PONE-D-20-04662

Phosphorus assimilation effects by root exuded compounds across plant developmental stages

PLOS ONE

Dear Dr. Vivanco,

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.

Generally both reviewers liked your paper and neither suggested or required new experiments. However both of them made comments about the presentation and language. Reviewer 1 brings up some fairly substantial conceptual points. Please consider these and revise your text accordingly . Reviewer 2 has a large number of small but interesting comments and suggestions. The more of these that you can attend to, the better your paper will be. 

In addition, I read parts of your paper and have a few comments of my own. 

Line 1. Title. Your title is difficult to parse. Please rewrite. For example: “On the role of root exudates on the assimilation of phosphorus in young and old roots”. 

Line 26, and elsewhere. Please spell out phosphorus everywhere in the text. Do not use “P”. I am aware that many authors use this abbreviation. However repeated use is a poor argument for validity. In fact, abbreviations and acronyms make a text difficult to read. Abbreviations rarely occur in newspapers or fiction. Any time a reader encounters a symbol (P is a symbol for phosphorus), they must translate that symbol into words. Translation takes mental energy away from comprehension. Translation slows down the reader and gives nothing in return. It is one thing to have to write about a chemical with a name that is 38 syllables long. In that case, the name is just as difficult to read as the acronym. However phosphorus is a good word of the English language. In fact, phosphorus literally means ‘carrier of light’. Rather beautiful. Spell it out. 

Line 26. “… have not been conducted…” This wording implies that you have read every paper ever published. Use less extreme wording, such as “…rarely if ever…”

Line 29. You are using the word ‘metabolome’, to refer to root exudates. This is misleading and confusing. The word metabolome refers to all of the metabolites in the cell or organism. However here, you are not measuring the metabolome. Instead you are measuring exudates. These compounds number about a dozen, far less than the threshold for ‘omics’.  Please remove the word ‘metabolome’ throughout the paper and instead talk about ‘total exudates’ or the equivalent.  For example, the sentence staring at line 29 can read: “The composition of root exudates changed in response…”

Lines 29 and 34. You write ‘in vitro’ conditions. What does this mean? Instead say what the conditions are. By the way, typically in vitro implies isolated components, cells or cell fragments. I have never seen in vitro used for whole organisms. Also you write ‘solid’ and ‘liquid media conditions’. Again, be specific, say what the solid and liquid media are. 

Line 31. What does ‘respond negatively’ mean? If you mean decreased in quantity, just say so. 

Line 66. Based on long-established rules for scientific nomenclature, the word “Arabidopsis” (Capital A, italic font) means the genus. No matter how many illiterate molecular biologists make this mistake, rules of taxonomic nomenclature remain in force until the international committee decrees otherwise. Longstanding practice allows “arabidopsis” (lower case a, Roman font) as the common name of our friendly lab weed; however, many journals will auto-correct this by adding a capital and italics. I am not sure about PLoS One. Thus, either use ‘arabidopsis’ and hope the journal lets it stand, or use A. thaliana(italics) (but Arabidopsis thalianaat first mention). 

Line 83. This should read “Arabidopsis thalianaL. (Heynh) wild-type Columbia seed…” It is customary in the Material and Methods to give the full Latin binomial along with the taxonomic authority. And unless you sequenced your Columbia line, you should drop the “0”.

Line 84, and elsewhere. Do not use circle-R, or TM, or other commercial symbols. Those marks exist to protect consumers (not manufacturers) from fraud. Thus if you sell a product that you say contains X circle-R, you are defrauding the consumer if you substitute something cheaper for X. There is no such issue here. In fact you are not selling anyone anything (you are paying). 

Line 98. I don’t understand the meaning of the word ‘power’ here. 

You embedded the figure legends into the results text but not the figures. I think this is awkward. Please put the figure legends with the figures, preferably at the end of the text. Also for the principal component analyses, please simply call your x and y axes “PC1” and “PC2” and put the percentages of explained variance in the figure legend. 

In figure 3A, the names of some compounds are capitalized but others are not. And “control” is in all caps. This is bizarre. I suggest that none of them should be capitalized, but whatever you do please do the same for all. And note that if you choose to capitalize the first letter, this should be done even for those compounds that start with a number (e.g., “5-Aminovaleric acid”). Also, please put the name of the horizontal axis below, next to the numbers (or put the numbers above, next to the name; the point is, the numbers and the name belong together. 

Also please remember that the abbreviation for liter is L (not l). And to always put a space between the number and the unit (except when the unit is the degree sign or the percent sign). 

In Figure 4, there are problems with the y-axes. Many of them have huge numbers. But no units. I think peak area is arbitrary so you could use 1, 2, 3. If it is needed to compare the quantity between states, then you could have the smallest numbers be 1, 2, 3 and the other states relative to that. Also many of the graphs do not start at zero. But some of them do. This is misleading. They should all start at zero. The names of the axes could be in a larger font. 

We would appreciate receiving your revised manuscript by Apr 27 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Tobias Isaac Baskin

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

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: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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: Two minor and three major critics

minor:

1. Root exudates depending on plant develpment were reported by Keerthisinghe et al. 1998, Plant Cell Environ., 21, 467- 478 and by Neumann et al., 1999, Planta, 208, 373- 382.

2. L 278 ff. Misleading since P and carboxylate concetrations must be clearly separated.

major:

1. Misleading: P starvation response 50 µM P. Cultivation in non buffered systems, agar or solution means that a solution threshald is only valid for a special volume. In soil P is buffered and P starvation values are below 2 µM P , see Föhse te al., 1988, Plant Soil, 110, 101- 109.

2. Decisive for the solubilizing ability of root exudates is the relation between soil solid P and the quatity of exudates (see in detail Gerke, 2015, cited in the ms). Solution concentrations are not an appropriate measure of exudate efficiency. Mesurement of pH changes as a central factor of P mobilization from Ca-P forms?

3. The relevance of the results with respect to P solubilization is questionable since the P form is rather unrepresentative and the exudate quantity is rather high.

Reviewer #2: General comments:

The manuscript “Phosphorus assimilation effects by root exuded compounds across plant developmental stages” characterises root exudates across growth stages and P-levels for Arabidopsis. Interesting contrasts were found. The authors select some important exuded compounds and go on to test their ability at solubilising P in soil-analogues individually and together. This was a well written manuscript, with well thought out experiments, executed well leading to interesting results. I really enjoyed reading this manuscript, I learnt a lot that is relevant to my work, and appreciated the work that went into it. I have some general comments that I think would improve the presentation and some of the discussion.

1) I think in the materials and methods the presentation of the PCA is sometimes over complicated, and could be made simpler. My detailed comments below would address this.

2) The presentation of the PCA results could be improved. I think showing all the data with no groupings used on the PC-axis (with different colours and marker styles and legends!) would make it much clearer an. This could come at the expense of some of the plots where treatments are grouped. Also make it clear if a PCA is done per grouping or not. Again, many of my detailed comments try and address this.

3) The discussion would be improved by quickly pointing out the agricultural/ecological significance (or lack of) of the results. For example 7mM of acids order of magnitude more than what is found in the rhizosphere. See the Eva Oburger papers I referenced. Hence the solubilisation seen here might not appear in the field. One of our group's papers found that P solubilisation by a single root exuding citrate at a realistic rate actually made no difference to P uptake by the root https://doi.org/10.1007/s11104-019-04376-4 (dont feel you have to reference this). It also might be worth pointing out how similar NBRIP media is to soil, solubilisation in this media might not be the same as soil. Also see my detailed comment regarding your proposed mechanism number 1.

Sorry, I have a lot of detailed comments, however many of them are pointing out the same thing and many are complimentary. I hope they aren’t too hard to deal with.

Detailed comments:

L25: roots->root

Abstract is clear and well written.

The paragraph starting line 67 doesn’t segway smoothly into that starting on 73 because root exudates have been shown to be less important for good P conditions (not to say that the paragraph on L67 isnt useful introductory information). Calling back to the ‘legacy P’ argument here would make it smoother.

L90: ‘gradient’ isn’t the best word here. It makes it seem like the growth media each plant is grown in has a gradient of P conditions in it. Is ‘The low P conditions used in this study….’ Better?

L129: ‘…which compounds had the highest correlation with these components through the magnitude of the variance’. ‘correlation’ is unclear here. Do you mean which two original basis vectors (compounds) explained the most variance? This would be an easier way of explaining it.

L133: ‘The largest…’ Is it not that these compounds explain the most variance? Its best to write these in terms of what percent of the original variance they explain (before dimension reduction).

L134: differential isn’t the right word here.

I think the end of this paragraph can be explained in a simpler way by saying: “We determined which compounds explained the most variance for each P level and growth stage. The contrasts in these ‘most principle compounds’ over growth stage and P level indicated differences in exudation depending on these factors” Or something like this.

L144: This needs more explanation this is quite a large range, how did you ‘adjust’ the concentrations? The reader needs to know how this is done to interpret the results. Did you take concentrations from these references to calibrate your values? Due to the variability in reported exudate concentration due to sampling technique (https://doi.org/10.1016/j.rhisph.2018.06.004) ‘exudate concentration’ is relative to the study. Probably needs a mention in the discussion.

Side note: Could be worth mentioning that you didn’t adjust the concentrations for the PCA, when I first read it I thought you had.

L152: What concentrations did you add to test the P solubilising ability of the compounds? Were they comparable to rhizospshere concentrations? Knowing this puts this into an ecological context for the reader. Is the visual inspection for solubility standard? Add a reference if so. Otherwise explain the reasoning.

Table 1: were they diluted to 100mM before PCA? Would this effect PCA? (see my side note above). 100mM is a really high concentration for most root exudates in the rhizosphere no? I know for most organic acids and amino acids I would expect micro molar concentrations in the rhizosphere, see https://doi.org/10.1016/j.envexpbot.2012.11.007

L171: again this a high concentration for the rhizosphere, I would expect a discussion on the ecological relevance later.

L181: I think ‘cumulative’ effect is better than ‘additive’. Additive makes it seem like you could add up the effects individually to determine to summed effect.

L200: unclear what ‘distribution’ means here. After looking at FigS1 we see it’s the groupings after plotting on the first two principle components. This needs to be made clearer. I think the author means the separation of growth stage and P treatment by PCA persists after throwing away the non-annotated compounds. Which implies the non-annotated compounds explained little of the variance, which I believe justifies the author’s conclusion. Were the principle components determined twice i.e. before and after throwing away the non-annotated compounds? FigS1 could use a legend saying what the other colours are.

Fig 1A also needs a legend.

In Fig 1A did you group by developmental stage, do PCA then plot? If this is the case, the % variance explained by the PCs reported in line 203 isnt correct.

Same goes for Fig 1B but by P level? The different percent of variance explained by components suggests this is the case. Need legend for Fig 1B also.

I would use colours to indicate developmental stage and stars/dots/crosses to indicate P-levels (or vice versa). This and a legend would make fig 1 a lot easier to understand.

Fig 1A caption is wrong “201 annotated compounds with proper identification detected using GC-MS are plotted on the Principal Component Analysis (PCA) graph”. The compounds aren’t plotted. The compounds are the 201-axis, the dimension is then reduced to 2-axis with PCA and the locations of the growth stages are plotted.

L205: “We determined that the plant’s developmental stage was responsible for

the separation of the compounds in three marked groups” to convince the reader that its not P level you would need to not group by anything and plot all the data with stars/dots/crosses and colors.

All these figures desperately need legends (Fig1, S1, S2) and clearly state if there is a PCA per grouping.

L230: explained the most variance.

L231: ‘correlation’ isn’t really defined here. Its more that the compounds which point in the most similar direction to the two principle components, I would get rid of the bit after the comment.

Change this sentence to: For each of these treatments, we found the five top compounds that explained the largest proportion of the variance.

Figs S3-S4 are really intresting! Great results

Same goes for Fig 2 and 3A

L254: does the 1.75 7 and 28 mM mean that the sum of the concentrations or each have that concentration? Fig 3 caption makes this clear but also make it clear in the manuscript.

L255: The 1.75M is really interesting, 7mM of them mixed does worse than 7mM of any individually

Again, really interesting results with Fig 4, very hard to interpret though there is lots going on.

L300: …’where optimum conditions are scare’ -> where nutrients are scarce ??

L304: These papers might be useful for this discussion on maize need for P early on with respect to yield: https://doi.org/10.1007/s11104-011-0814-y. https://www.nrcresearchpress.com/doi/pdfplus/10.4141/P00-093) Take them or leave them

L309: ‘similarity’ do you mean they grouped close together in the PSA? To talk about similarity you would need to use a norm between the treatments.

L323: I think your mention of other functions of exudates is a good explanation and something other authors overlook.

346: that ‘the’ shouldn’t be there

L351: new paragraph

L352: mechanism 1. Your results suggest there is no synergy (in fact the opposite) in terms of P solubilisation by root exudates, see my comment about L255, at least for the 4 acids you picked out. But this doesn’t disprove your mechanism: I think I read somewhere that certain exudates solubilise P from certain soil surfaces, Al oxides, Fe oxides etc (possibly a paper by Gerke about citrate and malate?) Maybe this is why they exude a mixture of acids? Did the soil-analogue you used have this variability in mineral surfaces which soil has?

Nonetheless, I think this deserves some more discussion.

L357. I like point 2, I had never heard of this before.

I don’t think Table S1 is mentioned in the manuscript

**********

6. 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. Jörg Gerke

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-04662R1

Role of root exudates on assimilation of phosphorus in young and old Arabidopsis thaliana plants

PLOS ONE

Dear Dr. Vivanco,

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.

We are almost there. 

First I apologize for the length of time this step has taken. In addition the reviewers being late, there was a peculiar piece of administrative weirdness that I had to resolve before sending you the decision. That’s all set now. 

As you can see, reviewer 1 is totally satisfied and reviewer 2 raises only a couple of minor points. In fact, reviewer 2 has a very long comment about the ‘additive’ effect but in the revised manuscript, you have downplayed this, so I think most of the comment is moot. You may consider it as you see fit. However, reviewer 2 writes:

“I note the paper “Does the combination of citrate and phytase exudation in Nicotiana tabacum promote the acquisition of endogenous soil organic phosphorus?” which you cite for the synergy of exudates for P solubilisation is an experiment done in soil not NRIB.” Clearly this needs your attention. 

and writes:

“I think when describing the method for the cumulative experiment, you should make it clear whether the mixtures were all  added to 5 mL of liquid NBRIP so that the same amount of P is the same as the individual experiment. Currently it is unclear.”  And this too should be fixed. 

I also read the paper carefully. I uploaded the pdf with my edits. Most of these are places where your English is non-idiomatic or other small problems of style. I also added a few comments in places where I simply could not understand what you meant or where there was some other small problem. Where I understood, I offered a solution. I am not going to repeat them here but please go through them carefully as you revise. 

Two of these comments though I want to bring up here for emphasis. 

This first is the difference between phosphorus and phosphate. You use ‘phosphorus’ almost everywhere but I think this obscures an important difference. In experiments, you are fertilizing the plants with phosphate. I think it is sloppy to talk about adding “1 mM phosphorus” to the plants when you added 1 mM phosphate. Plants take up phosphate, not phosphorus. Likewise, your compounds act to dissolve phosphate. They do not free elemental phosphorus. It seems misleading to describe them as phosphorus-solubilizing compounds. Instead, phosphorus is more of a concept. It is reasonable to write about ‘phosphorus deficiency’ or like that. In some places, the choice is a bit arbitrary. In my edits, I changed ‘phosphorus’ to ‘phosphate’ where the sense was the specific chemical involved, which actually it usually was. 

The second comment concerns figure 3B where you check the solubilizing activity of combinations of the key compounds. The problem here is that you test the single compounds at 7 mM (Fig. 3A). But you test the combinations at that concentration, and also at lower and higher concentrations. I do not see how the higher and lower combinations can be interpreted without also showing values for the single compounds at each concentration. Without that data, I suggest adding the 7 mM combined results as the last bar of figure 3 A and deleting the data for the other combinations (unless you happen to have data for single compounds at the alternate concentrations). By the way, given that the combination solubilized about the same amount of phosphate as did 3-hydroxyproprionate alone, I don’t see how you can even think about an additive effect.

When you submit your revised ms, please do NOT use track changes for any of my edits on the text that you accept with no change. Instead, track only those places where you either do not use my edit at all or you modify it in some way. Also track any changes you make in response to reviewer two. 

All of this should be very straightforward and I expect the next round should go very much faster. 

We would appreciate receiving your revised manuscript by Jun 22 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. The journal requires you to say something but you do not need to go thru all the small things. Just comment on anything where there is some disagreement, etc. 
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. Please see my note above about how to handle the tracking. 
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Tobias Isaac Baskin

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

**********

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: I Don't Know

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 Ms should now be published in the present form.

One answer to the question the authors made:

Ca- phosphates may account for a high proportion of P in calcareous soils. However even in these soils as well as in other soils often P adsorbed to Fe/Al- surfaces is the oprincipal form of available P. This may be the case because in many soils P desorption from the soil solid phase is konetically and not thermodynamically controlled (see Gerke, 2015 and reference there).

Reviewer #2: Comments to the authors and editor:

Thank you for considering my comments so thoughtfully in the response. I believe the authors have answered the vast majority of my comments, I only have one outstanding query regarding the following in the response document:

'''

L255: The 1.75M is really interesting, 7mM of them mixed does worse than 7mM of any

individually

Again, really interesting results with Fig 4, very hard to interpret though there is lots going on.

RESPONSE: Each compound has a different molarity; therefore we cannot say that the 4 compounds at

1.75 mM add up to 7mM. We conclude that 1) compounds in combination have an additive effect, and

that 2) the addition of one does not inhibit the effect of the other.

'''

I think concluding there is an ‘additive effect’ is a bit miss leading. Here is the standard definition of an additive affect https://www.dictionary.com/browse/additive-effect. The sum of 7mM individually is much more than 7mM combined (I use the 7mmol case now so there is no issues with the molarity. Both cases has 7mM of each.) thus it is not an additive system. This is of course because in the individual cases are done in separate assays and they solubilise the lightly-bound P pool 4 times, while the combined is done in one assay. I think if you want to convince the reader that they are ‘additive’ (and later ‘synergize’ L348 in the discussion) you need to define exactly what it means for the current system to be additive or synergize. Possibly the current experiments and media aren’t suitable to make this conclusion. As I mentioned before, I suspect the compounds ‘synergise’ (I define this to mean to do better than the sum of its parts) when the media has a range of minerals that buffer P and are attacked more efficiently by certain exudates. For example (this is entirely hypothetical) assume the soil is made up of 50% Al-oxide and 50% Fe-oxide and nicotinic acid solubilises P from Al-oxide very effectively but badly from Fe-oxide and malic acid vice-versa. In this case the two compounds would certainly synergise. As I mentioned before I think there is papers about this but I cannot remember, the other reviewer Prof Gerke is likely to know about this and can point you in the right direction. I note the paper “Does the combination of citrate and phytase exudation in Nicotiana tabacum promote the acquisition of endogenous soil organic phosphorus?” which you cite for the synergy of exudates for P solubilisation is an experiment done in soil not NRIB.

I do not think more experiments or analysis is needed as this is a minor result in the overall paper, but a more careful discussion of this is needed in liight of the results.

I do agree with conclusion that the addition of one does not inhibit the other which is a good result in itself.

Detailed comments:

L187 (on tracked document): Should ‘A phosphorus gradient’ here instead be a ‘compound gradient’?

I think when describing the method for the cumulative experiment, you should make it clear whether the mixtures were all added to 5 mL of liquid NBRIP so that the same amount of P is the same as the individual experiment. Currently it is unclear.

I would like to point out to the editor that when responding to one of my comments they also partly answered one of reviewer one’s, this wasn’t clear in the authors response. In particular, reviewers one’s comment:

‘The relevance of the results with respect to P solubilization is questionable since the P form is rather unrepresentative and the exudate quantity is rather high’

Is partly answered by the author’s response to one of my comments in the same vain:

We have added a paragraph that discusses the relevance and ecological significance of organic acids as phosphorus mobilizers and the relevance of the root exudate analysis in liquid cultures. The new paragraph reads as follows: ‘It has been estimated that organic acids constitute 5 to 10 % of the total organic carbon in the soil solution. The concentration of organic anions measured in the soil solution usually range from 100 nM to more than 580 uM in the rhizosphere of cluster roots (Jones, 1998). However, millimolar concentrations of organic anions are likely required in the soil solution to effectively increase soluble P concentration especially in calcareous soils (Strom et al. 2005; Ryan and Jones, 2001). Strom et al. (2005) tested three organic acids (citrate, malic and oxalate) and a wide range of concentrations (1 mM to 100 mM) to evaluate its effects on the mobilization of phosphorus in calcareous soil. The results showed that the phosphorus mobilization of the tested compounds had a low efficiency and its effect varied depending on the type of organic acid, compound concentration, and pH. Further, due to the low phosphorus mobilization efficiency of those compounds it is still argued if the benefit of releasing large amounts of organic acids into the soil will exceed the cost of carbon lost by the plant, which can be seen as an unnecessary trade-off (Strom et al. 2005). However, low efficiency organic acids can be particularly important in phosphorus mobilization for calcareous soils with a limited phosphorus availability for plants. Finally, our evidence supports the above-mentioned hypothesis, that plants release a combination of compounds with different phosphorus-solubilizing efficiencies, at specific stages of growth, to deal with particular phosphorous needs.

**********

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

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: PONE-D-20-04662_R1TBedts.pdf

Role of root exudates on assimilation of phosphorus in young and old Arabidopsis thaliana plants

PONE-D-20-04662R2

Dear Dr. Vivanco,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Tobias Isaac Baskin

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: