The Pentatricopeptide Repeat Protein MEF100 Is Required for the Editing of Four Mitochondrial Editing Sites in Arabidopsis

Round 1

Reviewer 1 Report

Paper summary:

In the article “The pentatricopeptide repeat protein BIR1 is required for the editing of four mitochondrial editing sites in Arabidopsis”, the authors describe the identification and function of the PPR BIR1 in mitochondrial RNA editing. BIR1 was identified in an EMS mutant screen as insensitive to BSO. Analysis of RNA-Seq data identified four major editing sites dependent on BIR1 function, and three that were impacted by a lack of BIR1 but likely as a secondary affect. In the absence of BIR1 editing, complex 1 accumulation decreases and there is a slow-growth plant phenotype, both of which are recovered in complemented lines where editing is restored. Overall this article does a good job of characterizing the function of a PPR protein, which is important to the field of organellar gene expression and RNA editing.

I have suggested a few major and minor edits to be completed prior to publication (see below). Additionally, this paper could be made stronger by including a few additional experiments. One suggestion is to include analysis of T-DNA insertion lines in the bir1 gene to further confirm that the phenotype is caused by the bir1 EMS mutant. As bir1 complementation lines returned the WT phenotype, this suggestion is not compulsory. A second suggestion is to include transcript and/or protein analysis of bir1 to show that either the RNA and/or protein level is reduced.

Major Comments:

1. Tables S3-S4 – more detail should be given, either in the text (methods 2.4) or in the table, regarding the different motifs and the meaning of the scores.
   1. g. – table S3 has a single column with a letter code and number. Ideally these would be in separate columns. Please describe the letter code and the meaning of the number, since in cases where the number is not 0 there seems to be several different numbers.
   2. Table S4 title - combinaison to combination
   3. Table S4 – clarify the heading by adding “BIR1 PPR motif name/number”
   4. Table S4 – clarify the editing site by adding the full description
   5. Table S4 – define what the ‘—‘ means
2. Line 129 – the complemented plant is referred to as having 522 nt upstream of the start codon, but in the methods it says that 1000 nt upstream of the start codon were used. Please reconcile this discrepancy
3. Methods
   1. Need to add a section on protein analyses including extraction of leaf membrane proteins, BN-PAGE, immunoblot, and in-gel NADH oxidase activity staining
4. Figure 3
   1. The legend refers to Figure 1A for full images of the membranes, but there are no full images of the membranes in any of the figures. At the end of the manuscript an Appendix is referred to, but I was not able to find this appendix (perhaps the authors meant Figure A1 instead of 1A). In any case, it would be suitable to add the full membrane images as a supplemental figure without the need for an appendix
   2. define the abbreviations CI and CIII
5. Discussion – This section should include some comments on the observed decrease in complex I accumulation and the root phenotype observed in the BSO screen mentioned in the introduction

Minor/Additional Comments:

1. Line 62 – delete “onto plates”
2. Line 69 – refer to Table S1 for the primers; refer to the paragraph below for construct generation to complement the plants
3. Line 75 – be consistent with terminology throughout the manuscript: Col-0 vs. Columbia-0
4. Line 80 – reference
5. Lines 91 and 111 – fix the numbers in these headings
6. Table 1 – table heading could include “…at the seven positions impacted in bir1 with percentage….”
7. Line 227 – missing ‘,’ after ccmFn2-356
8. Figure S1 – specify the difference between the top and bottom panel for each gene
9. Figure 5 – addition of a key would make reading this figure more straightforward
10. Figures S2 and S3 – specify that the WT Col-0 is at the top and bir1 is the bottom
    1. Also, the text for the figure legend in S3 is hard to read

Author Response

I have suggested a few major and minor edits to be completed prior to publication (see below). Additionally, this paper could be made stronger by including a few additional experiments. One suggestion is to include analysis of T-DNA insertion lines in the bir1 gene to further confirm that the phenotype is caused by the bir1 EMS mutant. As bir1 complementation lines returned the WT phenotype, this suggestion is not compulsory.

Response: We originally planned to analyse both the EMS line and a T-DNA insertion line, but the latter was so difficult to grow (very low germination rate, early development arrest, rapid loss of germination power) that we could only work with the EMS mutant.

A second suggestion is to include transcript and/or protein analysis of bir1 to show that either the RNA and/or protein level is reduced.

Response: The mutation being a point mutation, there is no reason to believe that transcript and protein levels might be affected in the EMS line, therefore we did not conduct any qPCR or western blot analysis.

Major Comments:

1. Tables S3-S4 – more detail should be given, either in the text (methods 2.4) or in the table, regarding the different motifs and the meaning of the scores.
1. g. – table S3 has a single column with a letter code and number. Ideally these would be in separate columns. Please describe the letter code and the meaning of the number, since in cases where the number is not 0 there seems to be several different numbers.

Response: It looks like that the scoring tables in Table S3 lost formatting during the conversion to Excel. They are now publicly available at https://github.com/ian-small/PPRmatcher in ‘scoring_tables/Millman’ and some explanations are given as to the contents of these tables.

1. Table S4 title - combinaison to combination
2. Table S4 – clarify the heading by adding “BIR1 PPR motif name/number”
3. Table S4 – clarify the editing site by adding the full description
4. Table S4 – define what the ‘—‘ means

Response: Points 2, 3 and 5 were addressed and this table is now labelled as Table S3. We are not sure what the reviewer means by ‘clarify the editing site by adding the full description’

1. Line 129 – the complemented plant is referred to as having 522 nt upstream of the start codon, but in the methods it says that 1000 nt upstream of the start codon were used. Please reconcile this discrepancy

Response: 522 nt upstream of the start codon is correct and has been added in the Methods section (line 74).

1. Methods
1. Need to add a section on protein analyses including extraction of leaf membrane proteins, BN-PAGE, immunoblot, and in-gel NADH oxidase activity staining

Response: This section was accidentally deleted from the manuscript and has been added (lines 111-127).

1. Figure 3
1. The legend refers to Figure 1A for full images of the membranes, but there are no full images of the membranes in any of the figures. At the end of the manuscript an Appendix is referred to, but I was not able to find this appendix (perhaps the authors meant Figure A1 instead of 1A). In any case, it would be suitable to add the full membrane images as a supplemental figure without the need for an appendix

Response: This figure was renamed Figure S2 and added to the supplementary data

1. define the abbreviations CI and CIII

Response: the abbreviations have been added to the legend

1. Discussion – This section should include some comments on the observed decrease in complex I accumulation and the root phenotype observed in the BSO screen mentioned in the introduction

Response: comments were added lines 290-293

Complex I has an important function in cellular redox homeostasis, and when its activity is reduced, the alternative NADH oxidizing enzymes are induced. We previously showed that mutants with a reduced Complex I activity (bir6, css1 and otp439 mutants) had a reduced sensitivity to BSO-induced inhibition of root growth, because they retained more GSH and had a better ability to detoxify reactive oxygen species than wild-type plants.

 

Minor/Additional Comments:

1. Line 62 – delete “onto plates”

Response: deleted

1. Line 69 – refer to Table S1 for the primers; refer to the paragraph below for construct generation to complement the plants

Response: done

1. Line 75 – be consistent with terminology throughout the manuscript: Col-0 vs. Columbia-0

Response: done

1. Line 80 – reference

Response: reference added

1. Lines 91 and 111 – fix the numbers in these headings

Response: done

1. Table 1 – table heading could include “…at the seven positions impacted in bir1 with percentage….”

Response: done

1. Line 227 – missing ‘,’ after ccmFn2-356

Response: ‘,’ was added (now line 245)

1. Figure S1 – specify the difference between the top and bottom panel for each gene

Response: the top panel shows the nucleotide conservation in the RNA target and the bottom panel shows the amino acid conservation in the PPR protein.

1. Figure 5 – addition of a key would make reading this figure more straightforward

Response: a key was added

1. Figures S2 and S3 – specify that the WT Col-0 is at the top and bir1 is the bottom
1. Also, the text for the figure legend in S3 is hard to read

Response: both issues were addressed

Reviewer 2 Report

The manuscript “The pentatricopeptide repeat protein BIR1 is required for the editing of four mitochondrial editing sites in Arabidopsis” by Gutmann and coworkers presents novel findings about a DYW-type PPR protein and its importance to the assembly of the mitochondrial Complex I. They show that in the bir1 mutant C-to-U editing of four sites in mitochondrial transcripts is drastically reduced leading to defects protein accumulation and complex assembly. The authors also demonstrate the usefulness of a computational tool they had previously generated in predicting PPR-binding sites. They find that plants lacking the BIR1 editing sites have divergence in BIR1 binding sites. The manuscript is generally well written but it is not always easy to understand what the authors are try to communicate because details of experimental approaches are sparsely described.

Major Comments

1. Although the origins of the name are clearly described in the Introduction, it should be noted that there is a well-characterized Arabidopsis gene with this name already. The better known BIR1 (BAK1-INTERACTING RECEPTOR-LIKE KINASE 1) is the product of the locus AT5G48380 and its roles in plant defense have been studied for at least 15 years. In order to avoir confusion, perhaps Gutman and co-workers should consider modifying the name of the PPR protein described here.
2. The Materials and Methods do not adequately describe the content of the manuscript. Details of the BN-PAGE, Western blots and in-gel NADH oxidase assay have been omitted and should be added.
3. The mutant characterization is very weak and appears to be almost an afterthought. In describing the effects of EMS mutagenesis, the authors state that “This mutation terminates the translation of BIR1 in the first helix of the P2 motif (Figure 1a)”. There is no experimental evidence provided to demonstrate this so this is a prediction or expectation and should be reported as such. Further, do the authors know if this is a null mutation or is transcript accumulation affected by this mutation? This can easily be assessed by RT-PCR or with the existing RNA-seq reads that the authors have generated. In addition, the mitochondrial localization of this protein is never tested or experimentally confirmed. This can easily be verified.
4. With regards to the growth defects shown in Figure 1b: is it really growth rate that is affected? Was the rate of cell and organ increased reduced? It is also possible that cells are smaller than wild type cells, and so the bir1 mutants are smaller than wild type plants. Based on the data shown, the authors can only conclude that the mutants are smaller.
5. The description of how mitochondrial editing sites were identified (lines 139-146) is very weak and uninformative. The authors do not state that this was done with NGS and that hundreds or thousands of reads were scored. This information would make it much easier to follow Figure 2a and Table 1.
6. The last section of the Results is written for an expert audience. The authors should add a few sentences explaining the ‘PPR code”, so that a broad audience can better understand the relationship between the PPR repeat structure and the RNA binding sites identified.

Minor comments

1. Figure 2b can be made easier to read if the edited residue is highlighted.
2. Is there a way to indicate to indicate there was equal sample loaded on the gels in Figure 3A?
3. The legend of Figure 3 says membranes can be see in Figure 1A but this should read Figure A1 according to the information provided in line 338.
4. The word ‘catalysed’ in lines 256-257 is a strong choice. It is not incorrect, but it is also not experimentally verified that BIR1 directly catalyses the editing at its 4 prime targets.

Author Response

Major Comments

1. Although the origins of the name are clearly described in the Introduction, it should be noted that there is a well-characterized Arabidopsis gene with this name already. The better known BIR1 (BAK1-INTERACTING RECEPTOR-LIKE KINASE 1) is the product of the locus AT5G48380 and its roles in plant defense have been studied for at least 15 years. In order to avoir confusion, perhaps Gutman and co-workers should consider modifying the name of the PPR protein described here.

Response: The PPR protein has been renamed MEF100 for Mitochondrial Editing Factor 100, and the mutant mef100.

1. The Materials and Methods do not adequately describe the content of the manuscript. Details of the BN-PAGE, Western blots and in-gel NADH oxidase assay have been omitted and should be added.

Response: This section was accidentally deleted from the manuscript and has been added (lines 111-127).

1. The mutant characterization is very weak and appears to be almost an afterthought. In describing the effects of EMS mutagenesis, the authors state that “This mutation terminates the translation of BIR1 in the first helix of the P2 motif (Figure 1a)”. There is no experimental evidence provided to demonstrate this so this is a prediction or expectation and should be reported as such.

Response: the text has been modified accordingly.

 Further, do the authors know if this is a null mutation or is transcript accumulation affected by this mutation? This can easily be assessed by RT-PCR or with the existing RNA-seq reads that the authors have generated.

Response: The mutation being a point mutation, there is no reason to believe that transcript and protein levels might be affected in the EMS line, therefore we did not conduct any qPCR or western blot analysis.

In addition, the mitochondrial localization of this protein is never tested or experimentally confirmed. This can easily be verified.

Response: We agree that it could be verified, but it did not seem necessary as the characterisation of the mutant clearly shows that this editing factor acts on mitochondrial transcripts, resulting in an obvious complex I deficiency phenotype.

1. With regards to the growth defects shown in Figure 1b: is it really growth rate that is affected? Was the rate of cell and organ increased reduced? It is also possible that cells are smaller than wild type cells, and so the bir1 mutants are smaller than wild type plants. Based on the data shown, the authors can only conclude that the mutants are smaller.

Response: Figure 1 was redone with better pictures showing that the 3 complemented lines look like the Col-0 at 22 days. A picture from another experiment was added (Figure 1c) which shows that to reach the same developmental stage, the mef100 mutant has to be sown 3.5 weeks before Col-0, attesting that mef100 grows more slowly, which is observed in most Complex I mutants.

 

1. The description of how mitochondrial editing sites were identified (lines 139-146) is very weak and uninformative. The authors do not state that this was done with NGS and that hundreds or thousands of reads were scored. This information would make it much easier to follow Figure 2a and Table 1.

Response: This section only describes the point mutation and the fact that it creates a premature stop codon. The actual editing sites affected are described in the next paragraph (3.2) using the RNA-seq data.

 

1. The last section of the Results is written for an expert audience. The authors should add a few sentences explaining the ‘PPR code”, so that a broad audience can better understand the relationship between the PPR repeat structure and the RNA binding sites identified.

Response: Two phrases were added lines 237-239

‘PPR editing factors recognize their target sites via interactions between the 5^th and last amino acids in each motif and the aligned RNA base [10,13]. These base preferences are to some extent predictable’

Minor comments

1. Figure 2b can be made easier to read if the edited residue is highlighted.

Response: The edited C is already highlighted in grey in Figure 2b.

 

1. Is there a way to indicate to indicate there was equal sample loaded on the gels in Figure 3A?

Response: The lower band in the anti-RISP blot, which is likely to result from cross reaction of the antibody with other Rieske proteins present in thylakoids, such as the cytochrome b6/f, could be used as a loading control. This protein is likely to not be altered in the mutants as it never appears when working with purified mitochondrial proteins.

1. The legend of Figure 3 says membranes can be see in Figure 1A but this should read Figure A1 according to the information provided in line 338.

Response: Sorry about the confusion, this figure was renamed Figure S2 and added to the supplementary data

1. The word ‘catalysed’ in lines 256-257 is a strong choice. It is not incorrect, but it is also not experimentally verified that BIR1 directly catalyses the editing at its 4 prime targets.

Response: As requested, the term “catalysed” has been replaced by a less strong term, “affected” (lines 274-275).

Reviewer 3 Report

The author of the current study present data to show that the PPR protein BIR1 is responsible for editing four mitochondrial editing sites in Arabidopsis. The work presented here is sound and fairly standard for the analysis of PPR proteins in plants. While not overly exciting it is as it is predominantly just another PPR protein and its targets. The most interesting part is the comparison of different species where in some cases one of the editing sites had been lost. While not essential it would have been interesting to see what would have happened had one of these PPR proteins been transformed into Arabidopsis to see if the editing would have been restored or not.

I think the manuscript could be accepted in its current form. But would suggest a few minor changes.

 

1. The last sentence of the abstract for me is a bit strange. The use of the word former seems out of place. I understand what the authors are saying but maybe this sentence could be improved upon.
2. In the materials and methods there is no mention of the BN-Gel analysis, what detergent etc... or the enzyme activity or antibodies as well. This could be improved.
3. I think the phenotyping could be improved. As it amounts to little more than growing some plants and taking a couple of picture. Possibly the growth defect could be assayed quantitatively to be more scientific than just reporting slightly delayed. To the complementation analysis. Is the complementation completely restored? As the authors say. To me the plants still look smaller and slightly delayed.
4. All of the work was performed on the ems generated mutant of bir1. Was the phenotype and editing deficiencies also confirmed in a T-DNA inertional mutant as well? Since the complementation appears not complete?
5. In figure 2 there are a number of editing sites which appear to have enhanced efficiencies in the mutant plants. Would the authors like to comment of these. Some sites are also 100% edited in the mutant but are below 10% in the wild type. Just curious as to what is going on here.
6. Also in Table 1 the amount of reads for the mutant appear to much higher than the wild type. Is this normal? Do editing mutants always display a greater amount of transcription of mitochondrial genes?
7. Figure 3. The NADH oxidase activity stain looks strange to me. Why so many other bands after staining? Complex I appears rather weak? Also what happened to the bir6-1 lane. Could this gel be improved upon. Also what is the lower band in the anti-RISP blot? 
8. In Figure 5 there would appear some contradictory data in that the non editable species PPR proteins have a higher affinity for the ccmFN site. Is this correct? I think this should discussed a bit more. Is it just an anomaly or is there something strange going on here.

As I said before this study is pretty standard for the field. And after address the comments above I see no reason why the manuscript cannot be accepted for publication

Author Response

I think the manuscript could be accepted in its current form. But would suggest a few minor changes.

1. The last sentence of the abstract for me is a bit strange. The use of the word former seems out of place. I understand what the authors are saying but maybe this sentence could be improved upon.

Response: the word ‘former’ has been deleted

2. In the materials and methods there is no mention of the BN-Gel analysis, what detergent etc... or the enzyme activity or antibodies as well. This could be improved.

Response: The missing section has been added (lines 111-127).

3. I think the phenotyping could be improved. As it amounts to little more than growing some plants and taking a couple of picture. Possibly the growth defect could be assayed quantitatively to be more scientific than just reporting slightly delayed. To the complementation analysis. Is the complementation completely restored? As the authors say. To me the plants still look smaller and slightly delayed.

Response: Figure 1 was redone with better pictures showing that the 3 complemented lines look like the Col-0 at 22 days. A picture from another experiment was added (Figure 1c) and shows that to reach the same developmental stage, the mef100 mutant has to be sown 3.5 weeks before Col-0, attesting that mef100 grows more slowly, which is very commonly observed for Complex I mutants.

4. All of the work was performed on the ems generated mutant of bir1. Was the phenotype and editing deficiencies also confirmed in a T-DNA insertional mutant as well? Since the complementation appears not complete?

Response: We originally planned to analyse both the EMS line and a T-DNA insertion line, but the latter was very difficult to grow (very low germination rate, early development arrest, rapid loss of germination power) so we decided to only work with the EMS mutant.

5. In figure 2 there are a number of editing sites which appear to have enhanced efficiencies in the mutant plants. Would the authors like to comment of these. Some sites are also 100% edited in the mutant but are below 10% in the wild type. Just curious as to what is going on here.

Response: The most striking event (0% edited in wild-type, virtually 100% edited in mef100) may be a SNP in the DNA. A few other transcripts are clearly more edited in mef100 than in wild-type. All of these events are in nad or ccm transcripts and we suspect that RNA turnover of these transcripts is slowed in the mutant (perhaps as part of a compensatory feedback mechanism to the loss of Complex I activity?). But as this is pure speculation, we prefer not to include this discussion in the manuscript.

6. Also in Table 1 the amount of reads for the mutant appear to much higher than the wild type. Is this normal? Do editing mutants always display a greater amount of transcription of mitochondrial genes?

Response: In general, RNA-binding protein mutants (not only editing mutants) altered in the expression of mitochondrial genes tend to compensate by having higher numbers of mitochondria, therefore higher levels of transcripts. In the RNA-seq experiment, the Col-0 sample contains 9906296 reads, of which 8895400 map to the chloroplast genome and 1010896 to the mitochondrial genome. The mef100 sample contains slightly more reads (10727091) but the big difference is in how they partition between the 2 organelle genomes, with 1757483 mapping to the chloroplast genome and 8969608 to the mitochondrial genome. A comment has been added to the text.

 

Figure 3. The NADH oxidase activity stain looks strange to me. Why so many other bands after staining? Complex I appears rather weak? Also what happened to the bir6-1 lane. Could this gel be improved upon.

Response: The experiment was done with a crude leaf membrane extract therefore thylakoid and mitochondrial protein complexes are visible. A colour version of the gel photograph has been substituted to the black and white version so the NADH oxidase activity bands corresponding to complex I and DLD are more obvious, and some of the thylakoid complexes (PSI and LHCII) have been labelled to help understand the pattern.

Also what is the lower band in the anti-RISP blot? 

Response: This band does not appear when working with purified mitochondrial proteins. Therefore, it is likely to result from cross reaction of the antibody with other Rieske proteins present in thylakoids, such as the cytochrome b6/f (note that it is present in the wtf9-2 mutant).

7. In Figure 5 there would appear some contradictory data in that the non editable species PPR proteins have a higher affinity for the ccmFN site. Is this correct? I think this should discussed a bit more. Is it just an anomaly or is there something strange going on here.

Response: We agree that this is a puzzling anomaly, and we do not have a good explanation. It is possible that editing of ccmFN at this site can be disadvantageous and that in plants where the site is edited, there is selection for mutants that decrease MEF100 binding.