PONE-D-22-01261Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and developmentPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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3. 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: No

Reviewer #2: Yes

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

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Reviewer #1: No

Reviewer #2: 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: The manuscript lacks the novelty component and there are several technical points that makes this study not suitable for publication in the present form. The function of WRINKLED1 gene in context of lipid biosynthesis and their effect on plant growth has been already reported by various researcher previously in different plants. Author only reported the role of rice WRINKLED1 gene lipid biosynthesis and their effect on plant vegetative growth.

My specific comments are as under:

• Information on the confirmation of the integration and homozygosity of transgene in transgenic rice plants is missing.

• Line 189-190:- The author reported that the transcripts level of OsWRI1a in transgenic rice was around 25-35 fold high in leaves tissue (Fig 4D) as compared to control plant leaves. The author generated the OsWRI1a overexpression transgenic rice plant under endosperm specific Bt2 P1 (PBt2P1) promoter (highly expressed in endosperm and lower in leave; line:-174-175). It is not clear as to why OsWRI1a overexpression lines showed elevated OsWRI1a transcript levels in leaves tissue when it is an endosperm specific promoter.

• There is no insight into why OsWRI1a overexpression lines showed retarded plant growth phenotypes.

Reviewer #2: Comments on the manuscript entitled, “Ectopic expression of Wrinkled1 in rice improves lipid biosynthesis but retards plant growth and development”

The authors looked at two rice gene calls with functional annotation with the transcription factor that acts as a global regulator of fatty acid biosynthesis wrinkled1 (Wri1). They demonstrated that as expected the OsWri1a is post translationally targeted to the nuclear compartment using an Arabidopsis GFP fusion platform. The authors then assembled two cassettes to ectopically express OsWri1 constitutively and in a seed preferred fashion. The rationale for doing this is found on line 163 of the manuscript: “Ectopic expression of the WRI1 gene in several plant species results in an increase in oil accumulation in leaves and other organs. To investigate whether ectopic expression of this gene exerts the same effect in rice, we overexpressed the WRI1a gene under the control of the CaMV 35S promoter.”

This is where the problem lies in their communication. There are previous publications relating to expression of the AtWri1 in rice, that include additional alleles to pull carbon towards TAG biosynthesis (AtDGAT/AtPDAT) and protect TAG turnover (AtOle) see Izadi-Darbandi et al 2020 Mol Biol Rep 47:7917, and ectopically express AtWri1 alone in rice see Yang et al 2019 Biosci, Biotechnol, Biochem 83:1807. In the previous publications, significant increase in vegetative tissue fatty acids, with some seed reserve changes, but no phenotypic off types observed. The two key differences between this manuscript and the previous published ones, are ectopic expression of the OsWri1 and the rice genotype. The authors cannot ignore these publications, and will need to address, or at least attempt to explain why the different outcomes. In addition, Mano et al 2019 Plants8, 207; doi:10.3390/plants8070207 communicate on much deeper in silico analysis on rice Wri1 alleles. This publication is also not mentioned in this manuscript.

Secondly, the rationale for targeting lipid biosynthesis in a grain feedstock would be to see if this approach can impact harvest index, and thus grain yield. The outcome of merely stating see what happens, was expected.

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Reviewer #1: Yes: Arun Kumar

Reviewer #2: Yes: Tom Elmo Clemente

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Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and development

PONE-D-22-01261R1

Dear Dr. Jiang,

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,

Tamar Juven-Gershon, Ph.D.

Academic Editor

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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 #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

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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

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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 #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: Authors have addressed my comments satisfactorily. Just found one typo mistake in Figure 3 (Please change Brihgt to Bright).

Reviewer #2: The authors likely observed relatively high miss expression of the Brittle2 promoter given the use of pCAMBIA1301binary vector, for whatever orientation the Brittle2-OsWRI1 cassette was cloned in, the Brittle2 and a 35S CaMV promoter element would reside 5' to 5', which will impact specificity of Brittle2 expression

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr. Arun Kumar

Reviewer #2: Yes: Tom Elmo Clemente