Review Reports - Leaf Developmental Stage at Harvest Affects Postharvest Senescence and Quality Parameters of Kale

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study addresses a parameter of importance to postharvest physiology: how leaf age at harvest influences senescence, green color retention, nutritional quality, and the expression of chlorophyll-catabolic genes in kale stored at 20°C. The study evaluates various parameters, including weight loss, chlorophyll content, phenolic compounds, antioxidant activity, sugars, proteins, and the expression of five genes related to chlorophyll degradation.

Upon reviewing this research paper, several questions arose, detailed below, that the authors need to address.

First, I consider the statistical analysis to be incorrect and incomplete; the analysis proposed by the authors compares the three leaf ages separately for each day using one-way ANOVA, whereas the experiment clearly involves two factors: leaf age and storage time. This approach precludes a formal assessment of the main effect of time and, crucially, the age × storage interaction, which is essential for determining whether senescence begins earlier or progresses differently depending on leaf age. Therefore, the authors should reanalyze the data using a two-way ANOVA or a linear mixed model that includes age, time, and their interaction. They must also clarify the actual experimental unit and avoid treating the three technical replicates as independent replicates.

Another issue arising from reading the paper concerns pseudoreplication and the description of replicates; the manuscript states that 60 leaves were used per stage, 15 leaves were sampled per stage and time point, and all analyses were performed using three biological and three technical replicates. However, there is no explanation of how the leaves were grouped to form each biological replicate, nor whether the 15 leaves came from independent plants or a single plant. I consider this information essential, as treating multiple leaves from the same plant as independent biological replicates would constitute pseudoreplication. Therefore, the authors must clearly state the number of plants, the number of leaves per plant, how the leaves were distributed among treatments, and exactly what constitutes a biological replicate versus a technical replicate.

Regarding the definition of leaf age, I find the distinction between age and position confusing. The categories "young," "intermediate," and "mature" were defined based on plant position and leaf size: inner leaves (approx. 20 cm), central leaves (20–30 cm), and outer leaves (35–40 cm). While this classification may be correct or practical, it conflates position, size, and presumed physiological age. Consequently, the study does not strictly demonstrate an effect of leaf age, but rather a combined effect of position, size, and developmental stage. The title, abstract, and conclusions should reflect this limitation, unless the authors provide an independent characterization of physiological age, such as days since leaf emergence, leaf area, fresh/dry weight, standardized leaf position, or photosynthetic parameters.

In a scientific paper, conclusions must be concrete and should not be conflated with the results and discussion sections. In this study, the conclusions regarding "nutritional quality" are overly extensive. While the study evaluates total phenols, flavonoids, antioxidant activity, sugars, and proteins, it fails to analyze glucosinolates, ascorbic acid, carotenoids, minerals, or specific key compounds found in kale. Consequently, the claim that young leaves exhibit "better nutritional quality" is too broad. The authors should revise this statement to align it with the parameters actually measured. Furthermore, the Folin–Ciocalteu method is not specific to phenols, and the colorimetric flavonoid assay yields only a general estimate; these limitations must be acknowledged.

Another interpretation I find excessive concerns the "molecular" aspect of the study. The expression levels of *NYC*, *NOL*, *SGR*, *PPH*, and *PaO* are used to infer that the chlorophyll degradation pathway is differentially activated. However, only transcript levels were measured via RT-qPCR; protein levels, enzymatic activity, and chlorophyll degradation metabolites were not determined. Therefore, phrases such as "activation of the chlorophyll degradation pathway" should be qualified, for instance, as "increased transcript abundance of selected chlorophyll catabolic genes." A decrease in expression at D4 or D6 does not necessarily indicate that the pathway is inactive, as it could reflect advanced cellular deterioration, RNA loss, or changes in the quality of the biological material. The authors need to address this point in their discussion.

Regarding qPCR normalization and validation, I consider the current details insufficient for publication in *Metabolites*. The manuscript merely states that *ACT* was used as the normalization gene and refers to a supplementary table for the sequences. Essential data are missing: efficiency for each primer pair, amplification specificity, Cq values, the number of biological and technical replicates, the exact relative calculation method, validation of *ACT* as a stable reference gene during senescence, and controls for the absence of RNA/cDNA contamination. The authors must include this information.

Furthermore, the authors should follow reporting guidelines such as MIQE and explain how they verified that *ACT* expression does not change across leaf ages and storage times. The stability of a single reference gene cannot be assumed during a senescence process.

On the other hand, there are inconsistencies in the results; the text states that young leaves exhibit lower expression of catabolic genes, yet *SGR* initially shows relatively higher expression in young and intermediate leaves, whereas it remains low or undetectable in mature leaves. Although this exception is mentioned, it is not adequately integrated into the general conclusion that young leaves show limited pathway activation. The authors need to discuss and rephrase this point.

Additionally, Table 2 shows chlorophyll degradation values with very high standard deviations for young leaves, indicating considerable variability. The authors should discuss this dispersion and ensure that their conclusions regarding differences between leaf ages are more robust.

Finally, reference 16 is incomplete. Moreover, the editorial template contains internal MDPI instructions within the manuscript, as well as a section for author contributions and statements that has not yet been replaced with the authors' specific information. These issues must be corrected prior to acceptance.

Author Response

Upon reviewing this research paper, several questions arose, detailed below, that the authors need to address.

First, I consider the statistical analysis to be incorrect and incomplete; the analysis proposed by the authors compares the three leaf ages separately for each day using one-way ANOVA, whereas the experiment clearly involves two factors: leaf age and storage time. This approach precludes a formal assessment of the main effect of time and, crucially, the age × storage interaction, which is essential for determining whether senescence begins earlier or progresses differently depending on leaf age. Therefore, the authors should reanalyze the data using a two-way ANOVA or a linear mixed model that includes age, time, and their interaction. They must also clarify the actual experimental unit and avoid treating the three technical replicates as independent replicates.

Thank you for this observation. We have reanalyzed all the experimental data using a two-way ANOVA, considering developmental stage and storage time as the two factors, including their interaction. For each parameter, we first assessed the significance of the developmental stage × storage time interaction. When the interaction was significant, in the case of superficial color, total sugar, phenolic content, chlorophyll content and CCGs gene expression determinations, Tukey’s multiple comparison test was performed to compare all combinations of developmental stages and storage times. When the interaction was not significant, but developmental stage was a significant factor, the statistical analysis and comparisons were presented considering the effect of developmental stage. The corresponding statistical analyses and results have been revised throughout the manuscript. We add to results section, the tables 1, 4 and 5. We changed graphics and the legends.

Another issue arising from reading the paper concerns pseudoreplication and the description of replicates; the manuscript states that 60 leaves were used per stage, 15 leaves were sampled per stage and time point, and all analyses were performed using three biological and three technical replicates. However, there is no explanation of how the leaves were grouped to form each biological replicate, nor whether the 15 leaves came from independent plants or a single plant. I consider this information essential, as treating multiple leaves from the same plant as independent biological replicates would constitute pseudoreplication. Therefore, the authors must clearly state the number of plants, the number of leaves per plant, how the leaves were distributed among treatments, and exactly what constitutes a biological replicate versus a technical replicate.

The paragraph corresponding to the Plant Material section was rewritten according to the reviewer’s suggestions.

“Leaves were categorized into three groups: (i) inner leaves (approx. 20 cm in length), representing the innerest, inmature developmental stage located at the apex; (ii) middle leaves (20–30 cm), representing the intermediate developmental stage; and (iii) outer leaves (35–40 cm), representing the fully expanded, outer stage located at the basal region of the plant. Sixty leaves from each of three developmental stages were harvested, only one leaf from each developmental per plant. After harvest, leaves were stored at 20 °C in darkness for six days at 90–95% relative humidity (RH). Samples were collected at 0, 2, 4, and 6 days of postharvest storage. At each sampling time, 15 leaves from each developmental stage were analyzed, corresponding to three biological replicates of five leaves each. After recording fresh weight and determining surface color, the leaves were frozen in liquid nitrogen, powdered, and stored for subsequent analyses”

Regarding the definition of leaf age, I find the distinction between age and position confusing. The categories "young," "intermediate," and "mature" were defined based on plant position and leaf size: inner leaves (approx. 20 cm), middle leaves (20–30 cm), and outer leaves (35–40 cm). While this classification may be correct or practical, it conflates position, size, and presumed physiological age. Consequently, the study does not strictly demonstrate an effect of leaf age, but rather a combined effect of position, size, and developmental stage. The title, abstract, and conclusions should reflect this limitation, unless the authors provide an independent characterization of physiological age, such as days since leaf emergence, leaf area, fresh/dry weight, standardized leaf position, or photosynthetic parameters.

We fully agree with the reviewer’s methodological distinction. Since we did not track the exact chronological age (e.g., days since emergence), our classification based on vertical position and size strictly reflects a combined effect of canopy stratification, architectural size, and physiological maturity. Following your valuable recommendation, we have comprehensively revised the manuscript to eliminate the potentially confusing use of "leaf age" as an independent variable. We have replaced it with "developmental stage" or "leaf position" throughout the manuscript. The categories have been redefined as inner/immature, central/intermediate, and outer/mature leaves. We believe this modification accurately reflects our experimental design and adds rigor to the study. All changes are highlighted in the revised manuscript.

Moreover, we added the following paragraph in the conclusions section to clarify this fact: “It is worth noting that the variations observed across the different leaf groups in this study represent a synergistic effect of canopy position, size, and physiological tissue maturity, rather than a strict chronological age per se. This combined effect of stratification is, however, highly representative of commercial harvesting practices for kale”.

In a scientific paper, conclusions must be concrete and should not be conflated with the results and discussion sections. In this study, the conclusions regarding "nutritional quality" are overly extensive. While the study evaluates total phenols, flavonoids, antioxidant activity, sugars, and proteins, it fails to analyze glucosinolates, ascorbic acid, carotenoids, minerals, or specific key compounds found in kale. Consequently, the claim that young leaves exhibit "better nutritional quality" is too broad. The authors should revise this statement to align it with the parameters actually measured. Furthermore, the Folin–Ciocalteu method is not specific to phenols, and the colorimetric flavonoid assay yields only a general estimate; these limitations must be acknowledged.

We rewrote the Conclusions according to the reviewer’s suggestion, and the discussion regarding the role of cytokinins was moved to the Discussion section.

We agree with the reviewer that "nutritional quality" is an overly broad term given that specific key compounds of kale, such as glucosinolates, ascorbic acid, or minerals, were not quantified in this study. Following your valuable correction, we have downscaled this claim in the Conclusions (and throughout the manuscript) to strictly align with the parameters actually measured. The sentence has been rephrased to emphasize the specific enrichment in antioxidant compounds, soluble proteins, and reducing sugars in the inner leaves, rather than making a generalized claim about overall nutritional quality.

On the other hand, we agree with the editor’s comments regarding the fact that the Folin–Ciocalteu method is not specific to phenolic compounds and that the colorimetric flavonoid assay may yield non-specific responses. However, we believe that this is well-established knowledge within the scientific community and, therefore, does not require further clarification.

Another interpretation I find excessive concerns the "molecular" aspect of the study. The expression levels of *NYC*, *NOL*, *SGR*, *PPH*, and *PaO* are used to infer that the chlorophyll degradation pathway is differentially activated. However, only transcript levels were measured via RT-qPCR; protein levels, enzymatic activity, and chlorophyll degradation metabolites were not determined. Therefore, phrases such as "activation of the chlorophyll degradation pathway" should be qualified, for instance, as "increased transcript abundance of selected chlorophyll catabolic genes." A decrease in expression at D4 or D6 does not necessarily indicate that the pathway is inactive, as it could reflect advanced cellular deterioration, RNA loss, or changes in the quality of the biological material. The authors need to address this point in their discussion.

We agree with the reviewer’s distinction between transcript levels and functional enzymatic activity. Measuring mRNA abundance via RT-qPCR provides insights into transcriptional regulation but does not automatically imply the literal activation or inactivation of the downstream metabolic pathway, as protein levels and enzymatic activities were not determined.

Following your guidance, we have made the following improvements in the revised manuscript:

We qualified all absolute claims regarding the "activation of the pathway" and replaced them with more accurate terms, such as "increased transcript abundance of selected chlorophyll catabolic genes (CCGs)"
We expanded the Discussion section to address the downregulation observed at D4/D6. We explicitly discussed that this drop in expression does not necessarily mean the pathway is inactive, but rather likely reflects advanced cellular deterioration, generalized RNA degradation, or that the critical window for de novo transcription had already passed while pre-existing enzymes remained stable.

Regarding qPCR normalization and validation, I consider the current details insufficient for publication in *Metabolites*. The manuscript merely states that *ACT* was used as the normalization gene and refers to a supplementary table for the sequences. Essential data are missing: efficiency for each primer pair, amplification specificity, Cq values, the number of biological and technical replicates, the exact relative calculation method, validation of *ACT* as a stable reference gene during senescence, and controls for the absence of RNA/cDNA contamination. The authors must include this information. Furthermore, the authors should follow reporting guidelines such as MIQE and explain how they verified that *ACT* expression does not change across leaf ages and storage times. The stability of a single reference gene cannot be assumed during a senescence process.

Thank you for this important observation. We have expanded the description of the qPCR methodology to provide additional information regarding primer performance, experimental replicates, normalization, and controls. The amplification efficiency for each primer pair has now been included in Supplementary Table 1. Three biological replicates and three technical replicates were performed for each experimental condition. In addition, three negative controls containing Milli-Q water instead of cDNA were included to verify the absence of contamination. Relative gene expression was calculated using the 2^−ΔΔCt method. We have also clarified the qPCR methodology in the revised manuscript and included the corresponding information regarding primer efficiency and experimental replication in the Supplementary Material.
Regarding the use of ACT as the reference gene, we agree that the stability of the reference gene should be experimentally assessed, particularly under conditions involving different leaf developmental stages and senescence. We therefore clarify that ACT was not selected arbitrarily. Prior to the gene expression analysis, we evaluated candidate ACT primer pairs and performed preliminary qPCR assays using samples representative of the different leaf developmental stages and storage times evaluated in this study. The resulting Cq values were subsequently analyzed using RefFinder.

On the other hand, there are inconsistencies in the results; the text states that young leaves exhibit lower expression of catabolic genes, yet *SGR* initially shows relatively higher expression in young and intermediate leaves, whereas it remains low or undetectable in mature leaves. Although this exception is mentioned, it is not adequately integrated into the general conclusion that young leaves show limited pathway activation. The authors need to discuss and rephrase this point.

We added a comment regarding SGR expression in the discussion, and another noting that young leaves show limited activation of only the NYC, PPH and PaO genes.

Additionally, Table 2 shows chlorophyll degradation values with very high standard deviations for young leaves, indicating considerable variability. The authors should discuss this dispersion and ensure that their conclusions regarding differences between leaf ages are more robust.

We would like to clarify that an error was identified in the calculation performed in the Excel spreadsheet used to estimate the chlorophyll degradation rates. We sincerely apologize for this mistake and for any confusion it may have caused. In the revised version of the manuscript, we have carefully recalculated and thoroughly checked all the corresponding calculations. Accordingly, we have replaced the previous table with the corrected version. We greatly appreciate the reviewer’s understanding and apologize once again for this oversight.

Finally, reference 16 is incomplete. Moreover, the editorial template contains internal MDPI instructions within the manuscript, as well as a section for author contributions and statements that has not yet been replaced with the authors' specific information. These issues must be corrected prior to acceptance.

Thank you for pointing this out. We have revised and completed the reference for Somogyi's method for sugar determination. The reference has been updated in the manuscript and reference list to provide the complete bibliographic information, including the journal, year, volume, issue, page range, and DOI. The corrected reference is: Somogyi M. A new reagent for the determination of sugars. J Biol Chem. 1945;160(1):61-68. doi:10.1016/S0021-9258(18)43097-9.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I have reviewed the manuscript tilted “Leaf developmental stage at harvest affects postharvest shelf life and nutritional quality of kale”. The following issues should be addressed to improve the quality of the manuscript:

The authors should provide representative photographs of kale leaves from each developmental stage (young, intermediate, and mature) at each postharvest storage time (D0, D2, D4, and D6) to clearly support the results of the study.
The title of the study mentioned “postharvest shelf life”, but shelf life data were not presented in the manuscript.
Supplementary Figure 2 is cited in the manuscript for flavonoid content, but the supplementary figure was not available for evaluation in the submitted manuscript file.
The manuscript says: “increased protease activity during senescence in Brassica species [31].”But reference 31 is: Wang W, et al. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 2004... That does not appear to be an appropriate citation for increased protease activity during Brassica senescence.
Please use italic font for gene name.
The conclusion should be considerably more concise. The current conclusion repeats many results/discussion points and then introduces cytokinin speculation.
The Discussion should be revised to better integrate the physiological, biochemical, and gene-expression results rather than mainly repeating the findings.
The authors should distinguish clearly between correlations and causal mechanisms, particularly when linking chlorophyll catabolic gene expression with postharvest senescence and yellowing. Additionally, The discussion needs stronger comparison with previous studies.
There are several writing/grammar problems. The manuscript needs substantial English editing.

Author Response

The authors should provide representative photographs of kale leaves from each developmental stage (young, intermediate, and mature) at each postharvest storage time (D0, D2, D4, and D6) to clearly support the results of the study.

Thank you for this helpful suggestion. We have prepared a representative photographic panel showing kale leaves from each developmental stage at each postharvest storage. The photographs have been included as Supplementary Figure 2 in the Supplementary Material to provide visual support for the results presented in the manuscript. Also was writing in Results in the superficial color section: The differences in color retention were visually evident in the representative photographs of the leaves at the different developmental stages and storage times. During postharvest storage, kale leaves progressively lost their green color and developed a more yellowish appearance, with these changes being more pronounced in middle and outer leaves than in inner leaves. At day 4, inner and middle leaves appear more green than outer leaves. By the end of storage (D6), inner leaves retained a greener appearance compared with middle and outer leaves (Supplementary Figure 2). These visual observations were consistent with the HUE angle measurements.

The title of the study mentioned “postharvest shelf life”, but shelf life data were not presented in the manuscript.

Thank you for this valuable comment. We agree that the original title could imply that postharvest shelf life was directly evaluated, whereas the manuscript primarily focuses on postharvest senescence and quality parameters. Therefore, we have revised the title to more accurately reflect the scope and results of the study. The new title is: “Leaf developmental stage at harvest affects postharvest senescence and quality parameters of kale.”

Supplementary Figure 2 is cited in the manuscript for flavonoid content, but the supplementary figure was not available for evaluation in the submitted manuscript file.

We add supplementary material in .ZIP

The manuscript says: “increased protease activity during senescence in Brassica species [31].”But reference 31 is: Wang W, et al. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 2004... That does not appear to be an appropriate citation for increased protease activity during Brassica senescence.

Thank you for pointing this out. We agree that the reference originally cited was not appropriate to support the statement regarding increased protease activity during senescence in Brassica species. We have corrected this citation and replaced the original reference Wang, Y. T., Yang, C. Y., Chen, Y. T., Lin, Y., & Shaw, J. F. (2004). Characterization of senescence-associated proteases in postharvest broccoli florets. Plant Physiology and Biochemistry, 42(7-8), 663-670. The corresponding reference has also been updated in the reference list.

Please use italic font for gene name.

Done.

The conclusion should be considerably more concise. The current conclusion repeats many results/discussion points and then introduces cytokinin speculation.

The conclusion was rewriting to be more concise and cytokinin speculation was replace to discussion section.

The Discussion should be revised to better integrate the physiological, biochemical, and gene-expression results rather than mainly repeating the findings.

Thank you for this valuable comment. Following the Editor’s request, we have separated the Results and Discussion sections. We have also thoroughly revised the Discussion to better integrate and interpret the physiological, biochemical, and gene-expression results.

The authors should distinguish clearly between correlations and causal mechanisms, particularly when linking chlorophyll catabolic gene expression with postharvest senescence and yellowing. Additionally, The discussion needs stronger comparison with previous studies.

In this new version of the manuscript, in line with the editor’s suggestions, we have separated the ‘Results’ and ‘Discussion’ sections, placing the results in a separate section from the discussion. Furthermore, in accordance with Reviewer 1’s suggestions, we have provided further clarification regarding the relationship between gene expression and chlorophyll degradation. In the discussion, we have included further comparisons with previous results.

There are several writing/grammar problems. The manuscript needs substantial English editing.

Thank you for this comment. The manuscript has been thoroughly reviewed and revised for English language, grammar, and writing quality. We have corrected the identified issues throughout the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, after reviewing the updated version, I confirm that all previously raised questions have been addressed and the pertinent changes made to the manuscript. The statistical analysis now employs a two-way ANOVA with a stage×time interaction, correctly documented in tables. Biological replicates have been justified, ruling out pseudoreplication. The terminology has shifted from "leaf age" to "developmental stage/position," explicitly acknowledging that the effect is a combination of factors (position, size, maturity) rather than strictly chronological age. Conclusions regarding nutritional quality have been qualified to reflect the specific parameters measured. The molecular interpretation now distinguishes between transcripts and actual enzymatic activity. The exception regarding SGR has been incorporated into the discussion, a calculation error in the chlorophyll degradation table has been corrected, and the missing reference has been added. These corrections have significantly improved the manuscript; therefore, I recommend that the work be accepted for publication.

Author Response

We greatly appreciate the positive assessment of the revisions and are pleased that the changes made have satisfactorily addressed the concerns raised during the review process. We are grateful for the reviewer’s recommendation to accept the manuscript for publication.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for providing the revised manuscript. The authors responded positively to all comments. But the following minor issues should be addressed to improve the quality of the manuscript:

Introduction:

Page 2, lines 5-7: please add related references for these sentences.

Discussion:

Page 13, lines 3-2 and 27-29, authors claimed that "previous studies" but provided only one reference for these sentences, so please add two or three more related references for these sentences. And please add related references to support line 7-8.

The phrase "determines" was used too frequently. Consider replacing it with "is associated with," "influences," or "may contribute to."

The phrase "senescence program" is somewhat broad. Since the molecular analysis is limited primarily to chlorophyll catabolic genes in this study, "senescence-associated chlorophyll catabolism" would be more precise.

The NYC/NOL comparison is interesting but currently receives disproportionate attention relative to the overall objective. It could be shortened.

The final paragraph in discussion section overlaps substantially with the conclusion. The discussion should end with the biological interpretation and limitations, while the conclusion can provide the concise practical implication.

Author Response

Thank you for providing the revised manuscript. The authors responded positively to all comments. But the following minor issues should be addressed to improve the quality of the manuscript:

Introduction:

Page 2, lines 5-7: please add related references for these sentences.

Thank you for this suggestion. Accordingly, we have revised the Introduction and added the suggested references, including Reda et al. (2021) and Łukaszyk et al. (2025), which provide relevant information regarding the nutritional quality, functional properties, and postharvest/shelf-life aspects of kale.

Reda, T., Thavarajah, P., Polomski, R., Bridges, W., Shipe, E., & Thavarajah, D. (2021). Reaching the highest shelf: A review of organic production, nutritional quality, and shelf life of kale (Brassica oleracea var. acephala). Plants, People, Planet, 3(4), 308-318.
Łukaszyk, A., Kwiecień, I., Kanik, A., Blicharska, E., Tatarczak-Michalewska, M., Białowąs, W., ... & Szopa, A. (2025). Nutritional, therapeutic, and functional food perspectives of kale (Brassica oleracea var. acephala): An integrative review. Molecules, 30(21), 4214.

Discussion:

The corresponding citations have been incorporated into the revised Discussion.

Lines 3-2

De Azevedo, C.H., Rodriguez-Amaya, D.B., 2005. Carotenoid composition of kale as influenced by maturity, season and minimal processing. J. Sci. Food Agric. 85 (4), 591–597. https://doi.org/10.1002/jsfa.1993.
Ashenafi, E. L., Nyman, M. C., Holley, J. M., Mattson, N. S., & Rangarajan, A. (2022). Phenotypic plasticity and nutritional quality of three kale cultivars (Brassica oleracea L. var. acephala) under field, greenhouse, and growth chamber environments. Environmental and Experimental Botany, 199, 104895.

Lines 7-8:

Koukounaras, A., Bantis, F., Karatolos, N., Melissas, C., & Vezyroglou, A. (2020). Influence of pre-harvest factors on postharvest quality of fresh-cut and baby leafy vegetables. Agronomy, 10(2), 172.
Chase, K., Belisle, C., Ahlawat, Y., Yu, F., Sargent, S., Sandoya, G., ... & Liu, T. (2024). Examining preharvest genetic and morphological factors contributing to lettuce (Lactuca sativa L.) shelf-life. Scientific Reports, 14(1), 6618.

The phrase "determines" was used too frequently. Consider replacing it with "is associated with," "influences," or "may contribute to."

Done

The NYC/NOL comparison is interesting but currently receives disproportionate attention relative to the overall objective. It could be shortened.

Done

All the changes in the MS are marked in red

Author Response File: Author Response.docx