Review History


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Summary

  • The initial submission of this article was received on December 9th, 2019 and was peer-reviewed by 3 reviewers and the Academic Editor.
  • The Academic Editor made their initial decision on January 29th, 2020.
  • The first revision was submitted on July 10th, 2020 and was reviewed by 2 reviewers and the Academic Editor.
  • The article was Accepted by the Academic Editor on July 30th, 2020.

Version 0.2 (accepted)

· Jul 30, 2020 · Academic Editor

Accept

Both reviewers agreed that all their critiques were adequately addressed and the manuscript was amended accordingly.

[# PeerJ Staff Note - this decision was reviewed and approved by Gerard Lazo, a PeerJ Section Editor covering this Section, who added:"This was a well developed manuscript which clearly developed a hypothesis and explained the approach and workflow for attempting to answer key questions. A good literature review for the introduction provided key references to guide the reader. The results were provided in a fashion which may lead to further work to be performed to address the conclusions. In a pan-genome context the authors noted key differences between monocots/dicots and propose differential roles in transit-peptides. I agree that the manuscript is in proper form to move forward." #]

Reviewer 1 ·

Basic reporting

No comment

Experimental design

No comment

Validity of the findings

No comment

Additional comments

The manuscript by Christian et al. entitled, “Plastid transit peptides - Where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution”, has been suitably revised. The authors have addressed all queries in the present form.

Reviewer 3 ·

Basic reporting

No comment

Experimental design

No comment

Validity of the findings

No comment

Additional comments

The authors have satisfactorily modified the manuscript

Version 0.1 (original submission)

· Jan 29, 2020 · Academic Editor

Major Revisions

Please carefully address all the critiques of the reviewers and conduct proposed experiments. Please also note that one of the reviewers indicated that they cannot recommend publishing a study that does not account for possible gene modelling biases.

Reviewer 1 ·

Basic reporting

No comment

Experimental design

no comment

Validity of the findings

no comments

Additional comments

In the manuscript by Christian et al. entitled, “Plastid transit peptides - Where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution”, the authors provide new insights into acquisition or loss of plastid targeting peptides during evolution using comparative genomics approach across 15 phylogenetically diverse genera and pangenomes from Arabidopsis and Brachypodium. It is a good piece of work however, it requires minor clarifications/changes. These are as follows:
1. All the legends need a relook. They need to be crisp, presently it appears as if the legends state results only.
2. Lines 132 to 150. Authors need to define the criteria chosen for significant difference. While the authors place threonine (+11.1%) in a highly enriched category, but categorise asparagine (-28.6%) in a moderate depletion category. It would be better to adopt similar standards (percent) as ‘low, moderate and high’ for both enriched and depleted categories and subsequently reassign the amino acid residues.
3. Line 139. Authors note that phenylalanine was not highly different (-7.9%). This is surprising. Can authors comment on the significance of this in evolution?

4. In Table 1, please make ‘cTP’ uniform as ‘CTP’. Also expand the same in legend.

5. For prediction of protein localization authors have used Target P-1, but didn’t mention the specificity cut-off values or parameters utilized for candidate selection. Authors should repeat this prediction by using an updated version of TARGET P (TARGET P-2, http://www.cbs.dtu.dk/services/TargetP/) to ensure robustness of the previous analysis.

6. line 196, “only on only” does not make any sense. Please correct.

7. lines 316-319. Reference to Figure 6 comes before Figure 5 in text. Renumber and rearrange.

8. Figure 7B is not cited anywhere in the main text. Kindly include it at appropriate place.

9. Figure 8 is cited as one figure while it’s actually have three parts, cite all parts A, B and C.

10. Figure 10A and 10B are not cited in main text include these in appropriate section.

Reviewer 2 ·

Basic reporting

Literature references and sufficient field background/context is provided, with the following exceptions:
1. Origin of targeting systems and targeting signals: I think that the origin and evolution of the targeting itself should be covered in the introduction as well, with respect to the origin of the organelles, of the targeted genes, and of the targeting systems, and mitochondria should be mentioned (see https://doi.org/10.1016/j.tcb.2016.07.001 for an overview, and https://doi.org/10.1111/tra.12446 for an interesting hypothesis)
2. Role of miss targeting in evolution of metabolic pathway distribution: One interesting hypothesis to explain the maintenance of the cellular metabolism after gene transfer or targeting signal gain or loss is, that a low amount of “constitutional miss-targeting” enables survival of the cell, and further evolution of the targeting signals (https://doi.org/10.1098/rstb.2009.0252) I think that this is also relevant to the genes with recently acquired targeting signals analysed in this study.
(I am not an author of any of the cited papers, and I don't mind if these or other papers on the topics would be cited.)

The manuscript has a professional structure, figures, and tables, the raw data is shared. The text is on the long side, and the numbers of figures is high. As outlined below, I think that much of the data needs to be re-analysed. I think that some of the analyses might be difficult to maintain, because a part of the data might not easily be analysed on the within-genome level. However, I think that a revised manuscript with possibly shorter lenght/lower amount of data analysed would be interesting nevertheless.

Smaller things:

Line 48, sentence complicated, also sense unclear, maybe delete "in"?

Lines 63-70, the Apple part in the introduction seems unnecessary to me, I understand that Apple data was examined, but I do not understand the connection between the plastid types and the evolution of targeting peptides.

Line 101: I think that the term "nascent plastid-targeted proteins" has unfortunate potential for being confused with plastid targeted proteins that are in the process of translation at the ribosome (the "ribosome-nascent chain complex"). Furthermore, it's the genes that mutate, not the proteins.

Table 1 caption: I think that "subject and "query" labels make sense for the "Taxa Bias" columns, but they are irritating for the "Transit Peptide Bias" and "Prediction Bias" columns, in which different sets of sequences are compared (in my understanding).

Line 377, "all of cases", missing article?

Line 482, "radiated" is not a good choice of a word here; I think it might be confused with what happens in an "adaptive radiation" in evolutionary biology.

Line 807, "second", missing article?

The conclusion could be shortened to the key findings; here, especially I did not understand the sentence lines 707-709. What are "random substitution events"? Do the authors suggest that "simple substitutions, insertions and deletions, or alternative start sites" (earlier in the sentence) are not random?

Experimental design

The authors present a variety of multi- or pan- genome analyses, based on which hypotheses on the variability in plastid proteomes and the mutational processes that lead to their evolution are proposed. This concept is promising, however, the authors work with predicted protein sets from a variety of species, without accounting for the fact that mutations occur in the genes, not in the proteins, and without accounting for differences in the methods used to collect the protein sets. I think that such within-genome analyses are a necessary first step of the project in order to conduct further comparative analyses. A couple of mechanisms that have no biological relevance, but that do have an influence on the results of this study are:
1. The investigated genomes have been sequenced at different time points, the sequencing, assembly and annotation methods during this time improved, so in the "oldest" genomes, plastid targeted genes might not have been recognized simply because the projects at that time were more pioneering.
2. Pre-sequences are less conserved than mature protein sequences. So they are hard to detect via homology based gene modelling. And, depending on lengths and settings, a gene model that excludes an existing targeting signal might attain a higher homology score than one that includes a targeting signal. So the gene modelling itself has an influence, but also the choice of competing models for a non-redundant gene catalog.
3. Transcriptomics methods also changed with respect to sequence quality and coverage. Furthermore, due to cDNA synthesis with the often used oligo-T primers, there is often (but not always) a bias against the 5' end of transcripts.
I think that such possible biases in pre-sequence detection need to be standardized between the different investigated organisms/genomes.

Dual targeting (or multiple targeting) should be discussed more carefully, including estimations of numbers of dually targeted proteins.

Accounting for the gene structures and sequences would also allow to assess how existing 5' UTRs could be transformed into targeting signals, in all three reading frames.

Figure 2 might be condensed to sequence logos (http://dx.doi.org/10.1093/nar/18.20.6097) and/or frequency plots of amino acids. There is a fair amount of circular reasoning in the comparison of predicted transit peptides with the other sets, because the predicted transit peptides are already predicted to be transit peptides due to their sequence. I think this should be discussed more carefully/explicitly.
Sequence logos might be a good way to present the data shown in Figure 1 and 3, as well (although this would slightly change their meaning).

Validity of the findings

As mentioned above, I don't think that the results in the current form can be taken at face value, due to differences in gene modelling and model selection that led to the various investigated protein sets.

Additional comments

In their manuscript with the title "Plastid transit peptides - Where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution", Christian et al. investigate the evolution of plastid targeting signals in plants, especially with respect to genes of which the product has recently acquired a new targeting signal. I think that the topic is highly interesting and I would be happy to know more about the mutational processes that lead to modifications of intracellular targeting. In the present form, the analyses solely deal with protein sequences, without regard of the DNA sequence and gene structures of the investigated genes. However, I think that before multi- or pan- genome analyses can be done, within-genome analyses need to be completed. Please see the other sections for details.

Reviewer 3 ·

Basic reporting

See comments to author.

Experimental design

See comments to author.

Validity of the findings

See comments to author.

Additional comments

The current manuscript describes a very comprehensive analysis of the evolution of plastid transit peptides in plant systems. The evolution has been studied at both intra- and inter-genera level. Residue level variations have been analyzed. In general the methodology is appropriate and results draw some interesting conclusions on the possible mechanism of the divergence of the plastid transit peptides. Thus in my opinion the manuscript is suitable for publication in the journal in its current form. There are only few points that the authors may address or clarify (as below).

Minor points/issues:

1. In conclusions, authors write “….peptides evolve via simple substitutions, insertions and deletions, or alternative start sites rather than from random substitutions events…” I am not able to understand the difference implied in the usage of the terms ‘simple substitution’ and ‘random substitution’. Authors may kindly elaborate.

2. Colored boxes (guides) in pie charts of figures 6a, 8a and 9a are not clear at all. Especially the shades of blue are not distinguishable. Maybe the size of the boxes can be increased and color more contrast.

3. Legends of figure 10c are not clear at least at the resolution available to me.

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