Physiological Responses to Fe Deficiency in Split-Root Tomato Plants: Possible Roles of Auxin and Ethylene?

Round 1

Reviewer 1 Report

In the paper “Physiological responses to Fe deficiency in split-root tomato plants: a possible crosstalk between auxin and ethylene?” Celletti et al., provide data suggesting that tomato plant sensing response to iron concentration might involve a crosstalk between auxin and ethylene phytohormones. The first and last authors have extensive experience in this subject.

The paper is beautifully written; therefore, I do not have any concern on the English language. The data are presented only once and the methods are described in a sufficiently clear way.

Nevertheless, I have some comments on the content and require at least one more experiment before accepting the manuscript for publication:

• Abstract: write the full name for SIFRO1 and put the abbreviation in brackets
• Introduction: in line 77 correct the sentence stating that auxin biosynthesis mainly occurs in aerial parts. This is not correct. Update also the references and take previous results on auxin biosynthesis in roots into consideration also in the discussion when interpreting your data.

In line 92 you say the “Fe itself can act as a signal..” in which chemical form? Mention that.

• Results: write the full name for SPAD in line 203 (or in the methods) and put the abbreviation in brackets. Figures 2 and 4 have too small fonts making impossible to read them. Line 22 explain what +Fe means. In Figure 3 legend, rephrase the sentence “corresponding to the closed bars” as it is not clear what the reader should look at. In Figure 5 legend, mention that the analysis is done on roots. Also, there is no need to write “quantitative reverse…” just write qRT-PCR.
• Discussion: line 294 correct in “these observations suggested that Fe acts as local…”; line 299: please double check that you do need to put the ref. number in brackets for Valentinuzzi et al., (2020). The pars discussing auxin (lines 314-330) lack final statement interpreting and discussing the data in more detail considering appropriate ref.s. I would never finish a manuscript with “we cannot exclude that” (line 358), but rather “the data presented here indicate that.. “ or similar. Also, no potential for improved tomato crop production is suggested. Please include a final sentence on that. 
• Experiment needed: besides SIFRO1 you should check by qPCR also few auxin and ethylene signalling genes in the same experimental conditions presented in Figure 5. Please also explain why you did not check other organs (like leaves) as you suggest a systemic signalling event is happening. You need more data to support that, also to justify the title of the manuscript.
• Experiments suggested: it would have been very informative for this manuscript to include some mutants for auxin and ethylene signalling and/or biosynthesis and also perform combined treatments with NPA and AVG or with exogenously supplied hormones.

Author Response

Dear Reviewer,

we have appreciated the revision you have done to our manuscript and we think that, thanks to your suggestions and advice, we could improve the quality of our work. A point-by-point response to the criticisms raised by you has been provided. The changes to the text have been highlighted as “Track Changes” and the reference additions are highlighted in yellow along the manuscript. Please see the attachments.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript “Physiological responses to Fe deficiency in split-root tomato plants: a possible crosstalk between auxin and ethylene?” follows local versus systemic responses of root system to heterogeneous availability of iron. This is very interesting topic comprising to a general understanding of root system plasticity, nutrient retranslocation within plant body, and underlying regulatory mechanisms. The manuscript is fits formal standards and presents interesting data. It has however some weaknesses that should be resolved prior the publication. Among others, I do not see any auxin x ethylene crosstalk being addressed in the experiments. Gained data are sometimes too generalized and conclusions are not always clearly supported by the data. I would suggest some modification of the text prior to the publication. The language seems to me good.

In summary, the manuscript can be accepted after a revision. Detailed comments are below.

Detailed comments:

Title, Abstract and Introduction: I really like the Introduction, which is comprehensive and clear. I however do not believe that the experiments really follow the crosstalk (or “integrated signalling) of auxin and ethylene. It is rather an involvement of auxin or ethylene in local versus systemic responses of root tissues to Fe deficiency. The effect of each phytohormone was tested separately and via an addition of the inhibitor (NPA or AVG) into the growth medium only. No other phytohormone analyses or media amendments were done. It should be stated more clearly in the title, abstract as well as in the introduction.

Methods: Methods are clearly described. I have some doubts about the experimental setup of the cultivations. Phytohormone inhibitors (NPA or AVG) were added to the growth media for one day only (it was last day of 30-days cultivation and last day of 7-days split root Fe supply). It seems to me too late. Authors should explain the reasons for choosing such cultivation scheme more thoroughly (in methods or introduction). Was same amount of DMSO added to the second growth chamber to minimize the effect of DMSO itself?

Results: Results are presented clearly. Please, increase the font size in fig. 2 and 4. Add numbers of repetition to fig. 2, 4, 5.

Discussion: Discussion needs to be adjusted. Some results are omitted, some are over-generalized. The higher root biomass in Fe-deficient compared to Fe-sufficient root chamber is a bit surprising to me. Authors should discuss this observation. Is this a result of growth stimulation in Fe-deficient chamber? Can authors prove that root system was really split into “equal” halves at the start of 7-days split-root cultivation period? Moreover, the absence of any growth response to applied inhibitors can simply be a result of too short period of application. This is not mentioned. If that was the intention of the experimental design, it should be explained. I also miss any explanation (at least hypothesis or possible mechanism) what observed effects of AVG or NPA on SlFRO1 gene expression and reductase activity really mean for plant ability to coordinate nutrient acquisition from heterogeneous soil. These results are just described and compared with the literature but it is not enough!

Author Response

Dear Reviewer,

we have appreciated the revision you have done to our manuscript and we think that, thanks to your suggestions and advice, we could improve the quality of our work. A point-by-point response to the criticisms raised by you has been provided. The changes to the text have been highlighted as “Track Changes” and the reference additions are highlighted in yellow along the manuscript. Please see the attachments.

Author Response File: Author Response.docx