Round 1

Reviewer 1 Report

Please find enclosed some suggestions and comments:

Line 73: You should add chromosome number of pineapple 2n = 

Lines 94 and 95: pI, INSTABILITY INDEX, GRAVY : you should define and add what each parameter is.

Line 124: at the begining of the sentence you have a coma; the number is missing here, SO YOU HAVE TO PUT the number you obtained INSTEAD OF THE COMA

Line 146: in the title of table 1 you should add the specie (Ananas comosus) at the end after pineapple

Line 157: you said 19 members ?  is that correct, you should verify this.

Line 162: Fig1 in Page 5: the resolution is very bad, the letters are not clear at all, So you have to put the details of the different genes and the details of the different motifs in lisible words. You showed 9 motifs, what represents ? could you add detail of each motif ??

Line 165 in the title of Fig1: for Gene structure you should add in this sentence the mean of yellow and green colors (intron and exon, respecively or what represents the legend UTR and CDS you have in the top right of the figure)

Line 181, Fig 2 in Page 6 you have in (c) : the pie chart is illisible, please change for a better resolution to be easy to read it. 

Line 195: you should put "17 chromosomes among the 25 chrms in pineapple" (you precise the total number of chrm)

Line 200: DELETE 'was'

Line 201: put the entire word 'maximum likelihood' instead of ML

Line 225: Figure 5 Page 8 you have to put pineapple (Ananas comosus)

Line 226: same here you should add the detail of the specie as follows: grape (V. vinifera), rice (O. sativa) and banana (M. acuminate)

Line 256: Figure 7 page 10: You have to add in the title the explication of each stage: Bud-1w-1, Bud-1w-2 and Bud-1w-2 ..etc as follows: 1w, 3w or 7w (weeks after ethephon treatment); 1,2 or 3 ( what represent?)...

Besides in the title of this figure 7: put "flowering process" instead of "flowering"

Line 274: why you did not present the functional protein association of AcTrihelix19 (It is highly expressed in sepal formation and at the late stage of flower bud development?

lines 290 and 291: remove this to discussion section; is is not a result !!!

Line 298: Figure 9: why AcTrihelix19  IT IS  NOT PRESENTED?

Line 309: 'different among various species' instead of 'difference to various species'

Line 323: I suggest to put the sentence as follow: "distributed on 17 chromosomes among a total  of 25 chromosome in pineapple" (it is important to add the detail of the hole number of chrm)

Line 359: when you mention Fig S1, so wHEN WE OPEN THE SUPPLEMENTARY MATERIAL - FIGURE S1 we don't find the title of the figure please add this and add in the title a 2sentences explananation of the fig S1.

Author Response

Response to Reviewer 1 Comments

Point 1: Line 73: You should add chromosome number of pineapple 2n = 

Response 1: Thank you for your suggestion. The chromosome number of pineapple is 50, which is diploid. We added the chromosome number of pineapple 2n=50 according to your suggestion.

Point 2: Lines 94 and 95: pI, INSTABILITY INDEX, GRAVY: you should define and add what each parameter is.

Response 2: We have supplemented the definition descriptions of pI, INSTABILITY INDEX and GRAVY according to your suggestion.

Point 3: Line 124: at the begining of the sentence you have a coma; the number is missing here, SO YOU HAVE TO PUT the number you obtained INSTEAD OF THE COMA.

Response 3: It's an extra comma, we have corrected this error.

Point 4: Line 146: in the title of table 1 you should add the specie (Ananas comosus) at the end after pineapple

Response 4: We have added the specie (Ananas comosus) at the end after pineapple according to your suggestion.

Point 5: Line 157: you said 19 members? is that correct, you should verify this.

Response 5: After verified the figure and text, we found that there was a problem with the way we're phrasing it. AcTrihelix family had 23 members, 4 members have no introns, and 19 members have contained 1-16 introns, of which AcTrihelix13 contains the most introns.

Point 6: Line 162: Fig1 in Page 5: the resolution is very bad, the letters are not clear at all, So you have to put the details of the different genes and the details of the different motifs in lisible words. You showed 9 motifs, what represents? could you add detail of each motif ??

Response 6: We have reconstructed this diagram, and adjusted the position of the Figure1 to Figure3, and added Figure S1 to show the detailed information of all motifs.

Point 7: Line 165 in the title of Fig1: for Gene structure you should add in this sentence the mean of yellow and green colors (intron and exon, respecively or what represents the legend UTR and CDS you have in the top right of the figure)

Response 7: We had reanalyzed and reconstructed this figure to make the icons clearer. What’s more, we explained the mean of icons in the figure legends.

Point 8: Line 181, Fig 2 in Page 6 you have in (c): the pie chart is illisible, please change for a better resolution to be easy to read it.

Response 8: We have reconstructed this figure according to your suggestion.

Point 9: Line 195: you should put "17 chromosomes among the 25 chrms in pineapple" (you precise the total number of chrm)

Response 9: We have revised the description of this sentence. “17 chromosomes, accounted for 68 percent of 25 pineapple chromosomes”.

Point 10: Line 200: DELETE 'was'

Response 10: We have fixed this error.

Point 11: Line 201: put the entire word 'maximum likelihood' instead of ML

Response 11: We found that the evolutionary tree was more clarity constructed by ‘Neighbor-Joining’ method than ‘Maximum Likelihood’ method. Therefore, we have replaced the evolutionary trees of new Figure1 and Figure2 by using Neighbor- Joining method.

Point 12: Line 225: Figure 5 Page 8 you have to put pineapple (Ananas comosus)

Response 13: We have changed it in Figure 5 according to your suggestion.

Point 13: Line 226: same here you should add the detail of the specie as follows: grape (V. vinifera), rice (O. sativa) and banana (M. acuminate)

Response 13: We have added Latin names of related species in Figure 5 according to your suggestion.

Point 14: Line 256: Figure 7 page 10: You have to add in the title the explication of each stage: Bud-1w-1, Bud-1w-2 and Bud-1w-2 ..etc as follows: 1w, 3w or 7w (weeks after ethephon treatment); 1,2 or 3 ( what represent?)

Response14: We have added the relevant captions for these figures. Additionally, we have added the descriptions of plant materials and treatments in Material and Methods section.

Point 15: Besides in the title of this figure 7: put "flowering process" instead of "flowering"

Response 15: We have revised this description in Figure 7.

Point 16: Line 274: why you did not present the functional protein association of AcTrihelix19 (It is highly expressed in sepal formation and at the late stage of flower bud development? lines 290 and 291: remove this to discussion section; is is not a result !!!

Response 16: There are relatively few studies on the function of Trihelix transcription factors, so the predictions of gene functions have not achieved some expected results. Therefore, we deleted this part of protein interaction prediction and added the experimental verification results of qRT-PCR.

Point 17: Line 309: 'different among various species' instead of 'difference to various species'

Response 17: We have revised this description according to your suggestion.

Point 18: Line 323: I suggest to put the sentence as follow: "distributed on 17 chromosomes among a total of 25 chromosome in pineapple" (it is important to add the detail of the hole number of chrm)

Response 18: We have revised this description according to your suggestion.

Point 19: Line 359: when you mention Fig S1, so wHEN WE OPEN THE SUPPLEMENTARY MATERIAL - FIGURE S1 we don't find the title of the figure please add this and add in the title a 2sentences explanation of the Fig S1.

Response 19: We have added the relevant titles and captions in the supplementary figure.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript “Identification and Characterization of Trihelix Transcription Factors and Expression Changes during Flower development in Pineapple” represents an interesting work in which the trihelix transcription factor (TF) family (also called GT), one of the earliest discovered in plants, is first described in the species. These TFs have been described participating in responses to different stresses, highlighting their role in salt-tolerance; also, these elements have been associated to different plant development processes.

Although “Introduction” section could be some more paused and clearer (compared with close similar previous works for other plant species) to describe the relevance of these factors, in my opinion, under the current presentation the section is enough to introduce the GT family and its relevance.

Methods section. As mentioned, the work is quite close to previous studies having the same “study pipeline”; i.e., relying on bioinformatics processing to generate adequate information associated to the identification of the Trihelix genes in the species, gene subfamilies in accordance with a phylogenetic tree analysis, chromosome distribution, and offering an analysis of gene replication events. In addition, response elements and gene structures are given. In that line, the work is clean, and give us a good piece of information about GT family in pineapple which, due to the previous descriptions for other species, is strong and accepted. Also, the methodologies indicated in the section, are well accepted by the previous art.

Said that, methodology section sounds adequate and are solid supporting the complete GT family analyses as offered in Results section, associated to these results. However, other results have problems associated to their methods. 

Results. Along its first part (´til point 3.5), the section is well-structured according to the indicated methodology. Some particulars arise from the previous results, and these can be associated to certain organization issues. For instance, one of the first results should consider the pineapple sequences “validation”, which certainly is given by the phylogenetic tree (currently in Figure 3). From this, authors will have the chance of giving a more organized presentation of results.

My point is for next paragraphs. Inclusion of gene subfamilies in Figures 1 and 2 could contribute to a clearer observation of the GT family “organization” (structural and/or functional). The composed Figure 1 covers information for A) phylogenetic tree, B) motifs´ distribution, and C) gene structures. I strongly recommend including in A the subfamily information for the clades. In this way, members in the same subfamily will be compared having similar conserved motifs and gene structures. In the same line, in Figure 2 (cis elements in promoters), members are organized by gene numbers avoiding the possibility of grouping/associating gene groups to biological responses in which they could be involved.

The critical situation for the work comes at point 3.6 and those after this one. Line 228 refers to transcriptome data sets used to obtain and organize the next sub-sections. However, I was not able to find supporting information in “Methodology” section for these data and results generation. Figure 6 indicates interesting results about tissue specific gene expression, and Figure 7 is focused on flowering events in the species. Whereas I was able to recognize the type of data used and get the association to the web sites/computer package used for the previous results, I found no information given for analyses and datasets regarding gene expression profiles in general; this makes my evaluation going complicated. Please, clarify this or correct it. Also, I couldn´t find information for the different keys used in Figure 7 regarding flowering. In the same line, captions for these figures are weak (I thought that some of the "missed" information could be indicated here, not the case)   

Discussion. As above, everything makes sense in paragraph regarding genes structure and (bioinformatics) characterization. However, problems start in next paragraphs when authors consider genes function based on functional relationships (flowering or tissue specific expression), which I couldn´t find supportting the data source and processing. I encourage author to make this clear due to the occurrence of other works in which gene expression is experimentally established.

I think that discussion is interesting according, for instance, to the use of flowering inducers and so, however it is difficult to accept the information/results with no support for these procedures.  

Author Response

Point 1: Inclusion of gene subfamilies in Figures 1 and 2 could contribute to a clearer observation of the GT family “organization” (structural and/or functional). The composed Figure 1 covers information for A) phylogenetic tree, B) motifs´ distribution, and C) gene structures. I strongly recommend including in A the subfamily information for the clades.

Response: Thanks for your suggestion. We have added the subfamily information for the clades of Trihelix family in Figure 2 and Figure 3. Meanwhile, we have also rearranged the order of Figure 3 and Figure 1 to make the logic clearer.

Point 2: In the same line, in Figure 2 (cis elements in promoters), members are organized by gene numbers avoiding the possibility of grouping/associating gene groups to biological responses in which they could be involved.

Response: We have supplemented the subfamily information for cis-elements in promoters according to your suggestion.

Point 3: Line 228 refers to transcriptome data sets used to obtain and organize the next sub-sections. However, I was not able to find supporting information in “Methodology” section for these data and results generation.

Response: We have uploaded the transcriptome data for heat maps into National Genomics Data Center database (https://ngdc.cncb.ac.cn/databases). The assigned accession of the submission is: CRA006826. Meanwhile, we have also supplemented treatment and sampling information in the Materials and Methods section.

Point 4: Figure 6 indicates interesting results about tissue specific gene expression, and Figure 7 is focused on flowering events in the species. Whereas I was able to recognize the type of data used and get the association to the web sites/computer package used for the previous results, I found no information given for analyses and datasets regarding gene expression profiles in general; this makes my evaluation going complicated.

Response: We have supplemented the treatment and sampling information of transcriptome data in Material and Methods section for a clearer presentation of tissue-specific gene expression. Additionally, we have added the associated FPKM values in the Table S1, Table S2 and Table S3.

Point 5: I couldn´t find information for the different keys used in Figure 7 regarding flowering. In the same line, captions for these figures are weak (I thought that some of the "missed" information could be indicated here, not the case).

Response: We have added the relevant captions in these figures and supplemented sample information in Material and Methods section.

Point 6: problems start in next paragraphs when authors consider genes function based on functional relationships (flowering or tissue specific expression), which I couldn´t find supportting the data source and processing. I encourage author to make this clear due to the occurrence of other works in which gene expression is experimentally established.

Response: This is very good advice. It is difficult to elucidate genes function with transcriptomic data alone. Under normal conditions, qRT-PCR experiments can effectively verify the transcriptome results. Therefore, we have added the experimental verification results of qRT-PCR.

In subsequent studies, we will conduct more detailed functional verification of these candidate genes, including genetic transformation, target gene screening and regulatory mode. Thanks for your suggestion.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The previous observations have been reviewed by the authors, thanks.

Methods section.

-Most of the points previously indicated were amended by the authors too.

Some issues remain:

- in Section 2.4, the project number (CRA006826) is available just by 2024. I don´t know Journal´s policies about this. Please check that up. In the same paragraph, line 123, is not clear the idea for “… of pineapple. And the transcriptome data have uploaded…” (you meant “have been uploaded”?). Please, check.   

- In the same line, expression data for the Ac Trihelix genes are indicated as taken from the pineapple database. Considering my previous point, the information given by the authors about source of data (i.e., web site for the Genomics Data Center database), the link seems some ambiguous, consequently, authors should help to the reader, giving a more specific address for the used data (I mean if there is some additional data set to those in CRA006826, which I found not available yet). In the current format, I just was able to access into the home page of the Center (based on the link). Authors need to consider that they will be asked by Journal´s Editorial Office to indicate even the date of accession to that platform.

Results.

Authors have improved the previous presentation and included all the suggestions to generate a more consistent presentation of their results.

In this presentation, RNA work methods and additional information (i.e., suppl. materials included as figures and tables) allow for a proper traceability of the ideas organized in the section´s text.  

Captions for Figures have also been improved and they are satisfactory now, allowing for a proper fast reading throughout just figures (Figs 6-8). Line 314, please correct “Figure 8. The expression of AcTrihelix genes in different floral organs by TBtools.” to “Figure 8. The expression of AcTrihelix genes in different floral organs. The particular methodology has been moved to the convenient level in the caption.

Section 3.9. Paragraph and Figure (9) are OK.

Discussion.

As indicated in my previous revision, I think that discussion is interesting and now, considering the current version of the MS, these paragraphs find support in both results and methods. In general, the section is quite interesting and well supported.   

Author Response

Response to Reviewer

Point 1：in Section 2.4, the project number (CRA006826) is available just by 2024. I don´t know Journal´s policies about this. Please check that up. In the same line, expression data for the AcTrihelix genes are indicated as taken from the pineapple database. Considering my previous point, the information given by the authors about source of data (i.e., web site for the Genomics Data Center database), the link seems some ambiguous, consequently, authors should help to the reader, giving a more specific address for the used data (I mean if there is some additional data set to those in CRA006826, which I found not available yet). In the current format, I just was able to access into the home page of the Center (based on the link). Authors need to consider that they will be asked by Journal´s Editorial Office to indicate even the date of accession to that platform.

Response 1：Thanks for your suggestion. This journal requires that new high-throughput sequencing datasets must be deposited in online databases, but there is no requirement for this data to be in the released state.

These RNA-seq data are being analyzed and have not yet been published, so we set the release time of this data. If you want to view these data, you can find the uploaded data through this link (https://ngdc.cncb.ac.cn/gsa/s/QV56UwNN). In addition, data on the expressions used in this manuscript have been provided in the attachments (Table S2-4).

Point 2: In the same paragraph, line 123, is not clear the idea for “… of pineapple. And the transcriptome data have uploaded…” (you meant “have been uploaded”?). Please, check.   

Response 2：We have modified the description of this sentence “And the transcriptome data of pineapple have been uploaded into National Genomics Data Center database”.

Point 3: Line 314, please correct “Figure 8. The expression of AcTrihelix genes in different floral organs by TBtools.” to “Figure 8. The expression of AcTrihelix genes in different floral organs. The particular methodology has been moved to the convenient level in the caption.

Response 3：We have corrected this legend according to your suggestion.

Author Response File: Author Response.docx