PONE-D-21-21441

Silencing GhJUB1L1  ( J UB 1-like 1 ) reduces cotton  ( Gossypium hirsutum ) resistance to drought stress

PLOS ONE

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Comments to the Author

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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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors have functionally validated the role of GhJUB1L1 in drought tolerance of cotton by transient down regulation using VIGS approach. GhJUB1L1 codes for a transcription factor belonging the NAC family and is shown to regulate stress responsive and secondary cell wall genes. Some of the comments to the authors are as follows:

Major comments:

In material and methods section: Please add reference for the protocols used for staining of cellulose and others. NO clarity of sample collection for qRT, staining etc. Mention after how many days of silencing symptoms (albino) the samples were collected for qRT, staining. After how many days of re-watering the phenotype was recorded. Drought stress imposition protocol is missing-after how many days of silencing the drought stress was imposed? Mention the % field capacity after 10days of withholding water.

Results section:

Need to provide physiological data eg. RWC or EC etc., to support that plant are experiencing stress and control plants are unstressed.

In Fig4 and other expression data-normalization against what?

Fig 4b- what is drought 1, 2 and 3?

What statistical parameter (ttest, ANOVA) was used is not mentioned.

It would be nice to have the staining of re-watered plants.

Gene name must be italicized throughout. It is better to use drought tolerance rather than drought resistance.

No discussion about why of the two genes expressed (heat map) in stem only one was chosen for study-criteria used?

In general, the language can be improved as it is not clear is most places. The sentences can be reframed to make it simple and clear.

Line 44-46-not clear

Line 56- remove and after wheat

Line 58- reframe as-which plays role in regulating longevity and stress tolerance

Line 70-reframe as-In addition, ectopic expression of AtJUB1 led to enhanced stress tolerance and reduced oxidative damage.

Line 73- growth and stress response interactor

Line 76- drought induced gene

Line 128- to silence

Line 130-remove and then,

Line 146-reframe- once the material turns red, cover the glass quickly and observe under a normal microscope.

Line 149- accorded to the intensity of pigmentation

Line 150-it is browning

Line 165-predominantly not predominance

Line 168-Based on above analysis, Gh…..was selected for further study which was cloned and renamed as GhJUB1L1

Line 209- the results indicated tthat expression level of GhJUB1L1 was higher in stem and leaf while it was lower in flowers and roots under non-stress conditions. The expression of this gene in these tissues increased when plants were subjected to drought.

Line 217- in/different

Line 225- in the nucleus

Line 276- not clear

Line 281, line 288-289- reframe

Line 353- a homolog instead of one homolog

Reviewer #2: Overall the design of the study is good and authors wrote conveyed the research findings very well. I have few suggestions for authors to improve the manuscript quality.

1. Please change all the figure legends (figure 1, 2, 3, 5, 6, 8 and 9). They were very hard to read during the review process. Also maintain same size and font for figure legends.

2. Other than qRT-PCR data of downstream targets. authors have not shown any direct evidence like yeast one hybrid assay or luciferase reporter assay with few downstream target gene promoters. It would add more strength to the research findings.

3. Please rename the GhCCOAOMT1 to GhCCoAOMT1 across the manuscript.

Thanks

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Reviewer #1: Yes: Geetha Govind

Reviewer #2: Yes: Shailesh Karre

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Silencing GhJUB1L1 (JUB1-like 1) reduces cotton (Gossypium hirsutum) drought tolerance​

PONE-D-21-21441R1

Dear Dr. Luo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Ramegowda Venkategowda, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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 #1: Yes

Reviewer #2: Yes

**********

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 #1: Yes

Reviewer #2: Yes

**********

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 #1: The expression data seems to be normalised with Histone 3, and the data for control (Unstressed) is mentioned in the graph. Therefore the 2-delta Ct is used, accordingly change in material and methods section.

Reviewer #2: (No Response)

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Reviewer #1: Yes: Dr. Geetha Govind

Reviewer #2: Yes: Shailesh Karre