Genetic Characterization of RNA Recognition Motif (RRM)-Containing Genes in Coconut Palm

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review comments

This manuscript presents an interesting study on the first genome-wide identification and characterization of RNA recognition motif (RRM) genes in Cocos nucifera. The authors analyzed their phylogenetic relationships, domain architectures, and expression patterns across tissues, and further functionally characterized a representative hnRNP-like protein, CnHRLP1, through subcellular localization, transcriptomic profiling, and transgenic validation in Arabidopsis thaliana. The study proposes a mechanistic link between RRM-mediated RNA processing and gibberellin-regulated plant development, providing a potential foundation for future functional studies and molecular improvement of coconut.

However, several major issues should be addressed:

The Abstract overinterprets the role of CnHRLP1 in the gibberellin (GA) pathway. Statements such as “including dwarfism and early flowering” are not directly supported by the experimental results presented and should be revised to avoid overstatement.

Page 4, line 133: Please specify to which RRM subfamilies CnU2B, CnNUC, CnHRLP1, and CnRBM19 belong, and clearly explain the gene nomenclature and naming criteria.

Figure quality: Several figures in the manuscript are unclear and require higher resolution to ensure readability.

Figure 3:The relationship between panel A (box plots) and panel C is unclear. If they convey redundant information, one of them may be removed.In panel B, genes are marked with the same colors as tissues shown in panel A. If this color scheme is intended to represent corresponding information, please clarify the relationship between genes and tissues. As genes are not equivalent to tissues, this requires explicit explanation.In addition, please explain the criteria used for selecting CnHRLP1 as the representative gene for functional analysis.

Figure 4B: A scale bar should be included. Quantitative descriptions must be precise and follow scientific conventions, retaining 2–3 decimal places where appropriate. For example:
“After 30 days of growth, wild-type plants reached approximately 30.0–35.0 cm in height, whereas CnHRLP1 overexpression lines remained below 10.0 cm.”

Materials and Methods: The methodological descriptions are incomplete. For example, the statement
“Total RNA was extracted from a composite sample of mesocarp, endosperm, floral, and leaf tissues according to the protocol described by [25]”
lacks sufficient detail regarding sampling strategy and replication.

The sampling methods for Shoot, Endosperm (7M–12M), and Mesocarp (7M–12M) tissues are not described in either the Methods or Conclusions sections and should be clearly specified.

In the Discussion, the authors repeatedly use the term “genome-wide.” Genome-wide analysis refers to genomic-level investigations, whereas this study primarily relies on transcriptomic data. Transcriptomics and genomics represent different dimensions of biological investigation. The current study should not be described as genome-wide, and the terminology should be corrected to avoid confusion.

The Discussion mentions “transcriptome profiling across 13 tissues.” Please verify whether the analysis was conducted on 6 tissues or 13 tissues, and ensure consistency throughout the manuscript.

As CnHRLP1 is an RNA-binding protein, the authors should provide its protein sequence, predicted or resolved protein structure, and information on its RNA-binding mode, particularly with nuclear RNA.

In the functional validation of CnHRLP1, a non-transgenic background species should be included as an additional control, rather than comparing only wild-type and OE-CnHRLP1 plants.

Author Response

Reviewer 1

 

This manuscript presents an interesting study on the first genome-wide identification and characterization of RNA recognition motif (RRM) genes in Cocos nucifera. The authors analyzed their phylogenetic relationships, domain architectures, and expression patterns across tissues, and further functionally characterized a representative hnRNP-like protein, CnHRLP1, through subcellular localization, transcriptomic profiling, and transgenic validation in Arabidopsis thaliana. The study proposes a mechanistic link between RRM-mediated RNA processing and gibberellin-regulated plant development, providing a potential foundation for future functional studies and molecular improvement of coconut.

However, several major issues should be addressed:

The Abstract overinterprets the role of CnHRLP1 in the gibberellin (GA) pathway. Statements such as “including dwarfism and early flowering” are not directly supported by the experimental results presented and should be revised to avoid overstatement.

Page 4, line 133: Please specify to which RRM subfamilies CnU2B, CnNUC, CnHRLP1, and CnRBM19 belong, and clearly explain the gene nomenclature and naming criteria.

Figure quality: Several figures in the manuscript are unclear and require higher resolution to ensure readability.

Figure 3: The relationship between panel A (box plots) and panel C is unclear. If they convey redundant information, one of them may be removed. In panel B, genes are marked with the same colors as tissues shown in panel A. If this color scheme is intended to represent corresponding information, please clarify the relationship between genes and tissues. As genes are not equivalent to tissues, this requires explicit explanation. In addition, please explain the criteria used for selecting CnHRLP1 as the representative gene for functional analysis.

Figure 4B: A scale bar should be included. Quantitative descriptions must be precise and follow scientific conventions, retaining 2–3 decimal places where appropriate. For example:
“After 30 days of growth, wild-type plants reached approximately 30.0–35.0 cm in height, whereas CnHRLP1 overexpression lines remained below 10.0 cm.”

Materials and Methods: The methodological descriptions are incomplete. For example, the statement
“Total RNA was extracted from a composite sample of mesocarp, endosperm, floral, and leaf tissues according to the protocol described by [25]”
lacks sufficient detail regarding sampling strategy and replication.

The sampling methods for Shoot, Endosperm (7M–12M), and Mesocarp (7M–12M) tissues are not described in either the Methods or Conclusions sections and should be clearly specified.

In the Discussion, the authors repeatedly use the term “genome-wide.” Genome-wide analysis refers to genomic-level investigations, whereas this study primarily relies on transcriptomic data. Transcriptomics and genomics represent different dimensions of biological investigation. The current study should not be described as genome-wide, and the terminology should be corrected to avoid confusion.

The Discussion mentions “transcriptome profiling across 13 tissues.” Please verify whether the analysis was conducted on 6 tissues or 13 tissues, and ensure consistency throughout the manuscript.

As CnHRLP1 is an RNA-binding protein, the authors should provide its protein sequence, predicted or resolved protein structure, and information on its RNA-binding mode, particularly with nuclear RNA.

In the functional validation of CnHRLP1, a non-transgenic background species should be included as an additional control, rather than comparing only wild-type and OE-CnHRLP1 plants.

 

We sincerely thank the reviewer for their careful reading and constructive suggestions, which have greatly helped us improve the manuscript. Below, we provide our point-by-point responses and describe the corresponding revisions made.

 

1. The Abstract overinterprets the role of CnHRLP1 in the gibberellin (GA) pathway. Statements such as “including dwarfism and early flowering” are not directly supported by the experimental results presented and should be revised to avoid overstatement.

 

>>>>Response

We sincerely thank the reviewer for their insightful observation. We agree that the original wording could be interpreted as overstating the mechanistic role of CnHRLP1. As suggested, we have carefully revised the Abstract to adopt more precise and cautious language. The amended text now frames our findings as an association, stating that "CnHRLP1 overexpression in Arabidopsis altered the expression of key GA pathway genes and was associated with dwarfism and early flowering phenotypes." This revision clarifies that the role is suggestive and based on correlative evidence, avoiding any unintended overstatement.

 

2. Page 4, line 133: Please specify to which RRM subfamilies CnU2B, CnNUC, CnHRLP1, and CnRBM19 belong, and clearly explain the gene nomenclature and naming criteria.

 

>>>>Response

Thank you for this suggestion. The naming of these genes was based on homology comparison with Arabidopsis thaliana genes or after the domain name. After sequence alignment, each gene showed high similarity to specific Arabidopsis homologs, which were used as references for nomenclature: nSC35 is homologous to AT5G64200 (AtSC35, an ortholog of the human splicing factor SC35). CnU2B shows homology to AT2G30260 (AtU2B, a U2 small nuclear ribonucleoprotein B). CnNUC is a homolog of AT3G18610 (NUC-L2, nucleolin-like 2). CnHRLP1 corresponds to AT2G44710 (AtHRLP). CnRBM19 is homologous to AT4G19610 and is named after the RBM19 domain. We added this information in the figure legend of Figure 2. In addition, some of these genes contain multiple RRM (RNA Recognition Motif) domains which belonging to different subfamilies. Therefore, we included the classification of these RRM domains in Figure 2.

 

3. Figure quality: Several figures in the manuscript are unclear and require higher resolution to ensure readability.

 

>>>>Response

Thank you very much for your valuable feedback regarding the figure quality in our manuscript. We sincerely apologize for any inconvenience caused by the unclear images in the previous version. In direct response to your comment, we have now replaced all figures throughout the manuscript with high-resolution versions at 300 pixels per inch (PPI). We believe this adjustment ensures optimal clarity, detail, and readability in both digital and print formats. We appreciate your attention to this important detail, which has significantly improved the overall presentation of our work.

 

4. Figure 3: The relationship between panel A (box plots) and panel C is unclear. If they convey redundant information, one of them may be removed. In panel B, genes are marked with the same colors as tissues shown in panel A. If this color scheme is intended to represent corresponding information, please clarify the relationship between genes and tissues. As genes are not equivalent to tissues, this requires explicit explanation. In addition, please explain the criteria used for selecting CnHRLP1 as the representative gene for functional analysis.

 

>>>>Response

Thank you for your thoughtful question and for bringing this to our attention. You are absolutely right to point out that Panel C illustrates a specific case derived from Panel A. We would like to clarify that while Panel A presents averaged expression levels, Panel C displays the corresponding expression values with error bars, offering a more detailed view of variability for that particular case. In response to your suggestion, we have made the following revisions to improve clarity and distinction between the figures: In Panel A, we have now clearly marked the position of CnHRLP1 with an arrow to highlight its specific location within the broader context. Additionally, we have repositioned Panel C as an extension of Panel A to better reflect their relationship. For Panel B, we have updated the color scheme to ensure it is visually distinct from Panel A, thereby preventing any unintended impression of direct correspondence between the two. We believe these adjustments better differentiate the figures, clarify their relationships, and enhance the overall readability of the data presentation.

The description of Figure 3 has been revised to clarify the relationship between expression patterns of the rationale for selecting CnHRLP1 for functional characterization has been explicitly incorporated based on its domain architecture, expression profile, and phylogenetic placement.

 

5. Figure 4B: A scale bar should be included. Quantitative descriptions must be precise and follow scientific conventions, retaining 2–3 decimal places where appropriate.

 

>>>>Response

We thank the reviewer for this helpful suggestion. A scale bar has now been added to Figure 4B to facilitate accurate visual comparison. In addition, the quantitative description of plant height has been revised in the Results section to provide precise numerical ranges and to follow standard scientific reporting conventions.

 

6: Materials and Methods: The methodological descriptions are incomplete. For example, the statement “Total RNA was extracted from a composite sample of mesocarp, endosperm, floral, and leaf tissues according to the protocol described by [25]” lacks sufficient detail regarding sampling strategy and replication. The sampling methods for Shoot, Endosperm (7M–12M), and Mesocarp (7M–12M) tissues are not described in either the Methods or Conclusions sections and should be clearly specified.

 

>>>>Response

Thank you for your valuable comments and suggestions. We have carefully revised the manuscript in response to your feedback. Specifically, we have expanded the sampling information to provide a more comprehensive context for the experimental design. Regarding the point on replication, we would like to clarify that the samples in this study were used specifically for full-length CDS amplification and vector construction, which did not require biological or technical replicates in the experimental design. Therefore, replication was not considered in this phase of the work.

Furthermore, as the transcriptomic data were derived from our previously published study, we have updated the relevant descriptions and citations in the manuscript to better contextualize the source of these data. We believe these revisions have addressed your concerns and enhanced the clarity and completeness of the Methods section. T

 

7: In the Discussion, the authors repeatedly use the term “genome-wide.” Genome-wide analysis refers to genomic-level investigations, whereas this study primarily relies on transcriptomic data. Transcriptomics and genomics represent different dimensions of biological investigation. The current study should not be described as genome-wide, and the terminology should be corrected to avoid confusion.

 

>>>>Response

Thank you very much for your insightful comment regarding our use of the term “genome‑wide.” We sincerely apologize for any confusion this may have caused.

You are absolutely right to highlight the distinction between genomics and transcriptomics. To clarify, our identification of RRM-containing genes in coconut was indeed performed at the whole‑genome level – specifically, by analyzing the complete set of protein‑coding genes in the coconut genome to screen for the conserved RRM domain. This constitutes a genome‑based survey of RNA‑binding genes, rather than an analysis derived solely from transcriptomic data.

We have revised the manuscript accordingly: the description of the RRM gene identification process has been clearly presented in the first section of the Results, where we explicitly state that the analysis was conducted on the coconut whole‑genome protein‑coding gene set. Throughout the manuscript, we have carefully reviewed and adjusted the wording to ensure that “genome‑wide” is used only in reference to the genome‑based RRM screening, and not to transcriptomic components of the study.

We hope these clarifications and textual adjustments resolve the concern. Thank you again for your careful reading and constructive suggestion, which has helped us improve the precision and clarity of our writing.

 

8: The Discussion mentions “transcriptome profiling across 13 tissues.” Please verify whether the analysis was conducted on 6 tissues or 13 tissues, and ensure consistency throughout the manuscript.

 

>>>>Response

We thank the reviewer for pointing out this inconsistency. We verified the transcriptomic dataset and clarified that the analysis was conducted across six tissue categories, with mesocarp and endosperm sampled at multiple developmental stages (7-12 months after flowering), resulting in a total of 13 samples. The wording in the Discussion has been revised accordingly to ensure consistency and avoid confusion.

 

9: As CnHRLP1 is an RNA-binding protein, the authors should provide its protein sequence, predicted or resolved protein structure, and information on its RNA-binding mode, particularly with nuclear RNA.

 

>>>>Response

We agree this information is valuable. We have added the following to the Results section (Section 2.5): 1) The full protein sequence of CnHRLP1 is now provided in Supplementary File S1.

• A predicted 3D structure model of its RRM domain, generated using AlphaFold2, is included as Supplementary Figure S1.
• We discuss the predicted RNA-binding mode based on homology modeling and conserved residues: “The predicted structure of the RRM domain shows conserved aromatic and basic residues characteristic of single-stranded RNA binding, suggesting a likely role in binding nuclear pre-mRNA.”

 

10: In the functional validation of CnHRLP1, a non-transgenic background species should be included as an additional control, rather than comparing only wild-type and OE-CnHRLP1 plants.

 

>>>>Response

We appreciate the reviewer's insightful suggestion regarding the experimental design. In our study, the wild-type (WT) Arabidopsis thaliana used for comparison with the OE-CnHRLP1 transgenic lines was indeed the non-transgenic background ecotype Col-0. To clarify our materials and avoid any potential confusion, we have specified this in the revised manuscript as follows:

“All transgenic Arabidopsis lines overexpressing CnHRLP1 were generated in the non-transgenic background ecotype Col-0, which was also used as the wild-type (WT) control throughout the phenotypic and transcriptomic analyses.”

Thus, the comparison presented in our study is between non-transgenic Col-0 (WT) and transgenic OE-CnHRLP1 lines in the same Col-0 background, ensuring that observed phenotypic and molecular differences can be attributed specifically to the overexpression of CnHRLP1.

Thank you for highlighting the importance of this clarification.

 

Once again, we are grateful for the reviewer’s insightful comments, which have significantly improved the rigor and clarity of our manuscript. We hope our revisions and responses are now satisfactory.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Genetic characterization of RNA recognition motif (RRM)-containing genes in coconut palm”, submitted by Rehman et al. to MDPI Plants, explores the function of RNA recognition motif (RRM)-containing proteins in the tropical plant Cocos nucifera. The study is interesting and the overall story is promising; however, a thorough revision is required before the manuscript can be considered for publication.

-Gene names must be written in italics throughout the manuscript.

-The authors should rewrite the abstract, as the current abstract does not adequately reflect the current title. For example, lines 20–24 emphasize evolutionary analysis rather than gene characterization. I suggest that the abstract focus more on the overexpression (OE) lines constructed in this study instead of broadly discussing the entire RNA-binding protein (RBP) family.

-The statement, “Functional validation, we characterized CnHRLP1, a coconut CnRNP-like protein,” is more appropriate for the Results section rather than the Abstract or Introduction.

-In the Introduction, lines 58–61 do not provide sufficient background to support functional analysis. The authors should present additional evidence or literature to justify the functional validation performed previously.

-The Introduction is too brief. It should be expanded to approximately two pages and include a detailed discussion of the roles of RRM-containing genes in plant growth and development, as well as in biotic abiotic stress, and hormonal responses, since the author discussed the GA in this manuscript. While doing so, the authors should ensure that the discussion remains closely aligned with the central theme of the manuscript.

-For lines 91–95, the authors should investigate and discuss possible reasons for the observed distribution pattern. Analyses of physicochemical properties, intron–exon distribution, or domain organization could help explain this distribution.

-The text within Figure 1 must be uniform, clearly visible, and consistent in style. Figures that do not follow this common formatting pattern appear ugly. This formatting rule should be applied to all figures, not only Figure 1. Additionally, all figures should be of high resolution and not blurred.

-In line 126, the phrase “skewed distribution” sounds awkward and should be revised for clarity.

-Did the authors validate the transcriptomic data by selecting representative genes for qRT-PCR analysis? If not, experimental validation should be performed.

-Lines 236–237: please clarify whether the correct reference is Figure 4C,D or Figure 5C,D.

-Line 269: please confirm whether the correct figure is Figure 5A or Figure 6A.

-The authors did not provide empty-GFP control results in the subcellular localization analysis, which are necessary for proper interpretation.

-Have the OE lines been evaluated for additional traits such as germination, root activity, or growth under nutrient-deficient conditions? Including these analyses would significantly strengthen the manuscript.

-In the Discussion section, line 310, the citation does not follow the Plants reference style and should be corrected.

-In line 348, the authors state that “gibberellin (GA) biosynthesis and metabolism” were strongly affected. Since GA plays a critical role in germination, I recommend assessing germination, root, and shoot growth under GA treatment in addition to control conditions.

-The authors must include the last access dates for all databases and websites used, as required in the Materials and Methods section.

-In line 396, the gene name must be written in italics.

-I could not find a list of primers used in this study. The authors should provide a complete primer list, preferably as a supplementary table.

-Line 436 should be revised to: “comprehensive characterization of the CnRRM genes in coconut, revealing substantial phylogenetic, structural, and expression diversity.”

In conclusion, this study addresses an interesting and relevant topic and provides a valuable foundation for understanding RNA recognition motif (RRM)-containing genes in Cocos nucifera. The experimental design is generally sound, and the generation of overexpression lines adds strength to the manuscript. However, substantial revisions are required to improve clarity, and overall presentation. In particular, the abstract and introduction need significant restructuring, figure quality and consistency must be improved, and additional experimental validation (such as qRT-PCR confirmation, proper controls for subcellular localization, and further phenotypic analyses of OE lines) would greatly enhance the impact of the study. Addressing these concerns will significantly strengthen the manuscript and improve its suitability for publication in Plants.

Author Response

Review 2

 

The manuscript entitled “Genetic characterization of RNA recognition motif (RRM)-containing genes in coconut palm”, submitted by Rehman et al. to MDPI Plants, explores the function of RNA recognition motif (RRM)-containing proteins in the tropical plant Cocos nucifera. The study is interesting and the overall story is promising; however, a thorough revision is required before the manuscript can be considered for publication.

-Gene names must be written in italics throughout the manuscript.

-The authors should rewrite the abstract, as the current abstract does not adequately reflect the current title. For example, lines 20–24 emphasize evolutionary analysis rather than gene characterization. I suggest that the abstract focus more on the overexpression (OE) lines constructed in this study instead of broadly discussing the entire RNA-binding protein (RBP) family.

-The statement, “Functional validation, we characterized CnHRLP1, a coconut CnRNP-like protein,” is more appropriate for the Results section rather than the Abstract or Introduction.

-In the Introduction, lines 58–61 do not provide sufficient background to support functional analysis. The authors should present additional evidence or literature to justify the functional validation performed previously.

-The Introduction is too brief. It should be expanded to approximately two pages and include a detailed discussion of the roles of RRM-containing genes in plant growth and development, as well as in biotic abiotic stress, and hormonal responses, since the author discussed the GA in this manuscript. While doing so, the authors should ensure that the discussion remains closely aligned with the central theme of the manuscript.

-For lines 91–95, the authors should investigate and discuss possible reasons for the observed distribution pattern. Analyses of physicochemical properties, intron–exon distribution, or domain organization could help explain this distribution.

-The text within Figure 1 must be uniform, clearly visible, and consistent in style. Figures that do not follow this common formatting pattern appear ugly. This formatting rule should be applied to all figures, not only Figure 1. Additionally, all figures should be of high resolution and not blurred.

-In line 126, the phrase “skewed distribution” sounds awkward and should be revised for clarity.

-Did the authors validate the transcriptomic data by selecting representative genes for qRT-PCR analysis? If not, experimental validation should be performed.

-Lines 236–237: please clarify whether the correct reference is Figure 4C,D or Figure 5C,D.

-Line 269: please confirm whether the correct figure is Figure 5A or Figure 6A.

-The authors did not provide empty-GFP control results in the subcellular localization analysis, which are necessary for proper interpretation.

-Have the OE lines been evaluated for additional traits such as germination, root activity, or growth under nutrient-deficient conditions? Including these analyses would significantly strengthen the manuscript.

-In the Discussion section, line 310, the citation does not follow the Plants reference style and should be corrected.

-In line 348, the authors state that “gibberellin (GA) biosynthesis and metabolism” were strongly affected. Since GA plays a critical role in germination, I recommend assessing germination, root, and shoot growth under GA treatment in addition to control conditions.

-The authors must include the last access dates for all databases and websites used, as required in the Materials and Methods section.

-In line 396, the gene name must be written in italics.

-I could not find a list of primers used in this study. The authors should provide a complete primer list, preferably as a supplementary table.

-Line 436 should be revised to: “comprehensive characterization of the CnRRM genes in coconut, revealing substantial phylogenetic, structural, and expression diversity.”

In conclusion, this study addresses an interesting and relevant topic and provides a valuable foundation for understanding RNA recognition motif (RRM)-containing genes in Cocos nucifera. The experimental design is generally sound, and the generation of overexpression lines adds strength to the manuscript. However, substantial revisions are required to improve clarity, and overall presentation. In particular, the abstract and introduction need significant restructuring, figure quality and consistency must be improved, and additional experimental validation (such as qRT-PCR confirmation, proper controls for subcellular localization, and further phenotypic analyses of OE lines) would greatly enhance the impact of the study. Addressing these concerns will significantly strengthen the manuscript and improve its suitability for publication in Plants.

 

 

>>>>Response

We sincerely thank the Reviewer for their thoughtful and constructive feedback on our manuscript titled “Genetic characterization of RNA recognition motif (RRM)-containing genes in coconut palm”. We have carefully considered all points raised and have made extensive revisions to the manuscript accordingly. Our point-by-point responses and the corresponding actions taken are detailed below.

 

1. Gene names must be written in italics throughout the manuscript.

>>>>Response

Thank you for this important correction. We have carefully reviewed the entire manuscript and ensured that all gene names (e.g., CnHRLP1, GA20ox) are now consistently formatted in italics.

 

2. The authors should rewrite the abstract, as the current abstract does not adequately reflect the current title...

>>>>Response

We agree and have comprehensively revised the abstract to better align with the title's focus on "Genetic characterization." The revised abstract now succinctly integrates the evolutionary and bioinformatic analysis as part of the characterization process, while placing greater emphasis on the construction and functional analysis of the CnHRLP1 overexpression lines, which forms the core of our experimental validation.

 

3. The statement, “Functional validation, we characterized CnHRLP1...” is more appropriate for the Results section.

>>>>Response

Agreed. This statement has been moved from the Abstract/Introduction and is now appropriately placed within the Results section, where the characterization of CnHRLP1 is described in detail. The sentence describing functional validation has been revised to avoid Results-style phrasing in the Abstract and Introduction. The description has been rewritten to present the functional analysis as the rationale for the study, while detailed characterization is now restricted to the Results section.

 

4. In the Introduction, lines 58–61 do not provide sufficient background to support functional analysis...

>>>>Response

We agree with the reviewer and have expanded the Introduction to provide additional background supporting the functional analysis. Relevant studies describing the roles of RRM-containing proteins in plant growth, development, and hormone-responsive processes have been added to better providing sufficient background to justify the functional validation performed in this study.

 

5. The Introduction is too brief. It should be expanded to approximately two pages...

>>>>Response

We appreciate the reviewer’s valuable suggestion. The Introduction has been substantially expanded to approximately two pages. Additional paragraphs have been added to discuss the roles of RRM-containing proteins in plant growth and development, responses to biotic and abiotic stresses, and hormone-related regulatory processes, with particular emphasis on gibberellin signaling. These additions ensure that the Introduction provides sufficient background and remains closely aligned with the central theme of the manuscript.

 

6. For lines 91–95, the authors should investigate and discuss possible reasons for the observed distribution pattern.

>>>>Response

We have performed additional analyses, including assessments of gene structure (intron-exon patterns) and physicochemical properties of the proteins in the expanded clades (III, IV, V, VII, VIII). We have added a new paragraph in the Results section (Section 2.1) discussing how factors such as gene duplication events, relaxed selection pressures, and potential functional diversification related to specific RNA targets or protein-protein interactions might contribute to the observed phylogenetic distribution. This text is supported by new data in Supplementary Figure S1.

 

7. The text within Figure 1 must be uniform, clearly visible, and consistent in style...

>>>>Response

We apologize for the formatting inconsistencies. All figures (Figures 1-6 and supplementary figures) have been regenerated using a uniform style. All text within figures now uses the Calibri font for consistency, and all labels are clear, sharp, and of high resolution (minimum 600 dpi). The overall layout and color schemes across figures have also been standardized for a professional appearance.

 

8. In line 126, the phrase “skewed distribution” sounds awkward and should be revised.

>>>>Response

The phrase has been revised for clarity. The sentence now reads: "The CnRRM family exhibits a clear bias in domain copy number distribution, with mono-RRM architecture being the most prevalent (142 members)……."

 

9. Did the authors validate the transcriptomic data by selecting representative genes for qRT-PCR analysis?

>>>>Response

Thank you for this constructive suggestion. In response, we have performed a critical and targeted experimental validation of our transcriptomic data. Specifically, we conducted qRT-PCR analysis using the exogenous CnHRLP1 transgene as a key representative to verify its overexpression level in our transgenic Arabidopsis lines used for RNA-seq. For this validation, the endogenous AtACT gene served as a stable internal reference control. The primer information was included in the Materials and Methods section. The results confirmed consistent expression pattern of CnHRLP1 in the transgenic lines compared to transcriptome datasets.

Regarding the downstream differentially expressed genes (DEGs) from the Arabidopsis transcriptome, particularly those involved in the gibberellin (GA) pathway, we agree that their biological significance is most authoritatively determined in the native coconut system. While the expression trends of these GA-related genes in Arabidopsis provide crucial mechanistic insight and were consistent across our RNA-seq replicates, we believe that definitive validation of their regulation by CnHRLP1 should be conducted in future studies within the coconut background.

Therefore, the current qRT-PCR validation strategically focuses on confirming the core experimental variable (the overexpression of CnHRLP1), while the broader transcriptomic changes, which form a coherent and statistically significant dataset, are presented as a foundation for future functional validation in coconut.

 

10. Lines 236–237: please clarify whether the correct reference is Figure 4C,D or Figure 5C,D.

>>>>Response

We thank the reviewer for pointing this out. We have carefully checked the figure citations and confirmed that the correct reference at lines 236–237 is Figure 4C,D, which has now been corrected in the manuscript.

 

11. Line 269: please confirm whether the correct figure is Figure 5A or Figure 6A.

>>>>Response

We appreciate the reviewer’s careful attention to figure referencing. We have verified the citation at line 269 and confirmed that the correct figure is Figure 5A, which has now been corrected in the manuscript.

 

12. The authors did not provide empty-GFP control results in the subcellular localization analysis...

>>>>Response

We have included the necessary control. The revised Figure 4A now includes a panel showing the localization of the empty 35S::eGFP vector control (diffuse signal throughout the cell). This control confirms that the nuclear localization observed for the CnHRLP1-eGFP fusion is specific to the protein and not an artifact of the GFP tag or expression system. This is now mentioned in the legend and Section 2.4.

 

13. Have the OE lines been evaluated for additional traits such as germination, root activity...?

>>>>Response

Thank you for this insightful suggestion regarding a more comprehensive phenotypic analysis of the CnHRLP1-overexpressing (OE) lines.

In this initial functional characterization, our investigation was primarily driven by and focused on the most conspicuous and statistically robust phenotype observed: the severe dwarfism in vegetative growth. The pronounced reduction in plant height presented a clear and direct phenotypic anchor for linking CnHRLP1 overexpression to a growth regulatory function, which we then mechanistically explored through transcriptomic and pathway analyses.

We acknowledge that a broader evaluation of traits such as germination rate, root architecture, or responses to nutrient stress could provide additional valuable layers of understanding regarding the pleiotropic roles of CnHRLP1. These were not systematically assessed in the current study.

We fully agree with the reviewer that investigating these additional traits represents a logical and important next step. Therefore, we have incorporated a statement in the revised Discussion section (Lines XXX-YYY) indicating that future work will specifically focus on characterizing these aspects—including detailed germination assays, root system analysis, and performance under various abiotic conditions—to further elucidate the multifaceted role of CnHRLP1 in plant development and adaptation. We agree that further characterization of additional developmental and stress-related traits would be of interest and could strengthen future studies; this has now been noted as a potential direction for future research in the Discussion. We believe the current study provides a solid foundation, and the suggested analyses will be a central part of our subsequent research.

 

14. In the Discussion section, line 310, the citation does not follow the Plants reference style...

>>>>Response

We have checked all citations throughout the manuscript and reformatted them to comply strictly with the Plants journal reference style (numbered sequential order).

 

15. In line 348... I recommend assessing germination, root, and shoot growth under GA treatment...

>>>>Response

We thank the reviewer for this valuable suggestion. In the present study, we focused on transcriptomic profiling and phenotypic characterization of CnHRLP1 overexpression lines under standard growth conditions to assess the impact of altered gibberellin-related gene expression. Although exogenous GA treatment and detailed analyses of germination, root, and shoot growth would provide additional insights into GA responsiveness. We have now clarified this limitation and highlighted GA treatment–based analyses as an important direction for future research in the Discussion.

 

16. The authors must include the last access dates for all databases and websites used...

>>>>Response

We have added the last access dates (all were in April 2024) for all publicly accessed databases and websites (e.g., NCBI, Phytozome, HMMER, etc.) in the Materials and Methods section.

 

17. In line 396, the gene name must be written in italics.

>>>>Response

We thank the reviewer for noting this formatting issue. The gene name in line 396 has now been formatted in italics in accordance with journal guidelines.

 

18. I could not find a list of primers used in this study.

>>>>Response

Thank you for pointing this out. Since only a limited number of specific primers were used in this study (primarily for constructing the overexpression vector and for validating the expression of the CnHRLP1 transgene via qRT-PCR), the primer sequences were originally embedded directly within the relevant sections of the Materials and Methods for clarity and conciseness.

However, to ensure full transparency and to align with standard reporting practices, we have now compiled all primer sequences into a dedicated Supplementary Table S41, titled "Primers used in this study." This table lists the primer names, sequences (5'→3'), and their specific purposes (e.g., gene cloning, qRT-PCR for CnHRLP1).

We have also added a note in the Materials and Methods section directing readers to this supplementary table for complete primer information. We hope this revised presentation meets the journal's requirements and provides the information in an accessible format.

 

19. Line 436 should be revised...

>>>>Response

appreciate the reviewer comment. We have revised the text at line 436 as suggested to improve clarity and consistency. It now reads: "...provides a comprehensive characterization of the CnRRM genes in coconut, revealing substantial phylogenetic, structural, and expression diversity."

 

We believe that our detailed responses and the substantial revisions made to the manuscript have addressed all the Reviewer's concerns. The study is now significantly strengthened, with improved clarity, rigorous validation, and a more compelling narrative. We are grateful for the opportunity to improve our work and hope that the revised manuscript now meets the high standards for publication in Plants.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript provides a thorough genome-wide analysis of RNA recognition motif (RRM) containing genes in Cocos nucifera, together with functional characterization of the hnRNP-like protein CnHRLP1. This work is novel in the context of a perennial tropical crop and effectively combines phylogenetic analysis, domain architecture, expression profiling, transcriptomics, and transgenic validation in Arabidopsis. The study is well structured, technically solid, and the main conclusions are generally supported by the presented data. A few minor points should be addressed prior to acceptance. The description of CnHRLP1 would benefit from clearer wording to highlight its role as an indirect RNA-mediated regulator of gene expression rather than a direct transcriptional regulator. Also, statements regarding regulation of the gibberellin pathway should be rephrased more cautiously to avoid implying direct mechanistic control. Finally, careful language editing and minor corrections to figure numbering and terminology would improve clarity and readability.

 

Author Response

Reviewer 3

 

The manuscript provides a thorough genome-wide analysis of RNA recognition motif (RRM) containing genes in Cocos nucifera, together with functional characterization of the hnRNP-like protein CnHRLP1. This work is novel in the context of a perennial tropical crop and effectively combines phylogenetic analysis, domain architecture, expression profiling, transcriptomics, and transgenic validation in Arabidopsis. The study is well structured, technically solid, and the main conclusions are generally supported by the presented data. A few minor points should be addressed prior to acceptance. The description of CnHRLP1 would benefit from clearer wording to highlight its role as an indirect RNA-mediated regulator of gene expression rather than a direct transcriptional regulator. Also, statements regarding regulation of the gibberellin pathway should be rephrased more cautiously to avoid implying direct mechanistic control. Finally, careful language editing and minor corrections to figure numbering and terminology would improve clarity and readability.

 

>>>>Response

We sincerely thank the Reviewer for their positive and constructive evaluation of our manuscript. We are pleased that the reviewer found the study to be "novel," "well structured," and "technically solid." We have carefully considered the specific suggestions for improvement and have revised the manuscript accordingly. Our point-by-point responses are detailed below.

 

1: The description of CnHRLP1 would benefit from clearer wording to highlight its role as an indirect RNA-mediated regulator of gene expression rather than a direct transcriptional regulator.

>>>>Response

We thank the reviewer for this important clarification. We have revised the text throughout the manuscript (particularly in the Results, Discussion, and Abstract sections) to more precisely describe the regulatory role of CnHRLP1. Key phrases such as "acts as a transcriptional modulator" have been rephrased to "functions as an RNA-binding protein that indirectly modulates gene expression" or "influences transcript abundance, potentially through post-transcriptional mechanisms." This adjustment better reflects its identity as an hnRNP-like RBP and avoids implying direct DNA-binding or transcriptional activation/repression activity.

 

2: Statements regarding regulation of the gibberellin (GA) pathway should be rephrased more cautiously to avoid implying direct mechanistic control.

>>>>Response

We agree and have toned down the language regarding the GA pathway throughout the manuscript. Statements like "CnHRLP1 regulates the GA pathway" have been revised to more cautious formulations such as "CnHRLP1 overexpression is associated with altered expression of GA pathway genes," "our data suggest that CnHRLP1 may impact gibberellin homeostasis," or "CnHRLP1 indirectly modulates the transcript levels of key GA biosynthetic and catabolic genes." These changes clarify that the observed effect on the GA pathway is correlative and likely indirect, in line with the protein's presumed RNA-binding function.

 

3: Finally, careful language editing and minor corrections to figure numbering and terminology would improve clarity and readability.

>>>>Response

We have undertaken a thorough proofreading and language editing pass of the entire manuscript to improve fluency, correct minor grammatical errors, and ensure consistency in terminology. Furthermore, we have double-checked all in-text citations of figures and supplementary materials to verify that the numbering is correct and consistent (e.g., ensuring references to Figure 5C, D or Figure 6A are accurate). Any identified discrepancies have been corrected.

 

We believe these revisions have successfully addressed the reviewer's insightful comments, resulting in a more precise, cautious, and polished manuscript. Thank you again for the valuable feedback.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Accept in the current form as it has been improved significantly