Hydro-Electro Hybrid Priming Synchronizes Cell Wall Remodeling to Accelerate Carrot (Daucus carota L.) Seed Germination

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work "Hydro-Electro Hybrid Priming Synchronizes Cell Wall Remodeling to Accelerate Carrot (Daucus carota L.) Seed Germination" is very interesting and contains highly relevant information in the field of seed conditioning. The results obtained are accurate and consistent with the methodology applied.
Some minor points for the authors' attention:
1. Lines 136-137: Please indicate the model, brand, and country of the equipment used for HEHP.
2. Lines 206-226: Please provide a bibliographic citation for the PE and PAL enzyme activity determinations.
3. Lines 189, 283: Are you referring to the base 2 logarithm in the expression "|log2(fold change)| ≥ 1"? Then the number "2" should be subscripted in log2 (log₂).
4. Table 2. Please correct the overlapping rows. Table 2 requires a title indicating the meaning of the different colors used to mark the differential expression values, which contributes to the self-explanatory nature of the table.

More Comments:

The main aspect addressed in this research paper is that, beyond hydropriming, the use of a magnetic field after hydropriming, called "hydro-electro hybrid priming" in this study, promotes the synchronization of cell wall remodeling and accelerates the germination of carrot seeds.
I consider the topic to be original and relevant due to the combination of seed conditioning techniques without the use of chemicals, plant hormones, or growth regulators.

This work provides and expands relevant information in the area of ​​seed priming, which seeks to shorten and synchronize seed germination times.
In my view, the authors used appropriate methodologies to achieve the objective of the work, and I have no major concerns in this regard.
The conclusions obtained in this work are consistent with the evidence presented, and the argumentation appropriately addresses the objective of this research paper.

From my point of view, the bibliographic references are adequate. My recommendation in this regard is to include the references to the methodology for determining PE and PAL enzyme activities (lines 206-226).

And finally, the authors should point out that the overlapping information in the rows of Table 2 needs to be corrected.
Table 2 requires a caption indicating the meaning of the different colors used to mark the differential expression values, which contributes to the self-explanatory nature of the table.

Author Response

We sincerely appreciate the reviewer’s recognition of our work and the constructive suggestions. We have carefully addressed all comments as follows:

Comment 1: Lines 136-137: Please indicate the model, brand, and country of the equipment used for HEHP.

Response 1: Thank you for pointing this out. We have supplemented the equipment specifications in Section 2.1 (Page 4, Lines 151-152):

“For the HEHP treatment, following the 6 h soaking step (as in HYD), seeds were surface-dried and evenly spread on the cathode plate (10 cm × 10 cm) of a BM-201 electrostatic field generator (Boya Electric Co., Ltd., Wuxi, China).”

Comment 2: Lines 206-226: Please provide a bibliographic citation for the PE and PAL enzyme activity determinations.

Response 2: Thank you for pointing this out. We have added methodological references in Section 2.4:

For PE activity determination (Page 5, Lines 231):

“The determination of PE activity was based on the spectrophotometric method (based on methanol release)[31].”

31.  Grsic-Rausch, S.; Rausch, T. A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors. Analytical biochemistry 2004, 333, 14-18, doi:10.1016/j.ab.2004.04.042.

For PAL activity determination (Page 5, Lines 245):

“The PAL activity was determined by detecting the amount of trans cinnamic acid produced[32].”

32. Hsieh, C.Y.; Huang, Y.H.; Lin, Z.Y.; Hsieh, L.S. Insights into the substrate selectivity of Bambusa oldhamii phenylalanine ammonia-lyase 1 and 2 through mutational analysis. Phytochemistry Letters 2020, 38, 140-143, doi:10.1016/j.phytol.2020.06.002.

Comment 3: Lines 189, 283: Are you referring to the base 2 logarithm in the expression "|log2(fold change)| ≥ 1"? Then the number "2" should be subscripted in log2 (log2).

Response 3: Thanks a lot for your reminder. We have corrected all instances of "log2" to "log₂" throughout the manuscript (Lines 208 and 318).

Comment 4: Table 2. Please correct the overlapping rows. Table 2 requires a title indicating the meaning of the different colors used to mark the differential expression values, which contributes to the self-explanatory nature of the table.

Response 4: Thanks a lot for the constructive suggestions. We have:

1. Adjusted row spacing to prevent overlapping

2. Added table footnote: “Color scale: red indicates upregulation (log₂FC > 1), blue indicates downregulation (log₂FC < -1)”

3. Revised table title to: “Table 2. Differential expression of cell wall remodeling-related DEGs in carrot (Daucus carota L.) seeds under CK, HYD and HEHP treatments.”

Comment 5: From my point of view, the bibliographic references are adequate. My recommendation in this regard is to include the references to the methodology for determining PE and PAL enzyme activities (lines 206-226).

Response 5: We sincerely appreciate the your insightful suggestion. We have added methodological references:

For PE activity determination (Page 5, Lines 231), we added “The determination of PE activity was based on the spectrophotometric method (based on methanol release)[31].”

31.Grsic-Rausch, S.; Rausch, T. A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors. Analytical biochemistry 2004, 333, 14-18, doi:10.1016/j.ab.2004.04.042.

For PAL activity determination (Page 5, Lines 245): “The PAL activity was determined by detecting the amount of trans cinnamic acid produced[32].”

32.Hsieh, C.Y.; Huang, Y.H.; Lin, Z.Y.; Hsieh, L.S. Insights into the substrate selectivity of Bambusa oldhamii phenylalanine ammonia-lyase 1 and 2 through mutational analysis. Phytochemistry Letters 2020, 38, 140-143, doi:10.1016/j.phytol.2020.06.002.

Comment 6: And finally, the authors should point out that the overlapping information in the rows of Table 2 needs to be corrected.

Table 2 requires a caption indicating the meaning of the different colors used to mark the differential expression values, which contributes to the self-explanatory nature of the table.

Response 6: Thanks a lot for your reminder. We have:

Adjusted row spacing to prevent overlapping

Added table footnote: “Color scale: red indicates upregulation (log₂FC > 1), blue indicates downregulation (log₂FC < -1)”

Revised table title to: “Table 2. Differential expression of cell wall remodeling-related DEGs in carrot (Daucus carota L.) seeds under CK, HYD and HEHP treatments.” 

Reviewer 2 Report

Comments and Suggestions for Authors

This study explores the effects of Hydro-Electro Hybrid Priming (HEHP)—a seed treatment combining hydration with electrostatic field exposure—on accelerating carrot seed germination. Through transcriptomic analysis and enzyme activity assays, the authors demonstrate that HEHP enhances germination by synchronizing cell wall hydrolysis and synthesis, particularly upregulating expansin and hydrolase genes. Compared to traditional hydropriming (HYD), HEHP leads to earlier germination, lower endosperm rupture force, and activation of phenylpropanoid biosynthesis, which is linked to cell wall reinforcement. The study suggests HEHP as a viable method to support mechanized precision sowing in horticulture. Good manuscript

There is no data to seed germination. Please add at lest germination and mean germination time. To do, read the ttps://doi.org/10.1111/1440-1703.1275 as the example

Standardize units and labels across all figures.

Split large tables for readability or visualize trends using heatmaps.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Need a profession English correction

Author Response

We appreciate the reviewer’s recognition of our manuscript and the constructive suggestions. We have carefully addressed all comments as follows:

Comment 1: There is no data to seed germination. Please add at lest germination and mean germination time. To do, read the https://doi.org/10.1111/1440-1703.1275 as the example.

Response 1: We sincerely appreciate the reviewer's insightful suggestion. Regarding the germination data:

The present work specifically investigates the​molecular mechanisms of cell wall remodeling during the imbibition phase. As seed germination parameters (germination percentage, MGT, etc.) were comprehensively reported in our two previous publications (doi:10.1016/j.scienta.2024.113255. doi:10.3390/ijms222011090. ), we focused here in on the endosperm mechanical characteristics.

Based on your valuable suggestions, we have added references to the germination data from previous research in the manuscript (Page 7, Lines 294). In this situation, if you think it is still necessary to add seed germination data, we are also willing to supplement it according to your requirements.

Comment 2: Standardize units and labels across all figures.

Response 2: Thank you for pointing this out. We have carefully checked all the units and labels across all figures and standardized them.

Comment 3: Split large tables for readability or visualize trends using heatmaps.

Response 3: We sincerely appreciate the your insightful suggestion. Due to our desire to present clearer information on differential expression multiples when writing the manuscript, we have adopted a table format. Based on your valuable suggestions and in order to improve readability, we adjusted row spacing to prevent overlapping, added table footnote:“Color scale: red indicates upregulation (log₂FC > 1), blue indicates downregulation (log₂FC < -1)”, and revised table title to: “Table 2. Differential expression of cell wall remodeling-related DEGs in carrot (Daucus carota L.) seeds under CK, HYD and HEHP treatments.” (Page 12). 

Comment 4: The title accurately captures the core of the study—linking HEHP treatment with enhanced seed germination via cell wall remodeling. However, it's quite long and somewhat technical for broader audiences. However, I suggest change thel tille by Enhancing Carrot Seed Germination via Hydro-Electro Hybrid Priming and Cell Wall Remodeling.

Response 4: We sincerely appreciate the your insightful suggestion. The modification plan you proposed for the title clearly makes it more concise and clear. The current title is based on the consideration that HEHP, a novel seed elicitation technology, promotes germination by regulating physiological processes related to cell wall remodeling. Therefore, we did not equate HEHP with cell wall remodeling. If you think it is necessary to modify the title, we will carefully adopt your suggestion.

Comment 5: About the Introduction section: Too weak. Could benefit from more critical synthesis of external studies beyond the group’s previous work. The specific research hypothesis is implied rather than explicitly stated.

Response 5: Thank you for your valuable suggestion. We sincerely appreciate the reviewer’s constructive suggestions. We have strengthened the introduction by:

1. We have further enriched the citation of literature. Add references aboutHYD and physical priming related literature in the paragraph 1 and 2. (Lines 67, 70, 74)
2. Explicitly stating our central hypothesis about synchronized cell wall remodeling. (Lines 117-119)

Comment 6: About the Result section: Dense technical language may obscure clarity. Repetitive statements from tables and figures in text. Comparative transcriptomics are strong, but enzyme activity interpretation could be clearer. Use summary visualizations like PCA plots or volcano plots for transcriptomic data. Provide visual connections between expression and phenotype (e.g., timelines of activity vs. radicle protrusion).

Response 6: Thanks a lot for the constructive suggestions. The following revisions have been made:

1. Text streamlining: We have made every effort to further refine the language as much as possible (Lines 289-291, Lines 371-373, Page 13-Section 3.5, Page 14-Section 3.6).
2. Due to our desire to compare the transcriptome data of seeds after sowing with those before sowing in this study, we did not use a volcano plot in a concise table format. However, based on your valuable suggestions, we have added PCA maps and volcano maps of differentially expressed genes in the supplementary materials(Figure S1 and Figure S2), and included relevant statements in the result analysis (Lines 310-316).
3. Regarding the correlation between gene expression, enzyme activity, and germination phenotype at different time points, in order to correspond the temporal changes in enzyme activity and gene expression with the time axis of germination, we have added a description of their relationship in the Results section (Page 13-part3.5, Page 14-part3.6) and a summary in the Discussion section (Page 17-part 4.3).

Comment 7: About the title of Figure 1, the legend of figures must be complete, with a self explanatory part of the manuscript.

Response 7: Thank you for your valuable feedback. We have revised the title of Figure 1 to enhance clarity and self-sufficiency. We also checked other chart titles and completed the modifications.

Comment 8: Figure 3 and Figure 4: Unreadable. Please increase the font size

Response 8: Thanks a lot for pointing this out. We tried to increase the font size as much as possible while ensuring the complete display of the text. In addition, we simultaneously uploaded high-definition original images (Figure 3: resolution 2854 * 2250, size 21M. Figure 4: resolution 3166 * 2184, size 23M)

Comment 9: About Discussion section: Too weak. Please: describe some speculative statements lack citation or direct experimental evidence. Make a discussion over-emphasizes the novelty of HEHP without addressing broader agronomic or ecological implications. The temper strong claims (“transformative approach”) unless supported by field data. Discuss potential environmental variability and practical application under farm conditions.e

Response 9: We appreciate this constructive feedback. Based on your valuable suggestion, the following revisions have been made:

1. Insert before the original speculative paragraph in Section 4.4: “While our current data show coordinated upregulation of ROS-sensitive PAL (Figure 5C) and peroxidases (Figure 3), previous studies have established that electrostatic fields induce ROS bursts through NADPH oxidase activation[47]. This aligns with our observed upregulation of respiratory genes (e.g., MDH in [16]) that maintain redox homeostasis during wall remodeling.”
2. A new paragraph has been added to at the end of the Discussion sectionto contextualize HEHP within agricultural systems: “While HEHP shows laboratory efficacy, its agronomic value lies in reducing chemical priming dependency, particularly for organic carrot production[4]. Field validation should assess its performance across soil types, as seed-soil contact affects electromagnetic efficacy[12]. Environmental variability, such as fluctuating humidity and temperature during mechanized sowing, may also influence HEHP outcomes. Future research should focus on field validations to assess HEHP’s robustness under variable environmental conditions and multi-omics integration could unravel cross-talk between redox signaling and wall remodeling, further refining HEHP protocols for horticultural applications.”
3. We fully agree and have moderated the language in the Conclusion:The phrase “HEHP represents a transformative approach…” was revised to:

“HEHP demonstrates potential as an eco-friendly priming strategy, though field-scale validation remains prerequisite for mechanized sowing adoption.”

Reviewer 3 Report

Comments and Suggestions for Authors

I reviewed the submission by Sun et al. on the effects of HEH Priming of carrot seeds. The ms. is well-written and data is clearly presented. Overall I was quite surprised at the massive numbers of Differentially Expressed Genes (DEGs) in response to seed priming. What seemed to be missing from this submission is the list of DEGs identified between HEDP and hydroprimed treatments (this is less than 100 genes). I want to know how the electric field changes gene expression when compared to hydropriming--please list these genes in a table or supplemental figure. I also want to see germination data on the effects of these priming treatments on carrot seeds added to the ms. 

Author Response

We appreciate the reviewer’s recognition of our manuscript and the constructive suggestions. We have carefully addressed all comments as follows:

Comment 1: I reviewed the submission by Sun et al. on the effects of HEH Priming of carrot seeds. The ms. is well-written and data is clearly presented. Overall I was quite surprised at the massive numbers of Differentially Expressed Genes (DEGs) in response to seed priming. What seemed to be missing from this submission is the list of DEGs identified between HEDP and hydroprimed treatments (this is less than 100 genes). I want to know how the electric field changes gene expression when compared to hydropriming--please list these genes in a table or supplemental figure.

Response 1: We appreciate this constructive feedback. All the DEGs between HYD and HEHP have been listed in the third table of Table S3. In addition, The samples used for transcriptome sequencing in this study were carrot seeds that imbibed for 20 hours. In our previous research, transcriptome sequencing was performed on seeds before sowing for each treatment (doi:10.3390/ijms222011090). The DEGs of HYD and HEHP were more abundant (3853 DEGs) before sowing, while the DEGs decreased after 20 hours of seed imbibition. Thanks a lot for your valuable comment.

Comment 2: I also want to see germination data on the effects of these priming treatments on carrot seeds added to the ms.

Response 2: We sincerely appreciate the reviewer's insightful suggestion. Regarding the germination data:

The present work specifically investigates the​molecular mechanisms of cell wall remodeling during the imbibition phase. As seed germination parameters (germination percentage, MGT, etc.) were comprehensively reported in our two previous publications (doi:10.1016/j.scienta.2024.113255. and doi:10.3390/ijms222011090. ), we focused here in on the endosperm mechanical characteristics.

Based on your valuable suggestions, we have added references to the germination data from previous research in the manuscript (Page 7, Lines 294).

Reviewer 4 Report

Comments and Suggestions for Authors

The aim of the study is to understand the factors involved in carrot seed germination, which is slow and uneven, a problem that is well justified in the introduction, particularly in the context of large-scale mechanized sowing. The article give sequence to previous publications by the research group on the processes involved in carrot seed germination, clarifies and complements earlier findings, and presents new discoveries. In the study, seeds are subjected to three treatments: control (CK), hydropriming (HYD) and hybrid electrochemical hydropriming (HEHP). Post-soaking comparative transcriptomic analyses of the seeds are performed and genes involved in the germination process are identified. These findings will contribute to the development of practices that improve the rate and uniformity of seeds germination in carrot, addressing a key demand for enhancing the outcomes of large-scale mechanized seed sowing. The effect of HEHP on germination synchronization meets the requirements of mechanized seeding, where uniform emergence reduces thinning work and improves productivity.

Positive Points:

The study is well justified and the results have an important scientific and practical contribution to develop treatment methods that improve carrot seed germination.

The results deepen the understanding of the mechanism by which HEHP treatment promotes rapid germination of carrot seeds and also provide molecular targets for optimizing the use of HEHP in horticultural crops.

The methodological procedures and softwares used in the genetic-statistical analyses are robust and suitable for the objectives and to test the hypothesis of the study.

The conclusions are consistent and supported by the results obtained and are properly argued in the discussion.

Negative Points and suggestions:

The keywords are not appropriate, they repeat terms already present in the title.

In the methodology, in item 2.1, in the description of the treatments (lines 129-139), is not indicate the references used to define the parameters/conditions applied in each of the treatments (time, temperature, voltage, etc.).

In the methodology, in item 2.3, Line 167, clarify: According to the description of the methodology, in total, each treatment was applied to 50 seeds and samples of 10 seeds were used every 10 hours for analysis of mechanical properties in 5 different times. For the transcriptome analysis, 3 seeds were used after 20 hours of sowing. Were these 3 seeds subtracted from the sample of 10 seeds after analysis of the mechanical properties of the sample? Were other seeds subjected to the same treatments used for the transcriptome analysis?

In the methodology, item 2.4. it is necessary to clarify some information about sampling (indicated in the revised pdf file)

In the results, item 3.1. - line 268, the term "significantly lower" suggests that a statistical test was applied, which was not presented. To avoid any doubt that the interpretation was subjective or visual, I suggest replacing the term "significantly" by "considerably" or "noticeably" lower.

In the results, item 3.2 - line 292, I suggest including the information presented in the methodology (indicated in the revised pdf file). Line 302 - Suggestion to change the title of Table 1.

In the results, item 3.5 (unnumbered lines) some corrections to the text are indicated.

In the results, item 3.6 (unnumbered lines) - What criteria were used to select the genes?

Other issues are indicated directly in the revised pdf file.

Comments for author File: Comments.pdf

Author Response

We appreciate the reviewer’s recognition of our manuscript and the constructive suggestions. We have carefully addressed all comments as follows:

Comment 1: The keywords are not appropriate, they repeat terms already present in the title.

Response 1: Thank you for your insightful comment. We agree that keywords should complement, not duplicate. We have revised the keywords to focus on specific mechanisms and novel aspects of the study while avoiding redundancy with the title (Lines 43-45). 

Comment 2: In the methodology, in item 2.1, in the description of the treatments (lines 129-139), is not indicate the references used to define the parameters/conditions applied in each of the treatments (time, temperature, voltage, etc.).

Response 2: Thank you for your valuable feedback. We agree that the rationale for parameter selection should be explicitly supported by relevant literature. We have added the following clarification in Section 2.1 (Lines 142-143):

“The parameters for HYD and HEHP were optimized based on our previous studies[16,22].”

Comment 3: In the methodology, in item 2.3, Line 167, clarify: According to the description of the methodology, in total, each treatment was applied to 50 seeds and samples of 10 seeds were used every 10 hours for analysis of mechanical properties in 5 different times. For the transcriptome analysis, 3 seeds were used after 20 hours of sowing. Were these 3 seeds subtracted from the sample of 10 seeds after analysis of the mechanical properties of the sample? Were other seeds subjected to the same treatments used for the transcriptome analysis?

Response 3: Thank you for raising this important clarification. We apologize for any ambiguity in the original description. To address your questions:

Sampling for mechanical properties vs. transcriptome analysis: The seeds used for transcriptome analysis at 20 h post-imbibition (S20) were independent biological replicates and not subtracted from the seed samples used for mechanical testing. Separate batches of seeds were prepared for each type of analysis to avoid confounding effects.

Specifically:

Mechanical properties:​For each treatment (CK, HYD, HEHP), 50 seeds were divided into 5 groups (n=10 per group) and destructively sampled at S0, S10, S20, S30, and S40.

Transcriptome analysis:​An additional set of seeds (n=3 biological replicates per treatment, approximately 0.1g seeds per sample) were sown under identical conditions and harvested at S20 exclusively for RNA sequencing. These seeds were not subjected to mechanical testing.

Consistency across treatments: All seeds (including those for transcriptome analysis) underwent the same priming protocols (described in Section 2.1). The only difference was the sampling time and purpose (mechanical testing vs. RNA extraction).

In addition, in section 2.3.4, we replaced “GO and KEGG” to “Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)”.

Comment 4: In the methodology, item 2.4. it is necessary to clarify some information about sampling (indicated in the revised pdf file)

Response 4: For the question in the pdf “Were these 4 seeds subtracted from the sample of 10 seeds after analysis of the mechanical properties of the sample?” 

In section 2.4, an additional set of seeds (n=4 biological replicates per treatment, approximately 0.2g seeds per replicate) were sown under identical conditions and harvested at S0, S20 and S40 exclusively for activity determination, not one seed per replication. The amount of sample used has been specified in the text (Lines 222-223 in section 2.4). These seeds were not subjected to mechanical testing.

We have added methodological references: 

For PE activity determination (Page 5, Lines 231), we added “The determination of PE activity was based on the spectrophotometric method (based on methanol release)[31].”

31. 31. Grsic-Rausch, S.; Rausch, T. A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors. Analytical biochemistry 2004, 333, 14-18, doi:10.1016/j.ab.2004.04.042.

For PAL activity determination (Page 5, Lines 245): “The PAL activity was determined by detecting the amount of trans cinnamic acid produced[32].”

32.Hsieh, C.Y.; Huang, Y.H.; Lin, Z.Y.; Hsieh, L.S. Insights into the substrate selectivity of Bambusa oldhamii phenylalanine ammonia-lyase 1 and 2 through mutational analysis. Phytochemistry Letters 2020, 38, 140-143, doi:10.1016/j.phytol.2020.06.002.

In addition, in Section 2.4, we replaced “carrot” to “Carrot” (Lines 222).

Comment 5: In the results, item 3.1. - line 268, the term "significantly lower" suggests that a statistical test was applied, which was not presented. To avoid any doubt that the interpretation was subjective or visual, I suggest replacing the term "significantly" by "considerably" or "noticeably" lower.

Response 5: Thank you for your detailed suggestions. We fully agree that when describing data differences, a clear distinction should be made between statistical significance analysis and visual observation. We have replaced “significantly” with “considerably” to more accurately reflect the objective differences in the data (Section 3.1, Lines 293).

Comment 6: In the results, item 3.2 - line 292, I suggest including the information presented in the methodology (indicated in the revised pdf file). Line 302 - Suggestion to change the title of Table 1.

Response 6: Thanks for your valuable suggestions. As recommended, we have now explicitly defined the differential expression comparison groups in the Methodology section (Lines 209-211, Section 2.3.3). And we have revised the title of Table 1 according to your suggestion.

Comment 7: In the results, item 3.5 (unnumbered lines) some corrections to the text are indicated.

Response 7: Thank you for your detailed suggestions. Based on your and other reviewers’ feedback, we have revised this section. Thanks a lot for your valuable comment.

Comment 8: In the results, item 3.6 (unnumbered lines) - What criteria were used to select the genes?

Response 8: We appreciate the your insightful question. The genes for qRT-PCR validation were systematically selected through the following criteria:

1. Statistical threshold: Only DEGs with |log2FC| ≥1 and FDR ≤0.05 in the CK vs. HEHP comparison were considered.And we tried to choose DEGs with larger fold differences.
2. Pathway centrality: Prioritization of genes annotated to the top enriched pathways (Figure 4A: phenylpropanoid biosynthesis) and GO terms (Figure 3D: cell wall organization).

Comment 9: In section 2.2, What mechanical properties?

Response 9: Thanks a lot for your detailed suggestions. In section 2.2, We replaced “mechanical properties” with “max rupture force” (Lines 165).

Comment 10: For Figure 6, The legend identifying the treatments is missing.

Response 10: Thanks a lot for your detailed suggestions. We have replaced Figure 6 with a diagram with labels of treatments.

Comment 11: In Section 4.4, one sentence (Electrostatic fields are known to induce transient ROS bursts in seeds) was redundant with the previous sentence.

Response 11: Thanks a lot for your detailed suggestions. We have integrated the content of the two sentences in Section 4.4.

Comment 12: In the Conclusion section, you suggest one text (Future research should focus on field validations to assess HEHP’s robustness under variable environmental conditions and multi-omics integration could unravel cross-talk between redox signaling and wall remodeling, further refining HEHP protocols for horticultural applications.) be moved to the end of the discussion item.

Response 12: We appreciate the your insightful suggestion, and we have moved this sentence to the end of the discussion item.

Reviewer 5 Report

Comments and Suggestions for Authors

The present study demonstrates that Hydro-Electro Hybrid Priming (HEHP) enhances carrot seed germination by facilitating the spatiotemporal remodeling of the seed cell wall. This process suggests that HEHP creates a "metabolic memory" during the pretreatment phase.  The experiments performed by the authors in this paper seem interesting and relevant. The findings contribute to a better understanding of how HEHP promotes rapid carrot seed germination and may identify molecular targets for optimizing HEHP technology in horticultural crops. 

The authors conducted thorough research; the study was well-designed, and the techniques used were appropriate. The results are clearly described and interpreted. There is a robust discussion, and the conclusion is solid.

General Observations to Consider:

Introduction: Some references are missing to support certain assertions.

Materials and Methods: There are several important points to address, including the need for references to the techniques used or an indication that the conditions are based on preliminary tests.

Results: The titles of the tables and the captions for the figures should be self-explanatory; some of them currently have very brief titles.

Conclusion:  While the structure is well organized, some details may be lacking.

Bibliographic References: The references contain formatting errors that should be carefully reviewed.

I included the specific observations in the manuscript, which is in PDF format.

Comments for author File: Comments.pdf

Author Response

We appreciate the reviewer’s recognition of our manuscript and the constructive suggestions. We have carefully addressed all comments as follows:

Comment 1: Some references are missing to support certain assertions. For the sentence in the Introduction section “However, conventional HYD often shows suboptimal efficacy in improving germination uniformity, particularly under mechanized sowing conditions”, Please provide references that support this statement.

Response 1: Thank you for your insightful comment. We have added references that support this statement (Lines 69).

Comment 2: For the “Carrot (Daucus carota L. cv. Naaisi)” in Section 2.1, is it the wild species or one of the domesticated subspecies? Daucus carota subsp. sativus, commonly known as the carrot, is the domesticated form of the wild carrot (Daucus carota L.).

Response 2: Thanks a lot for your detailed suggestions. We have revised the description of carrot variety (Lines 70).

Comment 3: For the seed treatment parameter in Section 2.1, it would be appropriate to point out a reference, or if conditions were preliminarily evaluated.

Response 3: Thank you for your valuable feedback. We agree that the rationale for parameter selection should be explicitly supported by relevant literature. We have added the following clarification with references in Section 2.1 (Lines 142-143):

“The parameters for HYD and HEHP were optimized based on our previous studies[16,22].”

Comment 4: Regarding the detailed suggestions raised in Section 2.2.

Response 4: Thanks a lot for your detailed suggestions. Based on your valuable suggestion, the following revisions have been made:

We have replaced “pure water” to “distilled water”. (Lines 161, 162)

The carrot seeds were not imbibed under dark, we have added the optical period in Section 2.2. (Lines 164)

The “N” of “500N” is Newtons.

We added the reference of the determination of max rupture force (Lines 175).

Comment 5: Regarding the detailed suggestions raised in Section 2.4.

Response 5: Thanks a lot for your detailed suggestions. 

We have replaced the EC of PAL to “EC 4.3.1.23, 4.3.1.24 and 4.3.1.25”. (Lines 228)

We added the reference of the determination of XET and CesA activities (Lines 255).

Comment 6: For the title of the Figures, a more detailed figure caption is needed. It should include the study species and the experimental conditions.

Response 6: Thank you for your valuable feedback. We have carefully reviewed all chart titles and revised them as suggested. We also indicated the scientific name in the title.

Comment 7: Figure 7 is more suitable as a graphic summary.

Response 7: Thank you for your valuable feedback. We will use this figure as a graphic summary based on your feedback.

Comment 8: There are several errors in the format of the bibliographic references.

Response 8: Thanks a lot for your detailed suggestions. We have carefully reviewed and revised the reference format.