Tissue-Specific Metabolic Changes During Postharvest Storage of Friariello Napoletano

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigates postharvest metabolic changes in Friariello Napoletano, revealing tissue-specific responses to different storage conditions. While the research addresses an interesting topic, there are significant concerns regarding experimental design that must be addressed before consideration for acceptance.

 

Major Concerns:

 

1. The term "reprogramming" suggests mechanistic insights into metabolic pathway regulation, which are not supported by the current data (only metabolite measurements without pathway analysis).

Recommendation: Change title to "Tissue-Specific Metabolic Changes during Postharvest Storage of Friariello Napoletano"

 

2. The study only examines two time points (2 and 20 days) and two temperatures (4°C and 10°C), making it impossible to establish reliable metabolic patterns or trends.Additional time points (e.g., 5, 10 days) and temperature conditions are needed to draw meaningful conclusions. If additional experiments cannot be conducted, the conclusions must be substantially moderated.

 

3. The claim to "identify critical control points for improving shelf life" (Line 105) is not supported by the limited data.

 

Other specific suggestions:

 

4. All subfigures (A, B, C, D) should have clearly labeled axes and legends. Significance markers should explicitly indicate which groups are being compared.

 

5. Consider combining Figure 5 (pathway map) with Figure 1 (key metabolites) for easier comparison.

 

6. All instances of "post-harvest" should be revised to "postharvest" (no hyphen), and "glucosinolate pools" should be changed to "glucosinolates" throughout the manuscript for consistency.

 

7. Remove space between "Woodrow et al." and "[27]" in Line 190.

 

Author Response

Reviewer 1

Comments and Suggestions for Authors

This study investigates postharvest metabolic changes in Friariello Napoletano, revealing tissue-specific responses to different storage conditions. While the research addresses an interesting topic, there are significant concerns regarding experimental design that must be addressed before consideration for acceptance.

Major comments

1. The term "reprogramming" in the title is misleading, as it suggests mechanistic insights that are not supported by the data. Recommendation: Change title to "Tissue-Specific Metabolic Changes during Postharvest Storage of Friariello Napoletano"

- We have changed the title according to the suggestion.

 

2. The study uses only two time points (2 and 20 days) and two temperatures (4 °C and 10 °C). This design does not allow reliable trend identification. More intermediate time points would be needed, or the conclusions should be moderated.

We thank the reviewer for this observation. The selected storage temperatures (4 °C and 10 °C) reflect commonly encountered postharvest conditions for fresh vegetables. The 4 °C setting corresponds to standard refrigeration in domestic and retail environments (Ref. 24). In contrast, 10 °C approximates the temperature typically found in vegetable drawers of household refrigerators and in commercial cold rooms where precise climate control is not maintained (Ref. 25). Importantly, 10 °C is considered the upper optimal limit for preserving the quality of climacteric vegetables such as tomatoes (Ref. 26) and is often adopted for leafy vegetables to avoid chilling injury or accelerated senescence (Ref. 27). In the Campania region, Friariello Napoletano is traditionally cultivated in succession to industrial tomato on the same fields, as a winter crop. Consequently, the same postharvest infrastructure, originally designed for tomatoes, is frequently reused, making 10 °C a practical and operationally relevant storage temperature in the local context. We have clarified these points in the revised Methods section, and we have softened the conclusions  and abstract where appropriate.

 

3. The conclusion claiming to “identify critical control points for improving shelf life” is not supported by the limited dataset.

- We thank the reviewer for this observation. In response, we have revised the relevant sentence at the end of Introduction.

 

4. All subfigures should have clearly labeled axes and legends. Significance markers should indicate the compared groups.

-We thank the reviewer for this valuable suggestion. In response, we have revised the color scheme used in the graphs to improve clarity and consistency across treatment groups. We have also updated the figure caption of all figures to explicitly explain the meaning and order of the bar colors. In the lower panels, we have added explanatory text below the x-axis to clarify the correspondence between treatments and colors. To avoid redundancy and preserve graphic balance, we chose not to repeat the full labeling under each individual subpanel. Instead, we clearly state the order of conditions in the caption, which is consistent across all panels. We have also changed the lettering in the figures.

5. Suggest combining Figure 5 (pathway map) with Figure 1 for better visual comparison.

-We have tried to do it. However, integrating these two figures into a single panel would result in an overly complex and visually overloaded composition, which may reduce clarity and interpretability. Instead, we chose to maintain the separation between quantitative data (Figure 1) and pathway-level interpretation (Figure 5) to preserve graphical readability. We have ensured that the correspondence between metabolic data and pathway components is clearly described in the caption.

6. Typographic issues: use "postharvest" (no hyphen); change "glucosinolate pools" to "glucosinolates" consistently.

-We thank the reviewer for pointing out these typographic inconsistencies. We have revised the manuscript to consistently use “postharvest” without a hyphen throughout the text. In addition, the term “glucosinolate pools” has been replaced with the more appropriate and concise “glucosinolates” in all instances.

7. Minor formatting: remove extra space in "Woodrow et al. [27]”.

-Thank you. It has been done.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents very interesting content. The introduction and methodology are appropriately and thoroughly addressed. The results are clearly presented, and the discussion is relevant and well-articulated. I have included several suggestions in the attached PDF; however, the most important one concerns the Tukey test lettering in both the figures and the supplementary material. The current labeling is somewhat unclear, as the order and hierarchy of the letters are not easily understood, which may lead to confusion for the reader. With these revisions, the manuscript would be suitable for acceptance.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

This manuscript presents very interesting content. The introduction and methodology are appropriately and thoroughly addressed. The results are clearly presented, and the discussion is relevant and well-articulated. I have included several suggestions in the attached PDF; however, the most important one concerns the Tukey test lettering in both the figures and the supplementary material. The current labeling is somewhat unclear, as the order and hierarchy of the letters are not easily understood, which may lead to confusion for the reader. With these revisions, the manuscript would be suitable for acceptance.

1. Tukey test lettering in figures and supplementary materials is unclear. The order and hierarchy of the letters are confusing.

We thank the reviewer for this valuable suggestion. We have changed the lettering respecting the hierarchy.

2. General formatting and clarity suggestions in the annotated PDF.

2.1 tissue or organ?

-We thank the reviewer for raising this semantic point. Although both inflorescences and leaves are plant organs in anatomical terms, we chose the term “tissue-specific” to emphasize the functional and metabolic distinctiveness between these structures, as commonly used in plant metabolomics and postharvest studies. Nonetheless, we have verified the manuscript to ensure internal consistency in terminology and clarified in the Introduction that the comparison is based on two distinct plant organs.

 

2.2 To include the importance of Friariello Napoletano.

-We thank the reviewer for the suggestion. The opening of the abstract has been revised to directly introduce Friariello Napoletano, its importance, and the objectives of the study, avoiding the previous generic phrasing.

2.3 References.

-We have added references supporting the nutritional and cultural importance of Friariello Napoletano, including studies on its glucosinolate profile, response to fertilization, and postharvest quality (refs. 1-3).

-To support the statement on the global recognition of Friariello Napoletano (marketed abroad as rapini or broccoli rabe), we have cited a relevant article from the Providence Journal (2019). This source highlights how the crop is widely appreciated for its nutritional value and is featured in lifestyle and culinary magazines, confirming its popularity beyond Italy (ref. 16).

2.4 Delete this information.

We appreciate the reviewer’s suggestion and agree that the sentence at the end of the Introduction was redundant. However, other reviewers emphasized the importance of explicitly defining the research scope within the Introduction. We believe that maintaining this sentence ensures clarity for the reader without detracting from the overall flow.

2.5 Which design? completely randomized? How many treatments? Tissue or organ?

We thank the reviewer for the valuable questions. The experiment followed a completely randomized design. Treatments consisted of two storage temperatures (4 °C and 10 °C) and two storage durations (2 and 20 days), for a total of four postharvest treatments, plus the freshly harvested control, applied to two tissue types (inflorescences and leaves). This resulted in ten experimental groups. The term 'tissue' was used consistently to refer to plant organs (e.g., inflorescences vs. leaves), as clarified in the last part of Introduction.

2.6 I suggest to include a comparison test between organs (inflorescences vs leaves).

We appreciate the reviewer’s comment regarding the need to compare organs (inflorescences vs. leaves). We clarify that all biochemical data were analyzed using a two-way ANOVA, with tissue type and postharvest treatment as main factors. The model now includes both interaction terms (Tissue × Treatment and Inflorescence × Leaves) to assess tissue-specific responses and overall differences between organs. While the statistical approach was correctly indicated in all figure legends and supplementary tables, we acknowledge that the Methods section incorrectly mentioned a one-way ANOVA. This has now been corrected in the revised manuscript.

2.7 Delete. This is methodology.

As requested, we have removed the introductory methodological sentence from the Results section. Only minimal adjustments were made in the Materials and Methods to ensure that all essential information is clearly stated without redundancy.

2.8 Please revise Tukey letters in table S1, some letters are wrong

Thank you very much for all your comments. The lettering has been corrected throughout both in tables and figures, and the interaction between tissues/organs is now reported in addition to the tissue × treatment interaction.

2.9 I'm not sure if you are analyzing it this way; the Tukey letters are confusing. Please check."

Thank you for your observation. We have clarified the statistical annotation in the figure legends. Specifically, we now state that after applying the two-way ANOVA: “When significant effects were detected, treatment means were compared within each tissue type using Tukey’s HSD post hoc test at a significance level of p < 0.05; significant differences are indicated by different lowercase letters. Tissue × treatment (T × t) and Inflorescence × Leaves (I × L) interactions were also tested, and significance is indicated by asterisks ( p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001), and non-significance by ns.”*

2.10 PHD – postharvest day

We thank the reviewer for the observation. In the revised version, we included the full term “postharvest days” before the abbreviation. The abbreviation (PHD) is then used consistently throughout the text.

2.11 Units – µmol

We thank the reviewer for the observation. We have corrected “µmo” to “µmol”.

2.12 To explain differences between temperatures, and to include the role of ethylene

-We thank the reviewer for this insightful comment. The rationale for selecting the two storage temperatures (4 °C and 10 °C) is detailed in the Materials and Methods section. We also appreciate the reviewer’s suggestion regarding the interpretation of chlorophyll degradation, which prompted us to highlight better that, particularly in leaves, the more severe pigment loss observed at 4 °C is likely due to cold-induced stress rather than ethylene alone. Nevertheless, as noted by Saltveit (1999), basal levels of ethylene can still contribute to chlorophyll degradation in non-climacteric vegetables, such as those in the Brassicaceae family, and this factor has now been acknowledged in the revised Discussion.

2.13 To compare with another studies

-We thank the reviewer for the suggestion. We have replaced the citation (El-Ramady et al., 2015) with a more appropriate and widely accepted reference (Smirnoff, 2000), which specifically addresses the role of ascorbate redox balance in oxidative stress during postharvest metabolism.

2.14 Reference

-Although classical nitrifiers like Nitrosomonas have not been identified, Proteobacteria (to which belong nitrifying bacteria, not only belonging to Nitrosomonas and Nitrobacter genera) represent about 75% of the Brassicaceae microbiota (Ref. 48) and indeed may include heterotrophic bacteria capable of ammonia oxidation during postharvest storage.

2.15 Tissues/Organs

-The last part of the discussion has been revised.

2.16 Update many references

-The references have been updated.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents the Tissue-Specific Metabolic Reprogramming during Postharvest Storage of Friariello Napoletano. The results showed that both inflorescences and leaves exhibited clear biochemical changes during postharvest storage at 4°C and 10°C, including pigment loss, antioxidant depletion, and amino acid turnover. These findings provide insights into the metabolic adjustments during postharvest senescence and highlight key control points for improving shelf life and maintaining the nutritional quality of this traditional Mediterranean vegetable. In general, this paper has a complete structure, precise figures and tables, but some details need to be corrected. I offered the major revision to the authors and questions and suggestions as follows:

1. Line 135: Why does the authors choose “……conditions at 4 °C and 10 °C, and sampled at 2 and 20 days after harvest.” in this work? Why is it stored for 2 and 20 days instead of other days?
2. Respiration rate and ethylene production are important indicators for evaluating the physiological metabolism of vegetables. I suggest the authors to determine the respiration rate and ethylene production of Friariello Napoletano during storage.
3. Line 159: Changes “13,000 rpm” and “minutes” to “13,000 × g” and “min”, respectively. Please check throughout the manuscript.
4. Line 166: Change “U/mL” to “U mL−1”, please check throughout the manuscript.
5. Line 167: Please provide reference.
6. Line 170: Change “mg g⁻¹” to “g kg−1”, the same below.
7. Line 185: Full name, “BSA” first time of usage, please check throughout the manuscript.
8. Line 189: Change “µmol g⁻1” to “mmol kg−1”; What DW means? (dry weight?).

The previous text used fresh weight, please unify throughout the manuscript.

9. Line 208: Please supplement the visual appearance of Friariello Napoletano during storage.
10. Line 204-208: Suggest deleting this paragraph.
11. The color indexes for lightness (L) and chromaticity (a and b) should be measured.
12. Line 323: What PHD means?
13. Line 458-460: Suggest providing a precise summary of important results. Please rewrite the conclusion section.
14. The format of references should be checked and revised according to the guide of author.

Author Response

Reviewer 3:

The manuscript presents the Tissue-Specific Metabolic Reprogramming during Postharvest Storage of Friariello Napoletano. The results showed that both inflorescences and leaves exhibited clear biochemical changes during postharvest storage at 4°C and 10°C, including pigment loss, antioxidant depletion, and amino acid turnover. These findings provide insights into the metabolic adjustments during postharvest senescence and highlight key control points for improving shelf life and maintaining the nutritional quality of this traditional Mediterranean vegetable. In general, this paper has a complete structure, precise figures and tables, but some details need to be corrected. I offered the major revision to the authors and questions and suggestions as follows:

1. Line 135: Why does the authors choose “……conditions at 4 °C and 10 °C, and sampled at 2 and 20 days after harvest.” in this work? Why is it stored for 2 and 20 days instead of other days?

• The selected storage temperatures (4 °C and 10 °C) reflect commonly encountered postharvest conditions for fresh vegetables. The 4 °C setting corresponds to standard refrigeration in domestic and retail environments (Ref. 24). In contrast, 10 °C approximates the temperature typically found in vegetable drawers of household refrigerators and in commercial cold rooms where precise climate control is not maintained (Ref. 25). Importantly, 10 °C is considered the upper optimal limit for preserving the quality of climacteric vegetables such as tomatoes (Ref. 26), and is often adopted for leafy vegetables to avoid chilling injury or accelerated senescence (Ref. 27). In the Campania region, Friariello Napoletano is traditionally cultivated in succession to industrial tomato on the same fields, as a winter crop. Consequently, the same postharvest infrastructure, originally designed for tomatoes, is frequently reused, making 10 °C a practical and operationally relevant storage temperature in the local context. We have clarified these points in the revised Methods section.

2. Respiration rate and ethylene production are important indicators for evaluating the physiological metabolism of vegetables. I suggest the authors to determine the respiration rate and ethylene production of Friariello Napoletano during storage.

• We thank the reviewer for this valuable suggestion. The determination of respiration rate and ethylene production is indeed important for understanding postharvest physiology and will be addressed in future studies. In the present work, while these parameters were not directly measured, we have expanded the Discussion to consider their likely contribution.

As now stated in the revised text: “Interestingly, this degradation was more severe at 4 °C than at 10 °C, suggesting that low-temperature stress may accelerate senescence in leaves. Although FN is a non-climacteric vegetable, basal levels of ethylene are still physiologically relevant in Brassicaceae, including broccoli and cabbage, where ethylene has been shown to accelerate chlorophyll degradation and senescence [36].”

We also appreciate the reviewer’s insight, which helped us clarify that, particularly in leaves, the more severe pigment loss observed at 4 °C is likely due to cold-induced stress rather than ethylene alone. Nevertheless, as noted by Saltveit (1999), basal ethylene levels can still contribute to chlorophyll degradation in non-climacteric vegetables such as those in the Brassicaceae family, and this consideration has been explicitly included in the revised Discussion.

3. Line 159: Changes “13,000 rpm” and “minutes” to “13,000 × g” and “min”, respectively. Please check throughout the manuscript.

• The rotor used in our Eppendorf centrifuge (model 5418 R) has a radius of 7.9 cm, and at 14,000 rpm this corresponds to approximately 15,000 × g. Accordingly, we have updated the manuscript to report relative centrifugal force (RCF × g) rather than rpm. We have also replaced “minutes” with “min” throughout the text, as requested.

4. Line 166: Change “U/mL” to “U mL−1”, please check throughout the manuscript.

• We have corrected as required.

5. Line 167: Please provide reference.

• We have included the reference.

6. Line 170: Change “mg g⁻¹” to “g kg−1”, the same below.

• We have corrected it in the text and in all the figures and tables

7. Line 185: Full name, “BSA” first time of usage, please check throughout the manuscript.

• “bovine serum albumin (BSA)” had already been introduced in the sub-paragraph 2 Reagents and Laboratory Materials

 

8. Line 189: Change “µmol g⁻1” to “mmol kg−1”; What DW means? (dry weight?).

The previous text used fresh weight, please unify throughout the manuscript.

• It was wrong. We have expressed all for FW. We have corrected it.

9. Line 208: Please supplement the visual appearance of Friariello Napoletano during storage.

• We thank the reviewer for the suggestion. Unfortunately, we do not have images documenting the visual appearance of Friariello Napoletano during storage. Due to internal safety protocols still in place until December 2023, access to confined spaces such as climate rooms was restricted to one person at a time. As a result, sample handling was performed individually, and immediate snap-freezing in liquid nitrogen was prioritized over photographic documentation. Nevertheless, we have included representative images of the plants in the field and of the harvested edible parts (inflorescences and leaves) also sused for analyses at time zero. These are now available in the Graphical Abstract.

10. Line 204-208: Suggest deleting this paragraph.

• As requested, we have removed the introductory methodological sentence from the Results section. Only minimal adjustments were made in the Materials and Methods to ensure that all essential information is clearly stated without redundancy.

 

11. The color indexes for lightness (L) and chromaticity (a and b) should be measured.

• We thank the reviewer for this valuable suggestion. Unfortunately, color measurements (L*, a*, b*) were not included in the current experimental design. However, we fully acknowledge their relevance in assessing visual quality during postharvest storage and plan to incorporate this analysis in future experiments that, due to the seasonal nature of the crop, will be conducted in future studies planned next Autumn.

12. Line 323: What PHD means?

• We thank the reviewer for the observation. In the revised version, we included the full term “postharvest days” before the abbreviation. The abbreviation (PHD) is then used consistently throughout the text.

 

13. Line 458-460: Suggest providing a precise summary of important results. Please rewrite the conclusion section.

• We thank the reviewer for the suggestion. The conclusion section has been revised to provide a more precise and structured summary of the main findings, as requested. The new version highlights tissue-specific responses, key biochemical changes, and potential implications for storage strategies.

14. The format of references should be checked and revised according to the guide of author.

• We revised the references as required.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has revised the manuscript and it is ready for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

After the revision, I think that it could be accept.