Review Reports - Morphological and Transcriptomic Analyses Provide New Insights into Linseed (<i>Linum usitatissimum</i> L.) Seedling Roots Response to Nitrogen Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript illustrates a well-structured and insightful study combining phenotypic and transcriptomic approaches to elucidate linseed root responses under nitrogen-deficient conditions, which is timely given the global push toward sustainable agriculture. The combined integration of morphological traits with RNA-seq data provides a comprehensive understanding of stress-induced molecular mechanisms, presenting the key gene pathways and transcription factors involved in nitrogen uptake and its metabolism. The experimental design is sound, and the data interpretation is generally credible. However, the author must minimize plagiarism similarities to at least below 15%. The following points should be taken into consideration:

Abstract:

The author writes the abstract based on their obtained findings.

Introduction: The author precisely addresses the research gap with proper justification. However, the author needs to address the previous research findings on nitrogen-responsive transcriptomic analysis on roots in different relevant crops.

Results:

Line 152: In Figure 2, why does the root length and growth much higher in the (N-) condition in N stress-tolerant accession O_IRL_C_CN98192?

Materials and Methods:

Line 380-384: The author needs to explain which amount of nitrogen application is considered low nitrogen or nitrogen stress and nitrogen sufficient.

Discussion:

The author discussed their obtained findings with proper justification.

Conclusion:

The author writes the conclusion perfectly. However, the author needs to mention some key findings, such as how many key responsive genes are detected.

References:

The author needs to check the reference lists those are cited in the text. Needs to write every scientific name as Italic.

Author Response

Response letter to Reviewer 1

Comments and Suggestions for Authors

Abstract:

The author writes the abstract based on their obtained findings.

Q1. Introduction: The author precisely addresses the research gap with proper justification. However, the author needs to address the previous research findings on nitrogen-responsive transcriptomic analysis on roots in different relevant crops.

R: Previous research findings on nitrogen-responsive transcriptomic analysis on roots in different relevant crops were included in Introduction lines: 99-110 of the “track changes” manuscript.

Results:

Q2: Line 152: In Figure 2, why does the root length and growth much higher in the (N-) condition in N stress-tolerant accession O_IRL_C_CN98192?

R: The N-stress tolerant genotype possesses a battery of adaptive mechanisms to N stress that the sensitive genotype does not. The superior NUE indicates that the tolerant genotype can absorb, translocate and metabolize more N, constructing more biomass. Moreover, It can better maintain its metabolism close to the homeostasis point between both N condition. This is reflected at the transcriptome level where most of DEGs up- or downregulated were observed in the transcriptome of the sensitive genotype. This type of genotypes are also known as N responder-N efficient because they perform well under both condition which is the ideal for breeding.

Materials and Methods:

Q3: Line 380-384: The author needs to explain which amount of nitrogen application is considered low nitrogen or nitrogen stress and nitrogen sufficient.

R: This is explained in lines 546-547 of the “track changes” manuscript.

Discussion:

The author discussed their obtained findings with proper justification.

Conclusion:

Q4: The author writes the conclusion perfectly. However, the author needs to mention some key findings, such as how many key responsive genes are detected.

R: This was improved in lines 617-620 of the “track changes” manuscript.

References:

Q5: The author needs to check the reference lists those are cited in the text. Needs to write every scientific name as Italic.

R: Scientific names were written as Italic throughout the reference list.

Reviewer 2 Report

Comments and Suggestions for Authors

Soto-Cerda et al., in their manuscript titled "Morphological and transcriptomic analyses provide new insights into linseed (Linum usitatissimum L.) seedling roots response to nitrogen stress," present a comparative analysis of morpho-physiological traits and RNA-seq data from two linseed genotypes grown under full nutrient and nitrogen-deficient conditions at the seedling stage. The goals are clear: to contrast how two selected, a nitrogen-tolerant and a nitrogen-sensitive genotype respond, and to link gene expression profiles with observed morpho-physiological diferences.

While the study is relevant in the field of plant/crop sciences and presents interesting data, I have a few major concerns related to missing information that is critical to complement the results and suport both the discussion and conclusions. For instance, in the sections describing the RNA-seq analysis and discussion, several genes associated with nitrogen acquisition, metabolism, and root development (lines 208–220 in the results section, and all cited genes in the discussion) are mentioned and linked to Supplementary Table 2, but their expression levels (e.g., fold change) are not shown. Including that information; ideally as a figure with graphs, would make it easier to visualize and interpret their expression patterns and would strenghten the overall analysis. Also, although the authors mention that qRT-PCR validation could be carried out in future research (line 449), I think it should be included in this study; at least for key genes involved in nitrogen transport, translocation, metabolism and root development, as this is essential to validate the RNA-seq results and support the findings. Moreover, Figure 4 only shows the KEGG pathway annotation for the DEGs identified in root tissue of the N-sensitive (NS) genotype; including the same data for the N-tolerant genotype would allow for a interesting comparison between genotypes, which could help identify pathways associated with nitrogen tolerance or sensitivity in linseed plants.

There are also some minor issues and suggestions that could improve the clarity of the manuscript. One is the inconsistent use of linseed genotype names; sometimes they are refered to as “N-tolerant” and “N-sensitive,” and in other places by their full identifiers (O_IRL_C_CN98192 and O_IND_C_CN98982). Keeping this consisten across the text, tables, and figures would help the reader follow more easily. Along the same lines, using “N-efficient” and “N-inefficient” instead of “N-tolerant” and “N-sensitive” might better reflect the focus on nitrogen use efficiency (NUE) rather than an stress response. The color scheme in the figures is also a bit confusing; for example, the NT genotype is shown in yellow in Figure 1 but appears as pink in Figure 3. It would be clearer to assign one color per genotype and keep it consistent across all figures.

In general, the study presented by Soto-Cerda et al. is interesting and provides valuable observations, but it would really benefit from including the missing gene expression data and making a few key ajustments to help strengthen the main message, support the findings, and make the manuscript easier to follow.

Author Response

Response letter to Reviewer 2

Comments and Suggestions for Authors

Q1: For instance, in the sections describing the RNA-seq analysis and discussion, several genes associated with nitrogen acquisition, metabolism, and root development (lines 208–220 in the results section, and all cited genes in the discussion) are mentioned and linked to Supplementary Table 2, but their expression levels (e.g., fold change) are not shown. Including that information; ideally as a figure with graphs, would make it easier to visualize and interpret their expression patterns and would strenghten the overall analysis.

R: Expression level was included for the 1,034 DEGs as Supplementary Table 3 and those DEGs highlighted in Results and Discussion sections were graphically shown as Supplementary Figure 1.

Q2: Also, although the authors mention that qRT-PCR validation could be carried out in future research (line 449), I think it should be included in this study; at least for key genes involved in nitrogen transport, translocation, metabolism and root development, as this is essential to validate the RNA-seq results and support the findings.

R: We are aware of and agree with the importance of validating key genes using qRT-PCR. However, the initial version of this project (March 2020 to March 2024) did not consider the transcriptome analysis. Then we requested to the Ministry of Sciences of Chile authorization to modify the budget and carry out this experiment. Unfortunately, the funds authorized were not sufficient to purchase the lab supplies for qRT-PCR, and we knew that this would be a weakness in our study. However, as the first N stress study in linseed we believe that still is a contribution to linseed genomics and molecular breeding. Now, we are applying to new funds to complete this experiment in the short term. Please, consider this particular scenario.

Q3: Figure 4 only shows the KEGG pathway annotation for the DEGs identified in root tissue of the N-sensitive (NS) genotype; including the same data for the N-tolerant genotype would allow for a interesting comparison between genotypes, which could help identify pathways associated with nitrogen tolerance or sensitivity in linseed plants.

R: We modified Figure 4 and now includes the KEGG results for the N-tolerant genotypes.

Q4: There are also some minor issues and suggestions that could improve the clarity of the manuscript. One is the inconsistent use of linseed genotype names; sometimes they are refered to as “N-tolerant” and “N-sensitive,” and in other places by their full identifiers (O_IRL_C_CN98192 and O_IND_C_CN98982). Keeping this consisten across the text, tables, and figures would help the reader follow more easily. Along the same lines, using “N-efficient” and “N-inefficient” instead of “N-tolerant” and “N-sensitive” might better reflect the focus on nitrogen use efficiency (NUE) rather than an stress response.

R: The names of the genotypes were renamed as N-efficient” and “N-inefficient” throughout the manuscript.

Q5: The color scheme in the figures is also a bit confusing; for example, the NT genotype is shown in yellow in Figure 1 but appears as pink in Figure 3. It would be clearer to assign one color per genotype and keep it consistent across all figures.

R: In Figure 1 panel A) what is shown and compared is the effect of the two treatments on the root and shoot traits assessed for both genotypes as an average effect (pink = N+; purple = N-). In panel B) it is shown and compared the effect of the two treatments for each genotype individually. Different colors for treatments and genotypes.

Q6: In general, the study presented by Soto-Cerda et al. is interesting and provides valuable observations, but it would really benefit from including the missing gene expression data and

R: Expression level was included for the 1,034 DEGs as Supplementary Table 3 and those DEGs highlighted in Results and Discussion sections were graphically shown as Supplementary Figure 1.

Q7: making a few key ajustments to help strengthen the main message, support the findings, and make the manuscript easier to follow.

R: Adjustments and improvements were included throughout the manuscript as also requested by other reviewers.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript investigates the phenotypic and transcriptomic responses of two linseed accessions (contrasting in nitrogen use efficiency) under N stress conditions. The study assesses 12 seedling-stage traits and uses RNA-seq analysis to identify 1,034 DEGs, including several TF families and nutrient transporters. The results reveal potential candidate genes and regulatory networks associated with improved NUE and RSA) laying the groundwork for future molecular breeding in linseed. The topic is timely and relevant to global concerns about fertilizer efficiency and environmental sustainability. While the study is promising and contributes significant new knowledge, it requires improvements in language, clarity of methodology, and some improved as mentioned below before acceptance

Line 24-28, please provide data on percentage increase/increase.

It should be number of tips

Please provide full name at the first mentioned place; ERF, MYB, NAC, and WRKY

19-21, its very general information, please make the first sentence more persuasive

RNA-seq, Candidate genes don’t fit to keywords, please change keywords

First sentence of introduction is quite strange, how Nitrogen fertilizers don’t have any direct effect on population feeding, rather it has indirect effect. Please be specific in the opening sentence

the consumption of N was nearly 46.3 million metric tons……. please change to global nitrogen fertilizer consumption was nearly…” to make clear this refers to fertilizer, not elemental nitrogen

when listing nitrate transporters, please note that NRT1 and NRT2 families are the major nitrate transporters, while CLC and SLAC/SLAH are involved in vacuolar nitrate storage and nitrate efflux.

Some sentences are overly long and packed with information. For example, the sentence from lines 84–85

The objectives could be more concise and formally stated

Discussion; Add clarification of gene function before mentioning their orthologous relationships

we phenotyped-----change to.. we evaluated

up regulated----upregulated

333- y transport) .....what does Y mean here

as previously reported in other crops…. Please crop examples

Author Response

Response letter to Reviewer 3

Comments and Suggestions for Authors

Q1: Line 24-28, please provide data on percentage increase/increase.

R: Data provided as percentage in lines 32-33 of the “track changes” version of the manuscript

Q2: It should be number of tips

R: It was modified as number of tips throughout the manuscript

Q3: Please provide full name at the first mentioned place; ERF, MYB, NAC, and WRKY

R: Full name provided at first time mentioned in Intro, Results and Discussion sections

Q4: 19-21, its very general information, please make the first sentence more persuasive.

R: First sentence was improved in lines 19-22 in the Abstract section of the “track changes” version of the manuscript.

Q5: RNA-seq, Candidate genes don’t fit to keywords, please change keywords.

R: RNA-seq was changed for Transcriptomics and candidate genes for Differentially expressed genes.

Q6: First sentence of introduction is quite strange, how Nitrogen fertilizers don’t have any direct effect on population feeding, rather it has indirect effect. Please be specific in the opening sentence

R: First sentence of introduction was rephrased as follow “Nitrogen (N) fertilizers have played an essential role in feeding a growing global population through an indirect positive effect on yield of main staple crops [1]. It is estimated that around 50% of the people alive today are dependent on synthetic N fertilizers for crop production (https://ourworldindata.org/fertilizers)”. Lines 49-52 of the “track changes” version of the manuscript.

Q7: the consumption of N was nearly 46.3 million metric tons……. please change to global nitrogen fertilizer consumption was nearly…” to make clear this refers to fertilizer, not elemental nitrogen.

R: Phrase was rewritten as follow “For example, in 1965, the global nitrogen fertilizer consumption was nearly 46.3 million metric tons and by 2021, this amount increased to 195.4 million metric tons, with N fertilizers accounting for 56% of the total fertilizer uses worldwide [2]”. Lines 52-55 of the “track changes” version of the manuscript.

Q8: when listing nitrate transporters, please note that NRT1 and NRT2 families are the major nitrate transporters, while CLC and SLAC/SLAH are involved in vacuolar nitrate storage and nitrate efflux.

R: CLC and SLAC/SLAH participation in vacuolar nitrate storage and nitrate efflux was clarified in lines 73-75 of the “track changes” version of the manuscript.

Q9: some sentences are overly long and packed with information. For example, the sentence from lines 84–85.

R: In general, sentences like that indicated were rephrased as also suggested by the other reviewers.

Q10: The objectives could be more concise and formally stated

R: The objectives were rewritten as follow “The objectives of the present study were to characterize the root and shoot morphological changes induced by N stress at the seedling stage in two linseed genotypes contrasting with NUE, and to identify differentially expressed genes associated with root responses under starved and normal N conditions using transcriptome sequencing and data analysis. Our results potentially will provide genetic and molecular resources to improve NUE in linseed”. Another reviewer asked us to include potential contributions of our research. Sometimes it is difficult to align the requests from different reviwers because they go in opposite directions.

Q11: Discussion; Add clarification of gene function before mentioning their orthologous relationships.

R: In the Discussion section all comparisons with ortholog genes were first clarified their function and then the potential role of the linseed version of the gene.

Q12: we phenotyped-----change to.. we evaluated.

R: Phenotyped was changed to evaluated in line 323 of the “track changes” version of the manuscript.

Q13: up regulated----upregulated.

R: Modified as suggested and down regulated to downregulated as well.

Q14: 333- y transport) .....what does Y mean here

R: Y removed.

Q15: as previously reported in other crops…. Please crop examples.

R: Sentence was rephrased as follow “Therefore, these three amino acids transporters found differentially expressed in our study could influence the allocation of the amino acids glutamine and glutamic acid to root and shoot meristems as precursors for the synthesis of other amino acids under N starvation in linseed as previously reported in plant species like Arabidopsis and V. vivifera [71, 73]”. Lines 519-523 of the “track changes” version of the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

Please see the attachment

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Some sections of the manuscript are verbose or repetitive, and there is a lack of logical connectors. It is recommended to appropriately combine similar paragraphs, streamline the writing, and increase the use of logical connectors (such as “such as”, “for example”, “this suggests that”, “in summary”, etc.) to make the text more coherent.

Author Response

Letter of response to reviewer 4

This manuscript offers valuable insights into the phenotypic and molecular responses of two contrasting linseed genotypes under nitrogen stress, employing both morpho-physiological and transcriptomic approaches. The study is timely and relevant, given the critical role of nitrogen use efficiency (NUE) in sustainable agriculture and flax breeding. However, the manuscript requires substantial revision to address significant concerns related to experimental rigor, data presentation, logical organization, and scientific depth. Major revisions are necessary to remedy these fundamental shortcomings. Without comprehensive improvements in experimental design, data analysis, presentation, and writing, the manuscript is not suitable for publication. Detailed comments and suggestions for improvement are provided below.

Materials and Methods

Representativeness of Materials: Only two linseed genotypes (one tolerant, one sensitive) were selected, and while it is stated that these were chosen from a pool of 150 accessions, the criteria and specific screening indices (e.g., nitrogen content, root traits) are missing. Please provide clear selection standards and background data to justify the choice and enhance the representativeness of your results.

R: The experimental conditions used to select the contrasting genotypes were the same as those described in the current manuscript. These experimental conditions were tested several times with groups of 20-25 genotypes before conducting the study using the 150 flax accessions, data that was later used to conduct a multi-locus GWAS (Int J Mol Sci. 2023 Dec 18;24(24):17624. doi: 10.3390/ijms242417624.

Nutrient Solution and Calcium Confounding: The use of Ca(NO₃)₂ for N+ and CaSO₄ for N- may cause confounding effects of Ca²⁺; the equivalency of Ca²⁺ concentration is not validated. Please clarify or provide supporting data for Ca²⁺ normalization, and discuss potential impacts on the results.

R: The purpose of using CaSO₄ for N- treatment is to avoid confounding effects for the lack of calcium, which is crucial for maintaining Ca²⁺ homeostasis and the membrane permeability for a normal trafficking of nutrients. Without adjusting calcium concentration is likely to generate stress responses not attributable to N deficiency. As indicated in response to previous comments this solution was tested at least 5 times with reproducible results.

Root Scanning and Standardization: The description of root scanning lacks detail, especially regarding sand removal and sample handling. Please specify your root cleaning, debris removal, and imaging standardization steps to ensure the accuracy and reproducibility of root data.

R: Details regarding root scanning and sand removal were extended as requested in lines 556- 565 of the “track changes” version of the manuscript.

4 Nutrient Concentration Rationale: The calculation of NO₃⁻ concentration (2.5 mM) is unclear. Please describe how this value was derived and whether it was confirmed as optimal through pre-experiments or literature.

R: The NO₃⁻ concentration of 2.5 mM represents thereal application of 100 Kg of N per hectare adjusted according to the volume of the pots used in this research. This amount of N is the recommendation for commercial production of linseed regardless of the source of N. We used this concentration because in parallele the 150 accessions were planted in the field in three locations in soils with low N (55 kg per hectare) and supplemented with 100 Kg of N. Thus, in future articles we could compare the performance of seedling versus adult plants and look for common/specific QTL and DEGs. As mentioned above this concentration was tested at least 5 times with small numbers of genotypes (20-25).

RNA Sample Design: The total of 16 RNA samples is described, but the allocation across genotypes and treatments is not explicitly detailed (e.g., 4 replicates per group). Please clarify the experimental design for transcriptomics to strengthen methodological transparency.

R: Experimental design for transcriptomics was clarified in lines 579-584 of the “track changes” version of the manuscript.

Introduction:

1 The background and significance are well described, but some sections are somewhat redundant. The introduction discusses the importance of nitrogen fertilizer in global agriculture and its environmental consequences in great detail; however, the background information before the flax-specific content is a bit too lengthy, which may distract readers. It is recommended to condense the section on global nitrogen fertilizer use, and shift the focus earlier to “flax NUE issues” and their practical significance, thus making the subject of the article more prominent.

R: Introduction section was reorganized as suggested incorporating other recommendations made by other reviewers.

2 The novelty and objectives of the research need further emphasis. Currently, the stated goals remain at the level of “comparing two genotypes” and “identifying differentially expressed genes,” but lack discussion on the anticipated scientific contributions and technological innovations. It is suggested to clearly highlight the innovation and potential application value of this study at the end of the introduction (e.g., providing genetic resources for molecular breeding to improve flax NUE, or revealing flax-specific molecular mechanisms for nitrogen adaptation).

R: The novelty and potential contribution of this research were emphasized at the end of the Introduction. Lines: 149-154 of the “track changes” version of the manuscript.

3 The definitions of key terms are not sufficiently clear. For example, terms such as “differentially expressed genes (DEGs)” are not defined upon first mention, and some technical terms should be presented with their English abbreviations. Please provide definitions for technical terms at first appearance, and maintain consistency between Chinese and English terminology to enhance professionalism.

R: Terminology and abbreviations were rewritten as suggested throughout the manuscript.

4 The introduction contains multiple sections of data-heavy description and the disciplinary logic is somewhat scattered, especially in the transitions from nitrogen utilization → molecular mechanisms → flax omics → research objectives, which appear abrupt. It is recommended to add more transitional language to emphasize the logical flow: global nitrogen fertilizer pressure → scientific challenges of NUE → economic value of flax and nitrogen bottleneck → existing research → significance and innovation of this study.

R: Introduction section was reorganized as suggested following the structure: global nitrogen fertilizer pressure → scientific challenges of NUE → economic value of flax and nitrogen bottleneck → existing research → significance and innovation of this study.

Discussion:

1 The discussion is overly descriptive and lacks structure and a clear main theme. The section is largely a compilation of observations, examples, and literature references, without a clear progression of ideas or elevation of scientific hypotheses. As a result, it is difficult for readers to grasp the core scientific contribution of this study. It is recommended to organize the discussion logically, for example, following the sequence: “main phenotypic findings — mechanistic explanations — comparison with previous studies — theoretical and practical significance — limitations and future directions.”

R: Discussion section was reorganized as suggested.

2 The novelty and scientific contribution are not sufficiently emphasized. Although the analysis covers multiple pathways and transcription factors, the discussion does not clearly highlight “what new knowledge has been discovered, extended, or validated for the first time” in this work, nor does it differentiate between previous studies and the present results. It is suggested to clearly specify how this study fills knowledge gaps in flax nitrogen response research, as well as its potential contributions to molecular breeding and theoretical research.

R: Previous transcriptomics studies on N stress in flax have not been carried out. However, stresses like drought. salinity, alkaline, there exist expression information. Thus, we try to use this information to compare the relevance of certain TF families in response to abiotic stresses and their contribution to potential genomic assisted breeding in flax.

3 The functions of transcription factor families are discussed in a general way, lacking specificity to flax or the current experiment. The discussion of families such as MYB, WRKY, NAC, and ERF is largely based on findings from model plants, without focusing on the unique characteristics or findings in flax (e.g., newly identified transcription factors or functional variants). It is recommended to select one or two transcription factors that show significant or novel patterns in flax, especially those

with limited prior reports, and analyze them in depth, or propose hypotheses based on your data (such as expression patterns or regulation of downstream genes).

R: Discussion regarding TFs was extended according to the literature available in flax to date.

4 The boundary between the results and discussion sections is somewhat blurred, with some paragraphs continuing to list data and numerical values. The discussion should focus primarily on the underlying mechanisms. Please minimize the repetition of specific result data and emphasize the elevation and interpretation of the scientific significance.

R: Excessive results were removed from the Discussion and were kept the necessary information to provide context to paragraphs as needed.

Language:

R: Some connectors were included to smooth the transition between paragraphs and sentences.

Additional issues:

1 Spelling errors: Words such as "Sentisitve" and "Diferentially" should be consistently corrected. For example, in the Abbreviation table, "Sentisitve" under N stress should be corrected to "Sensitive".

R: Spelling issues were corrected as suggested.

Figure 2 lacks a scale bar; it is recommended to add a 1 cm

R: Figure 2 has a 1 cm scale bar at the bottom-right corner. Now is emphasized in the legend.

The Materials section does not mention the year and season of the experiment, which affects the reproducibility and comparability of the results.

R: Year of the experiment now is mentioned in Material section, Lines 531-537 of the “track changes” version of the manuscript.

Figure 1 lacks significance indicators (e.g., a, b, c); please add statistical significance markers.

R: Significance indicators as asterisk were added to Figure 1A and 1B as suggested.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The effort by the authors (Soto-Cerda et al., in their manuscript titled "Morphological and transcriptomic analyses provide new insights into linseed (Linum usitatissimum L.) seedling roots response to nitrogen stress), to include DEG values and to plot the expression of the specific genes highlighted in the results and discussion sections (as Supplemental Table 3 and Supplemental Figure 1) is apreciated.

In this regard, I would recomend indicating directly in the graph (e.g., using asterisks to denote significance) under which conditions significant expression differences occur; whether the differences were observed between genotypes under only one condition (N0 or N1), or under both.

Accordingly, it would be usefull to classify the genes into functional categories. For example, those upregulated in the nitrogen efficient (NE) genotype and potentially associated with improved nitrogen use efficiency (e.g., ERF71, ZAT9, EZF2, NPF3.1, NPF1.1, GGCT2.1, and BAT1); those upregulated in the nitrogen inefficient (NI) genotype and possibly associated with reduced NUE; and those similarly regulated under N0 but potentially relevant for improving NUE under N1 conditions.

Based on such classification, it would also be valuable to include a schematic diagram (as an additional supplemental figure or integrated with the existing graphs), possibly using arrows and boxes to indicate the genes up or downregulation patterns and their potential positive or negative regulatory effects on key traits such as root development, nutrient uptake, nitrogen acquisition, nitrogen transport, or amino acid transport in the NE genotype. Such a classification and diagram would offer a clear visual summary and serve as a usefull reference for future studies focused on understanding gene regulation for improved NUE in linseed and other plants.

One additional suggestion: please ensure consistency in terminology throughout the manuscript, particularly regarding the use of “number of tips” versus “root tips,” and “normal nitrogen” versus “sufficient nitrogen” conditions.

Best regards

Author Response

Reviewer 2

Comments and Suggestions for Authors

Comment: In this regard, I would recomend indicating directly in the graph (e.g., using asterisks to denote significance) under which conditions significant expression differences occur; whether the differences were observed between genotypes under only one condition (N0 or N1), or under both.

Response: Significant differences in gene expression between genotypes were indicated by asterisk according to False discovery rate (FDR)-adjusted P value < 0.05.

Comment: Accordingly, it would be usefull to classify the genes into functional categories. For example, those upregulated in the nitrogen efficient (NE) genotype and potentially associated with improved nitrogen use efficiency (e.g., ERF71, ZAT9, EZF2, NPF3.1, NPF1.1, GGCT2.1, and BAT1); those upregulated in the nitrogen inefficient (NI) genotype and possibly associated with reduced NUE; and those similarly regulated under N0 but potentially relevant for improving NUE under N1 conditions. Based on such classification, it would also be valuable to include a schematic diagram (as an additional supplemental figure or integrated with the existing graphs), possibly using arrows and boxes to indicate the genes up or downregulation patterns and their potential positive or negative regulatory effects on key traits such as root development, nutrient uptake, nitrogen acquisition, nitrogen transport, or amino acid transport in the NE genotype. Such a classification and diagram would offer a clear visual summary and serve as a usefull reference for future studies focused on understanding gene regulation for improved NUE in linseed and other plants.

Response: DEGs were classified into categories, and they were grouped in a schematic diagram named Supplementary Figure 2.

Comment: One additional suggestion: please ensure consistency in terminology throughout the manuscript, particularly regarding the use of “number of tips” versus “root tips,” and “normal nitrogen” versus “sufficient nitrogen” conditions.

Response: Number of tips was replaced with “root tips” and sufficient N was replaced with normal nitrogen throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors significantly improved MS and I recommend it to accept now.

Author Response

Reviewer 3

Comments and Suggestions for Authors

Authors significantly improved MS and I recommend it to accept now.

Dear Reviewer,

We greatly appreciate the time and effort you dedicated to reviewing our manuscript and for sharing your critical perspective. Your comments have been carefully considered, and they have motivated us to thoroughly revise and substantially strengthen the paper.

We sincerely thank you again for your valuable input, which has undoubtedly helped us to refine and strengthen this work.

Reviewer 4 Report

Comments and Suggestions for Authors

I feel that the article lacks innovation. Even after major revisions, the quality of the paper has not significantly improved. I cannot accept it for publication. The content is unfocused and overall rather scattered. My recommendation is rejection.

Author Response

Reviewer 4

Comments and Suggestions for Authors

Comment: I feel that the article lacks innovation. Even after major revisions, the quality of the paper has not significantly improved. I cannot accept it for publication. The content is unfocused and overall rather scattered. My recommendation is rejection.

Response: We sincerely thank you for your careful reading of our manuscript and for sharing your concerns regarding innovation, focus, and the overall contribution of our study. We deeply value your feedback, as it has guided us to substantially strengthen the manuscript.

We respectfully acknowledge your perception that the manuscript lacked sufficient innovation in its earlier form. To address this, we have carefully restructured the paper to highlight more explicitly the novel aspects of our work:

Novelty and Contribution Clarified

The revised manuscript now emphasizes that this is the first integrative study combining morphological root traits and transcriptomic responses in flax seedlings under nitrogen stress.
Highlighted how our findings provide new insights into the mechanisms of nutrient uptake and stress adaptation in flax — a crop with increasing importance for both food and industrial uses.
Positioned our study as complementary to existing knowledge in cereals and model plants, but unique in its contribution to Linum usitatissimum, where such integrative analyses have not previously been reported.

Improved Focus and Structure

Results and Discussion were reorganized into a clear, hypothesis-driven framework, directly addressing how flax root morphology and gene expression interact under contrasting nitrogen conditions to sharpen focus on flax-specific insights.

Strengthened Presentation of Findings

Figures and tables were redesigned to visually highlight the most novel results, including key differentially expressed genes (DEGs) related to nitrogen transport, amino acids transport and root development.
Emphasized the integration of phenotypic and transcriptomic data, demonstrating how molecular changes align with observed morphological adjustments.

Broader Relevance Emphasized

Expanded the Conclusion to stress the agronomic importance of our findings, showing how they can inform breeding programs aiming to improve nitrogen-use efficiency in flax.
Connected the molecular insights (e.g., nitrogen transporter and transcription factor gene regulation) with their potential applications in developing more resilient cultivars under low-input agricultural systems.
Positioned our study within the global context of sustainable agriculture, where improving nitrogen-use efficiency is a key challenge.

We kindly ask you to reconsider your assessment of our work in light of these significant improvements requested by all reviwers. We remain confident that the revised manuscript makes a meaningful contribution and will be of interest to the readership of Plants.

Thank you again for your valuable feedback and for considering our response.