Review Reports - Conserved Cu-MicroRNAs in Arabidopsis thaliana Function in Copper Economy under Deficiency

Round 1

Reviewer 1 Report

The manuscript by Shahbaz and Pilon describes studies on the effect of inhibiting the action/reducing the levels of three miRNAs related to copper metabolism on the growth of plants under copper deficiency. They provide experimental evidence of the importance of the upregulation of these miRNAs under copper deficiency to allow the synthesis of essential copper proteins, mainly plastocyanin. The paper is clearly written and the experiments are straightforward. Although a rather small effect of inhibiting the miRNAs on plant growth was observed, it can be envisaged that their function is important to optimize plant performance. Further characterization of these lines will probably help to understand which processes, and under which conditions, require the action of these miRNAs.

I find that minor corrections are required:

-Please, check the references to the figures throughout the manuscript. They are mostly wrong.

-line 28: “cytochrome”

-line 45: “plastids and cytosol”

-line 89: “however” twice

-line 130: “targeted by the Cu-microRNAs”

-lines 134-138: “as expected” is used twice along these sentences. I would remove one.

-Supplementary table 2 is cited before suppl. table 1. Please, change the order.

-line 174: “All4-Cu miRNA mimicry lines”?

-line 183 and others: The letter “phi” before “PSII” does not appear in the pdf version of the manuscript I downloaded.

-line 184: “PSII in the three tandem”

-Please, cite the corresponding figures in the paragraphs starting in lines 195 and 207.

-line 272: “that” is used twice in this sentence.

-line 334: remove “of”

-line 338: remove the first “the” in this line.

-line 346: The list of antibodies used is not correct.

-Table S1 title: used “for” qRT-PCR

Author Response

Responses to reviewers.

We thank both reviewers and the editor for carefully checking the MS and the comments. All comments are addressed in the revision. Changes in text are highlighted in yellow in the revision. Figure references are highlighted in green. We edited the materials and methods as suggested by the editor. Responses to specific comments are below.

Reviewer 1.

-Please, check the references to the figures throughout the manuscript. They

are mostly wrong.

Reply: This was done, we sincerely apologize for the errors. We had re-organized figures shortly before submission because of journal formatting requirements, somehow I must have used the text that was not updated for the last figure revision. The figure references are corrected and are highlighted in the revision.

-line 28: “cytochrome” Reply: corrected

-line 45: “plastids and cytosol” Reply: corrected

-line 89: “however” twice Reply: corrected

-line 130: “targeted by the Cu-microRNAs” Reply: I was not sure which change was corrected here. But I carefully proofread the text.

-lines 134-138: “as expected” is used twice along these sentences. I would

remove one. Reply: corrected

-Supplementary table 2 is cited before suppl. table 1. Please, change the

order. Reply: corrected

-line 174: “All4-Cu miRNA mimicry lines”? Reply: corrected

-line 183 and others: The letter “phi” before “PSII” does not appear in the pdf

version of the manuscript I downloaded. Reply: corrected

-line 184: “PSII in the three tandem” Reply: corrected

-Please, cite the corresponding figures in the paragraphs starting in lines 195

and 207. Reply: corrected

-line 272: “that” is used twice in this sentence. Reply: corrected

-line 334: remove “of” Reply: corrected

-line 338: remove the first “the” in this line. Reply: corrected

-line 346: The list of antibodies used is not correct. Reply: corrected

-Table S1 title: used “for” qRT-PCR Reply: corrected

Reviewer 2.

-- However, the authors seem to overestimate their results,

claiming “strong” correlation of a clearly evident reduction in miRNAs and

increase of their mRNA target levels, with the phenotype, which rather is mild

(figures 3-5). This is not unexpected, since plants tend to cope with stressful

conditions based on more than one regulatory mechanism. Probably, at

different developmental stages or combined stresses the effect would have

been greater. Also, as mentioned in discussion the 4th†Cu-miRNA might

account for such mild effect.

Reply: We largely agree with the reviewer that the effects on phenotypes were mild in terms of size of effect. Nevertheless, even if an effect is mild, it can still show a strong correlation with the mimicry expression, which we believe is the case. We agree also with the reviewer that in nature other factors and stressors could affect the phenotype, in addition to an effect of th 4^th Cu-microRNA. All of these topics were/are discussed. We prefer to avoid further speculation. Also please note that we avoided developmental variations by focusing on the vegetative rosettes. We feel that because of this approach we were able to isolate a subtle (but we think important) effect.

-- Line 62, define the gene name SPL as done for PC or CSD. Reply: corrected

-- Line 63, space missing Reply: corrected

-- Line 81, do you mean PC1? Since according to a previous statement PC2

seems to be affected at protein level. Reply: Both PC1 and PC2. There is no microRNA site for either. It was shown before (Abdel-Ghany 2009) that the effect of low Cu on PC proteins is largely explained by post-translational processes. The text was slightly modified for clarity to read as follows: “To test this we aimed to inactivate the conserved Cu-microRNAs (miR397, miR398, and miR408) and to analyze, especially under impending deficiency, the effects on growth and Cu-allocation to abundant and essential Cu proteins such as PC1 and PC2, which are not down-regulated via a microRNA.”

-- Line 89, however was repeated (delete the second one). Reply: corrected

-- Lines 106-108, please clarify what the IPS sequence represents. In Figure

S1 it appears capped and polyadenylated, does it recruit ribosomes? Reply: We have better explained IPS1 towards the end of the introduction. The modified text is as follows” Target mimicry relies on the production of a “designed” RNA transcript, which is modified from INDUCED BY PHOSPHATE STARVATION1 (IPS1) a non-coding RNA in Arabidopsis that functions to sequester miR399, a microRNA that functions in the regulation of phosphate homeostasis [41]. The native IPS1 transcript contains a microRNA target site for miR399 that cannot be cleaved and that should sequester and inactivate the microRNA. The original mimicry target site in miR399 can be modified to inhibit other microRNAs [41]. “

IPS1 does not appear to have a possible coding sequence ORF beyond 4 amino acids (Franco-Zorrila et al, 2007). This is most likely not translated. Nevertheless there is no reason to assume that IPS RNA would not be capped and polyadenylated.

-- miRNAs recognizing the target mimicry are not depicted on RISC, why? Reply: it is unclear based on the literature. We think it makes no real difference for the conceptual model. The figure S1 was slightly simplified for better clarity and reduce possible controversy.

-- Also it is not clear why miRNA levels should be reduced by the mimicry if it acts

only by sequestering them. Reply: actually the same was found for other microRNAs before see: Todesco, M.; Rubio-Somoza, I.; Paz-Ares, J.; Weigel, D. A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana. PLoS Genet. 2010, 6 e1001031. We refer to this paper in the revised text as follows” Expression of target mimicry constructs caused a decrease in the microRNAs that were targeted (Figure 1). Such a decrease has been reported before for other microRNAs and indicates that target mimicry not just leads to target sequestration but also causes increased microRNA instability [59].”

-- The effect is much greater for miR397c, than for miR398 and miR408. What could be a possible explanation for this? Reply: actually it seems that the effect on miR397 is relatively a bit smaller. Clearly different mRNA targets experience different levels of silencing. This was extensively reported before. However, figure 2 really seems to show that the effect of mimicry on the target transcripts of the Cu microRNAs is comparable overall. We did not adjust the text except for referencing to figures correctly.

-- Lines 132-142, all the results presented here refer to figure 1, where one

could see the levels of mature miR397, miR398 and miR408. Figures 2-4

present the effect on miRNA targets, plant phenotype and effect on

photosynthetic electron transport and are not even mentioned in the

paragraph. Next paragraph also refer to figures 2-4, when only fig 2 is

showing the miRNA targets. Possibly the authors have changed the figure

organization without modifying the corresponding text. Reply: We apologize for these errors; they are corrected.

-- Lines 163-171, again the figure does not correspond to the presented

results. It should be figure 3. Reply: corrected (we apologize for the errors)

-- Lines 181-185, I could no see differences between Cu conditions for WT

plants in the PSII flux or 1-qP. The authors should better distinguish in the

text the only significant difference seen in measurements for the target

mimicry plants under -Cu (figure 4). Reply: we compared within a growth condition. We indicated this in the revised figure legend. When the WT is compared between the 3 growth conditions only a small effect of Cu depletion is seen albeit that this is statistically significant. This was reported before (Abdel-Ghany and Pilon, 2008)

--There is no figure 6. Reply: corrected

--Lines 195-206, please cite the corresponding figure (figure 5). Reply: corrected

--Lines 215-217, the conclusion is partially true since, according to figure 5,

reduction in plastocyanin and COXII levels detected on -Cu condition (not on

5 or 50 nM Cu), was not accompanied by an important accumulation of

CSD1 and CSD2 or enhancement of its activity (part B of figure 5).

Reply: there actually is a notable difference even on the lowest Cu concentrations in accumulation of CSDs and CCS due to mimicry. Because of this low Cu, the total Cu pools might be reduced this could have an impact for sure, we believe. In addition, the expression of laccase proteins cannot be measured due to lack of specific antibodies. We thus respectfully disagree with the reviewer on this point and believe that our conclusions are tentative enough as stated.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript by Shahbaz and Pilon aimed to demonstrate the role of three Cu-microRNAs, miR397, miR398 and miR408 in copper economy for Arabidopsis thaliana vegetative growth during deficiency of this element through inactivating simultaneously the three miRNAs by a tandem mimicry construct. The construct consisted of altered target sites for each miRNA in order to sequester them in inactive complexes. This work shows the relevance of the concerted action of Cu-miRNAs on copper proteins and how this impacts the on crucial energy-related enzymes in the plant also requiring Cu for their action. However, the authors seem to overestimate their results, claiming “strong” correlation of a clearly evident reduction in miRNAs and increase of their mRNA target levels, with the phenotype, which rather is mild (figures 3-5). This is not unexpected, since plants tend to cope with stressful conditions based on more than one regulatory mechanism. Probably, at different developmental stages or combined stresses the effect would have been greater. Also, as mentioned in discussion the 4^th Cu-miRNA might account for such mild effect.

In addition, there are several important aspects to be pursued before publication:

Line 62, define the gene name SPL as done for PC or CSD.

Line 63, space missing

Line 81, do you mean PC1? Since according to a previous statement PC2 seems to be affected at protein level.

Line 89, however was repeated (delete the second one).

Lines 106-108, please clarify what the IPS sequence represents. In Figure S1 it appears capped and polyadenylated, does it recruit ribosomes? miRNAs recognizing the target mimciry are not depicted on RISC, why? Also it is not clear why miRNA levels should be reduced by the mimicry if it acts only by sequestering them. The effect is much greater for miR397c, than for miR398 and miR408. What could be a possible explanation for this?

Lines 132-142, all the results presented here refer to figure 1, where one could see the levels of mature miR397, miR398 and miR408. Figures 2-4 present the effect on miRNA targets, plant phenotype and effect on photosynthetic electron transport and are not even mentioned in the paragraph. Next paragraph also refer to figures 2-4, when only fig 2 is showing the miRNA targets. Possibly the authors have changed the figure organization without modifying the corresponding text.

Lines 163-171, again the figure does not correspond to the presented results. It should be figure 3.

Lines 181-185, I could no see differences between Cu conditions for WT plants in the PSII flux or 1-qP. The authors should better distinguish in the text the only significant difference seen in measurements for the target mimicry plants under -Cu (figure 4). There is no figure 6.

Lines 195-206, please cite the corresponding figure (figure 5).

Lines 215-217, the conclusion is partially true since, according to figure 5, reduction in plastocyanin and COXII levels detected on -Cu condition (not on 5 or 50 nM Cu), was not accompanied by an important accumulation of CSD1 and CSD2 or enhancement of its activity (part B of figure 5).