Silicon Nanoparticles Modulate C:N:P Homeostasis and the Efficiencies of Nutrient Uptake, Translocation, and Use in Sugarcane Under Calcium Deficiency and Sufficiency

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Silicon is a very important and still somewhat underestimated element required for proper plant growth and development. I appreciate that the authors have addressed this topic, but…

First of all, I am not satisfied with the language of the manuscript. Many expressions (examples provided below):

• Despite these advances, mechanistic understanding of how nSiO₂ contributes to restoring C:N:P balance and to the integrated reconfiguration of nutrient concentrations, accumulations, and uptake, translocation, and use efficiencies in sugarcane under Ca restriction remains limited.
• but can induce a reprogramming of
• to the integrated reconfiguration of nutrient concentrations
• Incorporation into the system
• a modulator of nutritional functioning by intensifying Si integration into the system
• by promoting resource economy and improved nutrient-to-growth conversion
• reconfiguring carbon partitioning
• Mechanistically, this may reflect flux prioritization
• nSiO₂ exhibited a compensatory profile associated with performance recovery and rebalancing of limiting processes (this sentence sounds more like an advertisement for a supplement)

sound overly marketing-oriented, inflated, or imprecise for a scientific publication. The text is intellectually ambitious and stylistically modern; however, at times it falls into “grant language” or a “high-impact marketing tone.” It contains too many abstract nominalizations and elements of metaphorical systems-based language (e.g., reprogramming, reconfiguration, performance recovery).

Furthermore, as a reviewer, I have concerns regarding the experimental design. The study was conducted over a single growth cycle lasting three months, whereas standard field cultivation of sugarcane lasts at least 10–12 months. The authors performed the experiment under controlled conditions (which is understandable), but terminated it after three months. Why was the experiment concluded after three months and not, for example, six? If this decision was based on a specific developmental stage, this should be clearly stated. Do the authors have evidence that silicon affects nutrient uptake and translocation efficiency only at this particular stage of sugarcane development?

Please specify the basis on which the concentrations of 3 mmol Ca L⁻¹ and 1.77 mmol Si L⁻¹ were considered sufficient.

The manuscript should be supplemented with the number of plants per replicate, a clear description of the statistical methods used to analyse the data, and Table 1 (which appears to be missing). Units should also be standardized throughout the manuscript (µS cm⁻¹ vs dS m⁻¹; mmol L⁻¹ vs mM).

Figures 1–9 are difficult to read due to the excessive amount of information presented. I suggest including the legend explaining bar colours (absence or presence of silicon nanoparticle application) only once rather than repeating it in each figure. The descriptions in Figure 10 are completely illegible. However, Figure 10 and its discussion constitute the most valuable part of the manuscript.

Including elemental symbols (C, N, P, Si) in the Abbreviations section is unnecessary. If they are included, then consistency would require listing the remaining nutrients mentioned in the manuscript as well.

Additional minor comments are provided directly in the text.

Comments for author File: Comments.pdf

Author Response

Comment

The experiment was conducted over a single growth cycle of three months, whereas sugarcane cultivation in the field usually lasts 10–12 months. Why was the experiment concluded after three months?

Response: 

We thank the reviewer for raising this important point. The experiment was conducted under controlled greenhouse conditions with the objective of evaluating early physiological and nutritional responses of sugarcane plants to Ca deficiency and nSiO₂ application. The experimental period of approximately three months corresponded to the stage at which clear symptoms of Ca deficiency became established, allowing the assessment of changes in nutrient concentrations, stoichiometry, nutrient efficiencies, and biomass accumulation.

The purpose of the study was not to reproduce the full field production cycle of sugarcane, but rather to investigate nutritional and physiological adjustments during the early growth stage under controlled conditions, which is a common approach in plant nutrition studies. This clarification has been added to the Materials and Methods section of the revised manuscript.

Comment

Please specify the basis on which the concentrations of 3 mmol Ca L⁻¹ and 1.77 mmol Si L⁻¹ were considered sufficient.

Response:

We thank the reviewer for this important observation. The concentration of 3.0 mmol L⁻¹ Ca corresponds to 60% ionic strength of the Hoagland and Arnon (1950) nutrient solution, which was adopted as an adequate level to sustain plant growth under the experimental conditions used in this study. Preliminary assessments and nutrient solution management indicated that higher ionic strengths could negatively affect plant development, and therefore this level was adopted as the Ca-sufficient treatment.

The concentration of 1.77 mmol L⁻¹ Si was defined based on the stability of the silicon source in the nutrient solution. Higher concentrations favored polymerization of silicon species, which may reduce stability and availability of Si in solution. Therefore, this concentration was selected as a stable and feasible level for Si supply under the experimental conditions.

Comment

The manuscript should be complemented with the number of plants per replication.

Response:

We thank the reviewer for highlighting this important detail. The number of plants per replication has now been clearly specified in the Materials and Methods section, and the sample size (n) has also been indicated in the legends of all figures to improve clarity regarding the experimental design and statistical analysis.

Comment

A clear description of the statistical methods used to analyze the data should be included.

Response:

We appreciate the reviewer’s suggestion. The description of the statistical analysis has been expanded in the Materials and Methods section, including the statistical tests used to evaluate treatment effects and the criteria adopted for mean comparison. This information was added to ensure transparency and reproducibility of the analytical procedures.

Comment

Table 1 appears to be missing.

Response:

We thank the reviewer for this observation. Supplementary Table 1 has now been included in the revised manuscript and presents the composition of the Hoagland and Arnon (1950) nutrient solution used in the experiment.

Comment

Units should be standardized throughout the manuscript (µS cm⁻¹ vs dS m⁻¹; mmol L⁻¹ vs mM).

Response

We thank the reviewer for this suggestion. Units have been standardized throughout the manuscript to ensure consistency and clarity. In particular, electrical conductivity and nutrient concentration units were revised and harmonized across the text, tables, and figures.

Comment

Figures 1–9 are difficult to read due to the amount of information presented.

Response:

We appreciate this helpful suggestion. The figure presentation has been revised to improve readability. The explanatory legend regarding the presence or absence of nSiO₂ application was simplified and standardized to avoid unnecessary repetition across figures. These adjustments were implemented to improve the visual clarity of the results.

Comment

The descriptions in Figure 10 are completely unreadable.

Response:

We thank the reviewer for this observation. To improve readability and visual clarity, the original figure was reorganized. The panel previously presented as Figure 10a (shoot variables) was maintained as Figure 10, while the panel corresponding to root variables, previously shown as Figure 10b, was separated and is now presented as Figure 11 in the revised manuscript.

This modification allowed us to increase the font size and improve the clarity of labels and dendrogram structure, making the interpretation of the hierarchical clustering analysis clearer for the reader.

Comment

The inclusion of elemental symbols (C, N, P, Si) in the abbreviations section is unnecessary.

Response:

We appreciate the reviewer’s comment. The abbreviation section has been revised, and elemental symbols have been removed to avoid redundancy, since these symbols are widely recognized in plant nutrition literature.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a well-designed, carefully executed study that examines the role of silicon nanoparticles in modulating mineral nutrition, nutrient-use efficiency, and growth responses under contrasting calcium conditions. Overall, the manuscript is of high scientific quality and suitable for publication. The comments below are intended as minor suggestions to improve clarity, conceptual framing, and readability and do not affect the validity of the results or the main conclusions.

Considering the limited biological replication and the large number of variables analyzed, the Discussion could be further strengthened by providing additional conceptual context. In this regard, relevant foliar studies conducted in perennial crops - such as olive or grape - could be briefly introduced to emphasize the broader relevance of silicon-mediated modulation of nutrient composition across crop types, without implying identical physiological mechanisms (examples https://doi.org/10.3390/plants13243514; https://doi.org/10.3390/plants7020045). Moreover, it would be useful to clarify that silicon-driven modulation of mineral nutrition has also been reported with conventional (non-nanoparticulate) silicon sources, and that the nanoparticulate form used in the present study may primarily enhance reactivity and delivery efficiency rather than introduce fundamentally novel modes of action.

Several results, such as changes in nutrient stoichiometry and the lack of a biomass response despite improved mineral efficiency, suggest that underlying metabolic adjustments may be involved, although these were not directly examined in this study. So I suggest adding a sentence that future studies integrating metabolomic analyses could help to further elucidate the biochemical pathways underlying the observed silicon-mediated modulation of nutrient homeostasis and use efficiency.

Technical comments:

Latin names of plant species should be consistently italicized throughout the text.

Please ensure consistent use of terminology throughout the manuscript (e.g., Ca-sufficient vs. calcium-sufficient; nano-SiO₂ vs. nSiO₂)

The sample size (n) should be clearly indicated in all figures and table legends.

The hierarchical clustering analysis is appropriate and informative; however, the font size in the HCA figure is too small to be easily readable. Increase font size of labels.

Author Response

We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments. We appreciate the positive assessment of the study and the helpful suggestions aimed at improving the clarity, conceptual framing, and readability of the manuscript. All suggestions were carefully considered and the manuscript has been revised accordingly. Our responses are detailed below

Comment 1

Considering the limited biological replication and the large number of variables analyzed, the discussion could be strengthened with the addition of conceptual context. Relevant foliar studies conducted in perennial crops such as olive or grapevine could be briefly presented.

Response:

We thank the reviewer for this valuable suggestion. Following this recommendation, we expanded the conceptual context of the discussion by incorporating examples from perennial crops, including studies conducted in olive and grapevine systems. These references were added to highlight the broader relevance of silicon-mediated modulation of plant nutritional composition across different crop types. The suggested references (Polić Pasković et al., 2024; Laane, 2018) were incorporated and discussed in the revised manuscript. This information has been added at the beginning of Section 3.2 to strengthen the conceptual framing of the results.

Comment 2

It would also be useful to clarify that silicon-induced modulation of mineral nutrition has also been reported with conventional silicon sources.

Response:
We appreciate this important clarification. The revised manuscript now explicitly states that modulation of mineral nutrition has also been reported with conventional (non-nanoparticulate) silicon sources. We also clarified that the use of silicon nanoparticles in this study likely enhances the reactivity and delivery efficiency of Si rather than introducing fundamentally new physiological mechanisms. This clarification was incorporated in the introductory paragraph of Section 3.2

Comment 3
Several results suggest that underlying metabolic adjustments may be involved… Future studies integrating metabolomic analyses could help elucidate the biochemical pathways involved.

Response: 

We agree with the reviewer that metabolomic approaches could provide deeper insights into the biochemical pathways involved in silicon-mediated modulation of nutrient homeostasis and use efficiency. Following this suggestion, we added a statement in the discussion highlighting that future studies integrating metabolomic analyses may help elucidate the metabolic mechanisms underlying the observed responses.

Technical comments

Comment

Latin names of plant species should always be written in italics.

Response:

All Latin names of plant species were carefully revised and formatted in italics throughout the manuscript.

Comment

Please ensure consistent terminology throughout the manuscript (e.g., Ca-sufficient vs. calcium sufficient; nano-SiO₂ vs. nSiO₂).

Response:

The terminology used throughout the manuscript was standardized. In particular, we adoptedconsistent use of Ca-deficient/Ca-sufficient and nSiO₂ throughout the text to improve clarity and consistency.

Comment

The sample size (n) should be clearly indicated in all figures and table legends.

Response:

The sample size (n) has now been clearly indicated in all figure captions and table legends.

Comment

The hierarchical clustering analysis figure has very small font size.

Response:

We agree with the reviewer. The hierarchical clustering figure was revised and the font size of all labels was increased to improve readability.

 

We sincerely thank the reviewer again for the thoughtful comments and constructive suggestions, which helped us improve the clarity and quality of the manuscript.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you to the Authors for their comprehensive responses to the questions raised in the review and for implementing the suggested revisions. I still believe that the language of the manuscript could be simplified. However, this comment does not diminish the substantive value of the work.

Author Response

Dear Editor,

We sincerely thank you for the careful evaluation of the revised version of our manuscript and for the additional comments that helped improve its clarity and presentation. We have carefully addressed all the points raised and revised the manuscript accordingly. Our responses are provided below.

Commeter 1:

The sample size (n) should be clearly indicated in all figures and table legends

Response:

Thank you for this observation. We have revised all figure legends and table captions to clearly indicate the sample size (n). This information has now been included in each figure and table legend in the revised manuscript.

Comment 2:

The figures contain statistical treatment indicated by uppercase and lowercase letters above the columns. However, the method and software used were not described. Please add a brief subsection in Materials and Methods with this information

Response: 

We appreciate this important suggestion. A new subsection describing the statistical analysis has been added to the Materials and Methods section. In this subsection, we now clearly describe the statistical procedures used, including the analysis of variance (ANOVA), the post hoc test used to compare means, and the software employed for the analysis.

Comment 3:
The legends in the HCA figures were improved, but the font size is still very small.

Response: 

Thank you for pointing this out. We have revised the HCA figures and increased the font size of the legends and labels to improve readability. 

We believe that these revisions have improved the clarity and quality of the manuscript. We appreciate your careful evaluation and thank you again for your valuable comments