Genome-Wide Identification and Expression Analysis of AT-Hook Motif Nuclear Localized Gene Family in Birch

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Genome-Wide Identification and Expression Analysis of AT-2 Hook Motif Nuclear Localized Gene Family in Birch” by Chen et al. studies the AT-hook motif nuclear localized (AHL) gene family in birch, identifying 21 BpAHL genes that play essential roles in regulating growth, development, and responses to abiotic stress. Phylogenetic analysis classifies these genes into two main clades (A and B) and three types (I, II, and III) based on conserved PPC domains and AT-hook motifs, revealing variations in their structural characteristics and evolutionary relationships. Chromosomal mapping locates the BpAHL genes across nine chromosomes and one contig, with gene family expansion primarily driven by dispersed duplication events. Tissue-specific expression profiling demonstrates functional divergence, as type-I BpAHLs are mainly expressed in roots, while type-II and -III BpAHLs show higher expression in flowers and leaves.  In response to abiotic stresses, type-II and -III BpAHLs in leaves exhibit differential expression patterns under cold and heat stress, and type-I BpAHLs in roots are down-regulated under salt stress, indicating their specific roles in stress response mechanisms.

 

While the article presented holds promise, aspects of it would benefit from additional refinement to enhance their robustness and rigor. Specific areas for improvement are outlined below:

 

Introduction:

 

Lines 41-42: “....has since been extensively characterized across diverse terrestrial plant species….”. The sentence could be improved by indicating that they are plant species with sequenced genomes.

 

Lines 86-87: “Birch, a keystone, broad-leaved, deciduous hardwood tree species distributed in Northern Europe, Russia, and northern China, holds ecological and economic significance”. Including details about the plant's biology, taxonomy, ecological significance, and economic value is recommended at this stage. The authors should consider adding specific examples to emphasize its importance.

 

Materials and Methods:

 

Line 96: “The seeds of Betula platyphylla used in this research”. Please provide details on any notable biological traits (such as resistance or tolerance to specific factors) present in the plant species utilized in this study. Additionally, indicate if the plants belong to a particular variety of the species.

 

Lines 102-103: “When the seedlings reached 3 months, different plants tissues, including leaf, stem, and root, were collected in independent triplicates of each sample”.The manuscript's results section includes photographs and expression values for certain BpAHL genes under drought conditions. However, the methodology section does not mention this experiment. Please add the experimental details to the methodology.

 

Lines 106-108: “The hidden Markov model (HMM) of the HMMER 3.0 program [25] with PPC/DUF296 (PF03479) domain (https://pfam.xfam.org) [26,27] was employed to search for AHL proteins in the birch genome”. Was the chosen search method adequate for identifying all family members? Why were alternative methods, like BLAST alignments, not used to validate the HMM-based findings?

 

Lines 114-115: “Chromosomal localization, gene length, CDS length, protein length, and GO annotation of the BpAHL gene family were acquired from the annotation files of birch genome”. The authors cited the birch genome article, but should also specify the database from which the genomic resources for this species were obtained.

 

Lines 130-131: “AHL proteins across diverse plant species, including maize, soybean, sorghum, Arabidopsis thaliana”. Specify the database from which the sequences were sourced. If feasible, please provide a supplementary table listing their accession numbers.

 

Lines 132-134: “The analysis involved MUSCLE multi-sequence alignment, the Neighbor-Joining (NJ) method, and 1,000 bootstrap replicates to ensure the reliability of the tree topology”. For a more reliable phylogenetic analysis, the authors should justify their choice of the NJ method over more robust methods like maximum likelihood or Bayesian analysis. Additionally, they should employ an evolutionary model that better fits the analyzed data.

 

Lines 136-137: “The protein sequences involved in the phylogenetic tree and the Newick file obtained after MEAG software analysis are provided in the Table S11”. This information is not available in the article or as supplementary material. Also, correct MEAG.

 

Lines 185-186: “The ten most frequently occurring CREs were identified, classified, and visualized using the “Simple BioSequence Viewer” module in TBtools software”. Why were only the 10 most common CREs selected? Is there any reason for using this cutoff?

 

Lines 194-195: “The resulting BAM files were processed with StringTie software”. In this processing of BAM files, were reads that map to multiple locations excluded? In any differential expression analysis, certain criteria must be taken into account to avoid altered expression values.

 

Lines 219-220: “The bioinformatics procedures of these transcriptomes were performed as described in section 4.7”. Correct 4.7.

 

Lines 248: “The primers used for qRT-PCR are listed in Table S10”. This information is not available in the article or as supplementary material.

 

Results

 

Lines 265: Table 1. In this table it would be important to include a column that indicates on which chromosome each gene is located.

 

Lines 370-372: “Additionally, BpAHL21 is located in Contig1465, which remains unanchored to any specific chromosome”. Could comparing this gene to genes from other species suggest its potential location? What genes from other species exhibit high nucleotide and amino acid sequence identity with this gene? Please provide specific details regarding these similarities.

 

Line 415: “...as indicated in Table S14”. This information is not available in the article or as supplementary material.

 

Line 463: “Table S13”. This information is not available in the article or as supplementary material.

 

Lines 582-584: Please specify within the text the plant where this study was conducted.

 

Line 585: “We recorded the phenotype of birch under drought stress”. Please include the details of this experiment in the methodology section.

 

Line 593: Figure 8. Which of the evaluated genes exhibit the most substantial differences in expression? The provided figure suggests a lack of significant expression differences across the tested conditions. The authors could include the statistical analysis performed to include this information in the article.

Comments on the Quality of English Language

The manuscript requires revision by a native language specialist to address grammatical errors and improve paragraph structure.

Author Response

Response: We are deeply grateful for your thoughtful evaluation and generous comments on this manuscript. In response to your observation regarding our manuscript, we carefully modified certain expressions and also figures throughout the text to enhance clarity (see the revised manuscript with a yellow background). In addition, we thoroughly considered and addressed each of your comments. Thanks again for your time and constructive feedbacks, which have undoubtedly strengthened this work.

The revisions in this revised manuscript are marked with a yellow background, and detail response can be found in the attached file named 'Response to Reviewer1'.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Major Revision

The manuscript titled "Genome-Wide Identification and Expression Analysis of AT-Hook Motif Nuclear Localized Gene Family in Birch" presents a comprehensive study on AHL gene family characterization and stress response analysis in birch. While the study is well-structured, several sections require major revisions to enhance clarity, scientific rigor, and completeness.

The following comments are provided for improvement

1. Abstract – Line 22: Include specific abiotic stresses analyzed in this study. to clearly define the study’s scope.
2. Introduction: Begin the introduction by discussing the global significance of AHL gene family studies, particularly in the context of climate change and abiotic stress tolerance in economically important plants.
3. Lines 40-64: Rewrite this section in a simpler, more structured format. Divide into distinct paragraphs for: overview of AHL gene family functions, importance of studying AHLs in stress responses, relevance of birch as a study species.
4. Justification for Study – Line 46: Clarify the term "critical role" with specific examples. What specific functions of AHLs are considered critical in birch? Provide references to previous studies that support this statement.
5. Plant Selection Rationale: Clearly state why birch was chosen for this study. Is birch known to be particularly susceptible to abiotic stress?
6. How does this study address existing knowledge gaps?
7. Experimental Details – Methodology: Humidity Control: Specify the humidity conditions.
8. In which the experiments were conducted (e.g., greenhouse, laboratory, or growth chamber)? Include in the manuscript.
9. Seed Germination – Line 98: Rephrase the sentence.
10. Abiotic Stress Treatments: How were the temperature extremes (6°C and 35°C) selected? Justify the relevance of these temperature points in relation to birch growth conditions.
11. Include details about the drought treatment protocol.
12. Clarify the concentration and duration of the NaCl treatment (0.2 M).
13. Ensure treatment parameters are clearly defined and referenced for reproducibility.
14. Line 137: Confirm the correct spelling and version of MEGA software (not “MEAG”).
15. Formatting and Consistency: Ensure that all gene names, including AHLs and BpAHLs, are consistently italicized throughout the manuscript.
16. Replace all casual terms like “spotted” with precise scientific terminology throughout the manuscript.
17. Expand the conclusion to briefly discuss potential applications of BpAHL genes in birch breeding programs or genetic engineering for stress resistance.
18. Suggest future studies to explore the functional validation of Type-I, II, and III BpAHLs through gene editing, transgenic approaches, or field trials under multiple stress conditions.
19. The manuscript requires a thorough language review to address grammatical errors, awkward phrasing, and overly complex sentences. Consider professional editing for clarity and fluency.

Comments on the Quality of English Language

The manuscript requires a thorough language review to address grammatical errors, awkward phrasing, and overly complex sentences. Consider professional editing for clarity and fluency.

Author Response

Response: We sincerely appreciate your valuable evaluation and comments regarding our text. In response to your concerns, we thoroughly considered and addressed each of your concern. Thanks for your time and constructive feedbacks, which have undoubtedly strengthened this work.

The revisions in this revised manuscript are marked with a yellow background, and the detail responses can be found in the attached file named 'Response to Reviewer2'.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have reviewed the resubmission of the manuscript entitled "Genome-Wide Identification and Expression Analysis of AT-Hook Motif Nuclear Localized Gene Family in Birch". The authors answered in a satisfactory way the points that I have addressed in the first review. Thus this new version of the manuscript can be accepted for publication.

Author Response

Response to reviewer

I have reviewed the resubmission of the manuscript entitled “Genome-Wide Identification and Expression Analysis of AT-Hook Motif Nuclear Localized Gene Family in Birch”. The authors answered in a satisfactory way the points that I have addressed in the first review. Thus this new version of the manuscript can be accepted for publication.

Response: Thank you very much for your high appraisal of this revision and for your efforts in improving the quality of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made extensive and commendable efforts in addressing the major revision comments, significantly improving the manuscript. The revised version is much clearer and scientifically sound. Only a few minor corrections remain, such as ensuring that all gene names are italicized and removing unnecessary hyphens (“-”) throughout the text to maintain scientific rigor and consistency.

Author Response

Response to reviewer

The authors have made extensive and commendable efforts in addressing the major revision comments, significantly improving the manuscript. The revised version is much clearer and scientifically sound. Only a few minor corrections remain, such as ensuring that all gene names are italicized and removing unnecessary hyphens (“-”) throughout the text to maintain scientific rigor and consistency.

Response: We appreciate your suggestions and have accordingly verified and corrected all gene nomenclature throughout the text, ensuring that italicized ‘BpAHL’ or ‘AHL’ exclusively indicate genes while non-italicized forms represent proteins, and have additionally removed all hyphenated word breaks ‘-’ in the text to comply with formatting standards.