Integration of Illumina and PacBio HiFi Sequencing Reveals a Three-Linear-Molecule Mitogenome with RNA-Editing Sites and Phylogeny in Arrow Bamboo (Fargesia qinlingensis)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the reviewed MS, the authors give detail data on the structure of the mitogenome of a poorly investigated bamboo, Fargesia qinlingensis, from China. The Abstract does not reflect main achievements of the study and needs improvements. The Title is suboptimal, because “three mitogenomes” sounds like if the targeted plant has three types of homologous mitogenomes, whereas in fact the authors describe a single mitogenome that has complicated structural organization. The MS looks like a detail technical report. It gives many statistical and quantitative data and lacks strong conclusions, biological interpretations and comparison. The MS is quite wordy, it may be 2-3 times shorter. The Discussion repeats some data given in the Introduction. In general, the Discussion is poorly structured; it needs serious revisions. The Discussion could be subdivided into 3-4 subsections with distinct subheading corresponding to main findings of the research, e.g.: Diversity of mitogenomes in monocots, Unusual characteristics of the mitogenome of Fargesia qinlingensis; etc. Some additional remarks are listed below.

16: three overlapping linear molecule --- please consider rewording

26: revealed the close taxonomic status between --- please, consider rewording, taxonomic status is something followed from taxonomy, whereas your analysis revealed sister phylogenetic relationship

43: in most terrestrial plants --- please, specify, what is known about non-terrestrial plants

48-49: please, say this in other words, this may be unclear for a reader

50-57: this text sounds unnatural here, as if it was generated by AI or it is not connected with the context. Please, rewrite this.

58: tree-like grasses --- please, reconsider using this term

61: enormous tribes --- please, explain why do you call the tribes “enormous”

62: herbaceous bamboo --- please, compare with “tree like grasses” ( line 58)

87: “To date, very few high-quality bamboo mitogenomes have been reported in public databases, although it is known that they have undergone complicated and unusual evolutionary events in diverse families.” --- please, give references and be more precise: how many mitogenomes are known, and what are their main characteristics?

97: resolved --- ?

99: genome --- mitogenome?

101: “Our findings provide a genetic basis for elucidating the molecular mechanisms of mitochondrial genes underlying climate adaptation in subalpine bamboo” --- this is overgeneralization

106: harvested and sampled --- do you really need “harvested” here? Was the bamboo wild or cultivated?

108: Professor Tao Su and Dr. Yong Lijun identified ---- Usually persons who helped you are acknowledged in the Acknowledgment section, e.g. We are grateful to Dr… (country, university) for…

112: concentrations --- please, provide the obtained concentrations of the DNA and RNA samples

154: Section 2.5. --- please provide a brief explanation for “RNA editing”  otherwise it may be unclear for readers: what is this, why it is important and how it could be detected

202: Figure 1 --- please, explain the arrows, what do they mean? Additionally, please explain happens with DNA in the trifurcation areas? Please, specify 3’ and 5’ ends and positive and negative strands in M1, M2 and M3 fragments.

208: Table 1 --- please, explain what do the numbers given in the Table mean?

225: Figure 2 --- please consider revising this Figure it looks stylistically suboptimal

229: footnote is unnecessary, please use full protein name in the Table2

231: Figure 3 --- “Calculation of the” redundant

246: Figure 4,5: it looks really confusing when you give circular diagrams and say about linear molecules. Please, find a way to explain this better or avoid such misleading terminology

Section 3.5. and Figure 6 --- this part of the MS needs revising in order to make it easier to understand to a reader. Please, explain directly in the text why the “dotted line framed” are considered “RNA editing sites” but not nuclear polymorphism?

308: Figure 7 “the gray-curved ribbons” --- I do not see any gray ribbons in fig.7

337-353: this text corresponds to Introduction, repetition

370-389: this text looks unnatural here

478-483: this is just repetition from Results, no need to repeat

Discussion needs serious revision. Please, discuss the main results of your study in broader context and provide comparisons and interpretations. Currently, the Discussion repeats data from the Results and retells some general information that should be actually given in condensed form in the Introduction.

Table S1: please check all values of st. deviation, especially for insert size (1.5)

Comments on the Quality of English Language

minor

Author Response

In the reviewed MS, the authors give detail data on the structure of the mitogenome of a poorly investigated bamboo, Fargesia qinlingensis, from China. The Abstract does not reflect main achievements of the study and needs improvements. The title is suboptimal, because “three mitogenomes” sounds like if the targeted plant has three types of homologous mitogenomes, whereas in fact the authors describe a single mitogenome that has complicated structural organization. The MS looks like a detail technical report. It gives many statistical and quantitative data and lacks strong conclusions, biological interpretations and comparison. The MS is quite wordy, it may be 2-3 times shorter. The discussion repeats some data given in the Introduction. In general, the discussion is poorly structured; it needs serious revisions. The discussion could be subdivided into 3-4 subsections with distinct subheading corresponding to main findings of the research, e.g.: Diversity of mitogenomes in monocots, Unusual characteristics of the mitogenome of Fargesia qinlingensis; etc. Some additional remarks are listed below.

R1: We sincerely appreciate the reviewer’s time and efforts in extensively commenting on the manuscript. The title has been modified based on the comments of all reviewers. The discussion part was optimized appropriately. Nevertheless, variable structures and complexities of plant mitogenomes suggested that it might be impossible to conventionally consider them as a single/multiple linear/circled conformation like plastomes or mitogenomes from animals. Thus, the comprehensive dissection of mitogenome assembly “gives many statistical and quantitative data,” though it was plausible that it “lacks strong conclusions.” A native English researcher has further edited the manuscript and went through a double-check by a grammar-checking system. In addition, the manuscript has been edited by Springer Nature English editing service previously, as well as the novel MDPI system (certificate ID: english-82673).

16: three overlapping linear molecule --- please consider rewording

R2: Thanks. It has been reworded based on the reviewer’s comments.

26: revealed the close taxonomic status between --- please, consider rewording, taxonomic status is something followed from taxonomy, whereas your analysis revealed sister phylogenetic relationship

R3: This is a good comment, and the text has been revised by substituting the “taxonomic status” with “relationship.”

43: in most terrestrial plants --- please, specify, what is known about non-terrestrial plants

R4: This comment is quite interesting. The “non-terrestrial plants” have not been used in the text. We cannot see any problem with the description of  “in most terrestrial plants,” which was concluded from citation 6. Such words were presented commonly in the literature:

1. Franklin et al. Global change and terrestrial plant community dynamics. PNAS. 2016. https://doi.org/10.1073/pnas.151991111).

48-49: please, say this in other words, this may be unclear for a reader

R5: Thanks, but this comment is confusing. The detailed information and understandable background can be found in citation 8, which was used to summarize the sentence.

50-57: this text sounds unnatural here, as if it was generated by AI or it is not connected with the context. Please, rewrite this.

R6: Sorry, and we have never used AI or any app for scientific writing! How could it be possible? Please check the cited references 9 and 10 to obtain the detailed information. Such backgrounds in literature were naturally collected to generate these sentences that were typically used for bridging the identified DNA migration and RNA-editing events in the deciphered mitogenome of F. qinlingensis.

58: tree-like grasses --- please, reconsider using this term

R7: Thanks. This comment is quite interesting. The “tree-like grasses/grass trees/tree-grasses” bamboo refers to two large tribes of woody bamboo (Arundinarieae and Bambuseae). Such words can be used commonly in the exemplified literature:

1. Ma et al. Genome assemblies of 11 bamboo species highlight diversification induced by dynamic subgenome dominance. 2024. Nat Genet 56, 710–720. https://doi.org/10.1038/s41588-024-01683-0.

2. Lobovikov, M., Schoene, D. & Yping, L. Bamboo in climate change and rural livelihoods. Mitig Adapt Strateg Glob Change. 2024. 17, 261–276. https://doi.org/10.1007/s11027-011-9324-8.

61: enormous tribes --- please, explain why do you call the tribes “enormous”

R8: Thanks. This typo has been revised. Please check the revised manuscript in L238.

62: herbaceous bamboo --- please, compare with “tree like grasses” (line 58)

R9: Thanks and sorry. The herbaceous bamboo belongs to the large tribe of Olyreae. Such words can be used typically in literature:

1. Ruiz-Sanchez, E., Sosa, V., Ortiz-Rodriguez, A.E. et al. Historical biogeography of the herbaceous bamboo tribe Olyreae (Bambusoideae: Poaceae). 2019. Folia Geobot 54, 177–189. https://doi.org/10.1007/s12224-019-09342-7.

2. Ma et al. Genome assemblies of 11 bamboo species highlight diversification induced by dynamic subgenome dominance. 2024. Nat Genet 56, 710–720. https://doi.org/10.1038/s41588-024-01683-0.

87: “To date, very few high-quality bamboo mitogenomes have been reported in public databases, although it is known that they have undergone complicated and unusual evolutionary events in diverse families.” --- please, give references and be more precise: how many mitogenomes are known, and what are their main characteristics?

R10: This comment is quite essential. Hitherto, NO high-quality bamboo mitogenomes have been reported from the NCBI database and literature. The quality and structure complexity of the mitogenome deciphered were determined by the sequencing methods and covered the depth of the genome. Thus, we addressed that investigation of sequence variations and recombination events by using the complete mitogenome offers unique advantages in reconstructing phylogenies and classification, providing new insights into disease and stress susceptibility and lifespan extension during environmental and climate adaptation (detailed in ref 11).

97: resolved --- ?

R11: Thanks, but we cannot see any problem. The “resolved” means being imaged and processed clearly. Such words are used commonly in exemplified literature:

1. Zhou et al. The first mitochondrial genome of Melastoma dodecandrum resolved structure evolution in Melastomataceae and micro inversions from inner horizontal gene transfer. 2023. Industrial Crops and Products, 205, 117390.

2. Dorji, J et al. Recovery of mitogenomes from whole genome sequences to infer maternal diversity in 1883 modern taurine and indicine cattle. 2022. Sci Rep 12, 5582. https://doi.org/10.1038/s41598-022-09427-y)

99: genome --- mitogenome?

R12: Thanks, and it has been specified.

101: “Our findings provide a genetic basis for elucidating the molecular mechanisms of mitochondrial genes underlying climate adaptation in subalpine bamboo” --- this is overgeneralization

R13: Thanks. This sentence has been modified. Please check the revised manuscript in L156-157.

106: harvested and sampled --- do you really need “harvested” here? Was the bamboo wild or cultivated?

R14: Thanks. This comment is quite interesting. We addressed that fresh leaves of F. qinlingensis harvested and sampled from the Foping National Nature Reserve, Qinling Mountain, Hanzhong City, Shaanxi Province, at an altitude of 2,432 m (33°40′13′′ N, 107°49′56′′ E), suggesting that the bamboo wild growth habitat and sampling methods. Only N-frozen samples were qualified for delivery and were subsequently directly used for DNA isolation, mitogenome sequencing, and library construction. These general rules differed from plastome sequencing (damped or silicone-dried materials). If the response is not promising, we have revised the sentence to “Fresh leaves of F. qinlingensis were collected by using liquid nitrogen (N) from the Foping National Nature Reserve...”. Please check the revised manuscript in L160.

108: Professor Tao Su and Dr. Yong Lijun identified ---- Usually persons who helped you are acknowledged in the Acknowledgment section, e.g. We are grateful to Dr… (country, university) for…

R15: OK, and thanks. In fact, Tao Su and Lijun Yong are the coauthors who performed the current manuscript’s data analyses and experiments. How would it be possible for authors to acknowledge themselves?

112: concentrations --- please, provide the obtained concentrations of the DNA and RNA samples

R16: This comment is quite confusing. Does the reviewer request a list of concentrations determined for all DNA/RNA samples? Or do we need to provide the general DNA/RNA concentration criteria qualified for further analyses (sequencing and PCR)? If it is the latter, the “pure DNA has an OD 260 /OD 280 ratio of ~1.8 and pure RNA has an OD 260 /OD 280 ratio of ~2.0 were qualified…” has been provided. Please check the revised manuscript in L306.

154: Section 2.5. --- please provide a brief explanation for “RNA editing” otherwise it may be unclear for readers: what is this, why it is important and how it could be detected

R17: This is a good comment. The method in predicting RNA editing sites in all PCGs has been specified in L521-524.

202: Figure 1 --- please, explain the arrows, what do they mean? Additionally, please explain happens with DNA in the trifurcation areas? Please, specify 3’ and 5’ ends and positive and negative strands in M1, M2 and M3 fragments.

R18: Thanks. This comment is quite interesting. The black arrows (A, B, and C) generally indicate approaches from the initial draft of the mitogenome assembly (6 ctgs) to the resolved multi-branched conformation and preface of three linear molecules (M1, 2, and 3). Unfortunately, the positive and negative strands cannot be specified in a multi-branched conformation because they are assembled contigs/nodes with complicated configurations. The identified genes transcribed clockwise or counterclockwise were only exhibited in the circular molecules but not for presenting “3’ and 5’ ends and positive and negative strands”. 

208: Table 1 --- please, explain what do the numbers given in the Table mean?

R19: The number of nodes shown corresponds to Figures 1B and 1C. We provided an image with high resolution automatically compressed by the submitting system, prompting the concern.

225: Figure 2 --- please consider revising this figure it looks stylistically suboptimal

R20: Thanks. This comment is quite essential. Fig 2 is the typical schematic diagram of the mitogenome structure (three linear molecules) and gene distribution of F. qinlingensis, which are the critical findings of the current work. Unusual (actually, it is typical) mitogenome structures commonly occur in plants. Several recent reports (ref 22 and 48, see below) show that one linear combined two circular molecules mitogenomes were deciphered in V. leiocarpa and spearmint (M. spicata L.), which have been cited for the discussion.

22. Guo, S.; Li, Z.; Li, C.; Liu, Y.; Liang, X.; Qin, Y. Assembly and characterization of the complete mitochondrial genome of Ventilago leiocarpa. Plant Cell Rep. 2024, 43, 77.

48. Jiang, M.; Ni, Y.; Zhang, J.; Li, J.; Liu, C. Complete mitochondrial genome of Mentha spicata L. reveals multiple chromosomal configurations and RNA editing events. Int. J. Biol. Macromol. 2023, 251, 126257.

229: footnote is unnecessary, please use full protein name in the Table2

R21: Thanks, and it has been removed.

231: Figure 3 --- “Calculation of the” redundant

R22: Many thanks, and it has been deleted.

246: Figure 4,5: it looks really confusing when you give circular diagrams and say about linear molecules. Please, find a way to explain this better or avoid such misleading terminology

R23: Thanks. This comment is quite interesting. Three repetitive sequences predominantly occurred in plant mitogenome: SSRs, tandem repeats, and dispersed repeats (e.g., forward, reverse, complementary, and palindromic repeats). In Fig 4, three circular diagrams may clearly illustrate distributions and links of three types of repetitive sequences, including dispersed repeats (e.g., palindromic repeats, orange) and forward repeats, blue), tandem repeats (black lines, second ring), and SSRs (black lines, outer ring) in each single molecule rather than using more than eight bar graphs to show accounted numbers. The cyclic map would be optimized to avoid misleading and image redundancy.

Section 3.5. and Figure 6 --- this part of the MS needs revising in order to make it easier to understand to a reader. Please, explain directly in the text why the “dotted line framed” are considered “RNA editing sites” but not nuclear polymorphism?

R24: Thanks. This comment is very important. A detailed explanation for the validation of exemplified RNA-editing sites (atp9, cox2, nad1, and nad7) has been provided in the legend of Figure 6 and the text in L583-591. The Sanger sequencing determined the PCR amplified products, which used the cDNA and gDNA as templates. Based on the sequencing data, the changed bases (dotted line framed) were detected between the gDNA and cDNA PCR-amplified sequences, verifying the RNA-editing sites (A-T) and accounting for the consistency in the prediction (Figure S3).

308: Figure 7 “the gray-curved ribbons” --- I do not see any gray ribbons in fig.7

R25: Thanks. As the legend indicates, the gray-curved ribbons crossed with the pink-curved ribbons were presented in Fig 7A. It would be visible that Figure 7A had been thoroughly reviewed.

337-353: this text corresponds to Introduction, repetition

R26: Thanks. The referred overlapping sentences in the discussion have been modified.

370-389: this text looks unnatural here

R27: Many thanks. These sentences have been reworded in order to look natural.

478-483: this is just repetition from Results, no need to repeat

R28: Thanks, and after a double-check, the referred “repetition” has been removed.

Discussion needs serious revision. Please, discuss the main results of your study in broader context and provide comparisons and interpretations. Currently, the discussion repeats data from the Results and retells some general information that should be actually given in condensed form in the Introduction.

R29: Many thanks. The discussion has been revised and condensed based on the substantial results.

Table S1: please check all values of st. deviation, especially for insert size (1.5)

R30: Thanks for the comment. After double-checking the original report of three ctgs by using the qualimap analyses, we confirm that the SD values in the Table S1 are correct. Qualimap software was used to examine sequencing alignment data in SAM/BAM files based on mapped reads, providing an overall view that helps detect biases in the sequencing and mapping of the data and eases decision-making for further analysis. Nevertheless, the minimal typos have been corrected in the updated Table S1.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors wrote a manuscript that contributes to elucidating mitochondrial properties associated with climate resilience and stress acclimation. The authors' manuscript is technically excellent and contains exceptionally deep analysis.

I provide a very brief comment on the manuscript below:

 

1. The authors explain de novo assembly in Materials and Methods 2.2. However, the authors explain that they assembled the short leads first and then assembled the long leads later and then made the comparison.

When doing long read sequencing, isn't it common to assemble the long reads first and then map the short reads to the low quality parts of draft genome from long reads? Rather than assembling both sides separately and comparing them. Also, since there were too many repetitive sequences, I think it would be necessary to clearly describe how you confirmed whether there were three contigs or whether they were really composed of three linear genomes.

 

2. The authors briefly mention other plants with linear or combined linear and circular mitochondrial genome structures in lines 359-361 of the Discussion section. However, in this regard, I propose to emphasize the significance of the fact that, if it is true that the mitochondrial genome is properly assembled, the three linear mitochondrial genomes of which the authors have revealed their genome structure are the first to be discovered among plants belonging to the Bambusoidae.

Author Response

The authors wrote a manuscript that contributes to elucidating mitochondrial properties associated with climate resilience and stress acclimation. The authors’ manuscript is technically excellent and contains exceptionally deep analysis. I provide a very brief comment on the manuscript below:

1. The authors explain de novo assembly in Materials and Methods 2.2. However, the authors explain that they assembled the short leads first and then assembled the long leads later and then made the comparison. When doing long read sequencing, isn’t it common to assemble the long reads first and then map the short reads to the low quality parts of draft genome from long reads? Rather than assembling both sides separately and comparing them. Also, since there were too many repetitive sequences, I think it would be necessary to clearly describe how you confirmed whether there were three contigs or whether they were really composed of three linear genomes.

R1: We appreciate the reviewer’s time and efforts in commenting on the manuscript. Thanks. This comment is significant. The methods in mitogenome assembly and configuration analyses have been specified. Please check the revised manuscript in L310-320.

2. The authors briefly mention other plants with linear or combined linear and circular mitochondrial genome structures in lines 359-361 of the Discussion section. However, in this regard, I propose to emphasize the significance of the fact that, if it is true that the mitochondrial genome is properly assembled, the three linear mitochondrial genomes of which the authors have revealed their genome structure are the first to be discovered among plants belonging to the Bambusoidae.

R2: Many thanks and the answer is positive. Hitherto, no additional high-quality assembled mitogenomes have been released in bamboo. This poor situation could be caused by the complexity of bamboo’s genome, polyphyletic origin, and evolutionary diversity. Nevertheless, the multi-branched molecules/chromosomes mitogenome might be typical in most terrestrial plants but not animals. Thus, “one ring does not rule them all,” and representations of plant mitogenomes as simple, circular molecules are not accurate descriptions of their true nature and that, in reality, plant mitochondrial DNA is a complex, dynamic mixture of forms (detailed in the ref 7). As it was responded to other reviewers, some of the recent literatures are required and necessary for an in-depth understanding of complexity in the mitogenome assembly and conformation deduction:

1. Guo, S.; Li, Z.; Li, C.; Liu, Y.; Liang, X.; Qin, Y. Assembly and characterization of the complete mitochondrial genome of Ventilago leiocarpa. Plant Cell Rep. 2024, 43, 77.

2. Jiang, M.; Ni, Y.; Zhang, J.; Li, J.; Liu, C. Complete mitochondrial genome of Mentha spicata L. reveals multiple chromosomal configurations and RNA editing events. Int. J. Biol. Macromol. 2023, 251, 126257.

3. Hao, Z., Zhang, Z., Jiang, J. et al. Complete mitochondrial genome of Melia azedarach L., reveals two conformations generated by the repeat sequence mediated recombination. BMC Plant Biol. 2024, 24, 645.

Reviewer 3 Report

Comments and Suggestions for Authors

Review on “Integration of Illumina and PacBio HiFi Sequencing Revealed Three Linearized Mitogenomes in Arrow Bamboo (Fargesia qinlingensis) with RNA Editing and Implicated Phylogenies” for manuscript ID forests-3106910

 

In this manuscript the authors describe mitochondrial genome of Arrow bamboo, that consists of three linear molecules that overlap and make up multi-branched mitochondrial genome skeleton. The obtained data is valuable and could help to improve our knowledge on the evolution of plant mitochondrial genomes.

In the Introduction section authors describe plant MT genomes in general, ecological facts on different bamboo species, and known up to date information on sequenced bamboo MT genomes. Additionally, the authors discussed the role of RNA editing in mitochondrial genomes and its impact on plant development and function.

The paper is very interesting and results are indeed valuable, but English needs to be heavily revised, as well as some abbreviations usage.

My comments on Abstract section:

Authors should use “PCG” (Protein Coding Genes) abbreviation, instead of “PEG” that is widely used for “PolyEthylene Glycol”, and incorporate the abbreviation decoding in text. And if the text is about protein-coding genes, rRNA and tRNAs, they should not be summarized by PCG abbreviation, but by terms: genes, genomic features, annotation features, etc.

Line 22 – “dispersed repeats” – tandem repeats?

Line 23 – “forward” – direct.

Lines 27-29 “The primary features of the mitogenome of F. qinlingensis will help decipher the biological functions of the mitochondrial traits related to climate resilience and stress acclimation.” – needs to be rewrited.  

Lines 50-51 – “The horizontal transfer of homologous DNA fragments between plant genomes is critical for mitogenome evolution.” – the statement needs clarification. Horizontal transfer may mean in the context “Nucleus to Mitochondria” or “Mitochondria to Nucleus”. What “homologous” stands for? The Nucleus – to – Mitochondria DNA transfer is connected to MGE activity. The second variant – to unknown processes resulting in emergence of mtDNA fragments in nDNA. None of these share “homologous DNA fragments”. On the other hand – the homologous recombination occurs in mtDNA, and it is not horizontal gene transfer.

Lines 51-52 – The aim of RNA editing is indeed maintaining of correct mRNA maturation and translation by generating start, stop and splicing sites, but it is resulting in not “altered”, but “correct” length and function.

I also suggest to include recent work on the same species, which MT genome was released couple of weeks ago https://www.ncbi.nlm.nih.gov/nuccore/PP885440.1 (no paper is published yet).

 

My questions and comments about Results and Discussion:

You need to clarify the proposed model of mitochondrial genome in terms of its molecular structure. As from the Fig 1A it seen as three circles possessing two overlapped regions and how the second strand of the DNA is positioned is hard to understand – are there nicked DNA molecules? First DNA strand is near 1.5 times longer than second? And from the Fig 1C it is seen as two circular molecules with big light blue complementary region and second strand is…? How exactly the connection of three DNA strands are formed in your model?

Furthermore, latest findings on mitochondrial genome structure tells that it is highly subject to rearrangements during the lifetime of a single organism – the same direct repeats between gene groups allows to swap these groups, and inverted repeats allows the group to roll over to inverse direction, which results in multiple variants of mtDNA could be detected simultaneously in one sample. Comment these please in terms of your work. Is the 3 linear DNA molecules model only the one possible state? Or it is just one of possible states?

Line 247 – What linked palindromic repeats is?

Line 251 – there are two types of repeats – tandem and interspersed. What do dispersed stand for?

Figure 4 should be moved down, after it was mentioned in text.

Figure 7 – the same as Fig. 4.

Figure 7B – what is the purpose of that small tree just above the tree scale?

Lines 318-328 – You need to clarify either here or in Methods section – what approach did you used for phylogeny estimation. Did you used just MAFFT alignment of full mt-genomes? Or alignment of shared genes – concatenation and subsequent phylogeny estimation? Or the coalescence approach?

Line 351 – Why “structural” in particular? Or you meant nucleoid proteins? needs clarification.

Comments on the Quality of English Language

Author Response

Review on “Integration of Illumina and PacBio HiFi Sequencing Revealed Three Linearized Mitogenomes in Arrow Bamboo (Fargesia qinlingensis) with RNA Editing and Implicated Phylogenies” for manuscript ID forests-3106910. In this manuscript the authors describe mitochondrial genome of Arrow bamboo, that consists of three linear molecules that overlap and make up multi-branched mitochondrial genome skeleton. The obtained data is valuable and could help to improve our knowledge on the evolution of plant mitochondrial genomes. In the Introduction section authors describe plant MT genomes in general, ecological facts on different bamboo species, and known up to date information on sequenced bamboo MT genomes. Additionally, the authors discussed the role of RNA editing in mitochondrial genomes and its impact on plant development and function. The paper is very interesting and results are indeed valuable, but English needs to be heavily revised, as well as some abbreviations usage.

R1: We sincerely appreciate the reviewer’s time and efforts in extensively commenting on the manuscript. A native English researcher has further edited the manuscript and went through a double-check by a grammar-checking system. In addition, the manuscript has been edited by Springer Nature English editing service previously, as well as the novel MDPI system (certificate ID: english-82673).

My comments on Abstract section:

Authors should use “PCG” (Protein Coding Genes) abbreviation, instead of “PEG” that is widely used for “PolyEthylene Glycol”, and incorporate the abbreviation decoding in text. And if the text is about protein-coding genes, rRNA and tRNAs, they should not be summarized by PCG abbreviation, but by terms: genes, genomic features, annotation features, etc.

R2: Thanks. This comment is quite interesting. In summary, we addressed that “thirty-five unique PCGs were annotated in the complete mitogenome, including twenty-four structural genes, eleven nonstructural genes” with an additional three rRNA and sixteen tRNA genes, suggesting that rRNA and tRNA are not categorized into the PEG. Given that the PEG (protein-encoding gene) was personally regarded as a dehydration chemical, they have been replaced by the PCG (protein-coding gene). Nevertheless, the PEG is also typically used in exemplified literature:

1. Maik Pietzner et al. Mapping the proteo-genomic convergence of human diseases. 2021. Science 374, eabj1541. DOI:10.1126/science. abj 1541.

2. Rocca, J et al. Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed. 2015. ISME J 9, 1693–1699. https://doi.org/10.1038/ismej. 2014.252.

Line 22 – “dispersed repeats” – tandem repeats?

R3: Thanks! We confirmed that “dispersed repeats” is correct. Dispersed repeats (transposable elements) are a class of DNA sequences that can move their position on the genome, regulate gene expression, and influence plant phenotypic traits. The dispersed and tandem repeats were two types of long repetitive sequences that determine structural variations of the mitogenome, as indicated in L412-413. Such words are used frequently in a large number of scientific publications.

Line 23 – “forward” – direct.

R4: Thanks. The forward repeat is a typical type of the long repeative sequence, belong to the dispersed repeats.

Lines 27-29 “The primary features of the mitogenome of F. qinlingensis will help decipher the biological functions of the mitochondrial traits related to climate resilience and stress acclimation.” – needs to be rewrited. 

R5: Thanks, and it has been reworded and specified.

Lines 50-51 – “The horizontal transfer of homologous DNA fragments between plant genomes is critical for mitogenome evolution.” – the statement needs clarification. Horizontal transfer may mean in the context “Nucleus to Mitochondria” or “Mitochondria to Nucleus”. What “homologous” stands for? The Nucleus – to – Mitochondria DNA transfer is connected to MGE activity. The second variant – to unknown processes resulting in emergence of mtDNA fragments in nDNA. None of these share “homologous DNA fragments”. On the other hand – the homologous recombination occurs in mtDNA, and it is not horizontal gene transfer.

R6: Thanks. This comment is quite interesting. Horizontal gene transfer (HGT; also known as lateral gene transfer) is the non-sexual movement of genetic information between genomes, NOT just in the context of “Nucleus to Mit” or “Mit to Nucleus.” Incoming DNA or RNA can replace existing genes or introduce new genes into a genome. Several plant mitogenomes have acquired genes via HGT from external organisms, incorporating multiple plastid sequences derived from chloroplasts (or vice versa), a common phenomenon in plants. Such basic knowledge can be obtained in exemplified literature:

1. Keeling, P., Palmer, J. Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet 9, 605–618 (2008). https://doi.org/10.1038/nrg2386.

2. Soucy, S., Huang, J. & Gogarten, J. Horizontal gene transfer: building the web of life. Nat Rev Genet 16, 472–482 (2015). https://doi.org/10.1038/nrg3962.

3. Shannon C.K. Straub, Richard C. Cronn, Christopher Edwards, Mark Fishbein, Aaron Liston, Horizontal Transfer of DNA from the Mitochondrial to the Plastid Genome and Its Subsequent Evolution in Milkweeds (Apocynaceae), Genome Biology and Evolution, Volume 5, Issue 10, October 2013, Pages 1872–1885, https://doi.org/10.1093/gbe/evt140

4. Cruz Plancarte, D., Solórzano, S. Structural and gene composition variation of the complete mitochondrial genome of Mammillaria huitzilopochtli (Cactaceae, Caryophyllales), revealed by de novo assembly. BMC Genomics 24, 509 (2023). https://doi.org/10.1186/s12864-023-09607-8.

5. Hao, Z., Zhang, Z., Jiang, J. et al. Complete mitochondrial genome of Melia azedarach L., reveals two conformations generated by the repeat sequence mediated recombination. BMC Plant Biol 24, 645 (2024). https://doi.org/10.1186/s12870-024-05319-7.

Lines 51-52 – The aim of RNA editing is indeed maintaining of correct mRNA maturation and translation by generating start, stop and splicing sites, but it is resulting in not “altered”, but “correct” length and function.

R7: Thanks. This comment is very important. The sentence has been reworded to avoid misleading information.

I also suggest to include recent work on the same species, which MT genome was released couple of weeks ago https://www.ncbi.nlm.nih.gov/nuccore/PP885440.1 (no paper is published yet).

R8: Thanks, but this comment is quite confusing. How would it be logical to make a comparison with recently released data? It is not even derived from any published paper. The referred mitogenome annotation (PP885440.1, 06-JUN-2024) in NCBI database was submitted two years later than ours (OP324798.1/OP324799.1/ OP324800.1, 27-AUG-2022). In addition, the deciphered mitogenome in our present work was performed using hybrid sequencing platforms (SRR27909850 for Illumina and SRR29094227 for PacBio) employing two combined assembling methods (Getorganelle and Unicycler), which are entirely distinct from the referred mitogenome annotation.  

My questions and comments about Results and Discussion:

You need to clarify the proposed model of mitochondrial genome in terms of its molecular structure. As from the Fig 1A it seen as three circles possessing two overlapped regions and how the second strand of the DNA is positioned is hard to understand – are there nicked DNA molecules? First DNA strand is near 1.5 times longer than second? And from the Fig 1C it is seen as two circular molecules with big light blue complementary region and second strand is…? How exactly the connection of three DNA strands are formed in your model? Furthermore, latest findings on mitochondrial genome structure tells that it is highly subject to rearrangements during the lifetime of a single organism – the same direct repeats between gene groups allows to swap these groups, and inverted repeats allows the group to roll over to inverse direction, which results in multiple variants of mtDNA could be detected simultaneously in one sample. Comment these please in terms of your work. Is the 3 linear DNA molecules model only the one possible state? Or it is just one of possible states?

R9: Thanks. These comments are pretty critical. If the manuscript has been thoroughly reviewed (including the supplementary files), it is not hard to understand how the mitogenome was sequenced, assembled, and deduced. In Fig 1, three images categorized into two groups (Fig 1A and Fig 1B, C) were presented in Figure 1. Fig 1A illustrates the draft of the mitogenome assembly with six branch contigs (ctg1-ctg6). The text describes the detailed bridge between the original draft (Fig 1A) and the resolved mitogenome structure (Fig 1B, C) in Table S2 and Fig S2. The ctg5 (14,342 bp, red color) may mediate the mitogenomic recombination, resulting in four possible solution pathways (ctg4-ctg5-ctg6, ctg1-ctg5-ctg2, ctg4-ctg5-ctg2, and ctg1-ctg5-ctg6) and conformational changes. Based on the highest supported number (Table S3), the long-read collections of BLASTn were validated by mapping the reference sequence composed of ctg5 and its 500 bp flanking regions. Eventually, in contrast to the supported path of ctg1-ctg5-ctg2, the multi-branched mitogenome sketch with the ctg1-ctg5-ctg6 path was predicted and primarily obtained, prompting images of Fig 1B and 1C for the subsequent analyses.

The increasing number of reports implicated that the complexity of the multi-branched molecule/chromosome mitogenome might be the typical feature in most terrestrial plants but NOT in animals. Thus, “one ring does not rule them all,” and representations of plant mitogenomes as simple, circular molecules are not accurate descriptions of their true nature and that, in reality, plant mitochondrial DNA is a complex, dynamic mixture of forms (detailed in the ref 7). Therefore, the assembled mitogenome was resolved using bioinformatics tools to explain the optimization of the mitogenomic structure, recombination, and HGTs. If the response is not promising or uncomfortable, some of the latest literature would be required necessarily for an in-depth understanding of the complexity of the mitogenome configuration deduction:

1. Guo, S.; Li, Z.; Li, C.; Liu, Y.; Liang, X.; Qin, Y. Assembly and characterization of the complete mitochondrial genome of Ventilago leiocarpa. Plant Cell Rep. 2024, 43, 77.

2. Jiang, M.; Ni, Y.; Zhang, J.; Li, J.; Liu, C. Complete mitochondrial genome of Mentha spicata L. reveals multiple chromosomal configurations and RNA editing events. Int. J. Biol. Macromol. 2023, 251, 126257.

3. Hao, Z., Zhang, Z., Jiang, J. et al. Complete mitochondrial genome of Melia azedarach L., reveals two conformations generated by the repeat sequence mediated recombination. BMC Plant Biol. 2024, 24, 645.

Line 247 – What linked palindromic repeats is?

R10: Thanks. Three primary repeat sequences are abundant in the plant mitogenome: SSRs (monomeric, dimeric, and trimeric), tandem, and dispersed (forward, reverse, complementary, and palindromic repeats). The cyclic maps illustrate the distribution and link of the various repetitive sequences within one molecule. The linked palindromic repeats are colored in orange, as indicated in Fig 4. Please check the updated legend in Fig 4 from the revised manuscript (L825-827).

Line 251 – there are two types of repeats – tandem and interspersed. What do dispersed stand for?

R11: Thanks. This comment is very important. As it was responded in R3 and R10, three types of repetitive sequences are predominant in the plant mitogenome, including SSRs, tandem repeats, and dispersed repeats (e.g., forward, reverse, complementary, and palindromic repeats), whereas very few literature use “interspersed repeats” rather than “dispersed repeats”, or maybe both. Some literatures are exemplified as references:

1. Lu, G.; and Li,. Q. Complete mitochondrial genome of Syzygium samarangense reveals genomic recombination, gene transfer, and RNA editing events. Front. Plant Sci. 2024, 14:1301164.

2. Guo, S.; Li, Z.; Li, C.; Liu, Y.; Liang, X.; Qin, Y. Assembly and characterization of the complete mitochondrial genome of Ventilago leiocarpa. Plant Cell Rep. 2024, 43, 77.

3. Jiang, M.; Ni, Y.; Zhang, J.; Li, J.; Liu, C. Complete mitochondrial genome of Mentha spicata L. reveals multiple chromosomal configurations and RNA editing events. Int. J. Biol. Macromol. 2023, 251, 126257.

4. Hao, Z., Zhang, Z., Jiang, J. et al. Complete mitochondrial genome of Melia azedarach L., reveals two conformations generated by the repeat sequence mediated recombination. BMC Plant Biol. 2024, 24, 645.

Figure 4 should be moved down, after it was mentioned in text.

R12: This is a good comment. According to the journal instructions, the figure and table should be displayed close to the text.

Figure 7 – the same as Fig. 4.

R13: Thanks.

Figure 7B – what is the purpose of that small tree just above the tree scale?

R14: Thanks. Tree scale was used to calculate the evolutional distance from the original tree (small tree).

Lines 318-328 – You need to clarify either here or in Methods section – what approach did you used for phylogeny estimation. Did you used just MAFFT alignment of full mt-genomes? Or alignment of shared genes – concatenation and subsequent phylogeny estimation? Or the coalescence approach?

R15: Many thanks. The methods used in the collinearity and phylogenetic analyses have been specified. Please check the revised manuscript in L539-548.

Line 351 – Why “structural” in particular? Or you meant nucleoid proteins? needs clarification.

R16: Thanks. These texts were summarized from the review article (detailed in ref 7). Structural genes (or protein-coding genes) are commonly used in the literature, coding for synthesizing specific polypeptides, enzymes, or proteins. In prokaryotes, functional relations of structural genes usually occur adjacent to each other and are modulated by a single promoter and operator. While in eukaryotes, they are primarily identified in the coding regions. If the comment is requesting to clarify the importance of the structural genes in mitogenome, that would be interesting for their diverse functionalities in energy metabolism, metabolite transport, and ROS homeostasis.

Reviewer 4 Report

Comments and Suggestions for Authors

A well-written paper.

Below are some comments for consideration.

Title and Abstract

• Title: Change "Integration of Illumina and PacBio HiFi Sequencing Revealed Three Linearized Mitogenomes in Arrow Bamboo (Fargesia qinlingensis) with RNA Editing and Implicated Phylogenies" to "Revealing Three Linear Mitogenomes in Arrow Bamboo (Fargesia qinlingensis) through Integrated Illumina and PacBio HiFi Sequencing."
• Abstract:
  • Line 13: Replace "superior adaptive resilience to changing climates" with "remarkable climate resilience."
  • Line 19: Replace "Thirty-five unique PEGs were annotated" with "Thirty-five unique protein-encoding genes (PEGs) were identified."
  • Line 23: Replace "Among the 482 potential C-U/T RNA-editing sites predicted in 35 PEGs, ccmFn (38 times) and ccmC (36 times) were at the top." with "A total of 482 potential C-to-U RNA-editing sites were predicted, with ccmFn and ccmC being the most frequently edited genes (38 and 36 sites, respectively)."

Introduction

• Line 34: Replace "Plant cells maintain the integration of two energy-circulating systems coupled between mitochondria and plastids, which originate from prokaryotes of symbiotic ancestors [1]." with "Plant cells integrate two energy-circulating systems involving mitochondria and plastids, both derived from prokaryotic symbiotic ancestors [1]."
• Line 41: Replace "Unlike nuclear genes, the maternal inheritance mode frequently occurs in mitochondrial DNA in most terrestrial plants rather than genetically passing on through parents, suggesting population fitness and structural stability [6]." with "Unlike nuclear genes, mitochondrial DNA in most terrestrial plants is often maternally inherited, contributing to population fitness and structural stability [6]."

Materials and Methods

• Line 105: Replace "Fresh leaves of F. qinlingensis were harvested and sampled from the Foping National Nature Reserve, Qinling Mountain, Hanzhong City, Shaanxi Province, at an altitude of 2,432 m (33°40′13′′ N, 107°49′56′′ E)." with "Fresh leaves of F. qinlingensis were collected from the Foping National Nature Reserve, Qinling Mountain, Hanzhong City, Shaanxi Province, China (33°40′13′′ N, 107°49′56′′ E), at an altitude of 2,432 meters."
• Line 118: Replace "The Illumina short-read sequencing was first performed by GetOrganelle software (v1.7.7.1 2023.01.05) to obtain a draft mitogenome graph with default parameters [25]." with "Illumina short-read sequencing was initially conducted using GetOrganelle software (v1.7.7.1, 2023.01.05) to generate a draft mitogenome graph with default parameters [25]."
• Line 125: Replace "Using Geseq software (v2.03 2020.12.18), the Arabidopsis and barley (Hordeum vulgare L.) mitogenomes were selected as references for gene annotation [28]." with "Gene annotation was performed using Geseq software (v2.03, 2020.12.18), with Arabidopsis and barley (Hordeum vulgare L.) mitogenomes serving as references [28]."

Results

• Line 184: Replace "Among these genes, the ctg5 in the unitig graph, holding 14,342 bp repeat sequence, may mediate the mitogenome recombination, resulting in four different solution pathways (ctg4-ctg5-ctg6, ctg1-ctg5-ctg2, ctg4-ctg5-ctg2, and ctg1-ctg5-ctg6) and subsequent conformational changes (Table S2 and Figure S2)." with "The ctg5 unitig graph, containing a 14,342 bp repeat sequence, may facilitate mitogenome recombination, leading to four different solution pathways (ctg4-ctg5-ctg6, ctg1-ctg5-ctg2, ctg4-ctg5-ctg2, and ctg1-ctg5-ctg6) and resultant conformational changes (Table S2 and Figure S2)."
• Line 198: Replace "The complete mitogenome conformation was simplified into three linear molecules with overlapping connections, comprising 208,716 bp for M1 (GC=44.21%, red), 160,262 bp for M2 (GC=44.06%, yellow), and 73,390 bp for M2 (GC=43.57%, blue) (Table S4)." with "The complete mitogenome was simplified into three linear molecules with overlapping regions: 208,716 bp for M1 (GC=44.21%, red), 160,262 bp for M2 (GC=44.06%, yellow), and 73,390 bp for M3 (GC=43.57%, blue) (Table S4)."
• Line 232: Replace "The RSCU values (> 1.2) defined the specific codon usage preference among synonymous codons." with "RSCU values (> 1.2) indicated specific codon usage preferences among synonymous codons."

Discussion

• Line 330: Replace "Technological advances and innovations in genome sequencing and assembly have facilitated a meteoric rise in published organelles, revealing the significance of divergent evolutionary trajectories [48]." with "Technological advancements in genome sequencing and assembly have led to a surge in published organelle genomes, highlighting diverse evolutionary trajectories [48]."
• Line 341: Replace "Nevertheless, the molecular functions of the mitochondrial genes involved in stress acclimation and whether mitochondrial DNA enhances the evolutionary relationships and species-level classification in bamboo remains largely unknown." with "However, the molecular functions of mitochondrial genes in stress acclimation and the role of mitochondrial DNA in evolutionary relationships and species-level classification in bamboo remain largely unknown."
• Line 374: Replace "Mitogenomes are highly constrained and pivotal for cellular respiration and aerobic metabolism [55]." with "Mitogenomes are highly conserved and crucial for cellular respiration and aerobic metabolism [55]."
• Line 393: Replace "Many exogenous sequence insertions and repeats have been identified in plant mitogenomes, leading to gene imbalances, multiple copy numbers, and DNA rearrangements that mediate the formation of multi-branched conformations [8,63]." with "Exogenous sequence insertions and repeats in plant mitogenomes often lead to gene imbalances, multiple copy numbers, and DNA rearrangements, resulting in multi-branched conformations [8,63]."

Author Response

A well-written paper. Below are some comments for consideration.Title and Abstract Title: Change “Integration of Illumina and PacBio HiFi Sequencing Revealed Three Linearized Mitogenomes in Arrow Bamboo (Fargesia qinlingensis) with RNA Editing and Implicated Phylogenies” to “Revealing Three Linear Mitogenomes in Arrow Bamboo (Fargesia qinlingensis) through Integrated Illumina and PacBio HiFi Sequencing.”

R1: We sincerely appreciate the reviewer’s time and efforts in commenting on the manuscript. Many thanks. Based on the reviewer’s comments, the title has been revised.

Abstract:

Line 13: Replace “superior adaptive resilience to changing climates” with “remarkable climate resilience.”

R2: Thanks, and it has been replaced.

Line 19: Replace “Thirty-five unique PEGs were annotated” with “Thirty-five unique protein-encoding genes (PEGs) were identified.”

R3: Thanks, and it has been revised.

Line 23: Replace “Among the 482 potential C-U/T RNA-editing sites predicted in 35 PEGs, ccmFn (38 times) and ccmC (36 times) were at the top.” with “A total of 482 potential C-to-U RNA-editing sites were predicted, with ccmFn and ccmC being the most frequently edited genes (38 and 36 sites, respectively).”

R4: Thanks, and it has been revised.

Introduction

Line 34: Replace “Plant cells maintain the integration of two energy-circulating systems coupled between mitochondria and plastids, which originate from prokaryotes of symbiotic ancestors [1].” with “Plant cells integrate two energy-circulating systems involving mitochondria and plastids, both derived from prokaryotic symbiotic ancestors [1].”

R5: Thanks. The text has been revised.

Line 41: Replace “Unlike nuclear genes, the maternal inheritance mode frequently occurs in mitochondrial DNA in most terrestrial plants rather than genetically passing on through parents, suggesting population fitness and structural stability [6].” with “Unlike nuclear genes, mitochondrial DNA in most terrestrial plants is often maternally inherited, contributing to population fitness and structural stability [6].”

R6: Thanks. The text has been modified.

Materials and Methods

Line 105: Replace “Fresh leaves of F. qinlingensis were harvested and sampled from the Foping National Nature Reserve, Qinling Mountain, Hanzhong City, Shaanxi Province, at an altitude of 2,432 m (33°40′13′′ N, 107°49′56′′ E).” with “Fresh leaves of F. qinlingensis were collected from the Foping National Nature Reserve, Qinling Mountain, Hanzhong City, Shaanxi Province, China (33°40′13′′ N, 107°49′56′′ E), at an altitude of 2,432 meters.”

R7: Thanks and the methods have been modified.

Line 118: Replace “The Illumina short-read sequencing was first performed by GetOrganelle software (v1.7.7.1 2023.01.05) to obtain a draft mitogenome graph with default parameters [25].” with “Illumina short-read sequencing was initially conducted using GetOrganelle software (v1.7.7.1, 2023.01.05) to generate a draft mitogenome graph with default parameters [25].”

R8: Thanks and the methods have been revised.

Line 125: Replace “Using Geseq software (v2.03 2020.12.18), the Arabidopsis and barley (Hordeum vulgare L.) mitogenomes were selected as references for gene annotation [28].” with “Gene annotation was performed using Geseq software (v2.03, 2020.12.18), with Arabidopsis and barley (Hordeum vulgare L.) mitogenomes serving as references [28].”

R9: Thanks. The methods have been revised.

Results

Line 184: Replace “Among these genes, the ctg5 in the unitig graph, holding 14,342 bp repeat sequence, may mediate the mitogenome recombination, resulting in four different solution pathways (ctg4-ctg5-ctg6, ctg1-ctg5-ctg2, ctg4-ctg5-ctg2, and ctg1-ctg5-ctg6) and subsequent conformational changes (Table S2 and Figure S2).” with “The ctg5 unitig graph, containing a 14,342 bp repeat sequence, may facilitate mitogenome recombination, leading to four different solution pathways (ctg4-ctg5-ctg6, ctg1-ctg5-ctg2, ctg4-ctg5-ctg2, and ctg1-ctg5-ctg6) and resultant conformational changes (Table S2 and Figure S2).”

R10: Thanks. The methods have been revised.

Line 198: Replace “The complete mitogenome conformation was simplified into three linear molecules with overlapping connections, comprising 208,716 bp for M1 (GC=44.21%, red), 160,262 bp for M2 (GC=44.06%, yellow), and 73,390 bp for M2 (GC=43.57%, blue) (Table S4).” with “The complete mitogenome was simplified into three linear molecules with overlapping regions: 208,716 bp for M1 (GC=44.21%, red), 160,262 bp for M2 (GC=44.06%, yellow), and 73,390 bp for M3 (GC=43.57%, blue) (Table S4).”

R11: Many thanks. The sentence has been revised.

Line 232: Replace “The RSCU values (> 1.2) defined the specific codon usage preference among synonymous codons.” with “RSCU values (> 1.2) indicated specific codon usage preferences among synonymous codons.”

R12: Many thanks, and the sentence has been revised.

Discussion

Line 330: Replace “Technological advances and innovations in genome sequencing and assembly have facilitated a meteoric rise in published organelles, revealing the significance of divergent evolutionary trajectories [48].” with “Technological advancements in genome sequencing and assembly have led to a surge in published organelle genomes, highlighting diverse evolutionary trajectories [48].”

R13: Thanks and revised.

Line 341: Replace “Nevertheless, the molecular functions of the mitochondrial genes involved in stress acclimation and whether mitochondrial DNA enhances the evolutionary relationships and species-level classification in bamboo remains largely unknown.” with “However, the molecular functions of mitochondrial genes in stress acclimation and the role of mitochondrial DNA in evolutionary relationships and species-level classification in bamboo remain largely unknown.”

R14: Many thanks. The sentence has been revised.

Line 374: Replace “Mitogenomes are highly constrained and pivotal for cellular respiration and aerobic metabolism [55].” with “Mitogenomes are highly conserved and crucial for cellular respiration and aerobic metabolism [55].”

R15: Many thanks. The sentence has been revised.

Line 393: Replace “Many exogenous sequence insertions and repeats have been identified in plant mitogenomes, leading to gene imbalances, multiple copy numbers, and DNA rearrangements that mediate the formation of multi-branched conformations [8,63].” with “Exogenous sequence insertions and repeats in plant mitogenomes often lead to gene imbalances, multiple copy numbers, and DNA rearrangements, resulting in multi-branched conformations [8,63].”

R16: Many thanks. The sentence has been revised.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I would like to thank the authors for the improving the manuscript, but some concerns remain to be addressed.

The English language is fine and easy to read now, thank for your efforts.

I suggest to omit some general discussion (L379-L395, L417-421) about plant mt-genomes, paying more attention to the Bamboo mt-genome specifics. 

 

Some minor comments:

L352: reference is required

L366: there are many studies devoted to the multi-chromosomal mitochondrial genome structure, f.e.:

1) Bi C, Qu Y, Hou J, Wu K, Ye N and Yin T (2022) Deciphering the Multi-Chromosomal Mitochondrial Genome of Populus simonii. Front. Plant Sci. 13:914635. doi: 10.3389/fpls.2022.914635 

2) Yang H, Chen H, Ni Y, Li J, Cai Y, Ma B, Yu J, Wang J, Liu C. De Novo Hybrid Assembly of the Salvia miltiorrhiza Mitochondrial Genome Provides the First Evidence of the Multi-Chromosomal Mitochondrial DNA Structure of Salvia Species. International Journal of Molecular Sciences. 2022; 23(22):14267. https://doi.org/10.3390/ijms232214267

3) Yang, L., Liu, J., Guo, W. et al. Insights into the multi-chromosomal mitochondrial genome structure of the xero-halophytic plant Haloxylon Ammodendron (C.A.Mey.) Bunge ex Fenzl. BMC Genomics 25, 123 (2024). https://doi.org/10.1186/s12864-024-10026-6

L388: please avoid repetition ("DNA")

L469: ref. [79] doesn't cover plants' mitochondria

Author Response

I would like to thank the authors for the improving the manuscript, but some concerns remain to be addressed. The English language is fine and easy to read now, thank for your efforts.

I suggest to omit some general discussion (L379-L395, L417-421) about plant mt-genomes, paying more attention to the Bamboo mt-genome specifics.

R1: Many thanks, and these redundant sentences have been removed.

Some minor comments:

L352: reference is required

R2: Thanks, and the citation has been provided.

L366: there are many studies devoted to the multi-chromosomal mitochondrial genome structure, f.e.:

1) Bi C, Qu Y, Hou J, Wu K, Ye N and Yin T (2022) Deciphering the Multi-Chromosomal Mitochondrial Genome of Populus simonii. Front. Plant Sci. 13:914635. doi: 10.3389/fpls.2022.914635

2) Yang H, Chen H, Ni Y, Li J, Cai Y, Ma B, Yu J, Wang J, Liu C. De Novo Hybrid Assembly of the Salvia miltiorrhiza Mitochondrial Genome Provides the First Evidence of the Multi-Chromosomal Mitochondrial DNA Structure of Salvia Species. International Journal of Molecular Sciences. 2022; 23(22):14267. https://doi.org/10.3390/ijms232214267

3) Yang, L., Liu, J., Guo, W. et al. Insights into the multi-chromosomal mitochondrial genome structure of the xero-halophytic plant Haloxylon Ammodendron (C.A.Mey.) Bunge ex Fenzl. BMC Genomics 25, 123 (2024). https://doi.org/10.1186/s12864-024-10026-6

R3: Thanks for the comments. These recommended reports have been cited in the revised manuscript.

L388: please avoid repetition ("DNA")

R4: Removed.

L469: ref. [79] doesn't cover plants' mitochondria

R5: Thanks. The sentences have been reworded with the updated citation.