Role of Endogenous Hormones on Seed Hardness in Pomegranate Fruit Development

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Peer Review Report for Manuscript: "The Changes Relationship between Endogenous Hormones and Seed Hardness during Pomegranate Fruit Development"

 

The manuscript investigates the relationship between endogenous hormones and seed hardness in two pomegranate cultivars, focusing on lignin synthesis during seed development. It provides valuable insights into the roles of hormones such as IAA, JA, ABA, CTKs, and GAs in regulating lignification and seed coat hardness. This is an interesting work that falls within the scope of the journal. However, there are several areas where the manuscript can be improved in terms of clarity, structure, and grammatical accuracy.

Summary of Key Findings

The authors investigated two pomegranate cultivars (soft-seeded ‘Huazi’ and hard-seeded ‘Shandazi’) to determine the role of endogenous hormones (IAA, JA, ABA, CTKs, and others) in lignin accumulation and seed hardness. The study demonstrates:

1. A direct correlation between seed hardness and the levels of IAA and JA.
2. An inverse correlation between seed hardness and CTKs, ABA, GAs, SA, and SLs.
3. Significant differences in lignin deposition and hormonal profiles between the cultivars across three developmental stages.

 

Corrections to be made:

Abstract:

• The phrase "compare the differential changes in endogenous hormones" should be simplified as it is redundant and unnecessarily complex.
• The sentence structure is verbose in statements like "During the development and maturation of pomegranate seeds, the seed hardness is directly proportional to the content of endogenous IAA and JA, and inversely proportional to the content of CTKs, ABA, GAs, SA, and SLs." A more concise description is recommended.
• The correlation between seed hardness and hormonal content can be summarized more effectively to improve clarity and engagement.

Introduction:

• The introduction includes excessive detail about seed anatomy and historical context, which could be streamlined to maintain reader engagement.
• Long, complex sentences reduce readability and should be broken into shorter, focused points.
• Including a graphical abstract summarizing the study's key findings and objectives would make the research visually engaging and immediately understandable.

Methods:

• Statistical analysis is briefly mentioned but lacks detail. Specific methods used for significance testing (e.g., ANOVA, t-tests, or post hoc tests) and threshold values (e.g., p-values) should be explicitly stated.
• Definitions for terms like "DAB" (Days After Blooming) should be included upon first mention to aid comprehension for readers unfamiliar with the terminology.

Results:

• Figures, such as 1 and 2, lack sufficient captions to explain their contents comprehensively. For example, descriptions of axes, legends, and what the visualizations represent should be included for standalone interpretation.
• Repetition of findings in both the text and tables or figures is noticeable. For example, the text repeats numerical data already provided in tables.
• Phrases such as "As shown in Figure 3" could be omitted or rephrased to avoid redundancy.

Discussion:

• Certain sections, such as the role of ABA in soft-seeded varieties, lack sufficient evidence to support speculative claims. Statements should be backed with more robust references or clearly indicated as hypotheses.
• The discussion is overly lengthy in some parts, with repeated points about the correlation between lignin content and seed hardness. Condensing these sections would improve flow and readability.
• Emphasize critical findings and their implications while avoiding excessive theoretical elaboration.

 

 

This manuscript offers significant contributions to the field of horticultural science. However, before publication, the authors should address the following:

1. Revise the abstract and discussion for brevity and clarity.
2. Expand the statistical analysis and define key terms in the methodology.
3. Correct grammatical errors throughout the manuscript.

With these revisions, the manuscript will be a valuable addition to the literature on pomegranate seed development.

 

Author Response

Dear Reviewer#1,

Thank you for your valuable feedback on my article. My response to the question you provided is as follows.

 

1. Abstract:

The phrase "compare the differential changes in endogenous hormones" should be simplified as it is redundant and unnecessarily complex.

The sentence structure is verbose in statements like "During the development and maturation of pomegranate seeds, the seed hardness is directly proportional to the content of endogenous IAA and JA, and inversely proportional to the content of CTKs, ABA, GAs, SA, and SLs." A more concise description is recommended.

The correlation between seed hardness and hormonal content can be summarized more effectively to improve clarity and engagement.

Response: I completely agree with your opinion and have made modifications to it. The phrase “compare the differential changes in endogenous hormones” has modified to “compare changes in hormones”. The sentence "During the development and maturation of pomegranate seeds, the seed hardness is directly proportional to the content of endogenous IAA and JA, and inversely proportional to the content of CTKs, ABA, GAs, SA, and SLs." has modified to “The results showed that the hardness of pomegranate seeds was positively correlated with endogenous IAA and JA, while it was negatively correlated with CTKs, ABA, GAs, SA, and SLs”. Additionally, we have added the sentence “The contents of IAA and JA have the highest content 8.615 ng•g^-1 and 4.5869 ng•g^-1 respectively in hard-seeded variety. Among the soft-seeded variety, the maximum values of DZ, DHZ7G, ABA, GA₁, SA, and 5-DS are 281.82 ng•g^-1, 1542.889 ng•g^-1, 61.273 ng•g^-1, 5.2556 ng•g^-1, 21.15 ng•g^-1 and 0.4494 ng•g^-1 respectively.” to improve clarity and engagement.

 

2. Introduction:

The introduction includes excessive detail about seed anatomy and historical context, which could be streamlined to maintain reader engagement.

Long, complex sentences reduce readability and should be broken into shorter, focused points.

Including a graphical abstract summarizing the study's key findings and objectives would make the research visually engaging and immediately understandable.

Response: Thank you for your suggestion. We have made modifications to complex long sentences. For example, the sentence “Soft-seeded pomegranates are more preferred by most consumers because their seeds are easy to chewing swallow, while semi soft soft seeded and hard hard seeded pomegranates may experience significant difficulties in chewing and swallowing.” has modified to “Soft-seeded pomegranates are more preferred by most consumers profit by chewing easily, while semi soft soft-seeded and hard hard-seeded pomegranates have a poor experience.”

The sentence “The edible part is usually the aril developed from outer epidermal cells of the seed, which is rich in sugars, organic acids, and polyphenols and also had high nutritional value.” has modified to “As the edible part, the aril is rich in sugars, organic acids, and polyphenols developed from outer epidermal cells of the seed.” The sentence “The seed coat develops arised from the integument contains high lignin, cellulose, hemicellulose, which is the main component affecting the hardness of the seeds.” has modified to “The seed coat arised from the integument contains high lignin, cellulose, hemicellulose.”. The sentence “Observation of the microstructure of pomegranate seed coat reveals that the aril was composed of two layers of cells: parechymatous cell layer and palisade tissue layer, while the seed coat was composed of three layers of cells: cork layer, stereid layer, and parechymatous cell layer” has modified to “Observation of the microstructure of pomegranate seed coat reveals that the aril was composed of parechymatous cell layer and palisade tissue layer, while the seed coat was composed of cork layer, stereid layer, and parechymatous cell layer.”.

 

3. Methods:

Statistical analysis is briefly mentioned but lacks detail. Specific methods used for significance testing (e.g., ANOVA, t-tests, or post hoc tests) and threshold values (e.g., p-values) should be explicitly stated.

Definitions for terms like "DAB" (Days After Blooming) should be included upon first mention to aid comprehension for readers unfamiliar with the terminology.

Response: Thank you for your suggestion. We have supplemented the description of data statistical analysis. The content is “The data was calculated as mean ± standard deviation. One-Way ANOVA was used to detected differences between two cultivars. P-value< 0.05 was considered significant difference.”

We have supplemented the definition of DAB. The content is “Days after blooming (DAB) represents when the pomegranate bisexual flowers are slightly open, the calculation of days begins.”

 

4. Results:

Figures, such as 1 and 2, lack sufficient captions to explain their contents comprehensively. For example, descriptions of axes, legends, and what the visualizations represent should be included for standalone interpretation.

Repetition of findings in both the text and tables or figures is noticeable. For example, the text repeats numerical data already provided in tables.

Phrases such as "As shown in Figure 3" could be omitted or rephrased to avoid redundancy.

Response: Thank you for your suggestion. We have supplemented and modified the annotations of Figures 1, 2, 3, and 4. Figure 1. Morphology of two varities of pomegranate seeds in three fruit developmental stages. A, B and C represent the seeds of ‘Shandazi’ at 60, 90, and 120 DAB. D. E and F represent seeds of ‘Huazi’ at 60, 90, and 120 DAB. Figure 2. Safranin O-Fast greenstaining showed lignin deposition in cell walls of seed. The lignified cell wall appears red, with darker colors indicating more lignin deposition. The cellulose cell wall appears green. A, cell walls 60DAB of ‘HZ’ (soft-seeded); B, cell walls 90DAB of ‘HZ’; C, cell walls 120DAB of ‘HZ’; D, cell walls 60DAB of ‘SDZ’ (hard-seeded); E, cell walls 90DAB of ‘SDZ’;F, cell walls 120DAB of ‘SDZ’. Note: PL: Palisade layer; PC: Parechymatous cell; STL: Stereid layer; CK: Cork layer. Figure 3. A cluster analysis heatmap of phytohormones content in pomegranate seeds at 60, 90, 120 DAB between HZ and SDZ. Note: The horizontal axis represents the sample names from two varieties in three periods, the vertical axis (right side) represents hormone information, and the vertical axis (left side) represents classification of hormones. Different colors represent the values obtained after standardized processing of relative content, with red representing high content and green representing low content. H2JA, cZROG, OPC-6, BAP7G, BAPR, mT9G, IPR, ABA-GE, ABA, MEIAA, IAA-Glc, IAA-Asp, OxIAA, GA1, TRP, GA15, GA20, mT, tZR, JA, TRA, JA-VAL, JA-ILE, IAA-Glu, IAA, OPDA, GA19, ACC, DHZR, DHZROG, IAA-Ala, tZOG, iP9G, K9G, DZ, DHZ7G, iP7G, cZR, GA3, IP, tZ, oT, IAN, 2MeScZR, ICAld, IAA-Gly, MEJA, SAG, oTR, 5DS and SA present Dihydrojasmonic acid, cis-Zeatin-O-glucoside riboside, 3-oxo-2-(2-(Z)-Pentenyl)cyclopentane-1-hexanoic acid, N6-Benzyladenine-7-glucoside, 6-Benzyladenosine, meta-Topolin-9-glucoside, N6-isopentenyladenosine, ABA-glucosyl ester, Abscisic acid, Methyl indole-3-acetate, 1-O-indol-3-ylacetylglucose, Indole-3-acetyl-L-aspartic acid, 2-oxindole-3-acetic acid, Gibberellin A1, L-tryptophan, Gibberellin A15, Gibberellin A20, meta-Topolin, trans-Zeatin riboside, Jasmonic acid, Tryptamine, N-[(-)-Jasmonoyl]-(L)-valine, Jasmonoyl-L-isoleucine, Indole-3-acetyl glutamic acid, Indole-3-acetic acid, cis(+)-12-Oxophytodienoic acid, Gibberellin A19, 1-Aminocyclopropanecarboxylic acid, Dihydrozeatin ribonucleoside, Dihydrozeatin-O-glucoside riboside, N-(3-Indolylacetyl)-L-alanine, trans-Zeatin-O-glucoside, N6-Isopentenyl-adenine-9-glucoside, Kinetin-9-glucoside, Dihydrozeatin, Dihydrozeatin-7-glucoside, N6-Isopentenyl-adenine-7-glucoside, cis-Zeatin riboside, Gibberellin A3, N6-isopentenyladenine, trans-Zeatin, ortho-Topolin, 3-Indoleacetonitrile, 2-Methylthio-cis-zeatin riboside, Indole-3-carboxaldehyde, Indole-3-acetyl glycine, Methyl jasmonate, Salicylic acid 2-O-β-glucoside, ortho-Topolin riboside, 5-Deoxystrigol and Salicylic acid respectively. Figure 4. Changes of indole-3-acetic acid (IAA), L-tryptophan (TRP), dihydrozeatin (DZ), dihydrozea-tin-7-glucoside (DHZ7G), N6-isopentenyladenine (IP), trans-Zeatin (tZ), abscisic acid (ABA), 1-aminocyclopropanecarboxylic acid (ACC), gibberellin A1 (GA1), gibberellin A3 (GA3), jasmonic acid (JA), jasmonoyl-L-isoleucine (JA-ILE), salicylic acid (SA) and 5-deoxystrigol (5-DS) content in pome-granate seeds at three stages between HZ01, HZ02, HZ03and SDZ01, SDZ02, SDZ03. Note: The determination of the phytohormone contents is based on ultra high-performance liquid chro-matography-tandem mass spectrometry (UPLC-MS/MS) technology. The horizontal axis repre-sents the sample names from two varieties in three period, and the vertical axis represents hor-mones contents.

We have removed the following content to reduce duplication. The comparative analysis results showed that there were 28 significantly different phytohormones in HZ01_vs_SDZ01, of which 18 were upregulated and 10 were down-regulated (Table 2). TRA was upregulated in three combinations of HZ01_vs_SDZ01, HZ02_vs_SDZ02, HZ03_vs_SDZ03, while IAA-Ala，IAA-Asp were downregulated in that (Table 3). IAA was downregulated in the combinations between HZ03_vs_SDZ03. Besides, in the comparisons of HZ01_vs_SDZ01, HZ02_vs_SDZ02, HZ03_vs_SDZ03 DZ, IP, and tZ were downregulated. Also, ABA was downregulated in the comparisons of HZ01_vs_SDZ01 and HZ02_vs_SDZ02, which was upregulated between HZ01_vs_HZ02 and SDZ01_vs_SDZ02. In addition, GA1 and GA3 were downregulated in the groups of HZ01_vs_SDZ01, HZ02_vs_SDZ02 and HZ03_vs_SDZ03. JA, JA-ILE and MEJA were upregulated in the groups of HZ01_vs_SDZ01 and HZ03_vs_SDZ03, while JA-ILE and MEJA were downregulated in the group of HZ02_vs_SDZ02. Finally, in the groups of HZ01_vs_HZ02 and SDZ01_vs_SDZ02, ABA and 5-DS were upregu-lated, TRA, IAA-Glu, IAA, IP, tZ, JA-ILE and JA-VAL were downregulated.

We have modified phrases such as "As shown in Figure 3" to avoid redundancy. The sentence “3.4. The ABA content in HZ pomegranate seeds first increased and then slightly decreased, while the ABA content in SDZ gradually increased (Fig. 4G). Additionally, the content of salicylic acid (SA) in HZ pomegranate seeds increased first and then decreased, and the changes of SA in SDZ were also sim-ilar (fig. 4M).” have modified.

 

5. Discussion:

Certain sections, such as the role of ABA in soft-seeded varieties, lack sufficient evidence to support speculative claims. Statements should be backed with more robust references or clearly indicated as hypotheses.

The discussion is overly lengthy in some parts, with repeated points about the correlation between lignin content and seed hardness. Condensing these sections would improve flow and readability.

Emphasize critical findings and their implications while avoiding excessive theoretical elaboration.

Response: Thank you for your suggestion. We have removed the sentence “It is speculated that this may be the reason why the ABA concentration in soft-seeded variety of pomegranate is always higher than that in hard-seeded variety and remains at a high level in the later stage”, and added the sentence “It can be inferred that high concentrations of ABA inhibit lignin synthesis during the development of pomegranate seeds, while low concentrations promote it.”. At the same time, we have also provided corresponding data and literature support.

The sentences “Another study had found that the thickness of seed coat cell wall of hard seed varieties was significantly larger than that of soft seed cultivars.”, “Lignin was formed by the connection of three main monomers through various chem-ical bonds in palnts, namely coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol.”, “As the main component of the cell wall, lignin, cellulose and hemicellulose were con-nected to each other to form a network structure, which constituted the skeleton of plant cells.”, and “Besides, it was found that the seed hardness was significantly positively correlated with the lignin content.” have been removed.

We have removed the sentence “5. Conclusion In the later stage of maturity, the effect of IAA weakens and the effect of ABA gradual-ly increases, indicating a higher IAA/ABA ratio during growth and a lower ratio during maturity.” At the same time, we have also added some content to focus on our findings.

 

6. This manuscript offers significant contributions to the field of horticultural science. However, before publication, the authors should address the following:
7. Revise the abstract and discussion for brevity and clarity.
8. Expand the statistical analysis and define key terms in the methodology.
9. Correct grammatical errors throughout the manuscript.

With these revisions, the manuscript will be a valuable addition to the literature on pomegranate seed development.

Response: Thank you for your suggestion. I have made revisions to the abstract and discussion to ensure they meet the requirements. At the same time, a detailed description of the data analysis methods was provided, and key terms were defined. In addition, the article has been polished as necessary to ensure language standardization.

 

Thank you again for your suggestion again.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript: "The Changes Relationship between Endogenous Hormones and Seed Hardness during Pomegranate Fruit Development" represent a solid study that describes the hormonal status of pomegranate during fruit development. However, presented manuscript needs major revision in the following aspects:

 

Abstract

 

The phytohormones mentioned in the Abstract should be provided in their full names.

 

Introduction

 

Line 53: What type of polymerase is involved in polymerization of phenylalanine and tyrosine into lignin?

Line 53-54: “spp.” should not be in italic in “Crataegus spp.”

 

Materials and methods

 

Line 147-149: The gradient program for UPLC could be presented only with the percentage of one mobile phase (A or B).

All statistical tests/analysis, number of replicates, HCA, KEGG analyses and others should be provided in the separate subsection in Material and methods entitled as Statistical analyses.

 

Results

 

Line 177-179: The following statement should be revised, since there were not statistical significant differences between DAB: “The hardness of Huazi seeds decreased after 60 DAB and increased after 90 DAB; the seed hardness of the 90 DAB plants was the least, measured at 0.970 kg·cm^-2.”

Line 181-182: The following statement should be included in Discussion section: “Previous research showed a positive correlation between seed hardness and lignin content [14, 27].” The section results should include only results obtained in the current study.

Table 1: It should be emphasized in the footnote if the significant differences are regarded between different DAP of the samples (row) or different varieties at particular DAP (column).

Line 186: There is a typo: results.

Line 218-219: At first, the authors should present the total number of identified hormones, as well their categorization as auxins, cytokinins, gibberellins…

Figure 3: The full sample names should be presented in Figure caption. For the full names of identified phytohormones, the authors should indicate at Table 3.

Figure 4: Also, the full names of the hormones might be presented in the y-axis, while sample names could be presented in the Figure caption. Why statistical significant differences were not presented between samples?

Table 3: The abbreviations as CK, JA, SA etc should be provided in Table footnote.

Line 314-332: The results should be more clearly presented. Try to summarize the results and better organized the comparison between samples and DAP.

Figure 5: Try to present all figure parts (А, B, C) in same dimensions (width/height).

 

Discussion

 

The section Discussion should be revised, since it has too many general statements and citations that are barely connected to the present results.

Author Response

Dear Reviewer#2,

Thank you for your valuable feedback on my article. My response to the question you provided is as follows.

1. Abstract

The phytohormones mentioned in the Abstract should be provided in their full names.

Response: Thank you for your suggestion. I have already made the modifications. The results showed that the accumulation of lignin in the outer seed coat stereid layer of pome-granate seeds is the reason for the different seed hardness between soft-seeded variety and hard-seeded variety. The hardness of pomegranate seeds was positively correlated with endog-enous indole-3-acetic acid (IAA) and jasmonic acid (JA), while it was negatively correlated with cytokinins (CTKs), abscisic acid (ABA), gibberellins (GAs), salicylic acid (SA), and strigolactones (SLs). The contents of IAA and JA have the highest content 8.615 ng•g-1 and 4.5869 ng•g-1 re-spectively in hard-seeded variety. Among the soft-seeded variety, the maximum values of dihy-drozeatin (DZ), dihydrozeatin-7-glucoside (DHZ7G), ABA, gibberellin A1 (GA1), SA, and 5-deoxystrigol (5-DS) are 281.82 ng•g-1, 1542.889 ng•g-1, 61.273 ng•g-1, 5.2556 ng•g-1, 21.15 ng•g-1 and 0.4494 ng•g-1 respectively.

 

2. Introduction

Line 53: What type of polymerase is involved in polymerization of phenylalanine and tyrosine into lignin?

Line 53-54: “spp.” should not be in italic in “Crataegus spp.”

Response: Thank you for your suggestion. I have added the specific enzyme names and literature support. Ammonia-lyases (Als) is involved in the deamination of phenylalanine and tyrosine during lignin synthesis.

I have already made the modifications.

 

3. Materials and methods

Line 147-149: The gradient program for UPLC could be presented only with the percentage of one mobile phase (A or B).

All statistical tests/analysis, number of replicates, HCA, KEGG analyses and others should be provided in the separate subsection in Material and methods entitled as Statistical analyses.

Response: Thank you for your suggestion. I have already made the modifications. The gradient elution process was from 0 to 1 minute with 5% B, then with 95% B from 1 to 9 minute, finally with 5% B from 9.1 to 12 minute.

I have summarized all the content related to data analysis and set separate titles. Statisticalata analysis: Comparative analysis on seed hardness was completed using Microsoft Excel 2013 and SPSS (version 25.0). The data was calculated as mean ± standard deviation. One-Way ANOVA was used to detected differences between two cultivars. P-value< 0.05 was considered significant difference. MWDB database (Meatware, China) was structured based on standard samples for qualitative analysis of mass spectrometry data. Quantitative analysis of mass spec-trometry data was completed using the multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometry. Data processing was carried out using Analyst 1.6.3 software (AB Sciex, USA) and Multiquant 3.0.3 software (AB Sciex, USA). Data of phytohormone content was processed using unit variance scaling (UV). Heat maps were drawn using R software and hierarchical clustering analysis (HCA) was performed on the accumulation patterns of phytohormones among different sam-ples. Phytohormones were screened according to the requirement of Fold Change≥2 and Fold Change≤0.5, and were considered to have significant differences between each other. The detected phytohormones were annotated using KEGG database (http://www.kegg.jp/kegg/), the phytohormones with significant differences were clas-sified and enriched.

 

4. Results

Line 177-179: The following statement should be revised, since there were not statistical significant differences between DAB: “The hardness of Huazi seeds decreased after 60 DAB and increased after 90 DAB; the seed hardness of the 90 DAB plants was the least, measured at 0.970 kg·cm-2.”

Response: Thank you for your suggestion. I have already made the modifications.

 

Line 181-182: The following statement should be included in Discussion section: “Previous research showed a positive correlation between seed hardness and lignin content [14, 27].” The section results should include only results obtained in the current study.

Response: Thank you for your suggestion. I have already made the modifications. Both previous and current research showed a positive correlation between seed hardness and lignin content.

 

Table 1: It should be emphasized in the footnote if the significant differences are regarded between different DAP of the samples (row) or different varieties at particular DAP (column).

Line 186: There is a typo: results.

Response: Thank you for your suggestion. I have already added the note of “Huazi is soft-seeded variety, and Shandazi is hard-seeded variety.”

I have already made the modifications.

 

Line 218-219: At first, the authors should present the total number of identified hormones, as well their categorization as auxins, cytokinins, gibberellins…

Thank you for your suggestion. I have already added the content of “We detected a total of fifty-one endogenous hormones from pomegranate seeds, including two abscisic acid and its derivatives, eleven auxins, twenty-one cytokinins, one ethylene, five GAs, eight jasmonic acids, two salicylic acids, and one SLs.”

 

Figure 3: The full sample names should be presented in Figure caption. For the full names of identified phytohormones, the authors should indicate at Table 3.

Thank you for your suggestion. I have already I have already added “H2JA, cZROG, OPC-6, BAP7G, BAPR, mT9G, IPR, ABA-GE, ABA, MEIAA, IAA-Glc, IAA-Asp, OxIAA, GA1, TRP, GA15, GA20, mT, tZR, JA, TRA, JA-VAL, JA-ILE, IAA-Glu, IAA, OPDA, GA19, ACC, DHZR, DHZROG, IAA-Ala, tZOG, iP9G, K9G, DZ, DHZ7G, iP7G, cZR, GA3, IP, tZ, oT, IAN, 2MeScZR, ICAld, IAA-Gly, MEJA, SAG, oTR, 5DS and SA present Dihydrojasmonic acid, cis-Zeatin-O-glucoside riboside, 3-oxo-2-(2-(Z)-Pentenyl)cyclopentane-1-hexanoic acid, N6-Benzyladenine-7-glucoside, 6-Benzyladenosine, meta-Topolin-9-glucoside, N6-isopentenyladenosine, ABA-glucosyl ester, Abscisic acid, Methyl indole-3-acetate, 1-O-indol-3-ylacetylglucose, Indole-3-acetyl-L-aspartic acid, 2-oxindole-3-acetic acid, Gibberellin A1, L-tryptophan, Gibberellin A15, Gibberellin A20, meta-Topolin, trans-Zeatin riboside, Jasmonic acid, Tryptamine, N-[(-)-Jasmonoyl]-(L)-valine, Jasmonoyl-L-isoleucine, Indole-3-acetyl glutamic acid, Indole-3-acetic acid, cis(+)-12-Oxophytodienoic acid, Gibberellin A19, 1-Aminocyclopropanecarboxylic acid, Dihydrozeatin ribonucleoside, Dihydrozeatin-O-glucoside riboside, N-(3-Indolylacetyl)-L-alanine, trans-Zeatin-O-glucoside, N6-Isopentenyl-adenine-9-glucoside, Kinetin-9-glucoside, Dihydrozeatin, Dihydrozeatin-7-glucoside, N6-Isopentenyl-adenine-7-glucoside, cis-Zeatin riboside, Gibberellin A3, N6-isopentenyladenine, trans-Zeatin, ortho-Topolin, 3-Indoleacetonitrile, 2-Methylthio-cis-zeatin riboside, Indole-3-carboxaldehyde, Indole-3-acetyl glycine, Methyl jasmonate, Salicylic acid 2-O-β-glucoside, ortho-Topolin riboside, 5-Deoxystrigol and Salicylic acid respectively.”

We have added the full name of the hormones at Table 3.

 

Figure 4: Also, the full names of the hormones might be presented in the y-axis, while sample names could be presented in the Figure caption. Why statistical significant differences were not presented between samples?

Thank you for your suggestion. I have already added full names of hormones in fig.4. I have already added the sample names in fig. 4 caption. Figure 4. Changes of indole-3-acetic acid (IAA), L-tryptophan (TRP), dihydrozeatin (DZ), dihydrozeatin-7-glucoside (DHZ7G), N6-isopentenyladenine (IP), trans-Zeatin (tZ), abscisic acid (ABA), 1-aminocyclopropanecarboxylic acid (ACC), gibberellin A1 (GA1), gibberellin A3 (GA3), jasmonic acid (JA), jasmonoyl-L-isoleucine (JA-ILE), salicylic acid (SA) and 5-deoxystrigol (5-DS) content in pomegranate seeds at three stages between HZ01, HZ02, HZ03 and SDZ01, SDZ02, SDZ03. Note: The determination of the phytohormone contents is based on ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology. The horizontal axis represents the sample names from two varieties in three period, and the vertical axis represents hormones contents.

I have completed the significance analysis of the data and improved the fig.4 content.

 

Table 3: The abbreviations as CK, JA, SA etc should be provided in Table footnote.

Response: Thank you for your suggestion. I have added the full name in Table 3 footnote.

 

Line 314-332: The results should be more clearly presented. Try to summarize the results and better organized the comparison between samples and DAP.

Response: Thank you for your suggestion. I have made revisions to ensure clarity in the description.

 

Figure 5: Try to present all figure parts (А, B, C) in same dimensions (width/height).

Response: Thank you for your suggestion. I have resized the figure (A, B, C) in same dimensions.

 

5. Discussion

The section Discussion should be revised, since it has too many general statements and citations that are barely connected to the present results.

Response: Thank you for your suggestion. I have made comprehensive revisions to the discussion to ensure consistency with the results.

 

Thank you again for your suggestion again.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors failed to fully implement all the suggested comments. The paper is acceptable after minor revisions.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors improved the manuscript according to the suggestions.