Genome-Wide Identification of CmPOD Genes and Partial Functional Characterization of CmPOD52 in Lignin-Related Granulation of ‘Sanhong’ Pomelo (Citrus maxima)
, Wenqin She 1,2, Zhixiong Guo 1,2, Heli Pan 1,2 and Tengfei Pan 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsTitle
The title adequately reflects the focus of the study, although it could benefit from greater precision by indicating that it involves a partial functional characterization of CmPOD52. It is recommended to consider rephrasing it as:
“Genome-wide identification of CmPOD genes and partial functional characterization of CmPOD52 in lignin-related granulation of 'San hong' pomelo (Citrus maxima)”.
Abstract
The abstract presents a coherent synthesis of the work but omits key details about the experimental design and the nature of the results. It does not mention how many genes were evaluated or what criteria were used to select CmPOD52. It also does not specify whether negative controls were included or if causality was tested.
It is suggested to include:
How many CmPOD genes were identified and with what tools.
Which genes were selected for functional analysis and why.
What type of transient transformation was used (species, tissue, time).
Whether in vivo validation was performed or if functional inferences were based only on expression data.
What controls were included in the enzymatic assays with the recombinant protein.
Introduction
Although the context of granulation and its relationship to lignification is well established, the justification for the study is not sufficiently explicit. A clear scientific hypothesis or research question is not stated. Additionally, BPOD activity is mentioned without first establishing its differential relevance compared to SPOD in the context of lignification.
A clear explanation of the concept of "bound" peroxidases (BPOD) and their subcellular localization is lacking, especially since this aspect is central to the article.
It is recommended to clarify:
Why focus on CmPOD52 and not on other genes that also showed induced expression?
What is the relationship between GA₃-responsive cis-elements and functional regulation in the context of granulation?
What specific prior evidence exists that BPOD, and not SPOD, is more involved in secondary lignification in pomelo?
More recent and specialized literature on specific lignification mechanisms in citrus or on the direct relationship between CE structures and enzymatic function in PODs should be incorporated.
Materials and Methods
The overall experimental design is appropriate, but there is a lack of depth in technical details that ensure reproducibility and independent validation of results. The number of biological replicates used for each key experiment is not indicated, nor how environmental variables were controlled in functional assays.
In the transient transformation section:
Transformation efficiency is not described, nor whether negative controls with empty vector were performed.
It is not specified whether transient expression was quantified using qPCR or inferred only from GUS staining.
The use of E. coli to express a plant peroxidase with transmembrane domains may compromise activity, which needs to be discussed and justified.
It is unclear whether the refolding of the recombinant protein was validated through structural analysis (e.g., specific activity, conformation, UV spectroscopy, etc.).
The protocol for lignin quantification using a commercial kit does not include cross-validation with independent methods (e.g., thioglycolate, UV, histochemistry), which limits confidence in the lignification data.
In the bioinformatic analysis:
Average E-values of the selected PODs are not reported.
Specific versions of the databases used are not indicated.
The prediction of subcellular localization is not experimentally validated beyond the CmPOD52-GFP construct.
Questions that remain:
How many biological and technical replicates were conducted per treatment and time point?
How was transient transformation statistically evaluated? Was transcription confirmed or only GUS staining used?
Did the pCAMBIA1301-CmPOD52 construct include protein detection tags (e.g., His-tag, GFP)?
Were control assays conducted with recombinant proteins lacking activity (e.g., empty vector, mutated protein)?
Results
The results section is well organized, but in some cases, more is inferred than the data allow. The correlation between CmPOD52 expression, BPOD, and lignin is described as functional evidence, without demonstrating causality.
The genomic identification of CmPODs is comprehensive, but no analysis of gene duplication or evolutionary synergies with other species is included. The phylogenetic analysis is robust, but there is no discussion of segmental or functional duplication events.
In the functional validation of CmPOD52:
Lignin changes post-transient transformation are not quantified; inference is made only from expression of lignin-related genes.
The reported increase in expression of CmCOMT, CmC3H, and CmHCT is presented as functional evidence but is not confirmed enzymatically or through metabolite accumulation.
It is not shown whether the increase in BPOD after CmPOD52 expression is due directly to the activity of the recombinant enzyme or to a secondary cellular response.
In the enzymatic activity assays, there are no negative control data or structural confirmation of the dimeric product. The MS result is not compared with standards or validated through additional UV-vis spectra.
Missing results include:
Spatial variation in CmPOD52 expression across different fruit tissues.
In planta confirmation of the phenotypic impact of overexpression.
Gene silencing or editing assays to test loss-of-function.
Questions that remain:
Was lignin content quantified after transient transformation?
Were lignin intermediate metabolites analyzed in the transformed juice sacs?
Was the GA₃-induced increase in CmPOD52 expression transcriptional or also translational?
Discussion
The discussion is appropriate but lacks depth in critical analysis and exploration of the study’s limitations. The authors tend to confirm their results without questioning them or rigorously comparing with alternatives.
There is insufficient discussion of the possibility that CmPOD52 expression increase may be a consequence, not a cause, of lignification.
Other possible pathways involved in granulation, such as suberin accumulation, flavonoids, or changes in cell osmotic pressure, are not analyzed.
Comparison with Arabidopsis studies is useful, but it would be more relevant to contrast with citrus or other non-climacteric fruits. The possibility that multiple CmPODs act in functional redundancy is not explored experimentally.
Key technical limitations not addressed include:
Lack of whole-plant validation.
Absence of silencing or CRISPR-based loss-of-function tests.
Limited recombinant enzymatic activity due to inclusion bodies.
Recommended additions to the discussion:
The possibility that CmPOD52 has functions beyond lignification (e.g., defense, senescence).
Agronomic implications of manipulating CmPOD52 as a strategy to control granulation.
Need for more detailed localization studies (e.g., immunolocalization in lignified tissues).
Conclusions
The conclusions are consistent with the results but should be more precisely worded. Rather than stating that “CmPOD52 participates in lignin synthesis,” it would be more rigorous to say that “CmPOD52 is associated with increased expression of lignin pathway genes and BPOD activity during granulation, suggesting a possible role in lignification, although direct functional confirmation is still needed.”
It should be clarified that current findings are indicative, not conclusive, and that more in vivo functional studies are required to validate direct involvement in granulation.
Author Response
Thank you for taking the time to provide your valuable comments. These comments are crucial and have been instrumental in improving our work. We have prepared a detailed point-by-point response to all the comments, which has been uploaded as an attachment. We appreciate once again your diligent review of our work.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authorsplease see attached
Comments for author File: Comments.pdf
Author Response
Thank you for taking the time to provide your valuable comments. These comments are crucial and have been instrumental in improving our work. We have prepared a detailed point-by-point response to all the comments, which has been uploaded as an attachment. We appreciate once again your diligent review of our work.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe presented manuscript entitled ‘Identification of the CmPODs family and functional study of CmPOD52 expression during granulation in ‘San hong’ pomelo (Citrus maxima)’ focused on the effects of the peroxidase gene family (CmPOD) on the granulation of juice sacs of pomelo (Citrus maxima).
The main advantage of the presented research are multi-level analyses performed, starting from sequence identification of 97 genes and their chromosomal localization, phylogenetic analysis, as well as identification of 71 CmPOD proteins, its structure (akaline proteins), subcellular localization ( chloroplasts and the extracellular matrix mainly), and cloning.
However, there are some minor suggestions for the manuscript.
Introduction:
Line 43: Please include an interspace before the citation no. 3
Material and Methods:
Line 85-88 Please include how many trees were treated with GA3 and how many were control trees
Line 86 -87: Please include more details about the age of trees
Line 87-88: “we sprayed GA3 (200 mg·L-1) on the fruits and nearby leaves” please explain in more detail the spraying process ( what tools to spraying was used, the amount of solution per tree)
Line 87-88 “All the other management practices followed conventional procedures’ Please, what mean conventional procedures?
Line 92-93- please include the information about the juice sacs obtaining process
Line 99-101 “ At 175 days after flowering, the juice sacs were prepared from nine fruits collected from three healthy trees (three fruits from each tree), which were immediately used for transient transformation assays” the juice sacs was prepared separately from each fruit or as a bulk, including three fruits from each tree ?
Line 104: Please include an interspace before citation no. 25
Results:
Line:253-257 -Please include abbreviation meaning of ‘ns’
Discussion:
Line:445 Nicotiana Tabacum- ‘tabacum’ should be in small letters written
References:
Line: 531-531- please correct the references according to the template (surname of author at first)
Author Response
Thank you for taking the time to provide your valuable comments. These comments are crucial and have been instrumental in improving our work. We have prepared a detailed point-by-point response to all the comments, which has been uploaded as an attachment. We appreciate once again your diligent review of our work.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authorsno comments
Reviewer 2 Report
Comments and Suggestions for AuthorsAccept in present form
