Gibberellin-Treated Seedless Cultivation Alters Berry Fracture Behavior, Cell Size and Cell Wall Components in the Interspecific Hybrid Table Grape (Vitis labruscana × Vitis vinifera) ‘Shine Muscat’

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Gibberellin (GA)-induced seedless cultivation is widely used in grape production, yet the developmental basis of GA-associated textural changes remains unclear. In this study, the authors found GA treatment may shape‘Shine Muscat’berry texture through coordinated changes in flesh anatomy, pectin-centered cell wall traits, and related gene expression.the results could provide some ideas for understanding GA-associated textural changes. While there are some questions need to be attentioned.

1. Though it is popular for GA treated grape berry in production, it is better to display the picture in this study, especially for special sampling time points, such as 39 DAFB and 105 DAFB etc. Moreover, why this two time points was selected for sampling?
2. Line 81: What means for “a significantly lower fracture strain”, how is calculated in table 1?
3. In figure 2 and 3, all data was calculated according to inner wall and outer wall, I am wondering what is standard for division of inner wall and outer wall. Additionally, what is meaning for inner wall and
4. Additionally, it was recommended that figure 1 and figure 2 should be combined to one figure since both pericarp was described in these two figure.
5. In figure 3, the format and font sized of characters are different from other figures.
6. In figure 5, it was sampled at DAFB10, 24, and 105, which was different from anatomical sampling time, what is key reason for different sampling time? For Fig5D and E, it was only shown DAFB24 and DAFB 105.
7. Figure 6 and 7 should be combined together, since both of them are focused on transcriptome analysis, and the data for transcriptome analysis is not enough.
8. Figure 8 and 10, Figure 9 and 11 should be combined, respectively, and why these DEGs were selected? It is not clear in the MS. What are “Fir, Sec, MA” in figure 8 etc. represented?
9. Axis labels is unclear for all figures, and please standardize them in MS.
10. In 3.2, GA concentration should be used with a standard unit, and what kind of GA is used in the experiments? Such as GA3?

Author Response

Gibberellin (GA)-induced seedless cultivation is widely used in grape production, yet the developmental basis of GA-associated textural changes remains unclear. In this study, the authors found GA treatment may shape‘Shine Muscat’berry texture through coordinated changes in flesh anatomy, pectin-centered cell wall traits, and related gene expression.the results could provide some ideas for understanding GA-associated textural changes. While there are some questions need to be attentioned.

>Thank you very much for taking the time to review our manuscript despite the busy year-end season. We sincerely appreciate your constructive comments and suggestions. As you pointed out, several parts of the manuscript did not provide sufficient information. We greatly appreciate your valuable suggestions to improve the manuscript, and we provide our responses below.

 

1)Though it is popular for GA treated grape berry in production, it is better to display the picture in this study, especially for special sampling time points, such as 39 DAFB and 105 DAFB etc. Moreover, why this two time points was selected for sampling?

>We have provided photographs of the clusters in DAFB105 (Fig.1 ). Additionally, we have added an explanation regarding the sampling period (L100-102, L361-362, L372-375).

 

2) Line 81: What means for “a significantly lower fracture strain”, how is calculated in table 1?

>We apologize for the confusion caused by our mistake. We had inadvertently conflated fracture stress with fracture load; therefore, we have corrected the term to fracture load throughout the manuscript. In addition, we added a clarification that fracture load refers to the load at which fracture occurs. (L400~406.)

 

3) In figure 2 and 3, all data was calculated according to inner wall and outer wall, I am wondering what is standard for division of inner wall and outer wall. Additionally, what is meaning for inner wall and

> Thank you for the comment. We have clarified the definitions of outer wall and inner wall in the manuscript and added the relevant citation to previous report. (L121-137)

 

4) Additionally, it was recommended that figure 1 and figure 2 should be combined to one figure since both pericarp was described in these two figure.

> Thank you for the comment. We revised the manuscript accordingly, and the figures have been combined into Figure 2.

 

5) In figure 3, the format and font sized of characters are different from other figures.

>Thank you for your comment. The figure was created using R. We adjusted the format of Figure 3 to align it more closely with the other figures. (Figure 3)

 

6) In figure 5, it was sampled at DAFB10, 24, and 105, which was different from anatomical sampling time, what is key reason for different sampling time? For Fig5D and E, it was only shown DAFB24 and DAFB 105.

>Thank you for your comment. The sampling times used in Fig. 5 (DAFB10, 24, and 105) were selected to represent key developmental phases of grape berry growth: DAFB10 corresponds to the cell division–dominant stage, DAFB24 to the cell expansion–dominant stage, and DAFB105 to the mature/harvest stage. These time points allowed us to compare cell wall–related changes across major developmental transitions.

Regarding Figs. 5D and 5E, data for DAFB10 are not shown because we could not obtain reliable sugar content measurements from the fractionated cell wall materials at this stage. Specifically, after the starch digestion step (as a pretreatment for obtaining the KOH-soluble fraction), the remaining solids from DAFB10 samples became highly viscous, which likely interfered with proper handling and fraction recovery, resulting in missing/unstable measurements. Therefore, only DAFB24 and DAFB105 are presented for these panels.

In contrast, the anatomical (histological) sampling times were chosen for a different purpose: to compare berries around the initial of veraison period (DAFB39) and the harvest stage (DAFB105). We have added this reason to the revised manuscript. (L361-362, L372-375).

 

7) Figure 6 and 7 should be combined together, since both of them are focused on transcriptome analysis, and the data for transcriptome analysis is not enough.

> Thank you for the comment. We revised the manuscript accordingly, and the figures have been combined into Figure 6.

 

8) Figure 8 and 10, Figure 9 and 11 should be combined, respectively, and why these DEGs were selected? It is not clear in the MS.

>Thank you for the comment. After considering all reviewers' comments and reorganizing the figures, the figures you mentioned have been reorganized into Figure 7 and Figure 8, respectively. Furthermore, by restructuring the results and discussion, we have added Table S2-5 and Figure S2 related to these data.

 

9) What are “Fir, Sec, MA” in figure 8 etc. represented?

>We apologize for the unclear and inconsistent abbreviations. We have now unified the notation to DAFB throughout the manuscript (including Fig. 8), and revised the figures/text.

 

 

10) Axis labels is unclear for all figures, and please standardize them in MS.

>Thank you for your comment. We have revised the manuscript accordingly.

 

 

11) In 3.2, GA concentration should be used with a standard unit, and what kind of GA is used in the experiments? Such as GA3?

>Thank you for your comment. We have revised and described the information.

 

 

Following comments are commonly report and note for reviewers.

We apologize for the multiple corrections on the authors’ side and have listed the updates below for clarity.

 

＞We corrected the description of replication in the Materials and Methods.

The original text incorrectly stated that “three vines per treatment were selected.” For the compositional analyses and RNA-Seq, we used three clusters per treatment collected from different fruiting shoots within the same vine, and ten berries per cluster were pooled to generate one biological replicate (n = 3). The corresponding text has been revised in Section 3.1 (Plant material).

 

＞We had previously stated “fracture stress” but mistakenly used “fracture load” instead. We have corrected this to “fracture load” and converted it to the international standard unit, N, for inclusion in Table 1.

 

＞The DRA run accession number(s), which were under application at the time of submission, are now available and have been added to the Data Availability Statement (Section: Data Availability Statement).

 

>We have revised the Author Contributions statement following a reconfirmation among the co-authors to ensure that each author’s contributions are accurately described.

 

＞We have reconsidered the results and discussion in accordance with all reviewers' comments and added additional data. Additional data are included in parts of Figures 7-8, Tables S2-5, and Figure S2.

 

 

Following comments are commonly report and note for reviewers.

We apologize for the multiple corrections on the authors’ side and have listed the updates below for clarity.

 

＞We corrected the description of replication in the Materials and Methods.

The original text incorrectly stated that “three vines per treatment were selected.” For the compositional analyses and RNA-Seq, we used three clusters per treatment collected from different fruiting shoots within the same vine, and ten berries per cluster were pooled to generate one biological replicate (n = 3). The corresponding text has been revised in Section 3.1 (Plant material).

 

＞We had previously stated “fracture stress” but mistakenly used “fracture load” instead. We have corrected this to “fracture load” and converted it to the international standard unit, N, for inclusion in Table 1.

 

＞The DRA run accession number(s), which were under application at the time of submission, are now available and have been added to the Data Availability Statement (Section: Data Availability Statement).

 

>We have revised the Author Contributions statement following a reconfirmation among the co-authors to ensure that each author’s contributions are accurately described.

 

＞The cell density values in Figure 2h-i were listed as no./μm, so we have corrected this.

 

＞We have reconsidered the results and discussion in accordance with all reviewers' comments and added additional data. Additional data are included in parts of Figures 7-8, Tables S2-5, and Figure S2.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Journal Plants (ISSN 2223-7747)

Manuscript ID plants-4064423

Type Article

Title Gibberellin-induced seedlessness-associated changes in berry texture, cell architecture, and cell wall composition in ‘Shine Muscat’ grapes

Authors Hikaru Ishikawa , Kaho Masuda , Tomoki Shibuya *

Section Horticultural Science and Ornamental Plants

Special Issue Fruit Development and Ripening

 

Dear Editor,

The text of manuscript ID plants-4064423 requires substantive and editorial revision.

Please find my comments below.

Yours sincerely,

Reviewer

TITLE

1.

Please use the Latin name of the species in English.

2.

Please consider a topic that should correspond with the results.

Keywords

3.

Please eliminate terms that appear in the title of the manuscript.

Abstract

4.

Please use the Latin name of the species instead of the English name of the genus.

5.  

The first time an abbreviation is used, it should be explained. Please check this.

6.  

The broad terms “...fracture testing, histological analyses, cell wall fractionation,”

please specify what analyses were performed.

“...We integrated fracture testing, histological analyses, cell wall fractionation,...”

7.

Instead of abbreviations, please specify which cells in which tissues were analyzed “...texture and mesocarp traits...”.

8.

Please specify the physiological process influencing

“...berries exhibited higher fracture stress and lower fracture strain than non-treated seeded berries, suggesting enhanced resistance to fracture with reduced deformation capacity...”.

9.  

Please specify the names of these enzymes

“...together with pectin-modifying enzymes,...”

10.

Please analyze the abstract step by step to maintain the following structure:

1. a) place the introduction in a broad context,
2. b) emphasize the purpose of the study,
3. c) briefly describe the main methods used,
4. d) summarize the main findings,
5. e) and indicate the main conclusions.

INTRODUCTION

46. 33-46.

11.

Please avoid using the phrase “such as.”

12.

40. 33-40. Three sentences cannot form a thematic paragraph. Please change this.

13.

Please spell the varieties correctly in accordance with the accepted nomenclature for plant names.

14.

Please specify the physiological processes that increase flesh firmness and the biologically active compounds and pathways that improve eating quality [5].

“to increase flesh firmness and improve eating quality [5].”

15.

Please explain how and which anatomical and ultrastructural features of cells (specify which tissues) affect the relevant properties (specify which properties instead of a general statement).

“the physical properties of the flesh are affected by tissue microstructure and cell wall composition and architecture.”

 

 

16.

The structure of cell walls is very complex, ranging from cellulose particles to elementary microfibrils, macrofibrils, etc. Please supplement this information with reference to the topic of the manuscript instead of using a mental shortcut.

“the physical properties of the flesh are affected by tissue microstructure and cell wall composition and architecture.”

17.

Please provide examples of studies on species from the Vitaceae family or related species, as the Solanaceae and Rosaceae families are distant families in taxonomy in relation to the Vitaceae family.

56. 47-56.

18.

49. 47-49. Please describe this by citing the latest scientific publications related to the topic of the manuscript.

19.

53. 49-53. Please describe these processes in relation to the microstructure of cell walls.

20.

56. 53-56. Please explain this in close relation to the topic of the manuscript.

21.

60. 57-60. Modification of cell walls involves various types of compounds deposited in the process of incrustation and adcrustation. This is a broad concept, so please supplement the information related to the topic of the manuscript.

22.

60. 57-60. Please specify which chemical compounds and describe their role in relation to the topic of the manuscript.

23.

64. 60-64. Please describe this process in relation to the topic of the manuscript.

“suggesting that early ripening-associated cell wall reorganization contributes to flesh softening [16].”

24.

67. 64-67. Please explain the role of calcium at the cellular level in relation to the topic of the manuscript.

 

 

25.  
26. 64-67. Please supplement the role of uronic acids in the cell walls of the presented species in strict relation to the topic of the manuscript.
27.  
28. 68-70. Whose results, literature reports or those presented in the manuscript – this is unclear (if the presented results, then not in this place). Please describe the anatomical changes of selected cells of the given tissues instead of the mental shortcut

“texture through coordinated changes in flesh anatomy,”

27.  

What are the characteristics of cell walls? Please explain.

“pectin-centered cell wall traits, and related gene expression.”

28.

The mention of genes is out of place here; this is a very specific issue that cannot be signaled in this way. Please describe the expression of specific genes related to the presented topic.

“and related gene expression.”

29.  
30. 70-73. Please describe these changes.

“induces divergences in tissue architecture and cell wall traits that ultimately result in texture differences in ‘Shine Muscat’ remain limited.”

30.  

Justify the relevance of the research topic, justify the need to conduct it by indicating what has been done in this area and what gaps exist in the literature.

31.

77. 73-77. Formulate a specific objective for the work (main objective and specific objectives).

RESULTS AND DISCUSSION

89. 78-89.

32.

Please write the variety correctly (the variety should be written after the Latin name of the species in an apostrophe).

132. 89 – 132.

33.

Page 3, please remove the blank space.

 

 

34.

Figure 1a. What does “Outer wall” mean? “Inner wall” – do the authors mean the cell wall of the epidermis or selected cells of the pericarp? Please clarify.

35.

Figure 1b-c. Please select good quality photographs, and on good quality photographs, please add labels and indicate what the individual cells mean (the labels should be marked on the photographs).

36.

Figure 1b-c. Please insert numerical values for the bars to indicate how much a bar is.

37.

Figure 1. The last photograph does not have a letter label.

38.

Please describe in relation to Figure 1 in the text what the authors want to convey regarding Figure 1a.

39.  

Please cite Figure 1b, Figure 1c, and Figure 1d individually instead of citing (Figure 1) and include one sentence in the subsection describing Figure 1. Please complete the text for Figures 1a-d. Specifically, state what the authors want to convey or reduce the number of photographs to a smaller number but of good quality. The current ones are unacceptable for printing (the epidermis, parenchyma cells, etc. are not visible).

40.

Figure 2 a-f and Figure 3-5, 7-11. Please specify the X-axis (what the X-axis refers to; the graph should be understandable without referring to other parts of the manuscript).

41.

115. 110-115. It is good practice not to start a sentence with abbreviations; please change this.

42.

See comment above “Inner wall,” “outer wall.”

43.  

Please complete the discussion in subsections 2.1 and 2.2.

44.

146. 139-146. Please avoid the term “such as,” see comment above, please make the correction

“cations such as Ca2,” “changes such as molecular”

 

 

45.  

Please strengthen the discussion in subsections 2.3 and 2.4 using the latest scientific publications but in relation to the topic of the manuscript, maintaining the nature of the discussion: first your own achievements and then a comparison with the results of other authors. Maintaining a critical approach without adopting the conclusions of a given publication, this should be a discussion, not a literature review.

MATERIALS AND METHODS

46.  

For each methodological step, please cite the relevant literature confirming the validity of the methodological action taken.

47.

Preparing microscope slides is a complicated and very precise process. Please describe step by step how the semi-thin slides were prepared, including:

- fixation (what reagents, what concentrations, including fixative, buffer, specify concentrations)

- specify the concentrations in the process of dehydrating the sections

- specify the concentration of the resin used

- concentration of dyes

- what was used to cut the sections, etc., providing relevant scientific literature for each step of the method.

48.

Please check the thickness of the sections “9-12 μm thick”????

In most semi-thin preparations, the sections are 1 μm thick.

49.

Please complete the applicability of the obtained research results.

50.

Please complete the prospects for future research.

51.

Please complete the conclusions that should respond to the objective of the work and the scientific research theses.

References

52.

Please check each item of literature step by step to ensure that it complies with the guidelines for authors, e.g., items 3-7, etc.

INFORMATION FOR PROOFREADING THE MANUSCRIPT.

53.  

Please respond to each comment (black) and include the relevant section of the manuscript (red) below your response.

54.  

In the manuscript text, the text after correction (please mark all changes in red).

Comments on the Quality of English Language

Journal Plants (ISSN 2223-7747)

Manuscript ID plants-4064423

Type Article

Title Gibberellin-induced seedlessness-associated changes in berry texture, cell architecture, and cell wall composition in ‘Shine Muscat’ grapes

Authors Hikaru Ishikawa , Kaho Masuda , Tomoki Shibuya *

Section Horticultural Science and Ornamental Plants

Special Issue Fruit Development and Ripening

 

Dear Editor,

The text of manuscript ID plants-4064423 requires substantive and editorial revision.

Please find my comments below.

Yours sincerely,

Reviewer

TITLE

1.

Please use the Latin name of the species in English.

2.

Please consider a topic that should correspond with the results.

Keywords

3.

Please eliminate terms that appear in the title of the manuscript.

Abstract

4.

Please use the Latin name of the species instead of the English name of the genus.

5.  

The first time an abbreviation is used, it should be explained. Please check this.

6.  

The broad terms “...fracture testing, histological analyses, cell wall fractionation,”

please specify what analyses were performed.

“...We integrated fracture testing, histological analyses, cell wall fractionation,...”

7.

Instead of abbreviations, please specify which cells in which tissues were analyzed “...texture and mesocarp traits...”.

8.

Please specify the physiological process influencing

“...berries exhibited higher fracture stress and lower fracture strain than non-treated seeded berries, suggesting enhanced resistance to fracture with reduced deformation capacity...”.

9.  

Please specify the names of these enzymes

“...together with pectin-modifying enzymes,...”

10.

Please analyze the abstract step by step to maintain the following structure:

1. a) place the introduction in a broad context,
2. b) emphasize the purpose of the study,
3. c) briefly describe the main methods used,
4. d) summarize the main findings,
5. e) and indicate the main conclusions.

INTRODUCTION

46. 33-46.

11.

Please avoid using the phrase “such as.”

12.

40. 33-40. Three sentences cannot form a thematic paragraph. Please change this.

13.

Please spell the varieties correctly in accordance with the accepted nomenclature for plant names.

14.

Please specify the physiological processes that increase flesh firmness and the biologically active compounds and pathways that improve eating quality [5].

“to increase flesh firmness and improve eating quality [5].”

15.

Please explain how and which anatomical and ultrastructural features of cells (specify which tissues) affect the relevant properties (specify which properties instead of a general statement).

“the physical properties of the flesh are affected by tissue microstructure and cell wall composition and architecture.”

 

 

16.

The structure of cell walls is very complex, ranging from cellulose particles to elementary microfibrils, macrofibrils, etc. Please supplement this information with reference to the topic of the manuscript instead of using a mental shortcut.

“the physical properties of the flesh are affected by tissue microstructure and cell wall composition and architecture.”

17.

Please provide examples of studies on species from the Vitaceae family or related species, as the Solanaceae and Rosaceae families are distant families in taxonomy in relation to the Vitaceae family.

56. 47-56.

18.

49. 47-49. Please describe this by citing the latest scientific publications related to the topic of the manuscript.

19.

53. 49-53. Please describe these processes in relation to the microstructure of cell walls.

20.

56. 53-56. Please explain this in close relation to the topic of the manuscript.

21.

60. 57-60. Modification of cell walls involves various types of compounds deposited in the process of incrustation and adcrustation. This is a broad concept, so please supplement the information related to the topic of the manuscript.

22.

60. 57-60. Please specify which chemical compounds and describe their role in relation to the topic of the manuscript.

23.

64. 60-64. Please describe this process in relation to the topic of the manuscript.

“suggesting that early ripening-associated cell wall reorganization contributes to flesh softening [16].”

24.

67. 64-67. Please explain the role of calcium at the cellular level in relation to the topic of the manuscript.

 

 

25.  
26. 64-67. Please supplement the role of uronic acids in the cell walls of the presented species in strict relation to the topic of the manuscript.
27.  
28. 68-70. Whose results, literature reports or those presented in the manuscript – this is unclear (if the presented results, then not in this place). Please describe the anatomical changes of selected cells of the given tissues instead of the mental shortcut

“texture through coordinated changes in flesh anatomy,”

27.  

What are the characteristics of cell walls? Please explain.

“pectin-centered cell wall traits, and related gene expression.”

28.

The mention of genes is out of place here; this is a very specific issue that cannot be signaled in this way. Please describe the expression of specific genes related to the presented topic.

“and related gene expression.”

29.  
30. 70-73. Please describe these changes.

“induces divergences in tissue architecture and cell wall traits that ultimately result in texture differences in ‘Shine Muscat’ remain limited.”

30.  

Justify the relevance of the research topic, justify the need to conduct it by indicating what has been done in this area and what gaps exist in the literature.

31.

77. 73-77. Formulate a specific objective for the work (main objective and specific objectives).

RESULTS AND DISCUSSION

89. 78-89.

32.

Please write the variety correctly (the variety should be written after the Latin name of the species in an apostrophe).

132. 89 – 132.

33.

Page 3, please remove the blank space.

 

 

34.

Figure 1a. What does “Outer wall” mean? “Inner wall” – do the authors mean the cell wall of the epidermis or selected cells of the pericarp? Please clarify.

35.

Figure 1b-c. Please select good quality photographs, and on good quality photographs, please add labels and indicate what the individual cells mean (the labels should be marked on the photographs).

36.

Figure 1b-c. Please insert numerical values for the bars to indicate how much a bar is.

37.

Figure 1. The last photograph does not have a letter label.

38.

Please describe in relation to Figure 1 in the text what the authors want to convey regarding Figure 1a.

39.  

Please cite Figure 1b, Figure 1c, and Figure 1d individually instead of citing (Figure 1) and include one sentence in the subsection describing Figure 1. Please complete the text for Figures 1a-d. Specifically, state what the authors want to convey or reduce the number of photographs to a smaller number but of good quality. The current ones are unacceptable for printing (the epidermis, parenchyma cells, etc. are not visible).

40.

Figure 2 a-f and Figure 3-5, 7-11. Please specify the X-axis (what the X-axis refers to; the graph should be understandable without referring to other parts of the manuscript).

41.

115. 110-115. It is good practice not to start a sentence with abbreviations; please change this.

42.

See comment above “Inner wall,” “outer wall.”

43.  

Please complete the discussion in subsections 2.1 and 2.2.

44.

146. 139-146. Please avoid the term “such as,” see comment above, please make the correction

“cations such as Ca2,” “changes such as molecular”

 

 

45.  

Please strengthen the discussion in subsections 2.3 and 2.4 using the latest scientific publications but in relation to the topic of the manuscript, maintaining the nature of the discussion: first your own achievements and then a comparison with the results of other authors. Maintaining a critical approach without adopting the conclusions of a given publication, this should be a discussion, not a literature review.

MATERIALS AND METHODS

46.  

For each methodological step, please cite the relevant literature confirming the validity of the methodological action taken.

47.

Preparing microscope slides is a complicated and very precise process. Please describe step by step how the semi-thin slides were prepared, including:

- fixation (what reagents, what concentrations, including fixative, buffer, specify concentrations)

- specify the concentrations in the process of dehydrating the sections

- specify the concentration of the resin used

- concentration of dyes

- what was used to cut the sections, etc., providing relevant scientific literature for each step of the method.

48.

Please check the thickness of the sections “9-12 μm thick”????

In most semi-thin preparations, the sections are 1 μm thick.

49.

Please complete the applicability of the obtained research results.

50.

Please complete the prospects for future research.

51.

Please complete the conclusions that should respond to the objective of the work and the scientific research theses.

References

52.

Please check each item of literature step by step to ensure that it complies with the guidelines for authors, e.g., items 3-7, etc.

INFORMATION FOR PROOFREADING THE MANUSCRIPT.

53.  

Please respond to each comment (black) and include the relevant section of the manuscript (red) below your response.

54.  

In the manuscript text, the text after correction (please mark all changes in red).

Author Response

Thank you very much for your careful and thorough review. We sincerely appreciate you taking the time to provide detailed comments despite the busy year-end season. We apologize that several sections lacked sufficient information, and some explanations were too brief. We are deeply grateful for your valuable suggestions, which have helped us improve the manuscript. Below, we provide our responses to your comments and describe the revisions made accordingly. Thank you again for your constructive comments. We hope that our revisions satisfactorily address your concerns. Please excuse us for not indicating areas with a large volume of revisions or 2nd time or more, in red.

 

1. Please use the Latin name of the species in English.
2. Please consider a topic that should correspond with the results.

>Thank you for your comments. We have revised the title. Regarding naming conventions in title, we referenced the titling approach in prior research (Shirasawa et al., 2022).

Gibberellin-treated seedless cultivation alters berry fracture behavior, cell size and cell wall components in the interspecific hybrid table grape ‘Shine Muscat’

 

3. Please eliminate terms that appear in the title of the manuscript.

>Thank you for your comment. We have revised the manuscript accordingly.

 

4. Please use the Latin name of the species instead of the English name of the genus.

> Thank you for your comment. We have revised the manuscript. The description for ‘Delaware’ has been removed due to its complex Latin name and the use of Vitis Spp. in the text.

interspecific table grape (Vitis labruscana × V. vinifera) ‘Shine Muscat’

 

5. The first time an abbreviation is used, it should be explained. Please check this.

>Thank you for your comment. We have revised the manuscript accordingly.

 

6. The broad terms “...fracture testing, histological analyses, cell wall fractionation,”please specify what analyses were performed.
7. Instead of abbreviations, please specify which cells in which tissues were analyzed “...texture and mesocarp traits...”.

>Thank you for your comments. We have revised the manuscript accordingly these comments.

We integrated intact-berry fracture testing at harvest (DAFB105), quantitative histology of pericarp/mesocarp tissues just before veraison (DAFB39) and at harvest, sequential cell-wall fractionation assays targeting pectin-rich (uronic acid) and hemicellulose/cellulose-related pools at cell division period, cell expansion period and harvest, and stage-resolved RNA-Seq across the same three developmental stages.

 

8. Please specify the physiological process influencing

“...berries exhibited higher fracture stress and lower fracture strain than non-treated seeded berries, suggesting enhanced resistance to fracture with reduced deformation capacity...”.

> Thank you for these suggestions regarding the Abstract. In the revised manuscript, we comprehensively rewrote the Abstract to reflect the overall changes made throughout the manuscript. As a result, the specific wording/phrasing pointed out in this comment is no longer present in the current Abstract, and there is therefore no corresponding location for a direct, line-by-line revision. Instead, we have addressed the reviewer’s concern by replacing the Abstract in its entirety with a more concrete and streamlined version that is consistent with the revised Methods and Results/Discussion.

 

9. Please specify the names of these enzymes

“...together with pectin-modifying enzymes,...”

Thank you for your comment. We have revised the manuscript as suggested.

L28~31

...primary-wall remodeling genes, EXORDIUM and xyloglucan endotransglucosylase/hydrolases, at DAFB24 and suggested relatively enhanced ethylene-/senescence-associated transcriptional programs together with pectin-modifying related genes, Polygaracturonase/ pectate lyase and pectin methylesterase, ...

 

10. Please analyze the abstract step by step to maintain the following structure:
11. a) place the introduction in a broad context,
12. b) emphasize the purpose of the study,
13. c) briefly describe the main methods used,
14. d) summarize the main findings,
15. e) and indicate the main conclusions.

>Thank you for your comments. We have revised the manuscript accordingly.

L12-34

 

INTRODUCTION

11. Please avoid using the phrase “such as.”
12. 33-40. Three sentences cannot form a thematic paragraph. Please change this.
13. Please spell the varieties correctly in accordance with the accepted nomenclature for plant names.
14. Please specify the physiological processes that increase flesh firmness and the biologically active compounds and pathways that improve eating quality [5].

“to increase flesh firmness and improve eating quality [5].”

15. Please explain how and which anatomical and ultrastructural features of cells (specify which tissues) affect the relevant properties (specify which properties instead of a general statement).

“the physical properties of the flesh are affected by tissue microstructure and cell wall composition and architecture.”

16. The structure of cell walls is very complex, ranging from cellulose particles to elementary microfibrils, macrofibrils, etc. Please supplement this information with reference to the topic of the manuscript instead of using a mental shortcut.

“the physical properties of the flesh are affected by tissue microstructure and cell wall composition and architecture.”

17. Please provide examples of studies on species from the Vitaceae family or related species, as the Solanaceae and Rosaceae families are distant families in taxonomy in relation to the Vitaceae family.

47-56.

18. 47-49. Please describe this by citing the latest scientific publications related to the topic of the manuscript.
19. 49-53. Please describe these processes in relation to the microstructure of cell walls.
20. 53-56. Please explain this in close relation to the topic of the manuscript.
21. 57-60. Modification of cell walls involves various types of compounds deposited in the process of incrustation and adcrustation. This is a broad concept, so please supplement the information related to the topic of the manuscript.
22. 57-60. Please specify which chemical compounds and describe their role in relation to the topic of the manuscript.
23. 60-64. Please describe this process in relation to the topic of the manuscript.

“suggesting that early ripening-associated cell wall reorganization contributes to flesh softening [16].”

24. 64-67. Please explain the role of calcium at the cellular level in relation to the topic of the manuscript.
25. 64-67. Please supplement the role of uronic acids in the cell walls of the presented species in strict relation to the topic of the manuscript.
26. 68-70. Whose results, literature reports or those presented in the manuscript – this is unclear (if the presented results, then not in this place). Please describe the anatomical changes of selected cells of the given tissues instead of the mental shortcut

“texture through coordinated changes in flesh anatomy,”

27. What are the characteristics of cell walls? Please explain.

“pectin-centered cell wall traits, and related gene expression.”

28. The mention of genes is out of place here; this is a very specific issue that cannot be signaled in this way. Please describe the expression of specific genes related to the presented topic.

“and related gene expression.”

29. 70-73. Please describe these changes.

“induces divergences in tissue architecture and cell wall traits that ultimately result in texture differences in ‘Shine Muscat’ remain limited.”

30. Justify the relevance of the research topic, justify the need to conduct it by indicating what has been done in this area and what gaps exist in the literature.
31. 73-77. Formulate a specific objective for the work (main objective and specific objectives).

 

>Response for 11-31,

Thank you for the detailed and constructive comments. Based on these points, we substantially revised and reorganized the Introduction and then reviewed the entire section for clarity and consistency (Introduction, revised throughout). In the revised version, we refined wording and nomenclature, clarified which tissues and mechanical properties are relevant to intact-berry texture measurements, and expanded the background on tissue/cellular determinants of mechanical behavior in a grape-focused context. We also supplemented the description of cell-wall microstructure and the mechanistic basis of tissue mechanics, including primary-wall architecture, pectin-rich middle lamella–mediated cell-to-cell adhesion, and Ca²⁺-related pectin chemistry, supported by more recent literature. In addition, we clarified our intended meaning of “cell wall modification” in the context of this study and strengthened the rationale by explicitly stating the knowledge gap and the specific objectives of the work.

L37~97

 

 

RESULTS AND DISCUSSION

32. Please write the variety correctly (the variety should be written after the Latin name of the species in an apostrophe).

>Thank you for the comment. Because we already revised the name in Introduction, we have described only cultivar name in RESULTS AND DISCUSSION.

 

33. Page 3, please remove the blank space.

>Thank you for the comment. We have removed the space.

 

34. Figure 1a. What does “Outer wall” mean? “Inner wall” – do the authors mean the cell wall of the epidermis or selected cells of the pericarp? Please clarify.

> Thank you for the comment. We have clarified the definitions of outer wall and inner wall in the manuscript and added the relevant citation to previous report.

L121~137

The structure of a grape berry is illustrated in Fig. 2a. In this study, the term "pericarp" collectively refers to the exocarp, mesocarp, and endocarp (Fig. 2a). The exocarp is sometimes referred to as the skin. Within the pericarp, the region inside the vascular bundles was defined as the inner wall. The region outside the vascular bundles was defined as the outer wall (Fig. 2a). In addition, within the mesocarp, the region inside the vascular bundles was defined as the inner side. The region outside the vascular bundles was defined as the outer side (Fig. 2a). Thus, the inner wall comprises the endocarp and the inner side of the mesocarp. In contrast, the outer wall comprises the outer side of the mesocarp and the exocarp (Fig. 2a). Representative equatorial cross-sections of non-GA-treated seeded and GA-treated seedless berries at 105 days after full bloom (DAFB105) are also presented (Fig. 2b, c). Inner wall sides of pericarp are shown in Fig. 2b and outer wall sides of pericarp are shown in Fig. 2c. The pericarp is referred to as the inner wall and outer wall on the inner and outer sides of the vascular ring, respectively [26]. It has been reported that, in mature ‘Delaware’ grapes, the inner wall, outer wall, and placental tissue occupy 59.7%, 17.4% and 9.9% of the total cross-sectional area at maturity, respectively [26]. Based on previous studies, the mesocarp constitutes the majority of both the inner and outer walls [26], suggesting that the flesh is predominantly composed of the mesocarp.

 

35. Figure 1b-c. Please select good quality photographs, and on good quality photographs, please add labels and indicate what the individual cells mean (the labels should be marked on the photographs).

>Thank you for the comment. We have revised the figures. We replaced the section images of the same processing area with those possessing clear images, then performed grayscale-to-color conversion, enlargement, and segmentation processing to enhance visibility.

Figure 2 b-c. L121~137

 

36. Figure 1b-c. Please insert numerical values for the bars to indicate how much a bar is.

>Thank you for the comment. We have revised the figures.

Figure 2 b-c.

 

37. Figure 1. The last photograph does not have a letter label.

>Thank you for the comment. We have revised the figures.

Figure 2 b-c.

 

38. Please describe in relation to Figure 1 in the text what the authors want to convey regarding Figure 1a.

>Thank you for the comment. We revised the text to explicitly state the purpose of Fig. 1a and to clarify that our histological measurements were performed according to the schematic definitions shown in Fig. 2a (Results, Lines 121-122).

 

 Please cite Figure 1b, Figure 1c, and Figure 1d individually instead of citing (Figure 1) and include one sentence in the subsection describing Figure 1. Please complete the text for Figures 1a-d. Specifically, state what the authors want to convey or reduce the number of photographs to a smaller number but of good quality. The current ones are unacceptable for printing (the epidermis, parenchyma cells, etc. are not visible).

>Thank you for the comment. We revised the manuscript accordingly.

Representative equatorial cross-sections of non-GA-treated seeded and GA-treated seedless berries at 105 days after full bloom (DAFB105) are also presented (Fig. 2b, c). Inner wall sides of pericarp are shown in Fig. 2b and outer wall sides of pericarp are shown in Fig. 2c.

 

40. Figure 2 a-f and Figure 3-5, 7-11. Please specify the X-axis (what the X-axis refers to; the graph should be understandable without referring to other parts of the manuscript).

>Thank you for the comment. We revised the manuscript accordingly.

L175-178

Inner side, inner side of the mesocarp; Outer side, outer side of the mesocarp; GA, GA-treated berries; NT, non-treated berries; Transverse, circumferential on the equatorial plane of the berry; Longitudinal, radial on the equatorial plane of the berry.

 

41. 110-115. It is good practice not to start a sentence with abbreviations; please change this.

>Thank you for the comment. We revised the manuscript accordingly.

 

42. See comment above “Inner wall,” “outer wall.”

>Thank you for the comment. We revised the manuscript accordingly.

 

43. Please complete the discussion in subsections 2.1 and 2.2.

>Thank you for the comment. We have added a brief discussion.

L107-112, L184-201

 

44. 139-146. Please avoid the term “such as,” see comment above, please make the correction

“cations such as Ca2,” “changes such as molecular”

>Thank you for the comment. We revised the manuscript accordingly.

 

45. Please strengthen the discussion in subsections 2.3 and 2.4 using the latest scientific publications but in relation to the topic of the manuscript, maintaining the nature of the discussion: first your own achievements and then a comparison with the results of other authors. Maintaining a critical approach without adopting the conclusions of a given publication, this should be a discussion, not a literature review.

>Thank you for the comment. We revised the manuscript accordingly. Most of the additional considerations were added to Section 2.4 because they were necessary for examining the mechanism.

 

 

MATERIALS AND METHODS

46. For each methodological step, please cite the relevant literature confirming the validity of the methodological action taken.

>Thank you for the comment. We revised the manuscript accordingly.

L411-433 in 3.4. Histological analysis

 

47. Preparing microscope slides is a complicated and very precise process. Please describe step by step how the semi-thin slides were prepared, including:

- fixation (what reagents, what concentrations, including fixative, buffer, specify concentrations)

- specify the concentrations in the process of dehydrating the sections

- specify the concentration of the resin used

- concentration of dyes

- what was used to cut the sections, etc., providing relevant scientific literature for each step of the method.

>Thank you for the comment. We revised the manuscript accordingly.

L411-433 in 3.4. Histological analysis

 

48. Please check the thickness of the sections “9-12 μm thick”????

In most semi-thin preparations, the sections are 1 μm thick.

>We thank the reviewer for the comment on section thickness. We well understand your concerns. In our study, berry tissues embedded in Technovit 7100 were sectioned at 9–12 μm for light-microscopic quantitative analyses. We have rechecked the model number and year of manufacture for the microtome and revised the description（L428）. This model was manufactured in the 1960s and was not capable of consistently producing semi-thin sections as you pointed out; therefore, the thickness stated in the manuscript is not incorrect.

 

49. Please complete the applicability of the obtained research results.

>Thank you for the comment. We have described about the applicability of our results in conclusions.

L501-537

 

50. Please complete the prospects for future research.

>Thank you for the comment. We have described about the applicability of our results in conclusions.

L501-537

 

51. Please complete the conclusions that should respond to the objective of the work and the scientific research theses.

>Thank you for the comment. We added Conclusion.

L501-537

 

 

References

52. Please check each item of literature step by step to ensure that it complies with the guidelines for authors, e.g., items 3-7, etc.

>Thank you for the comment. We have checked and corrected all cited references.

 

53. INFORMATION FOR PROOFREADING THE MANUSCRIPT.

Please respond to each comment (black) and include the relevant section of the manuscript (red) below your response.

In the manuscript text, the text after correction (please mark all changes in red).

>Thank you for the comment. We’ve prepared the revised manuscript and responce accordingly.

 

 

Following comments are commonly report and note for reviewers.

We apologize for the multiple corrections on the authors’ side and have listed the updates below for clarity.

 

＞We corrected the description of replication in the Materials and Methods.

The original text incorrectly stated that “three vines per treatment were selected.” For the compositional analyses and RNA-Seq, we used three clusters per treatment collected from different fruiting shoots within the same vine, and ten berries per cluster were pooled to generate one biological replicate (n = 3). The corresponding text has been revised in Section 3.1 (Plant material).

 

＞We had previously stated “fracture stress” but mistakenly used “fracture load” instead. We have corrected this to “fracture load” and converted it to the international standard unit, N, for inclusion in Table 1.

 

＞The DRA run accession number(s), which were under application at the time of submission, are now available and have been added to the Data Availability Statement (Section: Data Availability Statement).

 

>We have revised the Author Contributions statement following a reconfirmation among the co-authors to ensure that each author’s contributions are accurately described.

 

＞The cell density values in Figure 2h-i were listed as no./μm, so we have corrected this.

 

＞We have reconsidered the results and discussion in accordance with all reviewers' comments and added additional data. Additional data are included in parts of Figures 7-8, Tables S2-5, and Figure S2.

 

 

Following comments are commonly report and note for reviewers.

We apologize for the multiple corrections on the authors’ side and have listed the updates below for clarity.

 

＞We corrected the description of replication in the Materials and Methods.

The original text incorrectly stated that “three vines per treatment were selected.” For the compositional analyses and RNA-Seq, we used three clusters per treatment collected from different fruiting shoots within the same vine, and ten berries per cluster were pooled to generate one biological replicate (n = 3). The corresponding text has been revised in Section 3.1 (Plant material).

 

＞We had previously stated “fracture stress” but mistakenly used “fracture load” instead. We have corrected this to “fracture load” and converted it to the international standard unit, N, for inclusion in Table 1.

 

＞The DRA run accession number(s), which were under application at the time of submission, are now available and have been added to the Data Availability Statement (Section: Data Availability Statement).

 

>We have revised the Author Contributions statement following a reconfirmation among the co-authors to ensure that each author’s contributions are accurately described.

 

＞The cell density values in Figure 2h-i were listed as no./μm, so we have corrected this.

 

＞We have reconsidered the results and discussion in accordance with all reviewers' comments and added additional data. Additional data are included in parts of Figures 7-8, Tables S2-5, and Figure S2.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

      How are you!

      In this research, through the comprehensive analysis in histology, plant chemistry of cell wall and RNA-sequence, an attempt had been made to determine the changes in grape fruit tissue structure and cell wall characteristics were induced at which stage of seedless development of grape fruits induced by GA, then ultimately leading to the texture changes of fruits. The study topic was innovated to some extent, and the results would do some help to the improvement in plant physiology theory. The experiment design was reasonable basically, and the results were determined.

       However it seems the analysis in RNA-sequence needs further analysing in-depth, and it had better be focused on the pathway of the cell wall components metabolism, so it isn't focused on the DEGs related  to ethylene synthesis and cell expansion only.

       Additionally the writing of the manuscript needs further modifications.

       1. The title could be simplified further.

       2. The symbol * symbolizing the significance of the difference could be followed the bigger value in the tables or stood above the higher bar in the figures, so the tables and the figures could be simplified.

       3. Section results & discussions from line 98 to line 115, the theoretical analysis of the results were lost, please add.

       4. In line 160, the words "marked significance" could be modified into the words "significant difference", so the full text needs checking avoiding this mistake.

       5. In figures 8~11, what the letters TPM means needs introducing at first.

       6. From line 307 to line 321, the molecular formulas weren't written standardized, please pay attention to the writing of the subscript.

       7. The unit ppm wasn't rigorous suggest to use the units mg/kg or mg/L.

       8. Please add the introduce of the data statistical analysis methods.

       9. section Abbreviations could be deleted, and the abbreviation could be noted when the full name appeared at first.

 

Author Response

In this research, through the comprehensive analysis in histology, plant chemistry of cell wall and RNA-sequence, an attempt had been made to determine the changes in grape fruit tissue structure and cell wall characteristics were induced at which stage of seedless development of grape fruits induced by GA, then ultimately leading to the texture changes of fruits. The study topic was innovated to some extent, and the results would do some help to the improvement in plant physiology theory. The experiment design was reasonable basically, and the results were determined.

However it seems the analysis in RNA-sequence needs further analysing in-depth, and it had better be focused on the pathway of the cell wall components metabolism, so it isn't focused on the DEGs related  to ethylene synthesis and cell expansion only.

Additionally the writing of the manuscript needs further modifications.

>Thank you very much for taking the time to review our manuscript despite the busy year-end season. We sincerely appreciate your constructive comments and suggestions. Given the large volume of RNA-seq data, we had presented only a limited subset of the results; nevertheless, your suggestions helped us improve both the reporting of the results and the depth of the discussion. Accordingly, we have revised the manuscript as outlined below. Thank you again for your constructive comments. We hope that our revisions satisfactorily address your concerns.

 

1. The title could be simplified further.

>Thank you for the comment. We have made the following revisions, according to all reviewer comments.

Gibberellin-treated seedless cultivation alters berry fracture behavior, cell size and cell wall components in the interspecific hybrid table grape ‘Shine Muscat’

 

2. The symbol * symbolizing the significance of the difference could be followed the bigger value in the tables or stood above the higher bar in the figures, so the tables and the figures could be simplified.

>Thank you for the comment. We revised the manuscript accordingly.

 

3. Section results & discussions from line 98 to line 115, the theoretical analysis of the results were lost, please add.

>Thank you for the comment. We added descriptions in Results and Discussions

L184-201

 

4. In line 160, the words "marked significance" could be modified into the words "significant difference", so the full text needs checking avoiding this mistake.

Thank you for the comment. We added descriptions in Results and Discussions

>Thank you for the comment. We revised the manuscript accordingly.

 

5. In figures 8~11, what the letters TPM means needs introducing at first.

>Thank you for the comment. We added description about TPM.

L264 Gene expression levels were summarized as transcripts per million (TPM), a gene length– and library size–normalized measure,

 

6. From line 307 to line 321, the molecular formulas weren't written standardized, please pay attention to the writing of the subscript.

>Thank you for the comment. We revised the manuscript accordingly.

L447,455,458,459

 

7. The unit ppm wasn't rigorous suggest to use the units mg/kg or mg/L.

>Thank you for the comment. We revised the manuscript accordingly in mg/L.

 

8. Please add the introduce of the data statistical analysis methods.

>Thank you for the comment. We revised the manuscript accordingly.

L484-499

 

9. section Abbreviations could be deleted, and the abbreviation could be noted when the full name appeared at first.

>Thank you for the comment. We revised the manuscript accordingly.

 

 

Following comments are commonly report and note for reviewers.

We apologize for the multiple corrections on the authors’ side and have listed the updates below for clarity.

 

＞We corrected the description of replication in the Materials and Methods.

The original text incorrectly stated that “three vines per treatment were selected.” For the compositional analyses and RNA-Seq, we used three clusters per treatment collected from different fruiting shoots within the same vine, and ten berries per cluster were pooled to generate one biological replicate (n = 3). The corresponding text has been revised in Section 3.1 (Plant material).

 

＞We had previously stated “fracture stress” but mistakenly used “fracture load” instead. We have corrected this to “fracture load” and converted it to the international standard unit, N, for inclusion in Table 1.

 

＞The DRA run accession number(s), which were under application at the time of submission, are now available and have been added to the Data Availability Statement (Section: Data Availability Statement).

 

>We have revised the Author Contributions statement following a reconfirmation among the co-authors to ensure that each author’s contributions are accurately described.

 

＞The cell density values in Figure 2h-i were listed as no./μm, so we have corrected this.

 

＞We have reconsidered the results and discussion in accordance with all reviewers' comments and added additional data. Additional data are included in parts of Figures 7-8, Tables S2-5, and Figure S2.

 

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript describes a detailed characterization of grape berries treated with gibberellin before ripening to determine the effect of GA on berry texture. This characterization included fracture testing, histological analysis, cell wall fractionation, and RNA sequencing. The authors showed that the pericarp of GA-treated berries thickens, cell size changes, the content of uronic acids/sugars decreases, and the berries become more “fragile”. Using RNA-seq, the transcriptional program associated with berry peel and cell wall modifications in response to GA was also characterized.

The Abstract should be updated by transcriptome data. Furthermore, the results do not mention the nutritional (eating) quality of GA-treated berries, although the Abstract does (L29).

In the Introduction, the authors mention that treating grapes with gibberellins not only suppresses seed formation but also affects various physiological and biochemical aspects of berry texture, including cell wall composition in different parts of the fruit. Given the authors' RNA-seq analysis of GA-treated berries compared to untreated ones, the Introduction needs to be supplemented with currently available data on the transcriptomic response of grapes to exogenous gibberellins (for example, doi 10.3390/ijms242216279, 10.3390/ijms231911108, 10.1371/journal.pone.0095634, something else, etc.).

Results and Discussion:

Section 2.2:  

Please clarify whether and how much the size of GA-treated berries has changed compared to untreated ones?

On Fig. 2, the term "length" in relation to pericarp parts might be better replaced by "width". (?)

Is it correct to understand that the width of the pericarp increased due to a decrease in the cell density?

L122-128: GA treatment promoted radial expansion of mesocarp cells and increased cell size heterogeneity compared to untreated berries. Is there any data on whether the mesocarp cell count changed in GA-treated berries?

Section 2.2 lacks a general conclusion regarding the effect of exogenous GA on berry size and firmness. For example: "In summary, GA treatment was shown to significantly increase pericarp thickness by reducing cell density and changing cell size and/or number, leading to increased berry size and damage ability" (Or something more accurate and correct from the authors' perspective).

Section 2.3:  

A more specific conclusion or hypothesis is missing, such as ‘the obtained results may indicate reduced mesocarp cell adhesion in GA-treated berries compared to untreated ones, which could explain the increased fracturing of these berries mentioned in Section 2.1 (if understood correctly from the author's text).

Also, it would be good to mention another consequence: the reduction in the amount of sugars in GA-treated berries should probably affect their taste, making them less sweet, right or not?

Figure 10, f: There is no indication of where NT is and where GA is; judging by the color of the bars, the samples have swapped places on this graph (?) Please make corrections.

Page 11: Taking into account the data on the expression of ethylene-response genes, it is advisable to clarify whether and to what extent the rate of berry ripening changed with GA treatment. That is, at 105 days after full bloom, the untreated berries were ripe. But did the treated berries meet the ripeness parameters (or were they perhaps underripe or overripe)?

The manuscript lacks a final conclusion.

The materials and methods are described in sufficient detail.

References cited in the work are used appropriately.

 

Author Response

This manuscript describes a detailed characterization of grape berries treated with gibberellin before ripening to determine the effect of GA on berry texture. This characterization included fracture testing, histological analysis, cell wall fractionation, and RNA sequencing. The authors showed that the pericarp of GA-treated berries thickens, cell size changes, the content of uronic acids/sugars decreases, and the berries become more “fragile”. Using RNA-seq, the transcriptional program associated with berry peel and cell wall modifications in response to GA was also characterized.

>Thank you very much for taking the time to review our manuscript despite the busy year-end season. We sincerely appreciate your constructive comments and suggestions. As you pointed out, several parts of the manuscript did not provide sufficient information. We greatly appreciate your valuable suggestions to improve the manuscript, and we provide our responses below.

 

 

1) The Abstract should be updated by transcriptome data. Furthermore, the results do not mention the nutritional (eating) quality of GA-treated berries, although the Abstract does (L29).

>Thank you for the comment. We revised the Abstract accordingly.

We agree with the reviewer that the term “eating quality” can imply changes in flavor-related components (e.g., sugars, acids, and aroma). Because the present study focuses on texture and cell-wall–associated traits, we have revised the text to limit the statement to texture-related quality (mouthfeel) and avoided implying changes in flavor composition . L43

 

 

2) In the Introduction, the authors mention that treating grapes with gibberellins not only suppresses seed formation but also affects various physiological and biochemical aspects of berry texture, including cell wall composition in different parts of the fruit. Given the authors' RNA-seq analysis of GA-treated berries compared to untreated ones, the Introduction needs to be supplemented with currently available data on the transcriptomic response of grapes to exogenous gibberellins (for example, doi 10.3390/ijms242216279, 10.3390/ijms231911108, 10.1371/journal.pone.0095634, something else, etc.).

>Thank you for the comment. We revised following sentence accordingly for all reviewer’s comments. L74-89

Collectively, previous studies suggest that GA-induced seedless cultivation can in-crease firmness-related mechanical indices of ‘Shine Muscat’ berries and is accompanied by changes in mesocarp cell wall composition, particularly in pectin-rich fractions. [4,5]. However, integrated studies that combine mechanical testing with quantitative tissue descriptors, cell wall compositional analyses, and transcriptome profiling remain limited in ‘Shine Muscat’. In particular, it remains unclear at which developmental stages GA treatment drives divergences in mesocarp cell expansion and wall-remodeling processes relevant to primary-wall extensibility and middle–lamella–associated cell adhesion. Transcriptomic studies indicate that exogenous gibberellin triggers coordinated gene-expression programs in grapevine tissues relevant to seedlessness/parthenocarpy and early berry development [23, 24]. Related transcriptome shifts have also been de-scribed under practical seedless-treatment regimes in ‘Shine Muscat’ [3]. Nevertheless, transcriptome evidence explicitly linking GA-treated seedless cultivation to berry me-chanical texture traits remains limited, particularly in studies that integrate mechanical phenotyping with quantitative histology and cell wall descriptors, sugar or uronic acid contents of hemicellulose or pectin-enriched fraction .

 

Section 2.2: 

3) Please clarify whether and how much the size of GA-treated berries has changed compared to untreated ones?

>Thank you for the comment. We added berry size data to address this point. Specifically, we included the equatorial diameter (maximum transverse diameter) of GA-treated and untreated berries in Table 1 and clarified this in the manuscript. L100-102)

 

4) On Fig. 2, the term "length" in relation to pericarp parts might be better replaced by "width". (?)

>Thank you for the comment. In Fig. 2, cell dimensions were quantified along more than one axis depending on the tissue/region. To prevent confusion, we revised the y-axis label from “Length of cell” to “Cell size” and specified the directionof measurement in the legend.

 

 

5) Is it correct to understand that the width of the pericarp increased due to a decrease in the cell density?

>Thank you for the comment. Pericarp thickness increased in GA-treated berries, and cell density decreased. We interpret these changes as being primarily associated with increased mesocarp cell size (cell expansion), rather than the reduced cell density directly causing the thicker pericarp.

 

6)L122-128: GA treatment promoted radial expansion of mesocarp cells and increased cell size heterogeneity compared to untreated berries. Is there any data on whether the mesocarp cell count changed in GA-treated berries?

> Thank you for the question. We did not measure mesocarp cell number (or layer number) in this study. Accurate cell counting was not feasible because mesocarp regions are difficult to define objectively across the pericarp (e.g., near vascular bundles, exocarp and endocarp) on our plastic section thickness, which would compromise reproducibility. Instead, we quantified cell size distributions/heterogeneity and cell density as consistent descriptors of mesocarp cellular organization.

 

7) Section 2.2 lacks a general conclusion regarding the effect of exogenous GA on berry size and firmness. For example: "In summary, GA treatment was shown to significantly increase pericarp thickness by reducing cell density and changing cell size and/or number, leading to increased berry size and damage ability" (Or something more accurate and correct from the authors' perspective).

>Thank you for the comment. We added the discussions and conclusions of the section.

L184-201

 

Section 2.3: 

8) A more specific conclusion or hypothesis is missing, such as ‘the obtained results may indicate reduced mesocarp cell adhesion in GA-treated berries compared to untreated ones, which could explain the increased fracturing of these berries mentioned in Section 2.1 (if understood correctly from the author's text).

>We apologize for the insufficient description. We have made substantial additions to the text, including the addition of relevant descriptions to the introduction and the addition of data to 2.4.

 

9) Also, it would be good to mention another consequence: the reduction in the amount of sugars in GA-treated berries should probably affect their taste, making them less sweet, right or not?

>Thank you for the comment. Our experiments exclude soluble sugars present in raw berry by 80% ethanol (L443-444). We do not measure indicators such as Brix that may relate to sweetness. We cannot answer the question based on our data.

 

10) Figure 10, f: There is no indication of where NT is and where GA is; judging by the color of the bars, the samples have swapped places on this graph (?) Please make corrections.

> Thank you for the comment. We apologize for the unclear and inconsistent abbreviations. We have now unified the notation throughout the manuscript and revised the figures/text.

 

11) Page 11: Taking into account the data on the expression of ethylene-response genes, it is advisable to clarify whether and to what extent the rate of berry ripening changed with GA treatment. That is, at 105 days after full bloom, the untreated berries were ripe. But did the treated berries meet the ripeness parameters (or were they perhaps underripe or overripe)?

>Thank you for the important comment. Honda et al. (2024) conducted a comparative study using a seedless-treatment regime with plant growth regulators broadly comparable to ours and assessed ripening progression in ‘Shine Muscat’ based on sugar accumulation and aroma profiles. They concluded that non-treated berries showed delayed ripening relative to treated berries. Considering their findings together with our harvest timing, we do not interpret our data as indicating that GA-treated berries were necessarily at a more advanced ripening stage at harvest. While your point is important, a detailed discussion of ethylene–ripening relationships is beyond the scope of the present study, particularly given that grape is a non-climacteric fruit.

 

The manuscript lacks a final conclusion.

>Thank you for the comment. We added Conclusion. L501-537

 

The materials and methods are described in sufficient detail.

>Thank you for the comment. We added description accordingly for other reviewer’s comments.

 

References cited in the work are used appropriately.

>Thank you for the comment. We added references

 

 

Following comments are commonly report and note for reviewers.

We apologize for the multiple corrections on the authors’ side and have listed the updates below for clarity.

 

＞We corrected the description of replication in the Materials and Methods.

The original text incorrectly stated that “three vines per treatment were selected.” For the compositional analyses and RNA-Seq, we used three clusters per treatment collected from different fruiting shoots within the same vine, and ten berries per cluster were pooled to generate one biological replicate (n = 3). The corresponding text has been revised in Section 3.1 (Plant material).

 

＞We had previously stated “fracture stress” but mistakenly used “fracture load” instead. We have corrected this to “fracture load” and converted it to the international standard unit, N, for inclusion in Table 1.

 

＞The DRA run accession number(s), which were under application at the time of submission, are now available and have been added to the Data Availability Statement (Section: Data Availability Statement).

 

>We have revised the Author Contributions statement following a reconfirmation among the co-authors to ensure that each author’s contributions are accurately described.

 

＞The cell density values in Figure 2h-i were listed as no./μm, so we have corrected this.

 

＞We have reconsidered the results and discussion in accordance with all reviewers' comments and added additional data. Additional data are included in parts of Figures 7-8, Tables S2-5, and Figure S2.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Journal Plants (ISSN 2223-7747)

Manuscript ID plants-4064423

Type Article

Title Gibberellin-induced seedlessness-associated changes in berry texture, cell architecture, and cell wall composition in ‘Shine Muscat’ grapes

Authors Hikaru Ishikawa , Kaho Masuda , Tomoki Shibuya *

Section Horticultural Science and Ornamental Plants

Special Issue Fruit Development and Ripening

 

Dear Editor

 

The manuscript ID plants-4064423

“Gibberellin-treated seedless cultivation alters berry fracture behavior, cell size and cell wall components in the interspecific hybrid table grape ‘Shine Muscat’”

in its present form may be proceeded with for further editorial stages. Please find below editorial comments to which I ask the Editor for approval without my opinion.

Best regards

Reviewer

1.

Please include the Latin name of the species in the manuscript title after the English name.

2.

Cultivars should always be written with the Latin name of the species; please correct "….originating from small-berry cultivars including ‘Delaware’ ……[1,2]."

3.

Figure 1. Please insert a space “5.0cm”.

4.

Another note from round one

Do not begin the description of the results in the RESULTS subsection by citing Fig. 2a. It is correct to describe the results first and then cite them (see round one review).

5.

Please eliminate repeating “…(Fig. 2a)…. (Fig. 6a)” four times in succession in adjacent sentences (please correct this throughout the subsection).

6.

Please check the lettering of the subsequent photographs in Figure 2; there are b and c, and the other two photographs are unlabeled.

7.

Please insert spaces next to the numerical values on the bars.

8.

Please note the terms "…..(b) inner wall and (c) outer wall side….." elsewhere in the text and in the figure (what the authors mean).

It is unclear whether this notation suggests a cell wall (a cell wall composed of a primary wall, a secondary wall, a middle lamella, and structurally, cellulose particles, fibrillar elements, microfibrils and macrofibrils, etc.) – see the first round of reviews. Please correct this – not everyone in the audience is a biologist and familiar with histology and ultrastructure to spot errors in the text.

9.

Please complete the X-axis label for graphs.

10.

Please check whether "100% ethanol" is usually used at a different concentration, but not 100% ethanol.

11.

Please eliminate the six-times repeated citation of "…[51]…" in adjacent sentences.

12.

There are still errors in the REFERENCES: "…(Japan) ...", "…Arabidopsis ...",

“RNA-sequencing reveals biological networks during table grapevine (‘Fujiminori’) fruit development.” P “ Etc.

Authors publish their manuscripts based on their own scientific judgment. I suggest checking the text step by step for content and editorial accuracy.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

 

Journal Plants (ISSN 2223-7747)

Manuscript ID plants-4064423

Type Article

Title Gibberellin-induced seedlessness-associated changes in berry texture, cell architecture, and cell wall composition in ‘Shine Muscat’ grapes

Authors Hikaru Ishikawa , Kaho Masuda , Tomoki Shibuya *

Section Horticultural Science and Ornamental Plants

Special Issue Fruit Development and Ripening

 

Dear Editor

 

The manuscript ID plants-4064423

“Gibberellin-treated seedless cultivation alters berry fracture behavior, cell size and cell wall components in the interspecific hybrid table grape ‘Shine Muscat’”

in its present form may be proceeded with for further editorial stages. Please find below editorial comments to which I ask the Editor for approval without my opinion.

Best regards

Reviewer

1.

Please include the Latin name of the species in the manuscript title after the English name.

2.

Cultivars should always be written with the Latin name of the species; please correct "….originating from small-berry cultivars including ‘Delaware’ ……[1,2]."

3.

Figure 1. Please insert a space “5.0cm”.

4.

Another note from round one

Do not begin the description of the results in the RESULTS subsection by citing Fig. 2a. It is correct to describe the results first and then cite them (see round one review).

5.

Please eliminate repeating “…(Fig. 2a)…. (Fig. 6a)” four times in succession in adjacent sentences (please correct this throughout the subsection).

6.

Please check the lettering of the subsequent photographs in Figure 2; there are b and c, and the other two photographs are unlabeled.

7.

Please insert spaces next to the numerical values on the bars.

8.

Please note the terms "…..(b) inner wall and (c) outer wall side….." elsewhere in the text and in the figure (what the authors mean).

It is unclear whether this notation suggests a cell wall (a cell wall composed of a primary wall, a secondary wall, a middle lamella, and structurally, cellulose particles, fibrillar elements, microfibrils and macrofibrils, etc.) – see the first round of reviews. Please correct this – not everyone in the audience is a biologist and familiar with histology and ultrastructure to spot errors in the text.

9.

Please complete the X-axis label for graphs.

10.

Please check whether "100% ethanol" is usually used at a different concentration, but not 100% ethanol.

11.

Please eliminate the six-times repeated citation of "…[51]…" in adjacent sentences.

12.

There are still errors in the REFERENCES: "…(Japan) ...", "…Arabidopsis ...",

“RNA-sequencing reveals biological networks during table grapevine (‘Fujiminori’) fruit development.” P “ Etc.

Authors publish their manuscripts based on their own scientific judgment. I suggest checking the text step by step for content and editorial accuracy.