Genome-Wide Identification of the PLATZ Gene Family and Its Roles in Stress Responses in Flax (Linum usitatissimum L.) Based on the Telomere-to-Telomere Genome

Response to Reviewer 1 Comments

1. Summary

Thank you for your professional opinion on our article. We greatly appreciate your comments and suggestions. After carefully considering your suggestions, we have revised our manuscript. In the following section, we have listed the reviewers' comments in italics and numbered the issues identified. Our responses are shown in normal font, while changes and additions made to the manuscript are highlighted in red for ease of reference. Please find the revised manuscript in agronomy’s manuscript center. The following modifications are based on the newly submitted manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Must be improved

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Yes

Are all figures and tables clear and well-presented?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The existing title is not appropriate to the manuscript content. Please revise the title.

Response 1: We sincerely thank the reviewer for the constructive comment. We agree that the original title was not fully aligned with the content of the manuscript. Accordingly, we have revised the title to more accurately reflect the scope of our study: “Genome-wide identification of the PLATZ gene family and its roles in stress responses in flax (Linum usitatissimum L.) based on the Telomere-to-Telomere genome” We hope that this revised title is more appropriate and consistent with the manuscript content.

Comments 2: Lines 44-86: The author must go into detail to describe the various categories of flax and their genomes. And contrast the T2T flax genomic information with the detailed description. Have any information about the identification of PLATZ gene family among the flax categories (linseed, fiber flax, and dual-purpose flax)?

Response 2: Thank you for your valuable suggestion. We agree that providing a more detailed description of the different categories of flax and their genomic resources will improve the clarity and completeness of the manuscript. We have expanded this section in lines 74–89. Regarding the identification of the PLATZ gene family, our current study is based on the T2T fiber flax genome, which provides the most complete reference to date. To the best of our knowledge, systematic genome-wide identification of PLATZ genes has not yet been reported in linseed or dual-purpose flax due to the lack of high-quality genome assemblies. We will clarify this point in the manuscript and emphasize that our study provides the first comprehensive characterization of the PLATZ gene family in flax using the T2T genome, which will serve as a valuable reference for future comparative analyses among different flax categories once additional high-quality genomes become available.

Comments 3: Line 93: For clear understanding for the readers, the author needs to clearly mention the seedlings’ age (how many-day-old seedlings) used in this experiment.

Response 3: We thank the reviewer for this suggestion. In the revised manuscript, we have clarified the seedlings’ age in the Materials and Methods section. Specifically, all experiments were conducted using three-day-old seedlings, and this information has been added to ensure clarity for the readers.

Comments 4: Lines 102-103: The meaning of this sentence is not clear.

Response 4: We thank the reviewer for this comment. The sentence in Lines 102–103 has been revised for clarity in the updated manuscript.

Comments 5: Lines 103-118: The authors mentioned they use 12 PLATZ protein sequences from the TAIR database. Did you check such relevant proteins in other databases for other important major crops?

Response 5: We thank the reviewer for the suggestion. The genome and protein sequence files of maize and wheat were obtained from the publicly available Phytozome database, and PLATZ gene family members in these species were identified using the same approach as for flax. We have added this revision to the Materials and Methods section.

Comments 6: Lines 163-169: The author needs to clearly explain the time frame (different hours) of the data collection.

Response 6: We thank the reviewer for this suggestion. The time frame for data collection has been clearly clarified in the revised manuscript. Specifically, leaf samples were collected at 0, 3, 6, 9, 12, and 24 h after stress treatments. This information has been added to the Materials and Methods section to ensure clarity for the readers.

Comments 7: Lines 382-383: Expression of both LuPLATZ14 and LuPLATZ21 was high for cold, salt, and drought treatment. Why do you mention here only drought tolerance?

Response 7: We thank the reviewer for the comment. Expression analysis revealed that LuPLATZ21 showed the highest expression under drought stress, while LuPLATZ14 was highly expressed under cold, salt, and drought treatments. Therefore, LuPLATZ14 and LuPLATZ21 were selected as candidate genes. Based on literature research, their functional roles under drought stress were validated using a yeast heterologous expression system, providing a reliable proof-of-concept for their involvement in abiotic stress responses.

Comments 8: Lines 404-408: You compared your (flax) identified genes with tomato and cucumber here. Why didn’t you check these genes using their database (cucumber, wheat, and tomato, or other family ‘Linaceae’ crops)?

Response 8: Thank you for your valuable comment. Within the Linaceae family, the genus Linum contains only Linum usitatissimum (flax), and no other species in this family currently have available genome sequences. Therefore, in this study, we compared the identified flax genes with those in tomato and cucumber, which are well-studied model species with comprehensive genomic information, to provide evolutionary and functional insights.

RE: agronomy-3858078

Response to Reviewer 2 Comments

1. Summary

Thank you for your professional opinion on our article. We greatly appreciate your comments and suggestions. After carefully considering your suggestions, we have revised our manuscript. In the following section, we have listed the reviewers' comments in italics and numbered the issues identified. Our responses are shown in normal font, while changes and additions made to the manuscript are highlighted in red for ease of reference. Please find the revised manuscript in agronomy’s manuscript center. The following modifications are based on the newly submitted manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Must be improved

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Must be improved

Are the conclusions supported by the results?

Must be improved

Are all figures and tables clear and well-presented?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The abstract starts with a general description of PLATZ genes but does not clearly emphasize why studying them in flax is important.

Response 1: Thank you for your feedback. I have made revisions to the abstract to clearly highlight the importance of studying PLATZ genes in flax. In the revised version, I explicitly outline how the expression patterns of PLATZ genes in flax are crucial for understanding aspects such as growth, development, and stress resistance, and discuss the value of this research for flax genetic improvement and molecular biology studies.

Comments 2: It is unclear whether expression data came from RNA-seq datasets or experimental validation, clarifying this distinction would strengthen credibility.

Response 2: Thank you for your insightful feedback. I have revised the manuscript to clearly indicate that the expression patterns of the flax PLATZ genes were analyzed based on RNA-seq data. This clarification should strengthen the credibility of the study and distinguish it from any experimental validation. I hope this addresses your concern. 

Introduction：

Comments 1: It is unclear whether expression data came from RNA-seq datasets or experimental validation, clarifying this distinction would strengthen credibility.

Response 1: Thank you for your insightful feedback. I have revised the manuscript to clearly indicate that the expression patterns of the flax PLATZ genes were analyzed based on RNA-seq data. This clarification should strengthen the credibility of the study and distinguish it from any experimental validation. I hope this addresses your concern.

Comments 2: Some references are duplicated or redundant: e.g., tomato and Solanum lycopersicum are the same species but cited separately [10] and [11]. This should be consolidated.

Response 2: Thank you for pointing that out. While I understand that tomato and Solanum lycopersicum refer to the same species, I have decided to keep the references separate in this case to maintain consistency with the genomic terminology used in the literature. I believe this distinction is relevant for clarity and precision in the context of the study. I hope this explanation is acceptable, but please let me know if further clarification is needed.

Materials and Methods:

Comments 1: Why PEG 10% and 100 mM NaCl? Are these physiologically relevant concentrations for flax? Adding a reference or rationale would improve credibility.

Response 1: Thank you for the valuable comment. We have added a rationale and supporting references in the Methods section to clarify that the use of 10% PEG and 100 mM NaCl is based on previous studies where these concentrations were shown to effectively induce drought and salt stress responses in flax.

Comments 2: Temperature/light conditions (26/18 °C, 16/8 h cycle) are provided, but humidity is missing, important for stress experiments.

Response 2: Thank you for your valuable comment. We have added the humidity conditions to the description of plant growth. The revised sentence now reads: “Plants were grown in a controlled growth chamber under a 16 h light/8 h dark photoperiod at 26/18 °C, with a relative humidity maintained at approximately 60%.”

Comments 3: Criteria for considering a sequence as a PLATZ gene should be stated more explicitly.

Response 3: We appreciate the reviewer’s valuable suggestion. We have revised the Methods section to explicitly state the criteria used to define PLATZ genes. Specifically, we now clarify that only sequences containing an intact and complete PLATZ domain (PF04640), verified by both SMART and CDD, were considered as PLATZ genes. This modification has been incorporated into the revised manuscript.

Comments 4: Mentioning the version of flax genome assembly is important for reproducibility.

Response 4: Thank you for the constructive comment. We have now added the version/source of the flax genome assembly (T2T flax, cv. Gsosi; PRJCA037526) in the Methods section to improve the reproducibility of our analysis.

Comments 5: Thresholds for duplication events (≥200 bp alignment, ≥85% identity, within 100 kb) need justification or references. Otherwise, the choice seems arbitrary.

Response 5: We thank the reviewer for this valuable comment. We have now added appropriate references to justify the thresholds (≥200 bp alignment, ≥85% identity, within 100 kb) used to define duplication events, so that the criteria are no longer arbitrary.

Comments 6: Please mention the number of replications in each of the analysis.

Response 6: Thank you for the helpful comment. We have now added the number of replications (three biological replicates and three technical replicates for each sample) in the Methods section to clarify the experimental design.

Results:

Comments 1: Phylogenetic analysis: the grouping into five clades is described, but no statistical support values (bootstrap scores) are reported in the text. These are important to validate the clustering.

Response 1: Thank you for this valuable comment. We have now included bootstrap support values (based on 1,000 replicates) on the phylogenetic tree and described them in the text to validate the clustering of the five clades.

Comments 2: The authors report 33 duplicated pairs, but there is no Ka/Ks analysis to infer whether duplication events were driven by purifying or positive selection. This is a major omission in evolutionary analysis.

Response 2: Thank you for this valuable comment. We have performed Ka/Ks analysis for the 33 duplicated gene pairs and included the results in the main text, with a corresponding statement added to the conclusion to discuss the selective pressures acting on these genes.

Comments 3: Methods/results mismatch: Methods section used pGD-GFP vector, but Results mention pCAMBIA1301. This inconsistency should be corrected.

Response 3: Thank you for your suggestion. We have made the revision accordingly and updated the vector in the Methods section to pCAMBIA1301.

Comments 4: No growth curve or viability quantification is presented, just colony spotting. A more quantitative assay (OD600 measurement, survival rates) would strengthen conclusions.

Response 4: Thank you for your valuable comments. We highly appreciate your suggestion. By referring to the methodologies used in previous studies, we also conducted only colony spot assays. Moreover, since clear phenotypic differences were already observed in this study, we did not perform further quantitative analyses.

Discussion:

Comments 1: Much of the discussion repeats results (e.g., gene counts, motif arrangements, exon–intron structures, RNA-seq/qRT-PCR results). Instead, authors should summarize briefly and focus on interpretation and biological significance.

Response 1: Thank you for your valuable comment. We have revised the discussion section to reduce the repetition of results and now provide only a brief summary of the key findings. We have also added more interpretation and discussion of their potential biological significance to improve the depth and focus of this section.

Comments 2: Did flax undergo specific whole-genome duplication events leading to expansion?

Response 2: Thank you for your insightful comment. We have added information regarding the evolutionary history of flax, noting that it is an ancient diploid plant that experienced three whole-genome duplication (WGD) events approximately 11.5, 53.5, and 114 million years ago. We also highlighted that these WGD events likely contributed to the expansion of the LuPLATZ gene family by providing redundant gene copies.

Comments 3: How does this expansion correlate with flax’s ecological adaptations (e.g., temperate climates, fiber vs oilseed types)?

Response 3: Thank you for your valuable comment. We have added a discussion suggesting that the expansion of the LuPLATZ gene family may have contributed to flax’s adaptation to temperate environments by enhancing its tolerance to abiotic stresses, and that the functional diversification of duplicated genes might have facilitated the divergence between fiber and oilseed flax types.

Comments 4: Could structural diversity contribute to alternative splicing under stress?.

Response 4: Thank you for your valuable comment. We have addressed this point by adding a discussion that the structural diversity of LuPLATZ genes, such as variations in exon–intron organization, may provide the genomic basis for alternative splicing under stress conditions, thereby contributing to their functional diversification.

Comments 5: Connect promoter elements (ABRE, MBS, LTR) with the qRT-PCR stress responses (e.g., drought-responsive LuPLATZ genes also containing MBS sites).

Response 5: Thank you for your valuable comment.We have addressed this comment by linking the presence of stress-related cis-elements (ABRE, MBS, LTR) in LuPLATZ promoters with their qRT-PCR expression patterns. For example, drought-responsive LuPLATZ genes often contain MBS sites, supporting their roles in stress responses.

Comments 6: Highlight which genes might be prime targets for genetic engineering based on cis-element patterns.

Response 6: Thank you for this valuable suggestion. We have revised the manuscript to briefly highlight that genes harboring multiple stress-responsive cis-elements (such as MBS, ABRE, and LTR) in their promoters may represent promising targets for future genetic engineering efforts aimed at improving stress tolerance in flax.

Comments 7: Suggest candidate LuPLATZ genes most likely regulated by miR167.

Response 7: Thank you for the helpful suggestion. We have addressed this by adding a statement in the Discussion section indicating that LuPLATZ1 and LuPLATZ4 are potential targets of miR167, suggesting that their expression may be post-transcriptionally regulated by this miRNA.

Comments 8: The authors only contrast with Arabidopsis and maize. They should discuss what this means for flax physiology (e.g., adaptation to abiotic stress in vegetative stages vs reproductive) and with other plants like tomato.

Response 8: We thank the reviewer for the suggestion. We have revised the discussion to relate our findings to flax physiology, highlighting their potential roles in adaptation to abiotic stress during the vegetative stage, and included comparisons with tomato to illustrate both conserved and flax-specific PLATZ functions.

Comments 9: Compare whether stress-inducible LuPLATZ genes overlap with orthologs functionally validated in other crops.

Response 9: Thank you for the suggestion. We have revised the discussion to show that several stress-inducible LuPLATZ genes cluster with functionally validated PLATZ orthologs (GmPLATZ11, GmPLATZ17, OsPLATZ7/9/11), indicating conserved roles in stress responses.

Comments 10: Authors should acknowledge this limitation.

Response 10: We appreciate the reviewer’s suggestion. We have revised the discussion to explicitly acknowledge this limitation, noting that the functional roles of LuPLATZ genes remain to be experimentally validated through approaches such as transgenic or mutant analyses.

Conclusion:

Comments 1: The conclusion does not connect back to the importance of flax as an oilseed/fiber crop. Authors could strengthen the impact by linking their findings to potential applications in crop breeding, stress resilience, or yield stability.

Response1: Thank you for the valuable suggestion. We have revised the conclusion to emphasize the importance of flax as an oilseed and fiber crop, and to highlight the potential applications of our findings in enhancing stress resilience, yield stability, and productivity through breeding strategies.

Comments 2: The conclusion should point to next research steps.

Response 2: Thank you for the insightful suggestion. We have revised the conclusion to include future research directions, such as functional validation of additional LuPLATZ genes and exploration of their roles in stress responses and breeding applications.

RE: agronomy-3858078

Response to Reviewer 3 Comments

1. Summary

Thank you for your professional opinion on our article. We greatly appreciate your comments and suggestions. After carefully considering your suggestions, we have revised our manuscript. In the following section, we have listed the reviewers' comments in italics and numbered the issues identified. Our responses are shown in normal font, while changes and additions made to the manuscript are highlighted in red for ease of reference. Please find the revised manuscript in agronomy’s manuscript center. The following modifications are based on the newly submitted manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Is the research design appropriate?

Yes

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Can be improved

Are all figures and tables clear and well-presented?

Must be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Title: Please correct your title.

Response 1: Thank you for your suggestion. We have revised the title of the manuscript as per your recommendation. We believe the new title better reflects the content of the study.

Comments 2: Abstract: The abstract is essential to your manuscript. It briefly summarizes your study. This version contains several grammatical errors. Here is the corrected version.

Response 2: Thank you for pointing out the grammatical errors in the abstract. We have carefully reviewed and revised the text to address the issues, ensuring that the abstract clearly and accurately summarizes the study. We appreciate your attention to this important section of the manuscript and believe the revised version is now much improved.

Comments 3: Line 37, 374: All text in Latin needs to be written in Italic script; please revise the entire text.

Response 3: Thank you for your comment. We have carefully reviewed the manuscript and ensured that all Latin text is written in Italic script, as per your recommendation. The necessary revisions have been made throughout the manuscript.

Comments 4: Line 89: The full botanical specification of your cultivar is missing. It is a type of oil- or fiber-producing linseed cultivar that you used in this study.

Response 4: Thank you for your comment. We would like to clarify that the cultivar "Gaosi" used in this study is a fiber-type flax (Linum usitatissimum) cultivar, not an oil-producing variety. We have now included this clarification in the manuscript.

Comments 5: Line 164: All techniques used must be supported by citations, as well as all products and equipment; they must include information about the supplier and manufacturer. Please add this information throughout the entire section M&M, e.g., Lines 170-179.

Response 5: Thank you for your valuable feedback. We have revised the Materials and Methods section and included citations for all techniques, products, and equipment used, along with details about the suppliers and manufacturers. These updates ensure that the section now meets the required standards and addresses your concerns regarding the completeness and clarity of the methods.

Comments 6: Figures 2, 3B, and 5 cannot be read. These figures present the essential results and findings of the authors. This part needs serious improvement. Currently, it is impossible to read these figures!

Response 6: Thank you for your valuable feedback. In response to your comment, we have revised Figures 2, 3B, and 5 to improve their readability. The resolution of these figures has been increased to ensure that all details are clear and legible. Additionally, the text and labels have been enlarged to make them more readable. We believe these changes will significantly improve the clarity of the figures and address your concern.

Comments 7: Figure 8: Clearly clarify the subcellular localization of the fluorescent signal and include this information in the figure caption.

Response 7: Thank you for your valuable suggestion. We have revised the figure caption to clearly specify the subcellular localization of the fluorescent signals, and we have included the use of RFP as the red fluorescence marker. We believe this revision addresses your concern and improves the clarity of the figure.

Comments 8: Figure 9: Out of focus, everything needs to be changed if you want to publish it!

Response 8: We sincerely apologize for the out-of-focus issue in Figure 9. We understand the importance of clear images, and we will pay closer attention to the quality of images in future experiments, especially regarding yeast-related work. Thank you for your valuable feedback.

Response to Reviewer 4 Comments

1. Summary

Thank you for your professional opinion on our article. We greatly appreciate your comments and suggestions. After carefully considering your suggestions, we have revised our manuscript. In the following section, we have listed the reviewers' comments in italics and numbered the issues identified. Our responses are shown in normal font, while changes and additions made to the manuscript are highlighted in red for ease of reference. Please find the revised manuscript in agronomy’s manuscript center. The following modifications are based on the newly submitted manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Must be improved

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

Are all figures and tables clear and well-presented?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Lines 37-38: Write all scientific names in Italics in the whole manuscript.

Response 1: Thank you for your suggestion. We have revised the manuscript to ensure that all scientific names are italicized throughout the text, including in lines 37-38, as per your recommendation. We appreciate your attention to detail, which has improved the clarity and consistency of our manuscript.

Comments 2: Lines 73-86: Explain some basic information of the T2T flax (Linum usitatissimum L.) genome.

Response 2: Thank you for your valuable suggestion. We agree that providing a more detailed description of the different categories of flax and their genomic resources will improve the clarity and completeness of the manuscript. We have expanded this section in lines 74–89. Regarding the identification of the PLATZ gene family, our current study is based on the T2T fiber flax genome, which provides the most complete reference to date. To the best of our knowledge, systematic genome-wide identification of PLATZ genes has not yet been reported in linseed or dual-purpose flax due to the lack of high-quality genome assemblies. We will clarify this point in the manuscript and emphasize that our study provides the first comprehensive characterization of the PLATZ gene family in flax using the T2T genome, which will serve as a valuable reference for future comparative analyses among different flax categories once additional high-quality genomes become available.

Comments 3: Lines 120-123: Why don’t you check some other crops under the ‘Linaceae’ family?

Response 3: Thank you for your valuable comment. Within the Linaceae family, the genus Linum contains only Linum usitatissimum (flax), and no other species in this family currently have available genome sequences.

Comments 4: Lines 153-162: The authors describe ‘Analysis of expression pattern of LuPLATZ Gene Family’ as publicly available RNA-seq data, not from the experimental analysis. That’s why authors need to clearly explain it as hypothetical analysis from a database. Otherwise, the readers will be confused.

Response 4: Thank you for your helpful suggestion. We have revised the manuscript to clearly indicate that the analysis of the expression pattern of the LuPLATZ gene family was based on publicly available RNA-seq data rather than experimental analysis. The updated version now explicitly states that the analysis is hypothetical and derived from a database, to avoid any potential confusion for readers. We appreciate your careful attention to this matter.

Comments 5: Lines 210-222: Although the authors searched and compared PLATZ proteins from 5 major crops, it is important to include some other crops from the same family ‘Linaceae’.

Response 5: Thank you for your valuable suggestion. While we agree that including other crops from the Linaceae family could provide additional insights, we would like to clarify that, to date, Linum usitatissimum (flax) is the only well-studied crop within this family with available genomic data. Although there are other species within the Linaceae, such as Linum usitatissimum’s wild relatives, their genomic resources are currently incomplete or not publicly available. Given these limitations, we focused on comparing PLATZ proteins in five major crops that offer well-characterized genomic data, which allows for more comprehensive analyses. We hope that future studies will expand the inclusion of other Linaceae crops as their genomic resources become available.

Comments 6: Lines 348-350: You mention the different time frame qRT-PCR data in the result. Are these real experimental data? Or retrieved from the public database? Please mention clearly.

Response 6: Thank you for your valuable comment. We have revised the manuscript to clarify that the qRT-PCR data mentioned in the results are original experimental data, not retrieved from any public database. The revised version of the text explicitly states that these data were generated in our study. We hope this clarification resolves any potential confusion.

Comments 7: The author needs to check the reference lists of those that are cited in the text. All the titles and other contents should be unique to the journal format.

Response 7: Thank you for your helpful feedback. We have carefully reviewed the reference list and ensured that all citations in the text comply with the journal's format. All titles and other content have been updated to match the required style. We appreciate your attention to this detail, which has helped improve the manuscript.