RNA Tailing by Nucleotidyltransferases in Plants: Mechanisms, Functions, and Biological Significance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I go through the review manuscript “plants-4150791” entitled “RNA tailing by Nucleotidyltransferases in plants: Mechanisms, functions, and biological significance” where the authors investigated the composition, evolution, and regulatory functions of the plant nucleotidyltransferase (NTP) gene family, which mediates the non-templated addition of nucleotides to RNA 3′ ends (RNA tailing). By synthesizing genetic and biochemical data from model plants and crops, the authors explain how NTPs select specific RNA substrates and how the resulting tail types and lengths influence RNA metabolism. Ultimately, the paper connects these molecular mechanisms to essential biological outcomes, including plant development, stress adaptation, antiviral immunity, and the enhancement of agronomic traits in crops. However, the given suggestions might further improve the manuscript.

• The title should be RNA tailing by Nucleotidyltransferases in plants: Mechanisms, functions, and biological significance rather then keeping the first letter of every word capital.
• Make correction to Arabidopsis in line 101, 268, 323, etc
• Keywords need to be acsending alphabetic order.
• Compositions of Plant NTP Gene Family should be Compositions of plant NTP gene family.
• Molecular Roles of NTPs in the Tailing of RNA Classes should be Molecular roles of NTPs in the tailing of RNA classes. Keep the same format throughout the manuscript in headings.
• I suggest add some tables (atleast 4 tables) summarizing key findings and insights. Also the number of only two figures about the phylogeny and motifs is not en ough to properly address the topic. Please add some more key topography.
• I suggest also add the roles of NTPs in the development and flowering of plants. That will further strengthen the review for potential roles of NTPs.
• The manuscript contains many minor typos that needs to be corrected.
• I also suggest to interlink all the citations to the references and the mention of figure and table in text to the correstponding figure and tables (Clickable citations and figure table in text).

Comments on the Quality of English Language

The manuscript contains minor typos that needs to be corrected. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study focused on the core post-transcriptional modification mechanism of NTP-mediated RNA tailing in plants, with the logical main line of "family composition and evolution → molecular functions → determinants of substrate selection → biological functions → future directions", it systematically integrates genetic, biochemical, and phylogenetic evidence from model plants and crops, constructing a complete knowledge framework that conforms to the academic norms of review articles. It not only covers the tailing regulation of multiple types of RNAs such as miRNA, mRNA, and rRNA, but also links to plant development, stress response, antiviral immunity, and crop agronomic traits (flowering period, 1000-grain weight), achieving a deep integration of molecular mechanisms with physiological functions, and basic research with crop applications, thus highlighting prominent academic value and application potential. It integrates key research results in this field in recent years, with cited literature covering multiple species such as Arabidopsis, rice, maize, and wheat, ensuring reliable data sources; the functional division and synergistic effects of core members (HESO1, URT1) are analyzed in detail, reflecting the systematic nature of the research. The perspective is novel and future-oriented: It emphasizes the emerging direction of NTP-mediated "non-degradative tailing", compares the functional differentiation of NTP families among different species, and the proposed regulatory mechanisms such as intrinsically disordered regions (IDR) and phase separation provide clear targets for subsequent research.

I just listed my comments here, please find them and fix them try your best.

1. 

Currently, the article mainly integrates consistent research results and does not mention controversial or contradictory cases in the field. For example, do the "repair function" and "degradation function" of NTP in tailing pre-miRNA in different studies have species-specific differences? The correlation between NTP gene expression and stress response in some crops has not been verified functionally; is there a false positive association? It is recommended to supplement the discussion on relevant controversial points to objectively present the current status of the field.

Regarding "non-degradative tailing", most of the existing evidence comes from Arabidopsis thaliana. Whether similar mechanisms exist in crops (such as uridylation stabilization of P4RNA) is still unclear. It is suggested to add a review of relevant studies in crops or point out the research gaps in this direction.

2.

The article mentions that NTP contains a conserved catalytic domain and IDR, but it does not incorporate the latest advancements in structural biology (such as how the crystal structure of URT1 affects substrate binding and tail length control). It is recommended to supplement the structure-function association analysis of core members (HESO1, URT1), for example, the impact of key amino acid mutations in the catalytic domain on enzyme activity, IDR-mediated protein interactions, and phase separation mechanisms, to enhance the depth of molecular mechanisms.

In the section on substrate selection, only factors such as RNA type and terminal structure are mentioned, without clarifying the molecular association between "tail length and functional outcomes" (e.g., regulatory differences between single U tails vs. oligouridine tails). It is suggested to analyze the regulatory code of tail types/lengths by combining specific cases (such as the single U tail of miR171a triggering phasiRNA synthesis).

3.

Current crop research focuses on rice, maize, soybean, and wheat, lacking analyses of the NTP family in other important crops (such as tomato, cotton, and rapeseed). It is suggested to supplement the research progress of these crops (if available) or compare the expansion characteristics and functional biases of the NTP family among different crops through a table to enhance the universality of the review.

In the section on functional differentiation of NTPs in different species, only differences in expression patterns are described, without in-depth analysis of the association between evolutionary driving forces (such as whole-genome duplication and environmental adaptive selection) and functional differentiation. It is recommended to combine the evolutionary history of species (such as the retention of wheat subgenomes and soybean whole-genome duplication) to analyze the molecular mechanisms of NTP family expansion.

4. 

Figure 1 (phylogenetic tree) does not label the specific range of bootstrap values for each branch, and the classification of NTP members in some species (such as wheat) is unclear; abbreviations such as "PTGS" and "RdDM" in Figure 3 (schematic diagram of RNA metabolism regulation) are not marked with their full names in the figure legend. It is recommended to supplement details to improve readability.

There is a duplicate numbering of Table S1 in the supplementary materials (simultaneously labeling NTP family members and an overview of Arabidopsis NTP functions), which needs to be corrected to Table S1 and Table S2; it is suggested to add a "Comparison Table of Core Characteristics of NTP Families in Different Plants" that integrates information such as the number of members, expansion mechanisms, and key functions to facilitate quick reference by readers.

6.

In the abstract, clearly specify the specific manifestations of "context-dependent regulatory outcomes" (such as degradation vs. stabilization, processing vs. clearance) to avoid vague expressions; supplement the keywords with "plant antiviral immunity" and "agronomic traits" to enhance search relevance.

In the introduction section, add the core differences in RNA tailing between plants, animals, and yeast (such as the antagonistic relationship between plant-specific HEN1 methylation protection and NTP tailing) to highlight the research characteristics in the plant field.

In the discussion section, strengthen the connection between "NTP-mediated RNA regulation and crop breeding" and provide specific examples (such as targeted editing of TaNTP6A/B/D to improve thousand-grain weight) to enhance application orientation.

Unify the reference format (such as abbreviation of author names, abbreviation of journal names, page number) to ensure consistency.

7. Please do a English editing due to I found many grammatical errors.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Review Plants 4150791

The review by Xu and colleagues submitted for publication in “Plants” examines the role of nucleotidyltransferase proteins (NTPs) in post-transcriptional modification of RNAs via the non-templated addition of nucleotides to RNA 3' ends. Based on the reports on several plant species, the authors systematically characterized the composition and evolutionary relationship of NTP family dividing its members into three major evolutionary clades. This diverse classification revealed both unity and diversity in the conservation and functional differentiation of the NTP members, providing framework for elucidating substrate specificity and biological functions. The authors describe diverse molecular roles of NTPs in the tailing of RNAs belonging to various RNA classes emphasizing how tail type and length confer context-dependent regulatory mechanisms.  According to the data presented, key functions of NTPs included, among others, directing mRNA stability and degradation through uridylation; controlling sRNA biogenesis, ensuring ribosomal RNA quality via adenylation-mediated turnover of precursors; and uridylation of polymerase IV RNA. The authors also examined the factors determining substrate choice by Arabidopsis NTP family members, which depend on RNA type, terminal structure, and subcellular localization, and showed that through integration into various RNA metabolic pathways, NTP family members are involved in vital biological processes such as growth, development, and stress adaptation.

Overall, the presented data provide a clear understanding of NTP-mediated RNA regulation and the potential for its use in crop genetic improvement. However, I have a number of technical comments.

The text needs editing, as some sentences do not meet stylistic standards and make it difficult to understand the meaning (lines 198-199, 210-213, 223-225).

Line 48. Please, add “C.” before elegans if you mean  Caenorhabditis elegans

 It is better to place the list of abbreviations at the beginning of the article.

The overall impression would benefit if the chapters4 and 5 were illustrated in the same way as the chapters 2 and 3.

Table S1 should be included in the main text.

 

 

Comments on the Quality of English Language

The text needs editing by native English speaker

Author Response

Please see the attachment.

Author Response File: Author Response.pdf