves, roots, buds, stems, and fruits!!! How many RNA-Seq analyses have been done with their number of replicates and how many RNA samples were obtained. You should clarify these technical details as you just said you froze the branches! What about the dormancy stage (Para, Endo Eco…) sampling??? Is the 0, 1, 3 and 5 days of treatment considered as dormancy stages? You need to clarify these precisely. Also, you should clarify how your RNA-Seq data was normalized! What was considered as control or base value? The non-treated buds? If yes in which stage? Or in every stage? We are talking about hundreds of RNA-Seq analyses if every treatment is analyzed 3 times?

 

#Reponse: Thank you for your comment. Here, we added some detailed information on the RNA-Seq analysis: Tissue-specific expression analysis of NF-Y gene family members in P. mume: The expression profiles of NF-Y genes in five different tissues (root, stem, leaf, flower bud, and fruit) were mapped using previous transcriptome data (accession number PRJNA172987) (Zhang et al., 2012). Each tissue had only one replicate, a total of 12 Gb clean data were generated, we use the Trimmed Mean of M-values (TMM) standardization method in edge R to standardize the RNA-Seq data.

 

Treatments (GA4; ABA; MeJA and control)

 

#Reponse: Thank you for your comment. Here we rewrite the section "Plant material and treatment" and give details on the treatments applied. In the manuscript you will see as written : “We selected branches at the endo-dormancy period for different hormones and low temperature treatment, Prunus mume have been completely dormant in this period, we want to further understand the effect on their dormancy through external hormones and low temperature treatment, and the non-treated buds were considered as control. The base of the branch was cut evenly and submerged at a height of 3 cm into 200 μmol/L GA4 solution; 200 μmol/L ABA solution; 200 μmol/L MeJA solution and distilled water respectively. After that they were immediately put in a light incubator for cultivation. The conditions of GA₄, ABA, and MeJA treatment were: day and night temperature 21 ± 3 ℃, light/dark hours 12/12 h, light intensity 55 mmol m^-2 s^-1, and relative air humidity 70% respectively. The conditions of 4℃ treatment are: day and night temperature 4 ± 3 ℃, light/dark hours 12/12 h, light intensity 55 mmol m^-2 s^-1, and relative air humidity 70% respectively. After every 3 days 2 mm base of the branches were cut to expose fresh injury in the media. After 0, 1, 3 and 5 days of treatment, the buds were collected from each treatment and stored at -80℃ for future use”

 

Dormancy stage (Para, Endo, Eco… and release)

 

#Reponse: Thank you again for your comment. Here, we added some detailed information about dormancy stage in the section of RNA-Seq analyses. Now we have : “Dormancy stage expression analysis of NF-Y gene family members in P. mume: The expression profiles of NF-Y genes in four different dormancy stages (para-dormancy, endo-dormancy, eco-dormancy and dormancy release) were mapped using previous transcriptome data (accession number PRJNA615074) (Gao et al., 2021). Each stage had three replicates, a total of 6.5 Gb clean data were generated, we use the Trimmed Mean of M-values (TMM) standardization method in edgeR to standardize the RNA-Seq data. The flower buds of Japanese apricot cv. ‘Taoxingmei’, a low chilling requirement cultivar, were collected from the National Field GenBank for Japanese apricot located in Nanjing, Jiangsu Province on 28th September and 7th November in 2017 and 2rd January and 2rd March in 2018, corresponding to the four stages of para-dormancy, endo-dormancy, eco-dormancy and dormancy release periods”

 

Line 161: what do you mean by ‘bioreactor?

 

#Response: Thank you for your comment! Here we rewrite the statement, and the new sentences is: “The collected flower bud materials were made up of three independent biological replicates. Three technical replicates were performed for each biological replicate.”

 

Line 375: In which tissue?

 

#Response: Thank you for your remark. We have revised and written a clear sentence focusing on the “tissue”. Here is the new statement: “Eight members of the PmNF-Y gene family from flower buds (in the endo-dormancy stage) showed distinct expression responses to various exogenous hormones and low-temperature treatment. The results are presented above.”

Author Response File: Author Response.docx