Transcriptomic Identification of Floral Transition and Development-Associated Genes in Styrax japonicus
Round 1
Reviewer 1 Report
Abstract
L20 source of nectar for whom?
Introduction
L39 . In Arabidopsis, there are…
L41 which process? Floral development? Please clarify
L41-46 this paragraph can be explained better, eg function and or involvement of the transcription factor
L51 56,58 60 126 and many more: species names in italics
The introduction can be improved by restructuring and explaining better the genes involved in flowering. Also mentioning if possible, genes involved in flowering in other tree species not only in Arabidopsis. Further, hormones are involved in many responses of the plant to environmental conditions, are the genes involved in flowering observed in arabidopsis also known in other tree species to be involved in flowering?
Material and Methods
Were the flowers collected from the same tree?
Results
L 126 this sentence belongs more to the discussion part. I would also rephrase the sentence since Vitis is a species from a completely different family. Most likely most overlapping sequences were from Vitis since it is a crop species.
Fig. 5 the resolution is not very good of this figure and it looks disproportioned. The letters are too close together to be readable
Discussion
4.1. paragraph could be shortened since it is mainly a summary of the results and repetition of the introduction
4.2. it is unclear if the genes listed here are from the differential expressed analysis or from the transcriptome deNovo assembly.
4.3. 4 I don’t understand the reason for this paragraphs. Did you investigate/controlled for photoperiodism and/or circadian clock in your experiment?
As you are mentioning in L238 circadian genes are included in the regulation processes of flowering, the last two sentences probably belong to 4.5
L244 do you mean here the 2606 unigenes differential expressed in all three analysis? (venn diagram)?
L266 please name these publications
Conclusion
Please rephrase the current version is a summary of the study again.
General
Citation style [1,2] not [1];[2] and other formatting mistakes (which might have happened during the conversion to pdf?)
In my opinion your research lead to nice and interesting results, although the description and connection to other studies needs to be improved. I expect that there are already studies on floral development on woody species. To compare the results with a species closer related to S. japonicas than Arabidopsis would be nice to see. There are a lot of interesting aspects you do not describe and discuss.
For example
which stages are more similar C1/C2 or C2/C3 or C1/C3 in terms of gene expression? In Figure 6 it seems that C2 (understandable) is an intermediate stage…
Are the most overlapping genes always upregulated or downregulated?
Is ABA,GA, cytokinin only differential expressed between C1 and C3? Are time wise differences ?
Author Response
Response to Reviewer 1 Comments
Point 1: L20 source of nectar for whom?
Response 1: Thank you for underlining this deficiency. This section was revised and modified. (See Page1, Line13)
Point 2: In Arabidopsis, there are…
Response 2: Regarding the suggestion, we changed this sentence. (See Page1, Line38)
Point 3: which process? Floral development? Please clarify
Response 3: Thank you for underlining this deficiency. This section was revised and modified as follows: In Arabidopsis, there are 6 key pathways regulating floral development: the photoperiod, autonomous, vernalization, gibberellin (GA), ambient temperature, and age pathways (See Page1, Line41)
Point 4: this paragraph can be explained better, eg function and or involvement of the transcription factor
Response 4: Thank you for underlining this deficiency. This section was revised and modified. (See Page2, Line42-Line46)
Point 5: and many more: species names in italics
Response 5: Thank you for underlining this deficiency. We have changed the species names in the text to italics.
Point 6: The introduction can be improved by restructuring and explaining better the genes involved in flowering. Also mentioning if possible, genes involved in flowering in other tree species not only in Arabidopsis. Further, hormones are involved in many responses of the plant to environmental conditions, are the genes involved in flowering observed in arabidopsis also known in other tree species to be involved in flowering?
Response 6: Thank you for underlining this deficiency. This section was revised and modified. (See Page1,2)
Point 7: Were the flowers collected from the same tree?
Response 7: Thank you for the suggestion, Samples were collected from three plants. we changed this sentence; it has been described as follows: “Three groups of samples were taken for each of these three periods and from three plants.” (See Page2, Line67)
Point 8: this sentence belongs more to the discussion part. I would also rephrase the sentence since Vitis is a species from a completely different family. Most likely most overlapping sequences were from Vitis since it is a crop species.
Response 8: Thank you for underlining this deficiency. We deleted this sentence from the original text because it was not rigorous. (See Page4)
Point 9: Fig. 5 the resolution is not very good of this figure and it looks disproportioned. The letters are too close together to be readable
Response 9: Thank you for underlining this deficiency. We modified Fig. 5 to make it clearer and transfer this diagram to supplementary material. (Supplementary Figure 4)
Point 10: paragraph could be shortened since it is mainly a summary of the results and repetition of the introduction
Response 10: The reviewer’s suggestion is very correct. We have re-organized the paragraph. (See Page6, Line198-Line210)
Point 11: it is unclear if the genes listed here are from the differential expressed analysis or from the transcriptome deNovo assembly.
Response 11: The reviewer’s explanation is very correct. We have re-organized the sentence as follows: “The 10 most dominant TF families identified during floral development induced basic helix-loop-helix, NAC, B3, MYB-related, bZIP, WRKY, ERF, FAR1, C2H2, and MYB families, with 626, 479, 45, 10, 58, 357, 347, 428, 25, and 422 members, respectively. found by transcriptome De Novo assembly” (See Page7, Line244-Line245)
Point 12: I don’t understand the reason for this paragraph. Did you investigate/controlled for photoperiodism and/or circadian clock in your experiment?
Response 12: In our study, we obtained a large number of unigene related to photoperiod, so we screened out functional genes related to photoperiod from the experimental data for differential expression analysis, in order to study whether there were photoperiod pathways in the process of plant development.
Point 13: As you are mentioning in L238 circadian genes are included in the regulation processes of flowering, the last two sentences probably belong to 4.5
Response 13: Thank you for underlining this deficiency. This section was revised and modified according to the information showed in the work suggested by the reviewer. We merged 4.4 and 4.3 into 4.3.
Point 14: L244 do you mean here the 2606 unigenes differential expressed in all three analysis? (venn diagram)?
Response 14: Thank you for the suggestion，2,606 genes in the Venn diagram indicate that these genes are expressed differently in all three stages of flower development, in other words, the amount of expression of these genes varies greatly in each stage.
Point 15: L266 please name these publications.
Response 15: Thank you for the suggestion. We have rewrite sections according to the suggestion. (See Page8, Line258)
Point 16: Please rephrase the current version is a summary of the study again.
Response 16: Thank you for underlining this deficiency. This section was revised and modified. (See Page9, Line292-Line301)
Point 17: Citation style [1,2] not [1];[2] and other formatting mistakes (which might have happened during the conversion to pdf?
Response 17: We are very sorry for our negligence of reference style. We have made correction according to the correct reference style.
Point 18: There are a lot of interesting aspects you do not describe and discuss.
Response 18: Thank you for the suggestion. We have rewrite sections according to the suggestion. Like (Line158-Line159), (Line171-Line173)
Reviewer 2 Report
This manuscript describes an RNAseq transcriptome study of three stages of flower development in the ornamental tree Styrax japonicus. They assemble a unigene set from the short reads and provide summary statistics for different categories of genes particularly those known to be involved in floral development and report on their differential expression over time.
The work appears to be technically sound although insubstantial. On the good side, they made the effort to sequence three biological replicates at each stage rather than pooling samples as is often the case on a budget, so the statistics for differential expression should therefore be more reliable. They have also made the data available in the Genbank Short Read Archive so that others may use it, although it would have been nice to have the assembled contigs available in a browser for others to use without having to reprocess the raw data. The analyses they provide are largely descriptive in nature and not all that exciting but does provide the beginnings of a genomic resource for this species that does have some value to other researchers interested in breeding within this genus as ornamental plants. I was surprised they didn’t mention their earlier paper in this same Journal from 2018 that looked at an RNAseq derived transcriptome from flowers (of unspecified age or stage) apparently from 9 different trees of this species (although the focus of that study was to identify SSR markers within ESTs) but there was also some ontology analysis in that paper – I assume that this is indeed a different set of data than reported in the earlier study. It is strange that there were also 9 libraries sequenced and the exact same number of unigenes 136,071, was reported, however that paper gave no SRA accession, BioProject or BioSample information, so either it is the same data now split by stage or there has been no real advance in the complement of expressed genes from this newer study at multiple bud stages during flower development. In essence, Fig 6, showing the expression changes of individual flower development related genes over time is really the only interesting bit of data coming out of this study and that is not particularly novel or meaningful. Just because Styrax has or doesn’t have a gene similar to one in Arabidopsis doesn’t really mean that floral development is regulated in the same way in these two species so in the end this is really just a catalogue of possible genes that might be worth looking at a little more closely. It would have been nice if they had looked at a number of the selected genes across a more extensive time series by Q-PCR and or did some hormone analyses on styrax bud development to add a bit more weight to their expression analysis observations.
Unfortunately, the delivery in this paper is not really up to scratch. There has been poor attention to detail throughout the preparation of the text – there are numerous typos and a number of unsupported and speculative observations made throughout without any real evidence. Statements like line 126-127 that because there were more hits to grape genes than some other species then grape and Styrax must be more closely related but this flies in the face of other phylogenetic analyses (Styrax is an Asterid and Grape in the Rosid clade, so worlds apart). In this respect Fig 2 adds nothing to the paper and should be dropped completely. Line 204-205, notes that there were many (almost half of them) genes that had no homologous gene in Genbank, which may be true (this value is lower than reported in other such studies in plants that often get 70-80% or more of the unigenes matching something in Genbank’s NR database), but does not support the notion that there are lots of unique or different genes in Styrax involved in floral development compared to other well characterised plants. More likely it is something to do with their assembly protocols or cut-offs used in assigning homologous genes. Line 253-4 talks about expression of the IAA3 homolog as not being consistent with IAA changes but as no hormone measurements have been done in Styrax – how can this be asserted?
The manuscript particularly highlights the changes in expression in hormone and hormone signalling pathways but this adds little other than to say that these pathways are changing during floral development which is nothing surprising. The specific observations drawn also don’t seem to be consistent with the colours shown in Figure 6. Z-scores scale the data for different genes and positive z-scores indicate number of standard deviations in expression above the mean across the samples and shown in green (according to their legend) and negative z-scores expression levels below the mean and shown in red. For example, they say PIN7 and MLO4 homologs decrease in expression over time, but these genes have different patterns on the heatmap with PIN7 going up (from red to green) and MLO4 rising then falling. They also say that GA3OX1 increased gradually but to me it looks to be falling(green to red to black)? Line 278 and 279 has two GA3OX1s but perhaps one is meant to be GA2OX6? They also say IAA3 rose over time, but it looks to have decreased over time?
As many people are red/green colour blind they may wish to change the colours used in the heat map to something more acceptable anyway.
Specific minor points
Line 22 – delete thaliana
Line 23 replace thaliana with flowering
Line 88 fragments not gragments
Line 89 – the SRA entry says that they used paired end sequencing so that should be noted here.
Line 113 japonicus not japonicas
Line 115 Supplementary Table 1 not Stable 1
Line 117 Following not Folowing – superscript for 10 to the 7
Line 124 annotated not annotate
Line 127 delete the part suggesting that the two plants are closely related.
Line 198 delete jasmine
Line 201 delete jasmine
Line 211 delete anti-aging
Line 217 NAC not MAC?
Line 218 ARF or ARR not AR?
Line 222 ageing
Line 224 The light signal not optical signal – is received by phytochromes
Line 236 FLC not FAC?
Line 249 is an early mediator
Line 257 ARF6 not Arf6
Line 267 ATPIN1 not Atpin1
Line 268 delete thaliana
Line 278-279 is GA3OX1 duplicated?
Line 347 – other Journal titles are not abbreviated – be consistent
Line 387 and elsewhere Bmc is usually all capitals BMC
Line 397 – are Chinese characters acceptable here in the references?
Author Response
Point 1: I was surprised they didn’t mention their earlier paper in this same Journal from 2018.
Response 1: Thank you for underlining this deficiency. The earlier paper and this paper were used the same material. The earlier paper only focus on the development the SSRs. We have modified the introduction and mention the earlier paper. (See Page1, Line33)
Point 2: It would have been nice if they had looked at a number of the selected genes across a more extensive time series by Q-PCR and or did some hormone analyses on styrax bud development to add a bit more weight to their expression analysis observations.
Response 2: Thank you for the suggestion. We screened some important genes related to flowering and performed Q-PCR validation. (See Page9, Lin189-195)
Point 3: Statements like line 126-127 that because there were more hits to grape genes than some other species then grape and Styrax must be more closely related but this flies in the face of other phylogenetic analyses.
Response 3: Regarding the suggestion, we changed this sentence, delete the part suggesting that the two plants are closely related.
Point 4: In this respect Fig 2 adds nothing to the paper and should be dropped completely.
Response 4: Thank you for underlining this deficiency. We transferred Fig2 to the supplementary material.
Point 5: Line 253-4 talks about expression of the IAA3 homolog as not being consistent with IAA changes but as no hormone measurements have been done in Styrax – how can this be asserted?
Response 5: Thank you for underlining this deficiency. We have modified and deleted this description in the original text. (See Page9, Line251)
Point 6: The specific observations drawn also don’t seem to be consistent with the colours shown in Figure 6.
Response 6: We are very sorry for our incorrect writing. We reversed the meaning of colors in the heat map. This section was revised and modified according to the data presented in the work (See Page8, Line255- Line282)
Point 7: As many people are red/green colour blind they may wish to change the colours used in the heat map to something more acceptable anyway.
Response 7: Thank you for the suggestion. We have modified it according to the comment. (Figure 6) Trends in gene expression are shown in blue and yellow, respectively.
Specific minor points
Point 8: Line 22 – delete thaliana
Response 8: We have modified it according to the comment.
Point 9: Line 23 replace thaliana with flowering
Response 9: We have used the “flowering” to replace “thaliana”
Point 10: Line 88 fragments not gragments
Response 10: We have modified it according to the comment.
Point 11: Line 89 – the SRA entry says that they used paired end sequencing so that should be noted here.
Response 11: We have modified it according to the comment (See Page3, Line88)
Point 12: Line 113 japonicus not japonicas.
Response 12: We have changed it according to the comment.
Point 13: Line 115 Supplementary Table 1 not Stable 1
Response 13: We have modified it according to the comment. (See, Line118)
Point 14: Line 117 Following not Folowing – superscript for 10 to the 7
Response 14: Thank you for underlining this deficiency. This section was revised and modified. (See, Line120)
Point 15: Line 124 annotated not annotate
Response 15: Thank you for underlining this deficiency. This section was revised and modified. (See, Line133)
Point 16: Line 127 delete the part suggesting that the two plants are closely related.
Response 16: Thank you for underlining this deficiency. This section was revised and modified.
Point 17: Line 198 delete jasmine
Response 17: we have omitted the word.
Point 18: Line 201 delete jasmine
Response 18: we have omitted the word.
Point 19: Line 211 delete anti-aging
Response 19: Thank you for underlining this deficiency. This section was revised and modified.
Point 20: Line 217 NAC not MAC?
Response 20: We are very sorry for our incorrect writing. We have used the “NAC” to replace “MAC” in our revised manuscript. (See, Line221)
Point 21: Line 218 ARF or ARR not AR?
Response 21: We are very sorry for our incorrect writing. We have used the “ARF” to replace “AR” in our revised manuscript. (See Page7)
Point 22: Line 222 ageing
Response 22: We have changed it according to the comment.
Point 23: Line 224 The light signal not optical signal – is received by phytochromes
Response 23: Thank you for underlining this deficiency. This section was revised and modified.
Point 24: Line 236 FLC not FAC?
Response 24: We are very sorry for our incorrect writing. We have used the “FLC” to replace “FAC” in our revised manuscript. (See Page7)
Point 25: Line 249 is an early mediator
Response 25: We have changed it according to the comment. (See, Line250)
Point 26: Line 257 ARF6 not Arf6
Response 26: We have changed it according to the comment. (See, Line257)
Point 27: Line 267 ATPIN1 not Atpin1
Response 27: We have changed it according to the comment. (See, Line264)
Point 28: Line 268 delete thaliana
Response 28: we have omitted the word.
Point 29: Line 278-279 is GA3OX1 duplicated?
Response 29: We are very sorry for our incorrect writing. We have re-organized the sentence as follows: “Interestingly, the expression of the GA biosynthesis genes GA3OX1 expression declined and GA2OX6 showed a trend of decreasing first and then increasing during the flowering process in this study, reaching peak expression during stage C3” (See Page8, Line276- Line277)
Point 30: Line 347 – other Journal titles are not abbreviated – be consistent
Response 30: Thank you for the suggestion. We have modified it according to the comment.
Point 31: Line 387 and elsewhere Bmc is usually all capitals BMC
Response 31: Thank you for the suggestion. We have modified it according to the comment.
Point 32: Line 397–are Chinese characters acceptable here in the references?
Response 32: Thank you for underlining this deficiency. We have removed the Chinese representation from the references.
Round 2
Reviewer 2 Report
I still have some concerns about the paper even though they have tried to address many of my earlier points – in their comments to reviewers the authors have confirmed that this is the exact same dataset as used in their previous paper published in Forests in 2018, where admittedly the focus was on the identification of SNPs for use in mapping. While this new paper focuses on genes related to flowering and flower development they still go over a lot of the characterisation of the transcriptome that they did in the previous paper including contig sizes, gene ontology classifications etc – in fact one of the Figures (Fig 2) is exactly the same as Fig 2 in their 2018 paper although with a different legend and some of the other supplementary figures and tables are just rehashes of the same data from tables or figures in the first paper. The need to more explicit in referencing the earlier work that generated these libraries and transcriptome assemblies - not just “However, few basic biological and genetics information was found in the present literatures. previous molecular biology studies on this plant only focus on the development of EST–SSRs[2]”. Sections 3.1-3.3 could probably be deleted or just summarised very briefly with a reference to the earlier manuscript. They have redrafted some sections that has improved the manuscript although some of the new changes introduce further bits of poor English. I would suggest they get a native English-speaking molecular biologist to review the manuscript for them before any resubmissions.
They have added some new data on expression confirmations of selected floral genes using Q-PCR (which is good) but they look to have just used the same three timepoints – presumably the same RNA samples they did the RNASeq on (which is not so good) rather than using the opportunity to do a more extensive time course over development, so I’m not sure it really adds a lot.
I don‘t really have time to go through and correct the English but a couple of other small points are:
Line 129 – should be 136,071 not 13,6071
Line 143 – Table 1 is this supposed to be >1000bp rather than >100bp?
Line 432 Not sure where this number 119368 unigenes comes from? 136,071 is indicated in Table 1?
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
