PONE-D-21-11562

Enhancement of diterpenoid steviol glycosides by co-overexpressing SrKO and SrUGT76G1 genes in <stevia rebaudiana=""> Bertoni.

PLOS ONE

Dear Dr. Nasrullah,

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.</stevia>

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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: 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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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: No

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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: No

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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 authors Nasrullah et al. presented their research entitled “Enhancement of diterpenoid steviol glycosides by co-overexpressing SrKO and SrUGT76G1 genes in Stevia rebaudiana Bertoni”. They have demonstrated the functional characterization of SrUGT76G1 and SrKO by over-expression in stevia via transient or stable transformation. The study is well designed and performed. However, there are some deficiencies which should be addressed to disseminate the detailed information to the scientific community.

Materials and Methods

1. In Materials and Methods headings 2.4 and 2.5 Transient transformation has been repeated twice. It should be incorporated into single heading.

2. For the Heading 2.6 Screening, in results section there is no figure of GFP in plant tissues?

3. Eight commonly used reference genes (RGs) (SrβTUB, SrARF1, SrαTUB, SrUBQ10, SrSAND, SrGAPDH, SrEF-1α, and SrACT7) were selected and their sequences were obtained from the TAIR database (http://www.arabidopsis.org). How the genes of stevia were taken from Arabidopsis database?

Results

1. In the results section “3.3 Molecular analysis of transformed and non-transformed stevia plants” the figure 3 which is a gel picture, is hard to understand. Arrows with PCR product size should be given. One gel picture contains 2 types of PCR products. The figures should be assigned with Lettering.

2. “3.5.9 Analysis of metabolites” It would be better if figure 9 presented as Fig. 9 A, Fig. B and Fig. 9C for the clarity of the results.

3. Under the heading of “3.5.10 Metabolic changes and correlation of metabolite levels” Figure 13 is mentioned. However, there is no figure in the manuscript.

In addition, there are some English grammar deficiencies those should be improved.

Reviewer #2: Introduction:

Introduction part was well written but still contains mistakes related to English grammar. Improve them. Authors should give page number and line number to ease the process of revision, so that errors/mistakes can be highlighted corresponding to specific lines. Some general comments in the introduction part are as follows:

• Authors must define the acronyms MVA and MEP pathways, at their first mention in the text such as Mevalonate and Methylerythritol Phosphate Pathways. (3rd paragraph of introduction)

• Likewise, please mention the acronym KO. The step in GA biosynthesis is catalyzed by ent-kaurene oxidase (KO) leading to the synthesis of…………….. (3rd paragraph of introduction).

Materials and Methods:

Materials and methods section was well elaborated with scientific reasonings; however, it needs some minor improvements as indicated below:

• In subsection 2.1. Chemicals for molecular research were obtained or purchased from….. (Use scientific style of writing).

• In sub section 2.2. Rephrase this sentence “The plants were grown in plastic pots (with a diameter of 31 and a height of 27 cm) in 1:1 ratio of soil and sand in poly house at Jamia Hamdard, New Delhi, India under controlled conditions, in an artificial climate chamber programmed for at 25°C/16°C 16 h/8 h day/night environment with a light intensity of 300 μmol/m2s-1 and relative humidity, 60%”

• Space should be given between a numerical value and unit like 100 mg. Please follow the Journal style for further rectification.

• GFP should be in capital letters. Please replace this in the complete manuscript.

• Why 2.4 and 2.5 subsections shared same heading “transient transformation”??

• It is mentioned in the text 1:1 ratio, which is wrong. The symbol “ : ” represents ratio, therefore no need to say 1:1 ratio. Remove the word ratio in the text.

Results:

This section needs improvements in following aspects as listed below:

• CAMVS35/SrKO ?? Is it CAMVS35 or CAMV 35S? In figure the authors have mentioned CAMV 35S whereas in text it is written CAMVS35. Please use the similar and standard format in the whole manuscript to avoid confusion (see section 3.1 and 3.3 etc..)

• Authors claimed that “The transformed leaves were confirmed by visualising the leaves under Leica TCS SP2 confocal microscope equipped with long-lasting solid-state lasers (Leica Microsystems, Germany), BUT NO GFP visualization of transiently expressed plant tissues has been given in the form of images in the manuscript? Please elaborate?? If GFP has been visualized through microscope, it must follow by proper images. Authors must give present appropriate images of GFP visualization (sec 3.1)

• Principal component analysis (PCA) of the stevioside and rebaudioside-A gave a clear cluster pattern (Tag this line with a relevant figure?) (sec 3.1)

• Relevant figures and tables should be mentioned and tagged in section 3.1. The results of increase and decrease of quantification studies and co expression results have been discussed without mentioning pertinent figures.

• Section 3.4 gave most of the details about How the morphological analysis of shoots and leaves were carried out. This is specifically a part of materials and methods section. Please focus on the results obtained in this section.

• The result presented in Fig. 5 does not correlate with these lines in the text (In the transformed leaf, SrUBQ10 has a minimum average Cq value of 15.62, while SrSAND has a maximum average Cq value of 22.83). Please check it again and took help of statistician.

• Fig.13: Correlation table shows the high correlation between steviol and rebaudioside A is not present in the manuscript.

Discussion:

In this section, please give more reasonings about why the co-overexpression of both genes resulted in decrease of stevioside content, whereas increases RebA amounts in comparison to individual expression studies. Since, no change in RebA to stevioside ratio was observed in overexpression of SrKO. Support your results by giving reasonings and explanations from the biosynthetic pathways of both genes involved or their homologs in other plants.

General Reviewers Comments:

Improve English grammar and sentence structure of the manuscript. Make all necessary corrections as indicated above in each section of the manuscript. Keeping in view the significance of stevia plant and originality of research conducted by the researchers, the study holds significance and is worthy enough to be accepted in the Journal after a major revision.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D 21 11562_Reviewer comments.docx

Enhancement of diterpenoid steviol glycosides by co-overexpressing SrKO and SrUGT76G1 genes in <stevia rebaudiana=""> Bertoni.

PONE-D-21-11562R1</stevia>

Dear Dr. Nasrullah,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Allah Bakhsh

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: (No Response)

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: Yes: Dr. Muhammad Naeem