Differential Effects of Melatonin on Nitrogen Metabolism and Growth in Capsicum chinense Jacq.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work makes an analysis of the effect of different exogenous melatonin levels on some growth and N parameters in seedlings of the hot pepper species C. chinense. The authors propose a priming strategy by treating seeds with melatonin and then they submit plantlets to diverse concentrations of such modulator. Results on growth, nitrate, amino acids and protein content are provided, and an analysis of diverse N-related genes are included.

The rationale of the work is good, but it can be framed within its preliminary steps. Thus, there are significant parameters that should be included to obtain a better picture of the effect of melatonin:

1. The specific contribution of this work is not really highlighted, considering that there are other works in the N metabolism of several solanaceae such as tomato, tobacco, and even other pepper species. What is the difference of this present work?
2. Total N should be also necessary as well as other macronutrients such as C (carbohydrates are fundamental to build new N-containing skeletons), P, K, Mg, Ca and micronutrients.
3. The phylogenetic analysis should also include other solanaceae especies.
4. The activity profile of some of the enzymes encoded by the genes investigated in this work is recommended.

Additionally, there are some minor comments, given below:

- Line 25: among instead of between.

- Lines 120-121: Please, indicate here results not shown.

- Fig. 1: Please, indicate if data in Figs 1A-1D from plants correspond to plants growing either in pots or petri dishes. Then, indicate in Figs. 1A-1D if data correspond to g shoot/root per plant.

- Fig. 22: Please, change scale in Figs. 2C, 2F and 2I, and indicate in the legend what Figs. 2J-2L depict.

- Table 1: Protein values are given but not genes as written in the text.

 

 

Author Response

Reviewer 1

This work makes an analysis of the effect of different exogenous melatonin levels on some growth and N parameters in seedlings of the hot pepper species C. chinense. The authors propose a priming strategy by treating seeds with melatonin and then they submit plantlets to diverse concentrations of such modulator. Results on growth, nitrate, amino acids and protein content are provided, and an analysis of diverse N-related genes are included.

The rationale of the work is good, but it can be framed within its preliminary steps. Thus, there are significant parameters that should be included to obtain a better picture of the effect of melatonin:

1. The specific contribution of this work is not really highlighted, considering that there are other works in the N metabolism of several solanaceae such as tomato, tobacco, and even other pepper species. What is the difference of this present work?

 

A.1.1 We appreciate the reviewer's comment, and in this new version of the manuscript, the specific contribution of this work has been highlighted (see lines 106-123, 423-435).

 

1. Total N should be also necessary as well as other macronutrients such as C (carbohydrates are fundamental to build new N-containing skeletons), P, K, Mg, Ca and micronutrients.

 

A.1.2 We greatly appreciate the reviewers' comments regarding the need to incorporate more experimental data for a more in-depth analysis. In this revised version of the manuscript, data on ammonium (see figure 2D-F, lines 166-169, section 3.1), proline (see figure 8B, lines 377-378, section 3.1), and total carbohydrate content (see figure 8A, lines 373-376, section 3.1) were added. Similarly, glutamate dehydrogenase enzyme activity values ​​were also included (see figure 7 lines 361-364, section 3.2). Furthermore, the principal component analysis (PCA) of nitrogen metabolites was performed again, now incorporating the ammonium data (see figure 3, section 2.3). A multivariate analysis and PCA of nitrogen metabolism gene expression were also conducted (see figure 6, section 2.6). Nitrogen content, as the sum of nitrate, ammonium, amino acids, and total protein in the root, leaf, and whole plant, was evaluated in greater depth in this study (see figure S1, lines 181-184). We believe that the addition of these data and analyses, together with those presented in the original manuscript, strengthens this study, and provides a better understanding of the effect of melatonin on primary nitrogen assimilation in habanero pepper.

Regarding the addition of macro- and micronutrient content, as the reviewer suggests, we believe that while it is of interest to know the effect of melatonin on these nutrients, this study should be addressed separately. In this case, the objective could be to understand the effect of melatonin on the absorption, assimilation, translocation, and remobilization of nutrients, in general, within the plant. In this manuscript, we chose to focus our study on the effect of melatonin on growth (dose-response), nitrogen metabolites, and transcript profiles of genes encoding nitrogen assimilation enzymes. Specifically, we included total carbohydrate content values, considering the reviewer's insightful comment that carbohydrates may play a direct role in amino acid synthesis during primary nitrogen assimilation.

 

1. The phylogenetic analysis should also include other solanaceae species.

 

A.1.3 As the reviewer suggested, we strengthened the phylogenetic analysis of the nitrogen metabolism enzymes GS, GOGAT, and GDH using proteins from four species of Solanaceae, in addition to C. chinense. The results obtained are shown in Figure 4 and discussed in the text (see section 2.4).

 

1. The activity profile of some of the enzymes encoded by the genes investigated in this work is recommended.

 

A.1.4 As the reviewer recommended, we included glutamate dehydrogenase activity in this study as a first step in understanding the effect of melatonin on the activity of nitrogen assimilation pathway enzymes in habanero peppers (see figure 7 lines 361-364, section 3.2). When principal component analysis of the nitrogen metabolism enzyme genes was performed, it was observed that GDH data, along with NiR data, were among the variables that had the greatest impact on the data variations; therefore, this enzyme was selected for activity evaluation. Unfortunately, we did not have the reagents to evaluate nitrite reductase activity; however, this enzyme's activity can be addressed in future studies. We believe that the data added to the study provided a clearer understanding of the effect of melatonin on nitrogen assimilation processes.

Additionally, there are some minor comments, given below:

- Line 25: among instead of between.

A.1.5 Thanks. This mistake was eliminated in the text (see Abstract).

- Lines 120-121: Please, indicate here results not shown.

A.1.6 Ok (see line 132)

- Fig. 1: Please, indicate if data in Figs 1A-1D from plants correspond to plants growing either in pots or petri dishes. Then, indicate in Figs. 1A-1D if data correspond to g shoot/root per plant.

A.1.7As mentioned in the Materials and Methods section, Petri dishes were used solely for seed germination. Once germinated, the seeds were transferred to pots (12-well trays) for plant growth. This information was added to the legend in Figure 1. The values ​​g per plant were included in the legend for the axes and in Figures 1A-1D (see figure 1 and figure 1 legend).

- Fig. 22: Please, change scale in Figs. 2C, 2F and 2I, and indicate in the legend what Figs. 2J-2L depict.

A.1.8 These comments were addressed in this new version of the manuscript. Figure 2 now includes the ammonium concentration data in the tissues. The y-axis scales in the graphs were modified, considering the highest values ​​for each metabolite per organ; this allows for a clearer observation of the variation in their concentrations in the different plant organs. The principal component analysis (Figures 2J-L, to which the reviewer refers) was redone, now incorporating the ammonium data, and is presented in a separate figure, as specified in the legend (Figure 3 and legend of Figure 3).

- Table 1: Protein values are given but not genes as written in the text.

Thanks. This mistake has been corrected in the text (see Line 260).

Reviewer 2 Report

Comments and Suggestions for Authors

 This study investigates the differential effects of melatonin applied via different methods (seed priming and root drenching) and at varying concentrations on seedling growth, nitrogen metabolite accumulation, and related gene expression in habanero pepper (Capsicum chinense Jacq.). The research topic closely aligns with the practical agricultural need to improve nitrogen use efficiency in Solanaceae crops. The experimental design, encompassing multiple concentration gradients and two application methods, demonstrates a certain degree of novelty and practical application value. However, appropriate revisions are still necessary to meet publication standards, as detailed below:

1. Please add experimental results instead of relying on speculation to get possible results.

2. The authors should provide clearer figures for readability. For instance, the titles of the x- and y-axes, as well as the letters indicating significant differences in Figure 1 (A, B, C, D), Figure 2 (A, B, C, D, E, F, G, H, I), and Figure 3, are not clear. It is recommended to adjust the font size and provide high-quality vector graphics.

3. In Figure 2, it is recommended to directly label the three parts: "roots, stems, and leaves" within the figure.

4. It is recommended to merge Figure 4 and Figure S1.

5. Nitrogen metabolism (such as nitrate reduction and ammonia assimilation) is a process highly regulated by post-translational modifications. The manuscript currently provides only data on metabolite contents and gene transcription levels (RT-qPCR). Transcript abundance is not entirely equivalent to enzyme activity. Without enzyme activity data, the conclusion that "melatonin promotes nitrogen assimilation" appears somewhat weak.

6. The authors observed an interesting phenomenon: melatonin promoted plant biomass growth but downregulated the expression of key nitrogen metabolism genes and total protein content in the leaves. Regrettably, the authors failed to provide a robust scientific explanation for this "paradox between phenotype and molecular mechanism" in the Discussion section.

7. Lines 333-338: The statement that leaf nitrate is utilized for synthesizing non-amino acid/non-protein nitrogenous compounds, such as polyamines, under the CP treatment is an unfounded deduction, as metabolites like polyamines and proline were not measured in this study. When drafting such descriptions, the authors should be mindful of their phrasing, as this conclusion is not derived from direct experimental measurements.

8. The main text mentions the evaluation of the GS3However, Table 1, Figure 4, and Figure S1 only list CcGS1.2, CcGS1.4, and CcGS2; there is no data or information regarding the GS3 gene.

9. The experiment utilized peat moss as the substrate, which was irrigated with 1/5 Hoagland nutrient solution. Peat moss intrinsically contains a certain amount of background nitrogen that is difficult to quantify. Would this obscure or interfere with the accurate assessment of plant nitrogen uptake and metabolism? 

10. Line 274: The "y" in "CcGS, CeGOGATy CcGDH" should be corrected to "and".

11. Line 229: Should "GHD2" be corrected to "GDH2"? 

12. Lines 130-132: "except for the 5 µM dose, which was reflected in a greater dry weight of both the shoots, and roots (Figure 1B, D)." Please pay attention to redundant punctuation; the comma in "shoots, and roots" should be deleted.

13. Please avoid excessive speculation in the Discussion section; kindly check and revise accordingly.

14. Lines 434-435: While providing the source of seeds is standard practice, the use of square brackets in "[Ashalim, M.P. Ramat Negev 8551200, Israel]" does not conform to common formatting norms. Please change them to parentheses or separate the information directly with commas.

15. Line 449: The nitrogen source ratio of the Hoagland nutrient solution is written as "0.80 mM mM Ca(NO₃)₂4H₂O". What is the meaning of "mM mM" here? 

16. Pay attention to the use of units throughout the text; for example, "ml" should typically be capitalized as "mL".

17. The Conclusion claims that "melatonin induces organ-specific and stage-specific regulation". However, this study did not conduct a temporal dynamic analysis (sampling was performed only at a single time point), making it impossible to draw a "stage-specific" conclusion.

18. The Conclusion section should focus on summarizing the findings of this study, pointing out its limitations, and suggesting potential directions for future research. In contrast, potential mechanisms that were not elucidated in this study should be presented in the Discussion section.

19. The authors should check the format of the references and revise them strictly according to the journal's guidelines. For instance, verify whether journal names should be abbreviated, and note that some cited references are missing their DOI numbers.

Author Response

Reviewer 2

 This study investigates the differential effects of melatonin applied via different methods (seed priming and root drenching) and at varying concentrations on seedling growth, nitrogen metabolite accumulation, and related gene expression in habanero pepper (Capsicum chinense Jacq.). The research topic closely aligns with the practical agricultural need to improve nitrogen use efficiency in Solanaceae crops. The experimental design, encompassing multiple concentration gradients and two application methods, demonstrates a certain degree of novelty and practical application value. However, appropriate revisions are still necessary to meet publication standards, as detailed below:

1. Please add experimental results instead of relying on speculation to get possible results.

A.2.1 We appreciate the reviewer's feedback. In this revised version of the manuscript, other experimental results were added. See A.1.2, 1.3 and 1.4 (answer to revisor 1):

“A.1.2 We greatly appreciate the reviewers' comments regarding the need to incorporate more experimental data for a more in-depth analysis. In this revised version of the manuscript, data on ammonium (see figure 2D-F, lines 166-169, section 3.1), proline (see figure 8B,  lines 377-378, section 3.1), and total carbohydrate content (see figure 8A,  lines 373-376, section 3.1) were added. Similarly, glutamate dehydrogenase enzyme activity values ​​were also included (see figure 7 lines 361-364, section 3.2). Furthermore, the principal component analysis (PCA) of nitrogen metabolites was performed again, now incorporating the ammonium data (see figure 3, section 2.3). A multivariate analysis and PCA of nitrogen metabolism gene expression were also conducted (see figure 6,  section 2.6). Nitrogen content, as the sum of nitrate, ammonium, amino acids, and total protein in the root, leaf, and whole plant, was evaluated in greater depth in this study (see figure S1,  lines 181-184).

“A.1.3 As the reviewer suggested, we strengthened the phylogenetic analysis of the nitrogen metabolism enzymes GS, GOGAT, and GDH using proteins from four species of Solanaceae, in addition to C. chinense. The results obtained are shown in Figure 4 and discussed in the text (see section 2.4)”.

“A.1.4 As the reviewer recommended, we included glutamate dehydrogenase activity in this study as a first step in understanding the effect of melatonin on the activity of nitrogen assimilation pathway enzymes in habanero peppers (see figure 7 lines 361-364, section 3.2). When principal component analysis of the nitrogen metabolism enzyme genes was performed, it was observed that GDH data, along with NiR data, were among the variables that had the greatest impact on the data variations; therefore, this enzyme was selected for activity evaluation”

 

 

The manuscript was also carefully reviewed, and all speculative phrases were removed (see the revised manuscript).

2. The authors should provide clearer figures for readability. For instance, the titles of the x- and y-axes, as well as the letters indicating significant differences in Figure 1 (A, B, C, D), Figure 2 (A, B, C, D, E, F, G, H, I), and Figure 3, are not clear. It is recommended to adjust the font size and provide high-quality vector graphics.

A.2.2 Thanks. We modified the figures according to the reviewer's recommendations (see figures 1-4).

3. In Figure 2, it is recommended to directly label the three parts: "roots, stems, and leaves" within the figure.

A.2.3 Now the new Figure 2 includes the labels that the reviewer suggested (see figure 2).

4. It is recommended to merge Figure 4 and Figure S1.

A.2.4 Figure 4 (Figure 5 in the current manuscript) and Figure S1 were merged (see figure 5).

5. Nitrogen metabolism (such as nitrate reduction and ammonia assimilation) is a process highly regulated by post-translational modifications. The manuscript currently provides only data on metabolite contents and gene transcription levels (RT-qPCR). Transcript abundance is not entirely equivalent to enzyme activity. Without enzyme activity data, the conclusion that "melatonin promotes nitrogen assimilation" appears somewhat weak.

A.2.5 We fully agree with the reviewer that the abundance of enzyme transcripts does not necessarily correspond to enzyme activity, and this was corroborated in this revised version of the manuscript with the inclusion of GDH enzyme activity data. We took the reviewer's comment into account when the discussion and conclusion of the paper were rewritten. See discussion section (lines 420-582) and conclusion (lines 767-782).

6. The authors observed an interesting phenomenon: melatonin promoted plant biomass growth but downregulated the expression of key nitrogen metabolism genes and total protein content in the leaves. Regrettably, the authors failed to provide a robust scientific explanation for this "paradox between phenotype and molecular mechanism" in the Discussion section.

A.2.6 We agree with and appreciate the reviewer's observation. The expansion of experimental data (ammonium, proline, carbohydrate content, and GDH activity) and the new analyses incorporated (PCA and multivariate) allowed us to propose a model to comprehensively explain the effect of the different types of melatonin application used in this study (see figure 9, and section 3.2).

7. Lines 333-338: The statement that leaf nitrate is utilized for synthesizing non-amino acid/non-protein nitrogenous compounds, such as polyamines, under the CP treatment is an unfounded deduction, as metabolites like polyamines and proline were not measured in this study. When drafting such descriptions, the authors should be mindful of their phrasing, as this conclusion is not derived from direct experimental measurements.

A.2.7 We appreciate the reviewer's feedback. The discussion has been rewritten. This section has been enriched with the newly added experimental data (e.g., data for ammonia, proline, and total carbohydrates), as well as new analyses. All speculative descriptions have been removed, as the reviewer noted (see discussion section lines 420-582).

8. The main text mentions the evaluation of the GS3. However, Table 1, Figure 4, and Figure S1 only list CcGS1.2, CcGS1.4, and CcGS2; there is no data or information regarding the GS3gene.

A.2.8 Thanks. This error has been corrected in the text; the correct nomenclature is CcGS1.2, CcGS1.2 and CcGS2 (see text in general).

9. The experiment utilized peat moss as the substrate, which was irrigated with 1/5 Hoagland nutrient solution. Peat moss intrinsically contains a certain amount of background nitrogen that is difficult to quantify. Would this obscure or interfere with the accurate assessment of plant nitrogen uptake and metabolism? 

A.2.9 Thanks for your comment. We believe that the use of peat moss as a substrate does not interfere with the effect of melatonin on the nitrogen metabolism parameters that were evaluated. Peat moss is an inert organic substrate that stands out for its high-water retention capacity and very low nitrogen content, generally less than 1.5%, making it not a source of fertilizer. Our group has always used this substrate, mainly because of its water retention capacity, which exceeds 20 times its weight, and its small percentage of total nitrogen. All our experiments have been conducted with this substrate to maintain consistent growing conditions. It is a substrate frequently used in plant physiology experiments worldwide.

10. Line 274: The "y" in "CcGS, CeGOGATy CcGDH" should be corrected to "and".

A.2.10 This mistake was eliminated when the manuscript rewrite.

11. Line 229: Should "GHD2" be corrected to "GDH2"? 

A.2.11 This mistake was corrected (see line 273)

12. Lines 130-132: "except for the 5 µM dose, which was reflected in a greater dry weight of both the shoots, and roots (Figure 1B, D)." Please pay attention to redundant punctuation; the comma in "shoots, and roots" should be deleted.

A.2.12 This mistake was corrected (see line 141)

13. Please avoid excessive speculation in the Discussion section; kindly check and revise accordingly.

A.2.13 The discussion has been rewritten. All speculative descriptions have been removed, as the reviewer noted (see Discussion section lines 420-582).

14. Lines 434-435: While providing the source of seeds is standard practice, the use of square brackets in "[Ashalim, M.P. Ramat Negev 8551200, Israel]" does not conform to common formatting norms. Please change them to parentheses or separate the information directly with commas.

A.2.14 This change was made as the reviewer suggested (see lines 585-586).

15. Line 449: The nitrogen source ratio of the Hoagland nutrient solution is written as "0.80 mM mM Ca(NO₃)₂4H₂O". What is the meaning of "mM mM" here? 

A.2.15 The repetition of mM was an error that was removed from the manuscript (see lines 600).

16. Pay attention to the use of units throughout the text; for example, "ml" should typically be capitalized as "mL".

A.2.16  We addressed this comment; all units were standardized and written according to the format (see the text).

17. The Conclusion claims that "melatonin induces organ-specific and stage-specific regulation". However, this study did not conduct a temporal dynamic analysis (sampling was performed only at a single time point), making it impossible to draw a "stage-specific" conclusion.

A.2.17 According to the reviewer. The conclusion was rewritten in this new version of the manuscript (see Conclusion, lines 767-782).

18. The Conclusion section should focus on summarizing the findings of this study, pointing out its limitations, and suggesting potential directions for future research. In contrast, potential mechanisms that were not elucidated in this study should be presented in the Discussion section.

A.2.18 According to the reviewer. The conclusion was rewritten in this new version of the manuscript (see Conclusion, lines 767-782).

19. The authors should check the format of the references and revise them strictly according to the journal's guidelines. For instance, verify whether journal names should be abbreviated, and note that some cited references are missing their DOI numbers.

A.2.19 Correct. The references section was carefully reviewed and adjusted to the journal's format (see References lines 799-978)

Reviewer 3 Report

Comments and Suggestions for Authors

The study addresses an interesting and relevant topic, particularly the role of melatonin in regulating nitrogen metabolism in Capsicum chinense. The integration of physiological, biochemical, and molecular analyses is commendable. However, several aspects of clarity, interpretation, and depth need improvement to strengthen the manuscript.

• Correct formatting issues caused by improper word breaks (e.g., “how- ever”, “metab- olism”) to improve readability.
• Ensure consistent italicization of the scientific name Capsicum chinense throughout the manuscript.
• Clearly define the abbreviation “SD” (substrate reinforcement) at its first mention.
• The Results section appears to contain limited data; consider adding more figures and/or tables in the main manuscript to strengthen data presentation and interpretation.
• The phenotypic observations are clear and well-presented, effectively supporting the study’s findings.
• Replace vague expressions such as “may lead to” and “likely resulting in” with more precise and definitive wording where possible.
• Improve sentence structure by simplifying long and complex sentences and refine the concluding statement by indicating more specific future research directions.

Author Response

Reviewer 3

The study addresses an interesting and relevant topic, particularly the role of melatonin in regulating nitrogen metabolism in Capsicum chinense. The integration of physiological, biochemical, and molecular analyses is commendable. However, several aspects of clarity, interpretation, and depth need improvement to strengthen the manuscript.

1. Correct formatting issues caused by improper word breaks (e.g., “how- ever”, “metab- olism”) to improve readability.

A.3.1 Thanks. These errors were corrected in the text (see text).

2. Ensure consistent italicization of the scientific name Capsicum chinensethroughout the manuscript.

A.3.2 Thanks. All scientific names of the species were reviewed and corrected (see text).

3. Clearly define the abbreviation “SD” (substrate reinforcement) at its first mention.

A.3.3 The abbreviation “SD” was defined initially in abstract (See abstract line 18) and results (See lines 128-129), a s reviewer suggested.

4. The Results section appears to contain limited data; consider adding more figures and/or tables in the main manuscript to strengthen data presentation and interpretation.

A.3.4 In this new version of the manuscript, the number of figures in the results was doubled (from 4 to 8). Data for ammonia, proline, total carbohydrates, and glutamate dehydrogenase activity were added. A principal component and multivariate analysis, along with a mechanistic diagram of the effect of melatonin, were also included, the latter as a ninth figure. All of these additions greatly contributed to a better interpretation and a more comprehensive discussion of these results. See Results (124-418) and Discussion (see lines 420-582) sections.

5. The phenotypic observations are clear and well-presented, effectively supporting the study’s findings.

A.3.5 We appreciate the reviewer's comment.

6. Replace vague expressions such as “may lead to” and “likely resulting in” with more precise and definitive wording where possible.

A.3.6 We carefully reviewed this version of the manuscript and considered the reviewer's comments, replacing vague phrases with more precise ones (see revised manuscript).

7. Improve sentence structure by simplifying long and complex sentences and refine the concluding statement by indicating more specific future research directions.

A.3.7 This comment was fully addressed, the sentence structure was improved where necessary, and the conclusion was rewritten, now containing suggestions for future research (see text and Conclusion lines 767-782)

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript by Leong-García et al. investigates the effects of melatonin priming (CP) alone and in combination with substrate drenching (CP+SD) on growth and nitrogen metabolism in habanero pepper. The study employs a robust physiological, biochemical, and molecular approach, including a well-executed phylogenetic analysis of key nitrogen assimilation enzymes. The findings that the application method influences nitrogen partitioning and that nitrite reductase (NiR) may play a central role are novel and contribute significantly to our understanding of melatonin's function in non-stressed plants. The manuscript is well-written and logically structured. However, several points require clarification and revision before it can be accepted for publication.

1. The nomenclature for experimental treatments is inconsistent throughout the manuscript. The treatments are referred to as "priming alone" and "in combination with substrate reinforcement" in the Abstract and Introduction, as "chemoprining, CP" and "CP + SD" in Section 2.1, and as "SP" and "SP+SD" in Figure 2, its legend, and Section 2.3. The terminology should be standardized across the entire manuscript. The use of "CP" (chemopriming) and "CP+SD" (chemopriming plus soil drench) is recommended and should be applied uniformly to all figures, legends, and text.
2. In Section 2.4, the description of gene expression analyses fails to distinguish clearly between the initial semi-quantitative RT-PCR screening (Figure S1) and the subsequent RT-qPCR quantification. The statement "RT-PCR analyses were performed only on root and leaf samples... These analyses revealed differential modulation..." is ambiguous. The authors should explicitly state that semi-quantitative RT-PCR was used as an initial screen, followed by RT-qPCR analysis of selected genes across the full concentration range.
3. In Section 2.2, whole-plant nitrate accumulation values are discussed (e.g., "15.3 mg g⁻¹ FW at 25 µM" and "19.5 mg g⁻¹ FW at 50 µM CP+SD"), yet these data are not presented in Figure 2A–C or in any other figure or table. The authors should either present the whole-plant nitrate data in a supplementary figure or table, or revise the discussion to refer exclusively to the organ-specific data shown in Figure 2.
4. The legend for Figure 2 does not include descriptions for panels J, K, and L, which display the principal component analysis (PCA) results. A description of these panels must be added to the figure legend.
5. In Section 2.3, a grammatical error occurs in the phrase "assigned bases on the closest phylogenetic relationship." The word "bases" should be corrected to "based."
6. Gene nomenclature in Section 2.4 is inconsistent with that presented in Table 1 and Figure 3. The text refers to "GS1, GS2, GS3," whereas the formal identifiers in Table 1 are CcGS1.2, CcGS1.4, and CcGS2. There is no "GS3" listed in Table 1. The nomenclature used in the main text must be revised to match precisely the gene identifiers provided in Table 1 and Figure 3.
7. In Section 3.1, the phrase "treatment of seeds Triticale hexaploide L." contains a grammatical error. It should be revised to "treatment of seeds of Triticale hexaploide L."
8. The reference list exhibits inconsistent formatting. The main text employs a numbered citation style (e.g., [16]), whereas several entries in the reference list lack numbering and do not adhere to a uniform format. The reference list must be reformatted to comply strictly with the journal's (MDPI) reference style guidelines.
9. In Section 3.3, the statement "Its role in nitric oxide production, vital for growth, development, and stress response, has been demonstrated" lacks a supporting citation. An appropriate reference should be provided.
10. In Section 2.4, the term "RT-PCR analyses" is imprecise when referring to the initial screening experiment. The phrase should be replaced with "semi-quantitative RT-PCR" to distinguish this experiment clearly from the quantitative RT-qPCR analysis.
11. In Section 4.1, the notation of nutrient solution concentrations is inconsistent. For example, "12.5 μM H₃BO₃" is followed by entries lacking units. All concentration values should be checked and presented in a uniform format.
12. The axis labels and grouping identifiers in panels J, K, and L of Figure 2 are rendered in a font size that may impede readability. It is recommended that the font size be increased for clarity.
13. Several non-standard abbreviations are introduced without definition upon first use. For instance, "FW" (fresh weight) appears for the first time in Section 2.2 without its full term. All abbreviations should be defined in full at their first occurrence in the main text.
14. Figure S1 is cited in the main text, but no corresponding figure legend is provided in the supplementary materials. A complete legend for Figure S1 should be included with the supplementary file.

Author Response

Reviewer 4

The manuscript by Leong-García et al. investigates the effects of melatonin priming (CP) alone and in combination with substrate drenching (CP+SD) on growth and nitrogen metabolism in habanero pepper. The study employs a robust physiological, biochemical, and molecular approach, including a well-executed phylogenetic analysis of key nitrogen assimilation enzymes. The findings that the application method influences nitrogen partitioning and that nitrite reductase (NiR) may play a central role are novel and contribute significantly to our understanding of melatonin's function in non-stressed plants. The manuscript is well-written and logically structured. However, several points require clarification and revision before it can be accepted for publication.

1. The nomenclature for experimental treatments is inconsistent throughout the manuscript. The treatments are referred to as "priming alone" and "in combination with substrate reinforcement" in the Abstract and Introduction, as "chemoprining, CP" and "CP + SD" in Section 2.1, and as "SP" and "SP+SD" in Figure 2, its legend, and Section 2.3. The terminology should be standardized across the entire manuscript. The use of "CP" (chemopriming) and "CP+SD" (chemopriming plus soil drench) is recommended and should be applied uniformly to all figures, legends, and text.

A.4.1 We appreciate the reviewer's comment; the CP (chemopriming) and SD (soil drench) nomenclature was standardized throughout the text, as the reviewer suggested. See text.

 

1. In Section 2.4, the description of gene expression analyses fails to distinguish clearly between the initial semi-quantitative RT-PCR screening (Figure S1) and the subsequent RT-qPCR quantification. The statement "RT-PCR analyses were performed only on root and leaf samples... These analyses reveal differential modulation..." is ambiguous. The authors should explicitly state that semi-quantitative RT-PCR was used as an initial screen, followed by RT-qPCR analysis of selected genes across the full concentration range.

A.4.2 We corrected the text according to the review suggestions, clarifying the distinction between the two methodologies, RT-PCR and RT-qPCR (see lines 264-270).

 

1. In Section 2.2, whole-plant nitrate accumulation values are discussed (e.g., "15.3 mg g⁻¹ FW at 25 µM" and "19.5 mg g⁻¹ FW at 50 µM CP+SD"), yet these data are not presented in Figure 2A–C or in any other figure or table. The authors should either present the whole-plant nitrate data in a supplementary figure or table or revise the discussion to refer exclusively to the organ-specific data shown in Figure 2.

A.4.3 The reviewer is correct. We removed this discussion from the results section. In this new version of the manuscript, we added the nitrogen values ​​(Supplementary Figure S1), as the sum of nitrate, ammonium, amino acids, and proteins by organ, and these values ​​were mentioned in the results and discussion sections (see lines 181- 184).

 

1. The legend for Figure 2 does not include descriptions for panels J, K, and L, which display the principal component analysis (PCA) results. A description of these panels must be added to the figure legend.

A.4.4 The results of the principal component analysis of the metabolite values ​​are now presented separately, and the description referred to by the reviewer was added to the figure legend (see figure 3 and legend)

 

1. In Section 2.3, a grammatical error occurs in the phrase "assigned bases on the closest phylogenetic relationship." The word "bases" should be corrected to "based."

A.4.5 This mistake was corrected (see line 245)

 

1. Gene nomenclature in Section 2.4 is inconsistent with that presented in Table 1 and Figure 3. The text refers to "GS1, GS2, GS3," whereas the formal identifiers in Table 1 are CcGS1.2, CcGS1.4, and CcGS2. There is no "GS3" listed in Table 1. The nomenclature used in the main text must be revised to match precisely the gene identifiers provided in Table 1 and Figure 3.

A.4.6 This mistake was corrected and now the nomenclature of the GS genes (CcGS1.2, CcGS1.4 and CcGS2) has been standardized in the text and figures (see text, table 1 and figure 5)

 

1. In Section 3.1, the phrase "treatment of seeds Triticale hexaploide L." contains a grammatical error. It should be revised to "treatment of seeds of Triticale hexaploide L."

A.4.7 In this new manuscript version, this phrase was eliminated (see section 3.1, lines 421-582).

 

1. The reference list exhibits inconsistent formatting. The main text employs a numbered citation style (e.g., [16]), whereas several entries in the reference list lack numbering and do not adhere to a uniform format. The reference list must be reformatted to comply strictly with the journal's (MDPI) reference style guidelines.

A.4.8 The entire reference list was carefully reviewed and corrected according to the journal's style (see references lines 800-977).

 

1. In Section 3.3, the statement "Its role in nitric oxide production, vital for growth, development, and stress response, has been demonstrated" lacks a supporting citation. An appropriate reference should be provided.

A.4.9 This comment was eliminated when discussion section was rewrite (lines 421-582).

 

1. In Section 2.4, the term "RT-PCR analyses" is imprecise when referring to the initial screening experiment. The phrase should be replaced with "semi-quantitative RT-PCR" to distinguish this experiment clearly from the quantitative RT-qPCR analysis.

A.3.10 We made the change that the reviewer suggested (see lines 264-270).

 

1. In Section 4.1, the notation of nutrient solution concentrations is inconsistent. For example, "12.5 μM H₃BO₃" is followed by entries lacking units. All concentration values should be checked and presented in a uniform format.

A.4.11 Thanks. This section was carefully reviewed and corrected (see section 4.1 lines 598-600).

 

1. The axis labels and grouping identifiers in panels J, K, and L of Figure 2 are rendered in a font size that may impede readability. It is recommended that the font size be increased for clarity.

A.4.12 Figure 2 was modified according to the reviewers' comments (see figure 2).

 

1. Several non-standard abbreviations are introduced without definition upon first use. For instance, "FW" (fresh weight) appears for the first time in Section 2.2 without its full term. All abbreviations should be defined in full at their first occurrence in the main text.

A.4.13 In this new version of the manuscript, all abbreviations were defined at their first appearance in the text (See text, and figure 2)

 

1. Figure S1 is cited in the main text, but no corresponding figure legend is provided in the supplementary materials. A complete legend for Figure S1 should be included with the supplementary file.

A.4.14 In this new version of the manuscript, Figure S1 referred to by the reviewer was merged with Figure 5, as suggested by reviewer 2 (see Answer 2.4), and the legend was added to the figure (see figure legend 5).

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors properly addressed the comments and have greatly improved the work.

Reviewer 2 Report

Comments and Suggestions for Authors

Have no comments