PONE-D-21-26095Transcriptome analysis of gibberellins and abscisic acid during the flooding response in Fokienia hodginsiiPLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Very nice paper on flooding tolerance! The data support is very clear. My main comments are about the grammar and wording structure, and to especially state the research gap this study fills.

Line 33: For “other three provenances” it may be helpful to specify the country of origin within introduction of the species.

Line 66: Missing a statement on the aim of this study at the end of the introduction section. What is novel about this research? Has physiological and molecular characterizations of F. hodginsii not been done before?

Line 78: Is the flooding assay done with some light or complete darkness?

Line 173: I feel the title “Different ABA- and GA-related response genes” is too simple and can be more elaborated, like “Transcriptomic analyses reveal different…”

Line 181: Missing hyphen “ABA related genes”

Line 228: Needs space between “1(“

Line 228: The jump between paragraphs from GA to SUB1 does not connect with the first sentence. It is more clear when the next sentence mentions GA. This may not be necessary, but perhaps there can be a better transition between the paragraphs.

Line 236: By the end of the SUB1 paragraph, it does not clarify how the SUB1 work relates to the data obtained. Perhaps a sentence can be added linking the connections between the studies, or how the SUB1 data can help support the current data.

Line 255: Before you mention the measurements of ABA and GA, you can highlight why you performed this work, the purpose, or how the research is novel. This is interpreted but not clearly stated.

Fig 1A: The black text in the map is hard to read and the font is small in the box.

Fig 1B: Since the squares are most strongly stacked and slightly bigger than the other symbols, perhaps showing a bar or box & whisker graph might make the data points more clear and easier to interpret.

Fig 2: Missing axis title for hours of submergence.

Fig 3A & C: It would help to draw a line under the provenance 1 & 4 samples in the graph.

Fig 3B & D: Missing axis title for hours of submergence.

Table S1: FJB91-R format needs to be centered.

More experimental support of course would be great, such as functional validation of the RNA-seq dataset, but for this level of publication I would not request any additional experiments.

Reviewer #2: The manuscript entitled 'Transcriptome analysis of gibberellins and abscisic acid during the flooding response in Fokienia hodginsii' describes the transcriptional and hormonal response of a Cupresaceaous tree to a 72h submergence period.

There are several critical aspects that need clarification:

1) In the M&M section says F. hodginsii seedlings were grown in hydroponics and subsequently (2 weeks after germination) completely immersed in water. The same hydroponic medium was used? Tap or clean water (potentially leading to the dilution of nutrients and a nutritional imbalance)? Seedlings were transplanted to soil prior to submergence? This needs to be further elaborated. O2 levels need to be measured in these conditions.

2) RNAseq data has to be deposited in a publicly available repository (e.g. NCBI SRA or similar) before manuscript is accepted for publication or upon acceptance, not later.

3) Authors recorded phenotypic data from F. hodginsii accessions collected from four different locations, but do not provide data that link these phenotypic traits with improved or decreased tolerance to anoxia. Moreover, phenotype responses to hypoxia were not measured or considered.

4) Typical responses to hypoxia are missing (ADH, PDC, SUSY upregulation) as well as induction of ERFVII homologs. This needs to be clearly present in the manuscript.

5) Statistical analysis is missing in Figures 1, 2 and 3

6) RNAseq results need to be provided as supplementary data, including annotation and statistical analyses.

7) Effects of flooding on GA and ABA signaling pathways needs to be supported also by transcriptional data. What GA was/were measured? What internal standards were used as surrogates?

Major aspects:

Ethylene has long been acknowledged as the main regulator of plants' responses to reduced O2 conditions, therefore, it cannot be excluded from the discussion. Interactions of ET with GA and ABA pathways in hypoxic conditions have been recently discussed (González-Guzmán et al. 2021).

A proper phenotypic evaluation of accessions in response to hypoxic conditions is required.

The proposal that SA signaling is involved is interesting but needs certain experimental backup. What happens with SA levels? What happens with typical SA-linked responses such as PR1 homolog overexpression? Could authors provide a tentative close relative of TRINITY_DN142_c0_g2 and TRINITY_DN7657_c0_g1?

L214-217 'suspect' is not scientific. Authors must provide plausible explanations well-grounded on empirical results.

L228-236 SUB1A is a non-canonical ERFVII which is not regulated by the N-end rule pathway and is present in rice. Does F. hodginsii has any potential ortholog? If not, these is out of context here. Other canonical ERFVII are better candidates to link ET and ABA or GA signaling (e.g. Arabidopsis RAP2.3, for instance).

For the sake of transparency and to allow replicability of results, transcriptional results must be presented in full, including annotation RPKM and statistics (FDR corrected) as supplementary.

To confirm that low O2 responses were activated under the experimental conditions described, expression of ET/O2 signaling ortholog elements must be presented along with typical anoxia survival responses (expression and/or enzyme activity of ADH, PDC and SUSY). Likewise, signaling pathways for GAs and ABA also need to be presented (expression of ABA receptors, PP2Cs, SnRKs and ABA-responsive genes such as ABI5, DREB2A, RD29, etc...).

Minor aspects:

L190 Where is Table 2, maybe authors refer to supporting Table S2. Please, amend.

Please, use the same coloring in Figures 3 and 4, as it stands is misleading.

Reviewer #3: Dear Dr. Tengfei Zhu,

I have read through the manuscript entitled "Transcriptome analysis of gibberellins and abscisic acid during the flooding response in Fokienia hodginsii”. The manuscript can provide new information in understanding the flooding tolerance mechanisms in non-model crops. With that in mind I would like to give the following comments and questions regarding the manusript.

1. Components of the PCR reaction volume doesn’t add up to 20. list them all. (Line 89)

Used GA standard/s (if used) not specified. There are various forms of GA available in the plants system, which one did you measure? (Line 95-102)

2. The gel electrophoresis will not tell you much/precisely the quality of the RNA. Have you run a the samples on the bioanalyzer? or Nano drop absorbance for these purposes? (Line 105)

3. what reference genome did you use to design the primers? (Line 109)

which qRT-PCR instrument did you use? (Line 108-113)

4. consistency in writing terminologies. For example qRT-PCR (Line 108) vs RT-qPCR (supporting information line 9. Look into the rest of the manuscript for consistency

5. How did you calculate the relative GA and ABA amounts in Fig. 2 and 3 (Line 14-19; 22-28)? it should be elaborated in the figure legends and method section.

6. I recommend to include the hormone analysis data, at least in the supporting information section of the manuscript.

7. Can the authors reanalyse the transcriptomic data considering the quality check used at the begining of the analysis were Q10. It should be at least Q30 with inferred base call accuracy of 99.99% rather than 90% in Q10 (Line 124).

8. How do you explain that you have supplemented the plants to be analyzed for RNAseq with GA3 but quantification from the other set of experiment stated only endogeneous GA. Can you reanalyze the hormone data by including other GA forms rather than stating indogenoeus GA, considering their que-specific responses?

Regards,

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Reviewer #1: Yes: Elaine Yeung

Reviewer #2: No

Reviewer #3: No

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PONE-D-21-26095R1Transcriptome analysis of gibberellins and abscisic acid during the flooding response in Fokienia hodginsiiPLOS ONE

Dear Dr. Zhu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As some former reviewers were unavailable to review the revised manuscript, a new reviewer (Reviewer 4) provided a review. Please address Reviewer 4’s comments. In addition, please address the following minor corrections (line numbers from track changes manuscript version):

• L121 space before comma should be deleted
• L145 should be “DNase I”
• L184 CRA005262 is currently duplicated

Please submit your revised manuscript by Feb 02 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

Reviewer #4: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: I have read the revised version of the manuscript entitled "Transcriptome analysis of gibberellins and abscisic acid during the flooding response in Fokienia hodginsii”. The manuscript is informative and can provide new insight in understanding the flooding tolerance mechanisms in non-model crops, mainly F. hodginsii.

Reviewer #4: 1. At no point in the text is cited the country where the provinces are located. It is implied but not explicit. Furthermore, the denominations of the provinces are ambiguous and change during the text, making the understanding of the reader from another country confusing.

2. In the introduction, in the paragraph beginning on line 71, the flooding conditions that may have triggered the plants' adaptation to excess water are mentioned. It would be interesting to inform the time needed for the studied species to complete a reproductive cycle, since the epigenetic changes triggered by stress might not have established themselves as marks transmitted between generations in a short period of time.

3. In lines 85-87 it is not clear whether the plants were grown under hydroponic conditions or in soil.

4. In line 91, it would be pertinent to add the justification for the growth to have occurred in the dark. Do the authors believe that the gene expression observed in the dark condition reflects the transcript pattern that would be expected for these plants under natural conditions? Furthermore, there is no information on acclimation to the dark condition or the growing temperature.

5. In line 100 there is no information on where the sequences of primers used in the experiment are found.

6. In line 122 there is not enough information about the analysis conditions in UHPLC.

7. In the “Simple Sequence Repeat (SSR) assay” section, there is no information on the methodology for extracting genetic material. How many grams of material were used? Did each sample come from different plants or from a pool of individuals? Couldn't the genetic variability of the species mask the results if different individuals make up the same sample?

8. In the section “Total RNA extraction, cDNA reverse transcription, and quantitative real-time (qRT) PCR” there is no information on the methodology used to extract the genetic material from the sample. How many grams were used in the extraction and which plant organs were collected for this trial? Did each individual compose a sample or was a pool of individuals performed during the extraction? Given the genetic variability of the species, would the pool represent a uniform condition of transcripts during the analyses?

9. In the section “RNA-Seq analysis and bioinformatics” an experiment is mentioned where the seedlings were treated with 1 µM GA3 and 20 µM ABA, but this methodology was not described in the text.

10. In line 159 the conditions for RNA sequencing were not described.

11. The “INITY_DN7657_c0_g1” gene is constantly written without “TR” (lines 36, 248, 250, 308 and 313 for example), and it was not clear if it was a typo or if the name really is “inity, since in the response to Reviewer #2 and in the caption of Figure 4 it was written as “TRINITY_DN7657_c0_g1”.

12. On line 291, correct O2 to O2

13. Between lines 289-296 the role of ethylene in flooding conditions was discussed. Did the authors analyze ethylene-responsive genes in transcriptomic analysis? The same can be pointed out in the discussion of salicylic acid (lines 297-305).

14. In the conclusion, it would be interesting to include the relevance of the results obtained in this work, as well as the future perspective for new experiments.

15. With regard to the images, in Figure 1B the data caption in the graph is in inverse order to the bars in the graphs (in the bars Province 4 appears first, although it is the last in the image caption).

16. It would be interesting to include the title of each graphic in the images (and not just in the caption).

17. Figures 4A and 4B are very small and difficult to read.

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Reviewer #3: No

Reviewer #4: No

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Transcriptome analysis of gibberellins and abscisic acid during the flooding response in Fokienia hodginsii

PONE-D-21-26095R2

Dear Dr. Zhu,

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