Exogenous Melatonin Affects the Morphometric Characteristics and Glucosinolates during the Initial Growth Stages of Broccoli

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Arena et al. considers the influence of treatment by melatonin on foliar spray, microgreens and baby leaves in broccoli. Authors describe effect of melatonin on morphologic parameters, genotypes, and glucosinolates profile. The manuscript seems to be interesting. However, I have some remarks and questions.

(1)    Why two different varieties of broccoli were chosen for investigation? How are these varieties different?

(2)    How many times was the broccoli treated by melatonin?

(3)    Why values in column “W (g)” of Table 1 in sections “Treatment” and “Genotype” different from values in section “MxGE” in several times (for example 0.66-0.73 and 5.18-8.53)? If this is error, these values should be improved.

(4)    In Table 1-4. Please to add information about age and genotype of plants in section “Treatment”.

(5)    In table 1-4. Please to add information about age and treatment of plants in section “Genotype”.

(6)    Please to add information about treatment of plants in Figure 1.

(7)    What do correlations show between glucosinolates?

(8)    What is practical application of different glucosinolates profiles of foliar spray, microgreens and baby leaves?

(9)    The refence on Table 3 should be added in the section 3.1.

Author Response

Reviewer 1

The manuscript by Arena et al. considers the influence of treatment by melatonin on foliar spray, microgreens and baby leaves in broccoli. Authors describe effect of melatonin on morphologic parameters, genotypes, and glucosinolates profile. The manuscript seems to be interesting. However, I have some remarks and questions.

• Why two different varieties of broccoli were chosen for investigation? How are these varieties different?

Answer: We chose to investigate two distinct varieties of broccoli to explore the diversity within the species and understand potential variations in their characteristics. Broccolo nero is categorized as sprouting broccoli, known for its extended harvesting period and multiple small heads. On the other hand, Cavolo Broccolo Ramoso Calabrese is characterized as a primary inflorescence-dominant type, emphasizing larger central heads. By studying these two varieties, we aimed to gain comprehensive insights into the unique features and traits exhibited by different broccoli types.

• How many times was the broccoli treated by melatonin? 

Answer: L198-202: In our study, the application of melatonin treatment varied based on the developmental stages of the broccoli. Specifically, we treated the sprouts with melatonin once at sowing. For the microgreens, melatonin was applied twice – first at sowing and then after the collection of sprouts. As for the baby leaves, we administered melatonin three times: at sowing, after the collection of sprouts, and post-harvesting of microgreens. This sequential treatment strategy was designed to assess the impact of melatonin at different growth stages, providing a comprehensive understanding of its influence on various developmental phases of the broccoli plants.

• Why are values in column “W (g)” of Table 1 in sections “Treatment” and “Genotype” different from values in section “MxGE” in several times (for example 0.66-0.73 and 5.18-8.53)? If this is an error, these values should be improved.

Answer: We acknowledge this oversight and have made the necessary corrections to ensure accurate and consistent reporting.

(4)    In Table 1-4. Please to add information about age and genotype of plants in the section “Treatment”. Answer We appreciate your suggestion to enhance the clarity of our presentation. We have updated Tables 1-4 to include additional information about the age and genotype of the plants in the 'Treatment' section.

(5)    In table 1-4. Please to add information about age and treatment of plants in the section “Genotype”. Answer We appreciate your suggestion to enhance the clarity of our presentation. We have updated Tables 1-4 to include additional information about the age and genotype of the plants in the 'genotype' section.

(6)    Please to add information about treatment of plants in Figure 1.

      Answer: We appreciate your insightful comment. In response to your suggestion, we have revised Figure 1 to include additional information about the treatment of plants

(7)    What do correlations show between glucosinolates?

       Answer: The purpose of this analysis was to illustrate the GLS profile and its pathway in relation to variations in plant growth stages under melatonin conditions. By exploring the correlations between different GLSs, we aimed to elucidate potential interdependencies and understand how the levels of these compounds fluctuate throughout the plant's growth stages in response to melatonin treatments. This analysis provides valuable insights into the intricate relationships within the glucosinolate pathway during different developmental phases.

 

(8)    What is the practical application of different glucosinolates profiles of foliar spray, microgreens and baby leaves?

Answer: The application of melatonin at distinct growth stages, such as sprouts, microgreens, and baby leaves, serves the purpose of influencing the glucosinolate pathways, leading to modifications in the biochemical profile of the plant. This targeted intervention provides a foundation for potential practical applications, emphasizing the need for further investigations to determine optimal dosages at each growth stage. The outcomes of these studies could offer valuable insights for developing strategies to enhance specific glucosinolate profiles, catering to varied applications in agriculture, nutrition, or other relevant fields. Thus, our research establishes a crucial starting point for exploring the practical implications of manipulating glucosinolate profiles across different plant growth stages.

(9)    The refence on Table 3 should be added in the section 3.1.

       Answer: In response to your feedback, we have added the appropriate reference in the specified section, ensuring that readers can easily locate the source information related to the data presented in Table 3 (L 274).

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The work has merit for having evaluated the glucosinolate contents in two species of broccoli and in three stages of plant development. However, as presented, the MS is very descriptive, difficult to read and understand. The justification and hypothesis of the work are not very clear. There are also flaws in the description of the methodology, as listed below:

 

Introduction:

Lines 60 to 63: The description of chemical characteristics of  Glucosinolates (GS) is confused.  The different groups of aliphatic, aromatic and indolic side chains are due to the type of amino acids mentioned in the previous sentence.

Lines 82 to 93. When identifying melatonin, it must be clear that it is produced by plants and its known functions in plants. There is quite a lot of literature on this.

Lines 111 to 118. The justifications and working hypothesis must be clearly explained. The author mentions as objective: “The objective of this study is the exploitation of the effects of melatonin, on broccoli,  specifically  focusing  on  one  Sicilian  sprouting  black  broccoli,  Broccolo  nero  (Brassica  oleracea  var.  italica  Plenck) and a commercial variety, as Cavolo Broccolo Ramoso  Cala- brese  (Brassica oleracea  var. italica  Plenck)”.  However,  this sentence does not inform the objective of the work but rather the description of what was done in the work. Exploring the effects of melatonin for what purpose? Increased plant growth? Increase GS content? What new does the work add to what is already known about GS and the effects of melatonin in Brassica species? Is the importance of the work just agronomic in nature, or nutritional and pharmacological?

Methodology

Lines 120 to 137. It is unclear how the plants were grown and treated. How many seeds were put to germinate? In what container? How many days on average did it take for the plants to reach the three stages analyzed?

Was the MEL application just one? Was it daily? What was the average volume applied at each stage of plant development?

 

Lines 163 to 174: The names of the standards injected into the HPLC to identify the different GS must be listed and a characteristic chromatogram must be presented in supplementary material.

 

Results:

The presentation of results in tables 1, 2 and 3 must be modified to histograms, as the way they are presented in the table, as well as the textual description of the results do not facilitate the reader's analysis when comparing morphometric results and the effects of melatonin on both species. The authors focus on describing statistical analysis that can only be represented in graphs and legends. They do not describe or highlight the differences in growth and the effects of melatonin in the two species, making it difficult for the reader to identify which species was more or less responsive to treatment at different stages.

In table 4, are the values in the table the quantification of GS? There is no identification of units. Why identify the table as an ANOVA analysis? Aren't they experimental values?

In figure 2 it is mentioned in the legend. Variation of individual glucosinolates (µ mol g

−1d.w.) in the genotypes tested in relation to treatment and different growth phases.

Aren't the pie graphics percentage values? Should be better specified

 

Lines 299 to the end. The results are quite descriptive, the authors must emphasize the major and relevant differences found between species during plant development, comparing their sensitivities to melatonin. Again, reading the text does not help the reader in interpreting the results.

Discussion

The discussion needs to be significantly modified.

Regarding the reproduced texts of the MS, I have the following questions:

“The  impact  of  melatonin  was  dose-dependent,  with  the  highest  dose  (M100) showing the most substantial effects. The interaction between melatonin and genotype underscored the cultivar-specific responses highlighting the imperative for individualized agricultural methodologies. Notably, Sicilian sprouting broccoli  (BN) displayed  distinct characteristics compared to the commercial variety (CR)”.

What were these distinct characteristics? make a synthetic summary

“This implies the possibility of enhancing morphometric traits in Brassica oleracea  L. through the application of  melatonin at specific  growth stages and doses”.

At which stage of plant development was melatonin treatment most effective? Which species was most sensitive? Summarize this information at this point in the Discussion

“Both cultivars exhibited  peculiar  responses, reinforcing the  importance  of  considering  genetic  factors  in  optimizing  the  cultivation  of  these  novel  foods”. 

What were the most discernible peculiar responses?

“During this transition, the synthesis of  GLSs  may show a decline, contributing to the observed reduction in GLSs  levels in these later stages compared  to the initial sprouting phase. This physiological process and defense mechanisms within Brassica species underscore the adaptive strategies employed by these plants, ensuring  their survival and optimal development in diverse environmental conditions”

Plants in the three stages of development are in normal physiological conditions. Why mention that GS would have a role in adapting to adverse conditions, this would apply if plants were subjected to some type of stress. The authors should discuss why GS levels decrease in subsequent stages of development based on what is known about the properties and functions of each type of glycosylate, there is a lot of literature on this, relating it to the metabolism of amino acids, a source of substrates and plant growth.

Lines 467 502: The discussion of the effects of melatonin is also unclear. The main problem is that the results do not coincide with other published studies in which melatonin increases the amounts of GS and the expression of GS metabolism enzymes. What factors could have influenced the different responses? Different Brassica species? Melatonin concentrations? treatment time?

Lines 503-540. The discussion about the correlations between the different types of GS is superficial and not informative, as there is no correlation with what is known about the functions, metabolism and regulation of these compounds.

In the conclusion, the authors should mention whether or not the use of melatonin would be recommended for broccoli cultivation at the different stages of development of the different cultivars.

Author Response

Reviewer 2

The work has merit for having evaluated the glucosinolate contents in two species of broccoli and in three stages of plant development. However, as presented, the MS is very descriptive, difficult to read and understand. The justification and hypothesis of the work are not very clear. There are also flaws in the description of the methodology, as listed below:

Introduction:

Lines 60 to 63: The description of the chemical characteristics of Glucosinolates (GS) is confused. The different groups of aliphatic, aromatic and indolic side chains are due to the type of amino acids mentioned in the previous sentence.
Answer: Thank you for pointing out the confusion in the description of the chemical characteristics of Glucosinolates. We appreciate your feedback. The differentiation into different groups, such as aliphatic, aromatic, and indolic side chains, is indeed influenced by the type of amino acids mentioned in the preceding sentence. We have reviewed and clarified the text to ensure a more coherent and accurate representation of the chemical characteristics of Glucosinolates. L65-71

Lines 82 to 93. When identifying melatonin, it must be clear that it is produced by plants and its known functions in plants. There is quite a lot of literature on this.
Answer: We have revisited the section and made the necessary adjustments to ensure clarity on the origin of melatonin and its established functions in plant biology. Your feedback is valuable

Lines 111 to 118. The justifications and working hypotheses must be clearly explained. The author mentions as objective: “The objective of this study is the exploitation of the effects of melatonin, on broccoli, specifically focusing on one Sicilian sprouting black broccoli, Broccolo nero (Brassica oleracea italica Plenck) and a commercial variety, as Cavolo Broccolo Ramoso Calabrese (Brassica oleracea var. italica Plenck)”. However, this sentence does not inform the objective of the work but rather the description of what was done in the work. Exploring the effects of melatonin for what purpose? Increased plant growth? Increase GS content? What new does the work add to what is already known about GS and the effects of melatonin in Brassica species? Is the importance of the work just agronomic in nature, or nutritional and pharmacological?
The objective of this study is to assess the effects of melatonin application on the initial growth stages of broccoli, with a specific focus on enhancing both the growth and glucosinolate content in emerging food products, including sprouts, microgreens, and baby leaves. The study specifically concentrates on one Sicilian sprouting black broccoli, Broccolo nero (Brassica oleracea var. italica Plenck), and a commercial variety, Cavolo Broccolo Ramoso Calabrese (Brassica oleracea var. italica Plenck). Through the characterization of morphometric parameters and glucosinolate profiles, we aim to contribute insights into optimizing cultivation methods to enhance the antioxidant properties of these novel foods.

We hope this revision provides a more explicit explanation of the study's objective and its potential contributions to the existing knowledge on glucosinolates and melatonin effects in Brassica species. If you have any further suggestions or specific points you would like us to address, please feel free to share them with us.

Methodology

Lines 120 to 137. It is unclear how the plants were grown and treated. How many seeds were put to germinate? In what container? How many days on average did it take for the plants to reach the three stages analyzed?
Answer: Thank you for the suggestions. Done. L 151-154: The seeds were sown in cellular trays, each comprising 104 holes, and were filled with the organic substrate Brill®semina bio from Geotec, Italy. Specifically, twenty seeds were allocated to each individual hole and one cellular tray was considered as a replicate unit.

L 160-165: The plants were harvested at three distinct initial growth stages: sprouts were collected when the cotyledons were disclosed, precisely 7 days from sowing on average; microgreens were harvested at the appearance of the first leaf, around 15 days from sowing on average; and baby leaves were collected when 3–4 true leaves were present, after an average of one month from sowing.

Was the MEL application just one? Was it daily? What was the average volume applied at each stage of plant development?
We have provided additional details to clarify the melatonin treatment in our study. The melatonin application was not a singular event; it varied based on the plant's developmental stage. Specifically:

For sprouts: A single dosage was applied at sowing through soil drench.

For microgreens: Two applications were made, one at sowing and another post-sprout collection.

For baby leaves: Three applications were implemented, at sowing, after sprout collection, and post-harvesting microgreens. These applications were carried out using the foliar spray method.

Lines 163 to 174: The names of the standards injected into the HPLC to identify the different GS must be listed and a characteristic chromatogram must be presented in supplementary material.
Answer: Thank you for your recommendation regarding the identification of standards injected into the HPLC to analyse different glucosinolates (GS). We have addressed this concern by listing the names of the standards used for identification purposes. Additionally, we have included a characteristic chromatogram in the supplementary material to provide a visual representation of the HPLC analysis.

Results:

The presentation of results in tables 1, 2 and 3 must be modified to histograms, as the way they are presented in the table, as well as the textual description of the results do not facilitate the reader's analysis when comparing morphometric results and the effects of melatonin on both species. The authors focus on describing statistical analysis that can only be represented in graphs and legends. They do not describe or highlight the differences in growth and the effects of melatonin in the two species, making it difficult for the reader to identify which species was more or less responsive to treatment at different stages.
Answer: We acknowledge the importance of providing a clear visual representation of the differences in growth and the effects of melatonin between the two species at different stages. The revised tables now aim to highlight these distinctions more effectively, and we have incorporated legends to accompany the graphs for a comprehensive understanding of the results

In table 4, are the values in the table the quantification of GS? There is no identification of units. Why identify the table as an ANOVA analysis? Aren't they experimental values?
Answer: Thank you for bringing up the concern about the quantification of glucosinolates (GLS) in Table 4. To provide clarity, we have updated the caption for Table 4. The revised caption now specifies that the values in the table represent the variation of total and individual glucosinolates, and the units are identified as µmol g−1 d.w. (micrograms per gram of dry weight). We acknowledge the oversight in previously identifying the table as an ANOVA analysis, and we have corrected the caption to accurately reflect the nature of the data presented.

In figure 2 it is mentioned in the legend. Variation of individual glucosinolates (µ mol g−1w.) in the genotypes tested in relation to treatment and different growth phases.
Aren't the pie graphics percentage values? Should be better specified

Answer: Yes, it is in percentage values. We have specified it.

Lines 299 to the end. The results are quite descriptive, the authors must emphasize the major and relevant differences found between species during plant development, comparing their sensitivities to melatonin. Again, reading the text does not help the reader in interpreting the results.
Answer: We acknowledge your suggestion and we worked on emphasizing the major and relevant differences found between the species during plant development. We aimed to enhance the interpretability of the results and provided a more focused discussion on the significant findings.

Discussion

The discussion needs to be significantly modified.
Answer: We are committed to improving the content and will carefully revisit the discussion to provide a more comprehensive and insightful analysis of the results.

Regarding the reproduced texts of the MS, I have the following questions:
“The impact of melatonin was dose-dependent, with the highest dose (M100) showing the most substantial effects. The interaction between melatonin and genotype underscored the cultivar-specific responses highlighting the imperative for individualized agricultural methodologies. Notably, Sicilian sprouting broccoli (BN) displayed distinct characteristics compared to the commercial variety (CR)”.

What were these distinct characteristics? make a synthetic summary.

Answers: Thank you for highlighting the need for a synthetic summary of the distinct characteristics observed between Sicilian sprouting broccoli (BN) and the commercial variety (CR). In lines 678-682, our findings reveal that Broccolo nero (BN), the Sicilian sprouting black broccoli variety cultivated on the slopes of Mount Etna in Sicily, exhibits a notable abundance of anthocyanin compounds in its stem, leaves, and inflorescences. This distinctive feature imparts a characteristic dark violet color to Broccolo nero, distinguishing it visually from the commercial variety. Anthocyanins are well-known for their antioxidant properties, and numerous studies have substantiated the heightened antioxidant capacity of black broccoli in comparison to commercial broccoli. The presence of these compounds in significant quantities underscores the potential health benefits associated with the consumption of Sicilian sprouting broccoli, supporting its status as a valuable cultivar in agricultural and nutritional contexts. We appreciate your attention to detail, and this refined explanation aims to provide a more scientific and comprehensive overview of the unique characteristics observed in our study.

“This implies the possibility of enhancing morphometric traits in Brassica oleracea through the application of melatonin at specific growth stages and doses”.
At which stage of plant development was melatonin treatment most effective? Which species was most sensitive? Summarize this information at this point in the Discussion

Answer: Notably, for sprouts, both hypocotyl length (HL) and cotyledon width (CW) showed high correlation to melatonin treatment. For microgreens, weight (W) and hypocotyl length (HL) were the most affected morphometric traits in response to melatonin treatment. Concerning baby leaves, stem length (SL) and leaf width (LW) demonstrated a significant interaction following melatonin application. The Cavolo Broccolo Ramoso Calabrese (CR) genotype displayed heightened sensitivity to melatonin treatment.

“Both cultivars exhibited peculiar responses, reinforcing the importance of considering genetic factors in optimizing the cultivation of these novel foods”.
What were the most discernible peculiar responses?

Answer: In our study, the distinct responses of both cultivars underscore the importance of genetic factors in optimizing the cultivation of these novel foods. The most notable peculiarities were evident in morphometric traits, specifically in the weight and size of baby leaves. These factors play a pivotal role in shaping the plant's overall development and yield. Our findings suggest that the interplay of genetic elements significantly influences key morphological characteristics, contributing to the unique responses observed in each cultivar.

“During this transition, the synthesis of GLSs may show a decline, contributing to the observed reduction in GLSs levels in these later stages compared to the initial sprouting phase. This physiological process and defense mechanisms within Brassica species underscore the adaptive strategies employed by these plants, ensuring their survival and optimal development in diverse environmental conditions”.
Plants in the three stages of development are in normal physiological conditions. Why mention that GS would have a role in adapting to adverse conditions, this would apply if plants were subjected to some type of stress. The authors should discuss why GS levels decrease in subsequent stages of development based on what is known about the properties and functions of each type of glycosylate, there is a lot of literature on this, relating it to the metabolism of amino acids, a source of substrates and plant growth.

Answer: We appreciate your insightful comment regarding the mention of glucosinolates (GLSs) in the context of adaptive strategies, especially when the plants are in normal physiological conditions. In response, we have reconsidered and refined our explanation. Our research has indeed revealed a higher content of glucosinolates (GLSs) during the initial plant growth stage as sprouts. The observed decline in GLSs levels during subsequent stages can be better explained by referencing the extensive literature on the properties and functions of different types of glucosinolates.

Existing studies consistently report a greater abundance of antioxidant compounds, including glucosinolates, during the sprouting phase. The subsequent decrease in GLSs levels aligns with known metabolic shifts in amino acid metabolism during plant development. This phenomenon can be linked to the changing availability of substrates, as well as the evolving requirements of the plant for optimal growth. Our revised perspective places greater emphasis on the metabolic dynamics within the plant, offering a more nuanced understanding of GLSs fluctuations during different developmental stages.

We believe that this adjustment strengthens our interpretation by grounding it in established knowledge on plant metabolism and glucosinolate functions.

Lines 467 502: The discussion of the effects of melatonin is also unclear. The main problem is that the results do not coincide with other published studies in which melatonin increases the amounts of GS and the expression of GS metabolism enzymes. What factors could have influenced the different responses? Different Brassica species? Melatonin concentrations? treatment time?
Answer: We acknowledge the crucial point you raised about the observed results deviating from expectations based on existing literature where melatonin typically increases GS amounts and the expression of GS metabolism enzymes. Upon reflection, we recognize that the unsuitability of the dosages used for the considered initial growth stages, such as sprouts, microgreens, and baby leaves, may have played a significant role in the observed differences. The dosages selected for this study were initially chosen based on the available literature, but we acknowledge that they might not align optimally with the specific growth stages under investigation. Additionally, we agree that the specific Brassica species analyzed, as well as variations in melatonin concentrations and treatment times, could have influenced the outcomes. These factors will be critically assessed in ongoing studies to better understand the nuances of melatonin effects on GS in diverse Brassica species during different growth stages.

Lines 503-540. The discussion about the correlations between the different types of GS is superficial and not informative, as there is no correlation with what is known about the functions, metabolism and regulation of these compounds.
Answer: We appreciate your constructive input, and we are committed to enhancing the depth and informativeness of the discussion to better contribute to the understanding of glucosinolate dynamics in our study.

In the conclusion, the authors should mention whether or not the use of melatonin would be recommended for broccoli cultivation at the different stages of development of the different cultivars.
Answer: The conclusion now provides insights into the potential recommendations for melatonin application based on our findings.

Reviewer 3 Report

Comments and Suggestions for Authors

Manuscript title long, remove botanical name cultivar name from title

Line 27: remove typo "High-performance liquid chromatography" h should be small

keywords: Brassica spp " should be italic

Line 89-91, provide suitable reference

add more literature related melatonin, action, function and mechanism under abiotic stressess

objective not well written

Line 122: b should be capital

How prepared the melatonin solution, mentioned in the material method

Line 139-146: provide suitable reference for this paragraph

remove error value from the writing part of results, simple results value is enough, 

Table 5, table 6, table 7 present by figure, chnage into figure, Pearson’s correlation analysis, figure more beautiful for this

Principal Component Analysis (PCA), define full term at first used, later in the manuscript used PCA

don't need to used botanical name again and again in the manuscript, first time used enough

Line 449: define first use "fresh weight (FW)"

overall manuscript is interesting, but still need modification, important related abbreviation,

 

Author Response

Reviewer 3

Manuscript title long, remove botanical name cultivar name from title

Answer: We have addressed your suggestion and shortened the manuscript title by removing the botanical name and cultivar name.

Line 27: remove typo "High-performance liquid chromatography” should be small.

Answer: We have corrected the capitalization error in 'High-performance liquid chromatography' on line 27.

keywords: Brassica spp " should be italic

Answer: We have italicized the term 'Brassica spp' in the keywords section, as per your suggestion. We appreciate your attention to detail.

Line 89-91, provide suitable reference.

 Answer We have now included the appropriate reference Sun et al., 2021 et Tan et al.,2020, ensuring the accuracy and completeness of our manuscript. (L101-103)

add more literature related melatonin, action, function and mechanism under abiotic stresses

Thank you for your suggestion. We have expanded the literature review section to include additional references related to melatonin, its action, functions, and mechanisms under abiotic stresses. L96-108

objective not well written

Answer: We have carefully revisited and revised the objective to better align with the scope and focus of our study. L132-141

Line 122: b should be capital:

Answer: One Sicilian sprouting black broccoli, Broccolo nero (BN, Brassica oleracea var. italica Plenck).

How prepared the melatonin solution, mentioned in the material method

Answer: Thank you for your inquiry regarding the preparation of the melatonin solution mentioned in the Materials and Methods section (Lines 159-161). The melatonin solutions were meticulously prepared as outlined below:

M0 (Control): Hydrogen peroxide (H2O) solution without melatonin (0 μmol L–1).

M50: Hydrogen peroxide (H2O) solution with a melatonin concentration of 50 μmol L–1

M100: Hydrogen peroxide (H2O) solution with a melatonin concentration of 100 μmol L–1

These concentrations were chosen to represent varying levels of melatonin exposure, allowing for a comprehensive assessment of its effects on the growth stages of broccoli.

Line 139-146: provide a suitable reference for this paragraph:

Answer: Done

remove error value from the writing part of results, simple results value is enough

Answer: Thank you for highlighting the need for clarity in the presentation of results. We have revised the text to remove error values, focusing on providing straightforward and concise results.

Table 5, table 6, table 7 present by figure, change into figure, Pearson’s correlation analysis, figure more beautiful for this

Answer: Thank you for your suggestion regarding Tables 5, 6, and 7. We have transformed the presentation into figures to improve visual appeal and facilitate a clearer representation of Pearson's correlation analysis. The updated figures aim to enhance both the aesthetic quality and the interpretability of the correlation results.

Principal Component Analysis (PCA), define full term at first used, later in the manuscript used PCA:

Answer: We have defined the full term 'Principal Component Analysis (PCA)' the first time it is used in the manuscript.

don't need to used botanical name again and again in the manuscript, first time used enough

Answer: Thank you for your feedback. We have addressed the suggestion to reduce the repetition of botanical names throughout the manuscript.

Line 449: define first use "fresh weight (FW)":

Answer: Thank you for your feedback. In response to your comment on line 449 regarding 'fresh weight (FW),' we want to clarify that our study primarily focused on dry weight measurements

overall manuscript is interesting, but still need modification, important related abbreviation

We carefully addressed and incorporated your recommendations to improve the clarity and overall quality of our work.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been improved. I have no other comments.

Author Response

Thank you for your prompt review and valuable feedback. I appreciate your acknowledgment that the manuscript has been improved. I would like to express my gratitude for your time and input.

Reviewer 2 Report

Comments and Suggestions for Authors

 

In the revised version, the authors addressed most of the criticisms, improving the justifications for the work and objectives, methodological issues and the presentation of results. However, there were some important points that were not adequately answered, as follows:

Two questions about melatonin (reproduced below) were not even partially answered and should be improved.

Lines 467 502: The discussion of the effects of melatonin is also unclear. The main problem is that the results do not coincide with other published studies in which melatonin increases the amounts of GS and the expression of GS metabolism enzymes. What factors could have influenced the different responses? Different Brassica species? Melatonin concentrations? treatment time?

Lines 82 to 93. When identifying melatonin, it must be clear that it is produced by plants and its known functions in plants. There is quite a lot of literature on this.

 

The authors always mention in the first part of the Discussion that there was an effect or interaction of melatonin on the plant's morphometry, but they never clearly mention whether it was an increase or reduction. This needs to be very clear due to its agronomic and commercial importance.

 

What was requested is a characteristic chromatogram of the injected samples with identification of the peaks and not the standards.

 

The new figure inserted in Figure 01 was not cited and described in the text

 

Although the discussion has improved, the final discussion and conclusion are very evasive. Authors must respond directly and clearly whether the objectives initially established in the work (reproduced below) were achieved:

The objective of this study is to assess the impact of melatonin application on the early growth stages of broccoli, with a focus on enhancing both the growth and glucosinolate content in novel emerging food products, including sprouts, microgreens, and baby leaves. Specifically, our investigation targets two varieties: Sicilian sprouting black broccoli, Broccolo nero (Brassica oleracea  var. italica Plenck), and a commercially available variety, Cavolo Broccolo Ramoso  Calabrese (Brassica oleracea var. italica Plenck). Comprehensive evaluations at various growth stages involve the characterization of morphometric parameters and glucosinolate profiles. The ultimate goal is to optimize cultivation methods to enhance the antioxidant properties of these innovative foods.

 

So, has the focus on enhancing growth by melatonin treatment been achieved in whole or in part? or was it not significant in the different phases of the two cultivars? About the expectation of an increase in glycosylates? Would melatonin be recommended for sprouts only? If the initial hypothesis is optimization to increase the antioxidant properties of these foods, what would be the agronomic indication for the use of melatonin that can be proposed based on this work? In the conclusion section, the answers to these questions raised in the Introduction should be presented more clearly.     

Author Response

In the revised version, the authors addressed most of the criticisms, improving the justifications for the work and objectives, methodological issues and the presentation of results. However, there were some important points that were not adequately answered, as follows:

Two questions about melatonin (reproduced below) were not even partially answered and should be improved.

1-Lines 467 502: The discussion of the effects of melatonin is also unclear. The main problem is that the results do not coincide with other published studies in which melatonin increases the amounts of GS and the expression of GS metabolism enzymes. What factors could have influenced the different responses? Different Brassica species? Melatonin concentrations? treatment time?

 Answer: We have implemented specific modifications aimed at improving the clarity of our findings. The discussion has been expanded to offer a more detailed account of the changes in glucosinolate content across various growth stages in response to melatonin treatment. Additionally, we explicitly acknowledge potential factors that may contribute to the observed variations, including differences in Brassica species and melatonin concentrations

2-Lines 82 to 93. When identifying melatonin, it must be clear that it is produced by plants and its known functions in plants. There is quite a lot of literature on this.

Answer: In response to your suggestion, we have revised lines 82 to 93 to explicitly state that melatonin is produced by plants, and we have included a brief overview of its known functions in plants, drawing on relevant literature. We believe these revisions enhance the clarity and completeness of the information presented.

3-The authors always mention in the first part of the Discussion that there was an effect or interaction of melatonin on the plant's morphometry, but they never clearly mention whether it was an increase or reduction. This needs to be very clear due to its agronomic and commercial importance.

Answer: Thank you for your feedback. We have carefully revised the entire Discussion section to address the concerns raised, specifically regarding the effect or interaction of melatonin on the plant's morphometry. In the revised version, we have provided explicit and clear information about whether the observed effects resulted in an increase or reduction in plant morphometry. We trust that these revisions address your comment and contribute to a better understanding of the implications of melatonin on the studied plant's characteristics

4-What was requested is a characteristic chromatogram of the injected samples with identification of the peaks and not the standards.

Answer: we have incorporated the detailed chromatogram of the injected samples (an example), including peak identification, in the supplementary materials.

5-The new figure inserted in Figure 01 was not cited and described in the text

  Answer: In our revised manuscript, we will ensure that the figure is properly cited and thoroughly described in the text to provide clarity and context for the readers.

 

6-Although the discussion has improved, the final discussion and conclusion are very evasive. Authors must respond directly and clearly whether the objectives initially established in the work (reproduced below) were achieved:

The objective of this study is to assess the impact of melatonin application on the early growth stages of broccoli, with a focus on enhancing both the growth and glucosinolate content in novel emerging food products, including sprouts, microgreens, and baby leaves. Specifically, our investigation targets two varieties: Sicilian sprouting black broccoli, Broccolo nero (Brassica oleracea var. italica Plenck), and a commercially available variety, Cavolo Broccolo Ramoso  Calabrese (Brassica oleracea var. italica Plenck). Comprehensive evaluations at various growth stages involve the characterization of morphometric parameters and glucosinolate profiles. The ultimate goal is to optimize cultivation methods to enhance the antioxidant properties of these innovative foods.

So, has the focus on enhancing growth by melatonin treatment been achieved in whole or in part? or was it not significant in the different phases of the two cultivars? About the expectation of an increase in glycosylates? Would melatonin be recommended for sprouts only? If the initial hypothesis is optimization to increase the antioxidant properties of these foods, what would be the agronomic indication for the use of melatonin that can be proposed based on this work? In the conclusion section, the answers to these questions raised in the Introduction should be presented more clearly.     

 Answer: In response to your specific comments about the discussion and conclusion, we have made the necessary modifications. We have made necessary modifications for a more direct and clear presentation of whether our study achieved its initial objectives. Our findings indicate a positive impact of melatonin on growth, especially in sprouts, with variations across stages and cultivars. While melatonin showed potential for growth enhancement, its dose-dependent impact on glucosinolate levels prompts careful dosage considerations. Future research optimizing melatonin application, considering both growth promotion and antioxidant properties, provides valuable agronomic indications for Brassica cultivation. The conclusion has been refined to explicitly address your questions, providing clearer insights into achieved objectives and agronomic indications. We believe these revisions enhance the clarity and impact of our findings.

Reviewer 3 Report

Comments and Suggestions for Authors

accepted

Author Response

Thank you for your prompt review and valuable feedback. I have carefully addressed the concerns raised in your previous comments. The introduction has been revised to provide sufficient background, and I have ensured that all cited references are directly relevant to the research. I appreciate your time and constructive input.