All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.
Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.
The issues raised are addressed satisfactorily.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
Please address all the comments carefully so that we may not need another revision.
There are still relatively minor issues with English in the revised version which should be able to be fixed with careful editing.
Otherwise, the manuscript is definitely of acceptable standard.
No comment.
The authors have revised their conclusions in accordance with previous comments by myself and Reviewer 2.
None
The changes made have clarified the manuscript significantly. Sections that lacked citations for supporting literature are now resolved with appropriate citations.
The changes made have clarified aspects of the experimental design that were unclear previously, and clarifications from the last review round that were missing from the manuscript have been added.
The changes made have clarified the interpretation of the results. My major remaining issue lies still with some of the interpretation of positive (or now, adaptive) selection. First, the change to "adaptive" instead of positive is not necessary. Second and more significantly, the authors still often interpret positive selection on a branch somewhere in the evolutionary past of SCC4 species as indicating that gene underwent positive selection associated with SCC4 function (e.g. line 370 "This represents the functional adaptation of CHUP1-like_a in the SCC4 species and it could be involved in the unique chloroplast localization mechanisms within the single cell"). If a gene underwent positive selection in the common ancestor of an entire large clade like the former Chenopodiaceae family, well before SCC4 photosynthesis evolved in Bienertia, it is unlikely that positive selection was related to SCC4 function. It's a bit like saying that positive selection on a gene in the common ancestor of all primates means that positive selection event was important for some feature unique to humans -- this couldn't be the case because humans (and SSC4 photosynthesis) would not exist until much later, in distant descendants of the ancestral species that experienced positive selection. For positive selection to be associated with SCC4 function it would have to be specifically on the SCC4 branch, not a deeper ancestral branch. A similar over-statement re: positive selection is made near the end, on line 434: "Finally, adaptive selection on SCC4 gives the primary clue about the independent evolution in the Caryophyllales clade." These statements should be clarified to changed to note that while positive selection was observed in some of the gene phylogenies, it was not specifically associated with SSC4 function, therefore if these genes are important for SCC4 function, their involvement in SCC4 photosynthesis did not require additional positively selected mutations, at least not at the amino acid sequence level. They very well could have experienced positive selection on regulatory sequences outside of the coding region, but it would take a population genetics-type of approach to find this, e.g. looking for signs of a "selective sweep" around a given locus.
Revise the paper carefully as per the reviewer's comments
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the response letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the response letter. Directions on how to prepare a response letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/. #]
The revisions the authors have made partially address some of the issues that I raised in my previous review regarding poorly-defined focus and context. I still find the focus on paralogs of CHUP1 a little odd as these have not, as far as the authors state or cite, been shown to actually be involved in chloroplast movement. The authors still state on lines 288-290 and in supplemental table S2 that these are nuclear-localized, which begs the questions, what do they have to do with chloroplast movement if they're in the nucleus, and why are they included so prominently in this paper? The authors note in the response to my review that CHUP1-like-b is "probably localized at the central cytoplasmic compartment" but the justification for this hypothesis is vague and the paper still fairly clearly states that they are predicted to localize to the nucleus.
The context has been improved somewhat, however a number of the additional statements in the introduction and discussion lack appropriate citations (e.g. lines 42-55 of the introduction, lines 94-95 of the introduction, and lines 389-393 of the discussion.
I'm thoroughly confused by the newly-added statement on lines 308-310 about CHUP1 being classified as mesophyll-associated and CHUP1-like-b being classified as bundle sheath associated -- what is the justification for this and where is it explained? There's hardly any discussion of differential M/BS cell expression of any of these genes in Kranz-type C4 plants, so if one is specific to M-cells and the other BS-cell specific, this is never explained or supported with citations. The only possible justification for this is the statement that in maize, CHUP1 is more highly expressed in M cells (lines 391-392), but as noted above there's no citation here, and nothing explaining why CHUP1-like-b is considered BS-associated. The authors restate that CHUP1-like-b has "characterized BS specificity" on lines 412-413 again with no apparent explanation. And, despite my raising this point in the previous review, CHUP1-like-b is still referred to as paraphyletic for some reason on lines 412-413.
The experimental design has been clarified somewhat by the revisions, but in a number of cases the authors responded to concerns I raised in their comments to the reviewers but did not actually add anything to the paper to address these -- for example, I noted that they did not explain how sub-cellular localization was predicted, and they explained and even provided a reference in their reviewer response, but did not add this explanation or reference to the paper (Sahu et al 2020, cited in the reviewer response but nowhere to be found in the manuscript). The authors state that "detailed information" about this has been added to the methods section, but is not there; tracked changes show only minor edits to the methods section that do not include anything about these predictions.
The authors have improved the validity of the findings by removing a number of mistakes that I previously pointed out, mostly centered around interpretation of the tests for positive selection and significance of which taxa have or lack certain conserved domains.
That said, the concluding remarks:
"Finally, positive selection on SCC4 gives the primary clue about the independent evolution in the Caryophyllales clade. Similarly, positive selection in CHUP1-like_b in the rice and maize showed that this species could also vary in the MS-specific chloroplast mechanism. .. The results of this study shed light on the functional and evolutionary details of CHUP1 proteins. In SCC4, CHUP1-like_a and CHUP1-like_b proteins could be vital for the unique spatial distribution of CCp and PCp."
Are a substantial over-statement. Positive selection unique to SCC4 species was not observed - the putative positive selection the authors showed occurred in the common ancestor of Chenopodiaceae, long before SCC4 photosynthesis evolved, in a paralog that the authors note lacks the domain associated with binding to the chloroplast membrane, and which they still state localizes to the nucleus. The authors go so far as to definitively state that "the absence of HD in the CHUP1-like_b showed that it is not involved in the chloroplast movement" earlier in the manuscript (lines 329-330), directly contradicting the conclusion that it "could be vital for the unique spatial distribution of [central chloroplasts] and [peripheral chloroplasts]."
An interesting hypothesis is raised by some of the new text added to the manuscript, which the authors unfortunately do not consider. Since CHUP1 contains domains that are thought to connect the chloroplast membrane to the plasma membrane (lines 94-95, though lacking citation) and given that CHUP1 expression in maize is higher in M than BS cells (lines 391-392, though again lacking citation), the down-regulation of CHUP1 that the authors observed (Fig. 7) may be important for establishing the central aggregation of chloroplasts in Bienertia by releasing chloroplasts from the cell wall. In Kranz-type C4 plants, BS chloroplasts are not associated with the cell wall, while M cell chloroplasts are. Higher express of CHUP1 in maize M cells and early developmental stages of Bienertia (both cases where chloroplasts are all at the periphery) may reflect its role in anchoring chloroplasts to the cell wall, and its down-regulation during development in Bienertia may be required to release some of those chloroplasts to form the central aggregation. Discussion of this potential role and maybe additional experiments to address this would substantially improve this manuscript.
Revise the paper as per the comments from both reviewers.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. #]
The basic reporting in this paper is at standard but there were some minor English expression issues to clear up.
The research aims and scope of this paper are clearly spelled out and the methods well described.
This is an interesting in silico analysis of the CHUP family of proteins across C3, C4, CAM and single cell C4 plants which leads to the authors to postulate a role for the CHUP1a-like proteins in chloroplast positioning in SCC4 species.
This paper is interesting and informative about the roles of CHUP1 and related proteins in the evolution of C4, CAM and SCCC4 species. However, the analysis is restricted to a very small data set of sequence and represents relatively few origins of C4 evolution. It would have been interesting in eh C4 grasses to include Switchgrass (NADME type C4) and Urochloa (PCK) which both have annotated genome sequence, in the analysis. In C4 plants, different decarboxylatino types have various chloroplast arrangements in the bundle sheath and there may be interesting variation when one looks out the NADPME types to which the current study is restricted. Likewise, for the dicots, inclusion of Flaveria (https://doi.org/10.1002/tpg2.20095) which represents C3, C4 and intermediates would be desirable.
The analysis of gene structure and motifs was interesting but I felt it did not link clearly enough to proposed function and predicted structure using up t date algorithms (alphafold?).
This work is interesting but would benefit from an extended sequence space in the in silico analytics
The focus of this paper is poorly defined. The title purports to examine one gene in one species, but then three paralogs are discussed roughly equally without very much focus on the specific single-cell C4 species that is the purported focus of the paper. The introduction is very short and doesn't set up the scope of the study appropriately, such that reading the methods it is unclear why many of the experiments described were conducted. Only the function of CHUP1 is described in the introduction while the rest of the paper focuses on many other paralogs as well, making it unclear why these are being discussed. The function of CHUP1 is poorly discussed, and the specific functions of the other two main paralogs discussed (CHUP1-like a and b) is not mentioned at all. Have these been studied with respect to chloroplast movement, in C4 plants or otherwise? If so, what do they do? Is their function similar to CHUP1? If not, why are they included in the focus of this paper? Discussion is frequently very superficial, with vague statements about what is known in other species and confusion or incorrect use of terms. Genus/species names are lacking italicization throughout, and C3/C4 are lacking subscripting. Interpretation of the data is frequently highly speculative or outright misinterprets the data, particularly with regards to the branches of the phylogeny showing positive selection.
The exact aim of this paper is unclear. The question appears to be something along the lines of "Is the structure and function of CHUP1 and its related paralogs unique in single-cell C4 plants?" But this is not particularly clear, and the results do not answer this question. Most of the results presented are descriptive, e.g. the exon and motif structures of genes and the gene phylogeny. The interpretations of these data are quite speculative and not well supported. The methods are mostly described in sufficient detail, with the exception of predictions of sub-cellular localization. In some cases, more detail than needed is provided - for example, the CTAB extraction protocol could probably just be cited.
The findings are vague and highly speculative. As far as I can tell, no definitive finding exists here that CHUP1 or its related genes may have a unique function in single-cell C4 plants relative to other plants (C4 or otherwise). The data are clearly provided, the problem is that no clear and well-supported interpretation is made from these.
Line by line comments:
Lines 41-42: Why does Bienertia kavirense have SCC4 anatomy like Sueada aralocaspica, not other Bienertia species, when Suaeda is a separate SCC4 lineage? What is the reference for different anatomy in B. kavirense?
Line 64: Amborella is not a common ancestor of the angiosperms, it is an extant species. Care should be used to not treat extant species as if they are ancestors just because they branch from a basal node in the phylogeny.
Line 82: Cacti represent an entire family, the authors should state which species, not simply "cactus"
Line 141: GAPDH seems like an unusual choice for control given that species differ in photosynthetic pathways and thus expeession of Calvin cycle enzymes (and thus the control) may differ.
Lines 157-160: The synapomorphy/pleisomorphy/apomorphy is unclear. I assume what is being referred to as synapomorphic etc. is presence/absence of these genes, but the phrasing here is overly convoluted.
Line 171: It's unclear what exactly is meant by motifs here
Line 187-188: It's likely this pattern just represents grass-specific changes in the three species that do not share these motifs. Same with motif 7, shared with Amborella and other dicots -- this is probably just a monocot-specific change.
Lines 195-196: Were these missing motifs confirmed to not be the result of missed exons? It would be helpful to BLAST these motifs against the entire gene region to determine if failed exon detection is the reason for their absence.
Line 203-204: The branch in Fig. 6 showing positive selection is actually for the common ancestor of Chenopodium, Suaeda, and Bienertia, so this would not be SSC4-specific positive selection. Also, the dN/dS ratio on this branch of 999 seems highly likely to be some kind of error. Is there division by zero causing this, e.g. one non-synonymous change and zero synonymous changes, so the reported value goes to infinity? If so this seems like weak evidence of positive selection.
Lines 204-206: This is incorrect use of the term diversifying selection, which refers to selection that pushes members of a population to extremes for a given trait, not separate instances of positive selection in different branches of a phylogeny.
Lines 220-221: It is unclear how subcellular localizations were predicted.
Line 225: How does CHUP1's function differ between C3 and C4 plants? The authors say this is well studied but do not discuss at all, which is needed to place any SCC4 functional interpretation in context.
Line 229-231: This statement is vague and, as written, seems self-evident, that gene numbers would be correlated with duplication and loss.
Line 232: What is MS? Mesophyll? And what is meant by mesophyll CHUP1 being a synapomorphy?
Line 233: How is CHUP1-like_b paraphyletic? Fig. 1 clearly marks a monophyletic group as CHUP1-like_b.
Line 234: What MS chloroplast proteins?
Line 237: This statement is vague and unsupported.
Line 245-246: "Amplified gene architecture" is unclear; I assume this means increasing intron / total gene size? There are many processes other than transposons that could increase intron size, and high transposon activity in a coding region would be expected to disrupt gene function and be selected against. Moreover, if the authors are interested in transposons, they could look for their sequences directly, rather than assuming that increasing intron size is due to transposons.
Line 247: Unsurprising given that these genes shared a very ancient common ancestral gene.
Lines 249-253: Very speculative, and again it's probably just a grass-specific change giving the appearance of similarity between pineapple and the dicots, particularly given that this pattern is shared with Amborella.
Lines 254-257: No, the branch showing high positive selection does not separate SCC4 plants from Chenopodium, it shows positive selection in the ancestor of all three (Chenopodium, Suaeda, Bienertia), and again the 999 value seems to strongly suggest something went wrong in the dN/dS calculations.
Lines 257-262: Again, wrong use of "diversifying selection" and highly speculative. Fig. 6 doesn't show any particular pattern of C4-specific positive selection. The closest it comes is in the Zea mays branch, but this should be one node deeper and shared with Sorghum bicolor if it was involved in the evolution of C4 photosynthesis, since Sorghum and maize are part of the same C4 lineage, Andropogoneae.
Lines 263-277, and Fig. 7: I think there may be a fundamental problem with the way gene expression was measured here. Was the GAPDH gene that was used as a control a photosynthetic GAPDH? If so, the declining relative expression levels of all CHUP1-related genes may simple be due to an increase in GAPDH expression as leaves become more photosynthetically active. Also, where is the information about nuclear-localization of genes coming from?
Lines 271-272: Why is lower expression correlated with nuclear localization? And why is a protein involved in chloroplast movement localizing to the nucleus?
Line 274: What is meant by structural interactions between plants?
Line 276-277: Again, if the paralogs are nuclear-localized, what is their involvement in chloroplast movement?
Line 283-284: The positive selection results do not show this.
FIGURES:
Fig. 1: The colored shapes are very difficult to interpret; gene names should be appended with some kind of species abbreviation so the reader doesn't need to keep checking the legend to see what everything is. Referring to Amborella as "extant" is strange; I assume this is another example of misinterpreting it as an ancestor.
Fig. 2: Comparing the photosynthetic functional types would be easier if the different paralogs were grouped together and photosynthetic type indicated, rather than grouping photosynthetic types and labelling the paralogs.
Figs. 3, 4, 5: The numbering of the conserved motifs doesn't seem to follow any particular logic. They're not numbered sequentially or by size. Sequential numbering would make it easier to follow the discussion of these in the main text. They could also be labelled in the figure if panel B were extended horizontally to give more space.
Fig. 6: The different panels are not described in the figure legend, leaving it to the reader to figure out from the sequence names that they represent the three different paralogs. The
Supplementary Files:
A document labelled supplementary "Fig" 7 is an Excel file.
All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.