lncRNA-mRNA-miRNA Networks in Arabidopsis thaliana Exposed to Micro-Nanoplastics

Round 1

Reviewer 1 Report

I reviewed the research article “ Interplay of lncRNA Genes with Protein-Coding Genes and miRNAs in Arabidopsis thaliana Exposed to Micro-Nanoplastics.” The article provides a thorough investigation of lncRNAs in Arabidopsis thaliana under nanoplastic stress, combining RNA-seq data, bioinformatics, and functional analyses. The author identified 281 novel lncRNAs whose differential expression under Tr-PET and BI-PET treatments adds value to the field. The Author also used multiple tools and statistical methods to strengthen the analysis. I suggest the paper is well-organized, with a logical flow from introduction to conclusion.

Comments:

1. The title is accurate but could be more concise.
2. Clearly explained your objectives at the end of the introduction.
3. In the introduction, the background on micro-nanoplastics is lengthy but lacks clarity on why PET (transparent vs. blue) was chosen.
4. L184, need space
5. Figure quality is not good
6. I suggest that the figure quality is not good, and it is also given in the supplementary material as an original full figure.

I reviewed the research article “ Interplay of lncRNA Genes with Protein-Coding Genes and miRNAs in Arabidopsis thaliana Exposed to Micro-Nanoplastics.” The article provides a thorough investigation of lncRNAs in Arabidopsis thaliana under nanoplastic stress, combining RNA-seq data, bioinformatics, and functional analyses. The author identified 281 novel lncRNAs whose differential expression under Tr-PET and BI-PET treatments adds value to the field. The Author also used multiple tools and statistical methods to strengthen the analysis. I suggest the paper is well-organized, with a logical flow from introduction to conclusion.

Comments:

1. The title is accurate but could be more concise.
2. Clearly explained your objectives at the end of the introduction.
3. In the introduction, the background on micro-nanoplastics is lengthy but lacks clarity on why PET (transparent vs. blue) was chosen.
4. L184, need space
5. Figure quality is not good
6. I suggest that the figure quality is not good, and it is also given in the supplementary material as an original full figure.

Author Response

We want to thank you so much for taking the time to review our manuscript. Please find the point-by-point answers below:

Comment 1. The title is accurate but could be more concise.

Response 1: Thank you for the comment. We have shortened the title accordingly.

Comment 2: Clearly explained your objectives at the end of the introduction.

Response 2: Thank you for your comment. We expanded the objectives of our study as required.

Comment 3: In the introduction, the background on micro-nanoplastics is lengthy but lacks clarity on why PET (transparent vs. blue) was chosen.

Response 3: Thank you for your guidance. We improved this information in the Introduction section, third paragraph, as required by the reviewer.

Comment 4: L184, need space

Response 4: Thank you for the comment. We corrected the missing spaces.

Comment 5: Figure quality is not good

Response 5: Thank you for your guidance. We have uploaded all figures in TIF format with 300 dpi resolution. However, in the peer-review process of IJPB, the MDPI provides the reviewers only figures with low resolution (non-original). For the end publication of this study, we think that the original figures (TIF 300 dpi) will be used by MDPI.

Comment 6: I suggest that the figure quality is not good, and it is also given in the supplementary material as an original full figure.

Response 6: Thank you for your guidance. Similar to question 5, we upload all figures in TIF format with 300 dpi resolution. However, in the peer-review process of IJPB, the MDPI provides the reviewers only figures with low resolution (non-original). For the end publication of this study, we think that the original figures (TIF 300 dpi) will be used by MDPI.

Reviewer 2 Report

The present manuscript by Galbo et al. is a study devoted to the identification of novel non-annotated lncRNAs in A. thaliana plants treated with micronanoplastics, namely, transparent (Tr-PET) and blue (Bl-PET) polyethylene terephthalate. The study is quite extensive and seems relevant as it addresses the impact of abiotic stress associated with negative environmental impacts.

At the same time, the manuscript needs some revision.

My main concern is the follows:

L. 26: “Our findings showed that Tr-PET changed the expression of 104 lncRNAs, while Bl-PET changed the expression of just 19”. In L. 582: “Overall, the Bl-PET versus unstressed control results suggest that Bl-PET induces less pronounced transcriptional changes compared to the Tr-PET treatment, with limited down-regulation of the target protein-coding genes.” This is an unexpected result of the work, since the blue plastic seems to be the same plastic as the transparent one, but with the addition of a dye. Firstly, what caused such a choice of the researchers, to separate these two types of plastic, secondly, why did they choose blue one and, thirdly, do they have any suggestions as to what such results of the study may be associated with?

L. 29: “Interestingly, lncRNAs usually regulate their neighboring protein-coding genes, and their promoters contain cis-acting regulatory elements responsive to ABA, light, MeJA, MYC/MYB, and other stress-related signals.” According to the Section 3.6, these are the data of the present Manuscript, but such statement in the Abstract gives an impression that these are some literature data.

L. 31 “Further, some of the miRNAs that participate in plant development and defense were also predicted to be sponged by the differentially expressed lncRNAs. Please, explain, which data show that. Besides, this statement in the Abstract also gives an impression that these are some literature data.

L. 73: “FLV gene”. Please, decipher the abbreviation.

Figure 2. Please, add to the legend the information, what yellow, blue, red sequences mean.

Table 1. “Differentially expressed genes: Kornienko plus novel identified lncRNA.” It looks weird if “Kornienko” is used as it should be the name of the gene group. Please, explain, why “Kornienko plus novel identified lncRNA” and “Novel lncRNA” are divided into separate groups.

L. 322: “The predicted interactions between lncRNAs regulated by Tr-PET and Bl-PET and mRNAs were determined similarly to protein-coding genes.” If these interactions were “predicted”. There should be some references, unless it is unclear, how “predicted interactions” “were determined”.

Overall, English is rather fine, but in L. 459: “In addition, were identified 14 protein-coding genes targeted by the lncRNAs differentially modulated...” the subject comes before the predicate.

There are no references to your own Figures and Tables in the entire Discussion section. Please add them, as readers should not have to guess what data is being discussed.

L. 612-615. This information should be excluded from the text.

The section Conclusion looks like it is part of the Introduction. The Conclusion should summarize the most important and valuable results of this study. This section should be completely rewritten.

Author Response

We would like to sincerely thank the reviewer for taking the time to review our manuscript. Kindly,you can find below the point-by-point responses related to each comment:

Comment 1: L. 26: “Our findings showed that Tr-PET changed the expression of 104 lncRNAs, while Bl-PET changed the expression of just 19”. In L. 582: “Overall, the Bl-PET versus unstressed control results suggest that Bl-PET induces less pronounced transcriptional changes compared to the Tr-PET treatment, with limited down-regulation of the target protein-coding genes.” This is an unexpected result of the work, since the blue plastic seems to be the same plastic as the transparent one, but with the addition of a dye. Firstly, what caused such a choice of the researchers, to separate these two types of plastic, secondly, why did they choose blue one and, thirdly, do they have any suggestions as to what such results of the study may be associated with? 
Response 1: 

The choice of performing an in silico analysis and de novo annotation of lncRNA regulated by these two types of micro-nanoplastics (including Bl-PET) was due to previous findings published in Cucurbita pepo showing significant differences in plant responses against both Tr-PET and Bl-PET, such as on plant growth and photosynthesis [72] (Colzi et al., 2022). In addition, these two micro-nanoplastic types showed severe effects on gene regulation in Arabidopsis thaliana, as previously shown by [54] (Dainelli et al., 2024). Both types of plastic material induced a root length reduction, while only Bl-PET reduced the rosette area. While Tr-PET-induced genes encoding proteins involved in xenobiotics signaling, Bl-PET has only a few effects at the transcriptomic profile, upregulating only a few genes involved in abiotic stresses. In addition, only Tr-PET has profound effects on genes involved in hormone biosynthesis and signaling such as brassinosteroid and abscisic acid (ABA).  Furthermore, Tr-PET and Bl-PET have received the most attention among xenobiotic compounds because they represent the majority of micro-nanoplastics found discarded and decomposing in nature [72]. Indeed, based on the significant differences in phenotypic, physiological, and gene expression levels previously shown in literature, in our opinion, it was important to show how these two treatments might have important effects on gene modulation at the post-transcriptional level through the action of new key identified lncRNAs.  A paragraph was added in the Introduction in this regard, in the third paragraph of the Introduction section. 

Comment 2: L. 29: “Interestingly, lncRNAs usually regulate their neighboring protein-coding genes, and their promoters contain cis-acting regulatory elements responsive to ABA, light, MeJA, MYC/MYB, and other stress-related signals.” According to Section 3.6, these are the data of the present Manuscript, but such a statement in the Abstract gives an impression that these are some literature data.

Response 2: Thank you for your comment. We changed the abstract section to be more clear about the mentioned results.

Comment 3: L. 31 “Further, some of the miRNAs that participate in plant development and defense were also predicted to be sponged by the differentially expressed lncRNAs. Please, explain, which data show that. Besides, this statement in the Abstract also gives an impression that these are some literature data.

Response 3: Thank you for your comment. We revised the abstract section to clarify the corresponding results. The data supporting our findings is provided in Additional File S12.

Comment 4: L. 73: “FLV gene”. Please, decipher the abbreviation.

Response 4: Thank you for your comment. We added the whole meaning of the abbreviation.

Comment 5: Figure 2. Please, add to the legend the information, what yellow, blue, red sequences mean.

Response 5: Thank you for your comment. We added a description of the legend colors for gene models to the Figure 2 caption.

Comment 6: Table 1. “Differentially expressed genes: Kornienko plus novel identified lncRNA.” It looks weird if “Kornienko” is used as it should be the name of the gene group. Please, explain, why “Kornienko plus novel identified lncRNA” and “Novel lncRNA” are divided into separate groups.

Response 6: Thank you for your comment. We clarified the “Kornienko” and “novel lncRNA” terms in the Table’s caption.

Comment 7: L. 322: “The predicted interactions between lncRNAs regulated by Tr-PET and Bl-PET and mRNAs were determined similarly to protein-coding genes.” If these interactions were “predicted”. There should be some references, unless it is unclear, how “predicted interactions” “were determined”.

Response 7: Thank you for your comment. We clarified the methods used to make the prediction in both  3.2.1 and 3.3 sections.

Comment 8: Overall, English is rather fine, but in L. 459: “In addition, were identified 14 protein-coding genes targeted by the lncRNAs differentially modulated...” the subject comes before the predicate.

Response 8: Thank you for your comment. We adjusted the sentence accordingly.

Comment 9: There are no references to your own Figures and Tables in the entire Discussion section. Please add them, as readers should not have to guess what data is being discussed.

Response 9: Thank you for your comment. We have added citations for all Tables and Figures used in the Results sections. In the Discussion section, we only discussed the previous results presented in the Results section. The standard is to present and cite Figures and Tables only in the Results section, while in the Discussion section, no citation but discussion.

Comment 10: L. 612-615. This information should be excluded from the text.

Response 10: Thank you for your comment. It was a typographical error left over from the manuscript preparation and has been removed from the manuscript.

Comment 11: The section Conclusion looks like it is part of the Introduction. The Conclusion should summarize the most important and valuable results of this study. This section should be completely rewritten.

Response 11: We appreciate the reviewer's comment. We agree, and we have eliminated redundant parts of the conclusions and relocated some sections to the Introduction to focus the Conclusion section solely on the main results of this study.

Comment 12: Does the title describe the article's topic with sufficient precision? No “Interplay of lncRNA Genes...”. At that in L. 46: “In plants, lncRNAs are typically transcribed by RNA polymerases I to V from intronic regions of transcriptional units of coding-protein mRNAs (intronic sense lncRNAs), negative-strand mRNAs (anti-sense lncRNAs), and intergenic regions (long intergenic ncRNAs)”. Thus, lncRNAs are not coding by the specific genes.

Response 12: Thank you for pointing out that, in the previous form, the sentence could be misleading. We modified the period to clarify the point.

Comment 13: Does the introduction provide a comprehensive yet concise overview about the state of knowledge in the area of research? No. The Introduction section some more specific information on how exactly lncRNA function for the activation or repression of responsive genes and how miRNAs interact with lncRNA.

Response 13: We thank the reviewer for the guidance. Some sentences were added in the Introduction in this regard, such as in the third paragraph of the Introduction section.

Comment 14: Is the research design appropriate and are the methods adequately described? L. 121: “After acclimation, ten-day-old plants…” If you used only ten-day-old plants, how did you check, if acclimation took place? That is very important question when studying any stress effects. Usually, some markers demonstrating the process of acclimation are used. L. 131: “(n = 3 treatments x 8 plants each)”. At hat, in Fig. 1 “n = 1”.

Response 14: We thank the reviewer for the comment. Although we have not performed any analysis of stress biochemical markers, we have looked at the plant phenotype during the 10 days of acclimatization in hydroponics conditions and we have not seen any stress symptoms. Plants look completely similar and for these reasons, they were randomly selected in the two three groups (control without micro-nanoplastics, in the presence of Tr-PET, and in the presence of Bl-PET). We followed the same procedure used in studies published by Colzi et al. 2022 (https://doi.org/10.1016/j.jhazmat.2021.127238) and Dainelli et al. 2025 (https://doi.org/10.1016/j.plaphy.2024.109409).

 

 

Reviewer 3 Report

This study systematically investigates, for the first time, the regulatory effects of micro-nanoplastics (Tr-PET and Bl-PET) on long non-coding RNAs (lncRNAs) in Arabidopsis thaliana, filling a research gap on the impact of environmental pollutants on plant non-coding RNAs and providing critical data for understanding molecular mechanisms underlying plant responses to emerging contaminants. However, the following revisions are required。

1. Keywords: The manuscript contains an excessive number of keywords; limit them to 5 or fewer.
2. Introduction: Emphasize the uniqueness of this study by comparing how physicochemical properties of differently colored PET (e.g., surface charge, adsorption capacity) may drive lncRNA expression differences, thereby deepening mechanistic insights.
3. Only 3 biological replicates per treatment were used, which may inadequately account for biological variability. Justify the sample size selection or increase replicates to enhance statistical power.
4. Include data on PET particle physical properties (e.g., particle size, dispersion) to rule out confounding effects from aggregation or physical factors.
5. Clarify whether multiple testing correction was applied to high-interaction lncRNAs to address potential false positives.
6. Explore mechanisms underlying lncRNA expression divergence (e.g., light absorption properties linked to color, chemical additive differences) beyond descriptive observations.
7. Biological Significance in Discussion: Strengthen explanations of key findings (e.g., expression changes in specific genes) by linking them to existing literature to highlight novelty and importance.
8. Figure 4 (Venn Diagram): Label exact numbers of overlapping lncRNAs in each region instead of only percentages.
9. Reference Formatting: Ensure consistent DOI formatting across references per journal guidelines.

Author Response

We want to thank you so much for taking the time to review our manuscript. Please find the point-by-point answers below:

Comment 1: Keywords: The manuscript contains an excessive number of keywords; limit them to 5 or fewer.

Response 1: Thank you for your comment. We have reduced the number of keywords as suggested.

Comment 2: Introduction: Emphasize the uniqueness of this study by comparing how physicochemical properties of differently colored PET (e.g., surface charge, adsorption capacity) may drive lncRNA expression differences, thereby deepening mechanistic insights.

Response 2: We appreciate the reviewer's guidance. Some sentences were added in the Introduction in this regard, such as in the third paragraph of the Introduction section.

Comment 3: Only 3 biological replicates per treatment were used, which may inadequately account for biological variability. Justify the sample size selection or increase replicates to enhance statistical power.

Response 3: Thank you for your comment. In this study, we followed the same procedure used in studies published by Colzi et al. 2022 (https://doi.org/10.1016/j.jhazmat.2021.127238) and the same experimental design by Dainelli et al. 2025 (https://doi.org/10.1016/j.plaphy.2024.109409). Therefore, we consider that the number of plants used per treatment is sufficient, as they present quite contrasting phenotypes when comparing the three treatments (Figure 1), and the RNA-Seq approach is very sensitive and accurate (https://doi.org/10.1186/s13059-016-0881-8).

Comment 4: Include data on PET particle physical properties (e.g., particle size, dispersion) to rule out confounding effects from aggregation or physical factors.

Response 4: Thank you for your comment. In this study, we focused on the bioinformatics analysis of lncRNAs in A. thaliana, and we cite a previous study by our research group that describes in detail the production process and physicochemical characteristics of the Tr-PET and Bl-PET micro-nanoplastics. Subsection 2,1: “For that, micro-nanoplastics were produced from commercial blue and transparent bottles previously cleaned following the procedure described by Ekvall et al. [89] and Dainelli et al. [58].”. Additionally, in a previous article (https://doi.org/10.1016/j.jhazmat.2021.127238), our research group detailed the specific characteristics of micro- and nanoplastics and their effects on plants. This study is being widely used and cited in the current manuscript.

Comment 5: Clarify whether multiple testing correction was applied to high-interaction lncRNAs to address potential false positives.

Response 5: Thank you for the comment. We specified why we aren’t using multiple test corrections in the predictions of lncRNA interactions.

Comment 6: Explore mechanisms underlying lncRNA expression divergence (e.g., light absorption properties linked to color, chemical additive differences) beyond descriptive observations.

Response 6: Thank you for the comment. In subsection 3.5, we presented the functional role data of protein-coding genes targeted by the differentially expressed lncRNAs, showing that Tr-PET impacts different biological processes compared to plants exposed to Bl-PET and that both Tr-PET and Bl-PET modulate several lncRNAs that target remarkable protein-coding genes. Additionally, in Figure 5, we presented the main interaction networks that are impacted by these differentially expressed lncRNAs, which are discussed in the subsequent section.

Comment 7: Biological Significance in Discussion: Strengthen explanations of key findings (e.g., expression changes in specific genes) by linking them to existing literature to highlight novelty and importance.

Response 7: Thank you for the guidance. Our discussion focused on the identification and expression of lncRNAs and their targets in A. thaliana exposed to or in response to micro-nanoplastics. The literature still requires studies in plants to expand the knowledge in this area and relate lncRNA, target protein-coding genes, plant defense response, and plant phenotype in response to micro-nanoplastics. However, all studies related to our study were used in this section.

Comment 8: Figure 4 (Venn Diagram): Label exact numbers of overlapping lncRNAs in each region instead of only percentages.

Response 8: Thank you for your comments, but in Figure 4 both absolute numbers and percentages were already reported.

Comment 9: Reference Formatting: Ensure consistent DOI formatting across references per journal guidelines.

Response 9: Thank you for your comment. We have now formatted all the references in accordance with ACS style.

Round 2

Reviewer 2 Report

The Authors have made all necessary improvements to the manuscript.

 

I have only a comment concerning the answer about the references to their own data in the Discussion.  It is unlikely that a reader could have memorized all the data presented in the Results section, and it is good manners on the part of the Authors to remind, which results are discussed in each point of the Discussion. This makes it easier for readers to understand your data and increases the possibility of citation of the future paper.