Responsivity of Two Pea Genotypes to the Symbiosis with Rhizobia and Arbuscular Mycorrhiza Fungi—A Proteomics Aspect of the “Efficiency of Interactions with Beneficial Soil Microorganisms” Trait
, Alexander Tsarev 3, Anton S. Sulima 4, Oksana Y. Shtark 4, Christian Ihling 2, Alena Soboleva 1, Igor A. Tikhonovich 3,4 and Vladimir A. Zhukov 4,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study focuses on characterizing the alterations in the root proteome of highly responsive pea genotype K-8274 and low-responsive genotype K-3358 grown in non-sterile soil, which were associated with root colonization by rhizobia bacteria and arbuscular mycorrhizal fungi compared to proteome shifts caused by soil supplementation with mineral nitrogen salts. The results show that supplementation of the soil with mineral nitrogen-containing salts switched the root proteome of both genotypes to assimilate the available nitrogen, while processes associated with nitrogen fixation were suppressed. The most pronounced response was observed in the highly responsive K-8274 genotype inoculated simultaneously with rhizobial bacteria and arbuscular mycorrhizal fungi. This response involved activation of proteins related to redox metabolism and suppression of excessive nodule formation. In contrast, the low-responsive genotype K-3358 demonstrated a pronounced inoculation-induced suppression of protein metabolism and enhanced diverse defense reactions in pea roots under the same soil conditions. This is a well-designed research study. However, many details should be revised.
Line 144 should include a sentence indicating that the result was based on proteins selected for PCA.
Line 155 should show the 0-coordinate point for the x and y axes.
Line 222 should use genetic names such as K-8274 or K-3358 to clearly present these results.
Line 162 states that PCA accomplished for all samples obtained from the K-3358 plants revealed the separation of the four treatment groups with 53.4% and 13.4% of the total variance for PC1 and PC2, respectively. Where is the 2.1%?
Author Response
We thank the Reviewer for thoughtful review and highly appreciate the valuable comments and suggestions to improve the manuscript. Following these advices we performed all required changes in corresponding sections, as indicated in the following rebuttal addressing each aspect. We uploaded also all raw data in the PRIDE repository and provided appropriate data availability statement. The quality of language is checked with a native speaker.
All changes are marked in the text with blue and the corresponding remarks are specified with comments to the remarks of Reviewer 1.
Remarks
Remark 1
Line 144 should include a sentence indicating that the result was based on proteins selected for PCA
Answer: No, there was no selections in the experiments meant in the line 144. We are sorry for being not enough precise. We correct it here: “…which were applied to the whole annotated proteome”.
Remark 2
Line 155 should show the 0-coordinate point for the x and y axes.
Answer: Unfortunately, we could not find the quantitative table to reproduce the figure 2. We don’t have time for new processing of the raw data (which are available now also in repository) we decided to remove the PCA part from the manuscript. All other quantitative data is represented in supplementary information and remains available. We are convinced, that this did not affect the quality of the manuscript and the conclusions done.
Remark 3
Line 222 should use genetic names such as K-8274 or K-3358 to clearly present these results.
Answer: We have added genetic names to make the text clearer
Remark 4
Line 162 states that PCA accomplished for all samples obtained from the K-3358 plants revealed the separation of the four treatment groups with 53.4% and 13.4% of the total variance for PC1 and PC2, respectively. Where is the 2.1%?
Answer: These 2.1% could be attributed tot he differences in other proncipal components, starting from PC3. We complemented the text to make it more clear: “…while the rest 2.1% could be attributed to further principal components”.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authorsplease see paper for sticky notes
the paper could be edited for better reading
but the work is in an area that has wanted sound molecular analyses for a long time
can you add data on the biology of the response- you know the outcomes the reader does not
a summary diagram would be useful
why do you think the two cultivars have these differences
suggestions are made to shorten the extensive lists of enzymes /proteins that change and putting them into functional groups ie it would help to do more discussion earlier
the supplemental data were not available
for me the discussion v results ratio were not optimal
any inferences of what might be happening with P?
discussion of phenolics as signalling molecules gating the processes of recognition and then symbiosis again summary diagrams useful
Comments for author File: Comments.pdf
the paper requires an edit and i think simplification of data sets otherwise there is a huge overload of facts
Author Response
We thank the Reviewer for thoughtful review and highly appreciate the valuable comments and suggestions to improve the manuscript. Following these advices we performed all required changes in corresponding sections, as indicated in the following rebuttal addressing each aspect. We uploaded also all raw data in the PRIDE repository and provided appropriate data availability statement. The quality of language is checked with a native speaker.
All changes are marked in the text with green and the corresponding remarks are specified with comments to the remarks of Reviewer 2.
Remarks
General
General remark A
can you add data on the biology of the response- you know the outcomes the reader does not
Answer: We are absolutely agree with the reviewer - we extended the beginning of the Discussion part and added some relevant information- In the referred publication (Afonin et al) our earlier biological data is provided
General remark B
a summary diagram would be useful
Answer: We absolutely agree with the reviewer! Moreover, we are convinced that it must be a graphical abstract. So, now such a summary figure - a graphical abstract, it is provided
General remark C
why do you think the two cultivars have these differences
Answer: we added the explanation to the Conclusion part. We believe that these differences reflect different ecological strategies of plants that can be more or less beneficial depending on the environmental conditions and/or biotic/abiotic stress factors.
General remark D
suggestions are made to shorten the extensive lists of enzymes /proteins that change and putting them into functional groups ie it would help to do more discussion earlier
Answer: We agree with the reviewer - it would be much more convenient. Please, see Supplementary information 4 for all proteins functional groups summarized
General remark E
the supplemental data were not available
Answer: We checked it - now supplementary information provided
General remark F
for me the discussion v results ratio were not optimal
Answer: The Discussion part is extended as suggested by the Reviewer
General remark G
any inferences of what might be happening with P?
Answer: We did not detect signs of the phosphorous starvation in any of the studied proteomes, so probably the amount of the phosphorus in the soil was sufficient for pea plants (note that this non-sterile soil contained indigenous microorganisms, including many rhizobial strains and many AM fungal isolates). Therefore we think that the effect of inoculation, at least at the sample collection stage (4 weeks after planting) may be connected with induction of plant defense rather that with nitrogen and/or phosphorus supply of plants with microsymbionts.
General remark H
discussion of phenolics as signalling molecules gating the processes of recognition and then symbiosis again summary diagrams useful
Answer: We added some information to the Discussion section, particularly about high- and low-EIBSM pea genotypes that differ in expression of genes related to flavonoid biosynthesis.
Other remarks
Remark 1
Replace “we decided for” with “we used”
Answer
Changed accordingly
Remark 2
Remove lines 87 - 89
Answer
Changed accordingly
Remark 3
Don’t understand explain what you mean by group
Answer
Here we mean the dispersion within one treatment group – i.e. dispersion within one group of plants grown under the same soil conditions. The appropriate text is provided: “i.e. dispersion within one group of plants grown under the same soil conditions”
Remark 4
Replace “could be” with were
Answer
Changed accordingly
Remark 5
Provide as Venn diagrams with all groups
Answer
We agree with the reviewer, such presentation is quite helpful - the corresponding Venn diagrams are presented now as Figure 1, S1-3 and S1-4
Remark 6
Change to italic
Answer
Changed accordingly
Remark 7
“Proteins” instead “protein”
Answer
Changed accordingly
Remark 8
You mean noduline 7 or what?
Answer
We mean here annotation as it given by the database search –“early noduline 7”
Remark 9
Replace “thereby” with “the”
Answer
Changed accordingly
Remark 10
Check throughout for unneeded - b etween words several of them
Answer
Changed accordingly
Remark 11
Replace with “used”
Answer
Changed accordingly- the text is changed: “For the functional annotation of the differentially expressed P. sativum proteome Mercator 4 v.2 software was used.”
Remark 12
Bins of??
Answer
Here we mean the word “bins”
Remark 13
Could these long lists be put into a table-to compare contrast between bacterial types-then the text could be used to express highlights of the findings
Answer
We are absolutely agree with the Reviewer. The requested tables are provided as Supplementary materials 3 and 4- Due to their comprehensive character we decided for supplementary, but not the main text
Remark 14
ie its the whole cell that is responding
not unexpected though since many metabolites nip in and out of organelles etc
Answer
We thank the Reviewer - this consideration is incorporated- The text is provided: “The results demonstrated that the whole cell that responded due to strong metabolic interconnection of the organelles.”
Remark 15
What is missing in text for me is a comparison table of the extent of nodulations and the amount of AMF colonization. There should be some discussion of potential suppression or activation of host defense responses. Rhizobia and AMF both will enhance P in the plant too. See Jeff Dangls review of how P levels affect defence responses
Answer
Unfortunately, no data on mycorrhization intensity were obtained in the experiment, therefore we can only hypothesize about the suppression or activation of the host defense responses due to inoculation. We added the discussion of possible activation of plant defense priming in k-8274 plants due to inoculation with AM, and cited and discussed several additional papers, including the review of Jeffery Dangl.
Remark 16
Specify the source of mineral nitrogen
Answer
Mineral nitrogen was used as ammonia nitrate - see now specified in the text
Remark 17
Please add data of nodulation and colonization
Answer
The data on nodulation has already been published (Zhukov et al., 2017; we cite this paper several times). The data on mycorrhization were not obtained in that experiment due to technical reasons.
Remark 18
Check spelling throughout
Answer
Spelling is checked
Author Response File: Author Response.pdf
