PONE-D-20-34002

Quantitative analysis of the seven plant hormones in Lotus japonicus using standard addition method

PLOS ONE

Dear Dr. Akashi,

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2This work was supported by Toyota Motor Corporation and the National BioResource

Project (NBRP) of the Japan Agency for Medical Research and Development (AMED)."

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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: 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: In this work, Hashiguchi and colleagues present a methodological manuscript in which they describe the quantitative analysis of seven plant hormones in Lotus japonicus roots, shoots and stems, with high-resolution mass spectrometry (HR-MS).

They showed an important technical analysis about the impact of matrix effect in the purification and quantification of hormones that can vary between 10 and 87%, therefore ion suppression can largely decrease the peak area of phytohormones. And among the main results, they demonstrated that there were no significant differences between standard addition and stable isotope addition methods, therefore allowing to quantify hormones without any treatment with stable isotopes.

Altogether the work is technically sound and the approaches are of wide interest for the community.

A further validation of the methods on a perturbed system would add some extra value to the work. For example the Authors could challenge the plants with one relevant hormone-inducing stress (just as an example drought stress to induce ABA) and further compare ABA concentration with a physiological condition. Another option would be to test a Lotus japonicus known mutant for any hormonal biosynthetic pathway.

In addition to this, I have a few specific comments:

Figure 3: Please do not use bar graphs but show all the measurements (refer to Weissgerber et al., 2015, Plos Biology, https://doi.org/10.1371/journal.pbio.1002128): this would

Finally to better validate the method, a calculation of repeatability (accuracy and precision) obtained during the validation of the method could constitute an added value. By repeating the same measurements on the same starting material how much variance are you observing? Such as done in the cited work by Trapp et al., Frontiers in Plant Science 2014. I think this is particularly important if the Authors are not adding any experiments on Lotus mutants and/or stress conditions that are inducing different hormonal changes.

As a suggestion, to improve the readability, consider to change the paragraph title by stating the key results obtained in that paragraph. For example instead of: “Quantification of plant hormones by standard addition and comparison of the two quantification methods” you could state that there is no significant difference between the two methods.

line 45-46: There are more than 8 groups of hormones: please, at least, also consider strigolactones

line 340-343: repetition of a full sentence, please correct it.

Reviewer #2: The paper “Quantitative analysis of the seven plant hormones in Lotus japonicus using standard addition method” is an interesting methodological manuscript which aims to compare standard addition and stable isotope dilution method to quantify plant hormones in different plant tissues. The authors have utilized leaves, roots and stems of Lotus japonica to test if the standard addition method could be a robust and accurate method to quantify plant hormones in plant tissues. The work was conducted utilizing a rigorous methodology and furnish a validated method to quantify plant hormones, in particular when it is difficult to find the appropriate internal standard. However, I have two main comments:

- the method is more complex, from a methodological point of view respect to the method of internal standard. I think this should be mentioned in the discussion (in addition to highlight the strong points of this method);

- the discussion should be revised in a more logical way, starting from the role of hormones and their importance in AMF- and bacterium-plant interaction (line 314-329) and then moving to the discussion of the results presented in the paper (validation of the standard addition method and comparison with the internal standard method) (line 295-313 and 330-345).

I suggest to revise also some minor points:

-line 26 “THEIR content levels vary depending on the species, and THEY also change...” I think this sentence is referred to plant hormones.

-line 55 “ionizing hydrophobic analytes”..not all plant hormones are hydrophobic substances (ex. salicylic acid, abscisic acid)..I suggest to revise this point

-line 107 “Extraction for plant hormones has BEEN CONDUCTED UTILIZING...”

-line 136: the authors have already described that extracts were purified using SPE. Here it can be said “Purified extracts were separated ....”

- line 328-329: the last sentence of the paragraph is very speculative. I suggest to revise this sentence.

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Reviewer #1: No

Reviewer #2: Yes: Cecilia Brunetti

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Quantitative analysis of seven plant hormones in Lotus japonicus using standard addition method

PONE-D-20-34002R1

Dear Dr. Akashi,

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,

Raffaella Balestrini

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: Please consider to correct line 351. You wrote: "the method would be evaluated using L. japonicus" but it could be better to write that it will be important to validate the method with using...

Reviewer #2: In this revised version of the paper, the authors have addressed all the concerns and suggestions. I am satisfied by all changes carried out and endorse the publication of the manuscript.

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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: Marco Giovannetti

Reviewer #2: Yes: Cecilia Brunetti