PONE-D-20-35417

Cloning and Functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.)

PLOS ONE

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Keqiang Wu, Ph.D

Academic Editor

PLOS 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: Yes

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

Reviewer #1: N/A

Reviewer #2: I Don't Know

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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: Yes

Reviewer #2: Yes

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4. 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

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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: Overall, this is a good experimental design. Here are some comments for the authors’ consideration -

1. The identified AhLEC1A is a homolog of Arabidopsis LEC1 – has this been supported by sequence alignment in this report or supported by appropriate references?

2. It is a logical alternative to use Arabidopsis for the promoter:GUS analysis, given that peanut was a difficult option to produce transgenic plants. However, the promoter;GUS analysis could be corroborated by tissue-specific RT-PCR to confirm the presence of the respective mRNA. Or, at least the expression of the similar Arabidopsis LEC1 promoter:GUS deletion constructs could be used as a control for a comparative expression analysis of the AhLEC1A promoter components. In absence of either of these supporting studies, the author needs to acknowledge the shortcomings of the promoter:GUS analysis alone in claiming spatial and temporal expression of AhLEC1A. (Ref #35 that claims to have shown expression of AhLEC1 is not available for non-Chinese readers and also it seems to support the Q7 GUS expression only)

Reviewer #2: In the current study, the authors have cloned a 2707 bp upstream sequence of the AhLEC1A gene from peanut and confirmed its transcription start site by 5' RACE. In addition, this study dissected the functions of the different regions of the AhLEC1A promoter under normal and dark/light conditions by using a promoter::GUS transgene approach in Arabidopsis. GUS staining showed that the 2300-bp AhLEC1A promoter (containing 82 bp of 5' UTR and 2228 bp promoter) was a seed-specific promoter and the expression pattern of this promoter may be controlled by several positive seed-specific regulatory elements and other negative regulatory elements. These findings will enhance the understanding of the regulatory mechanisms of the 2300-bp AhLEC1A promoter. I recommend it for publication in the journal after minor revisions.

Some points for the authors to consider in revision,

1. Research on the expression pattern of the LEC1 genes or functional analysis of the LEC1 promoters in different plants should be added in introduction. This will help readers understand the progress of the LEC1 promoter research.

2. The description of the transgenic lines is not detailed enough. The authors have not analyzed whether the transgenic Arabidopsis lines are single-copy or not. The expression and regulation of the transgene is correlated with the copy number of inserted transgenes.

3. The number of the transgenic lines in GUS staining analysis is a little small. Are the expression patterns of most of these transgenic lines (the major expression of the transformants) the same or similar?

4. The authors have cloned a 2707-bp upstream sequence of the AhLEC1A gene from peanut. However, they have not analyzed the activity of this longer promoter. The shorter 2300-bp AhLEC1A promoter (containing 82 bp of 5' UTR and 2228 bp promoter), not the longer 2707-bp promoter, was identified as the full length promoter of the AhLEC1A gene (Page 18, line 246, full-length AhLEC1A promoter (Q7)). Why?

5. The authors are suggested to add the positions of the deleted promoters in Figure 3, which would be more convenient to understand the paper.

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Reviewer #1: No

Reviewer #2: No

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Cloning and Functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.)

PONE-D-20-35417R1

Dear Dr. Shan,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Keqiang Wu, Ph.D

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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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 #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

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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 #1: Dear Authors: thanks for addressing the comments/ feedback. The phylogenetic analysis and the qRT-PCR figure could be a part of the main manuscript. Good work.

Reviewer #2: (No Response)

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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: No

Reviewer #2: No