Influence of Nanoparticles on Morpho-Physiological, Growth and Yield Traits of Rice (Oryza sativa L.) Cultivars Under Early Seedling Cold Stress at Different Developmental Stages

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please read the document

Comments for author File: Comments.pdf

Comments on the Quality of English Language

English needs improvement

Author Response

Response to Comments of Reviewer #1:

Comment 0:

The use of nanoparticles is a modern and effective approach to increasing plant resistance to adverse factors. Indeed, the arsenal of recommended and applied nanoparticles is wide today. And it is important to study which of the nano-preparations will be effective in increasing the cold resistance of rice.

Response 0:

Thank you for your careful evaluation of our manuscript and for providing us with the opportunity to revise it. We appreciate the constructive comments from you and the reviewers, which have helped us improve the quality and clarity of our work. All modifications in the manuscript are highlighted red, so that any changes can be easily reviewed by editors and reviewers.

Comment 1:

Please specify in the title of your work whether you used already stable rice seedlings or whether you studied the development of cold resistance.

Response 1:

We thank the reviewer for this suggestion. We have revised the title to clarify that we studied cold stress tolerance in rice seedlings at different developmental stages (14, 21, and 28 days after emergence). The revised title now reads: "Influence of nanoparticles on morpho-physiological, growth and yield traits of rice (Oryza sativa L.) cultivars under early seedling cold stress at different developmental stages"

Comment 2:

In the materials and methods section, I suggest making a table on the characteristics of the rice varieties that you used in your work.

Response 2:

We agree with this valuable suggestion. We have now included a new table (Table 1) summarizing the key characteristics of the four rice cultivars used in this study.

Comment 3:

The Abstract of the article is completely uninformative. Please list the rice varieties used, explain their differences, and indicate why you chose these particular varieties. Separately, list the parameters measured after exposure to the stress factor and explain what you mean by the recovery period. Indicate that you studied three types of cold stress duration and examined the plants 7 days after exposure to the stress factor. The current version of the abstract is difficult to read and does not match the information provided in the Materials and Methods section. You have studied the effect of cold on different stages of rice growth, and this should be emphasized. It is also important to specify the growth period for each variety. This can be found in section 2.9.

Response 3:

We greatly appreciate this critical feedback. We have completely rewritten the Abstract to address all these points.

Comment 4:

In the Materials and Methods section, section 2.6, provide the formulas that used the data from the three wavelength measurements of 450, 532, and 600 nm.

Response 4:

Thank you for your helpful suggestion. We have revised Section 2.6 by adding the mathematical formulas used to calculate the results from the absorbance values measured at 450, 532, and 600 nm. The equations and definitions of all variables have been included in the revised manuscript for clarity and reproducibility.

Comment 5:

It is necessary to distinguish between controlled and field conditions in the Abstract (Introduction) and this is unclear. Clarify that 14 degrees of cold were provided.

Response 5:

Thank you for this valuable comment. We have revised both the Abstract and the Introduction to clearly distinguish between the controlled experimental conditions and the field conditions. We have also explicitly stated that the cold treatment was applied at 14°C under controlled conditions, thereby improving the clarity of the experimental design.

Comment 6:

Table 1 is confusing. Make it logical. Separate growth parameters and pigment content. Provide figures for stress related indicators only and the use of nanoparticles. Nothing is clear. Please redo this section.

Response 6:

We thank the reviewer for this helpful suggestion. Table 1 has been completely reorganized to improve clarity.

Comment 7:

Figures 2 and 3—you indicate in the captions that, from left to right, these are the stages of rice development. Please include these designations in the figure itself. This will make things clear.

Response 7:

We thank the reviewer for this valuable suggestion. Figures 2 and 3 have been revised by adding the corresponding rice developmental stage labels directly within each figure. This modification improves clarity and makes the figures easier to interpret.

Comment 8:

In the Discussion section, I believe that we should first discuss the harmful effects of cold. Then, we can discuss the protective effects of nanoparticles.

Response 8:

We thank the reviewer for this valuable suggestion. The Discussion section has been reorganized to improve its logical flow. We now begin by discussing the detrimental effects of cold stress on early rice seedlings, including its impacts on growth, photosynthesis, oxidative stress, and cellular metabolism. This is followed by a discussion of how nanoparticle application mitigates these adverse effects and enhances cold tolerance. These revisions provide a clearer progression from stress-induced damage to the protective mechanisms of nanoparticles.

 

Reviewer 2 Report

Comments and Suggestions for Authors

1. I did not find any Supplementary Materials referenced in the manuscript (lines 122 and 124).

2. The Introduction and Discussion sections do not mention information on the effect of gold nanoparticles (the "standard" for nanoparticles) on wheat under low temperature conditions, including articles in the Plants.

3. Add a rationale for the choice of titanium oxide nanoparticles.

4. Add a rationale for the nanoparticle concentrations used.

5. For what purpose did the authors conduct (Subsection 2.3) and present the results of meteorological measurements (Section 3.1) if the plant cultivation conditions were mentioned in the description of the experimental design (Subsection 2.2)?

6. Explain why the maximum temperatures in Figure 1a were lower than the minimum temperatures for some days.

7. The subsections describing the effect of nanoparticles on rice seedlings begin with duplicate sentences. Carefully check the text for redundancy.

8. Is the manuscript missing information on the comparison of the responses of four varieties to nanoparticle treatment? Was the effectiveness of nanoparticles the same or different for all varieties?

9. A significant shortcoming of the manuscript is the lack of experimental data or discussion of the effect of the nanoparticles used by the authors on the growth, development, and yield of rice without cold stress.

10. In Figure 2, use "µmol" (according to the SI system) as unit of measurement.

11. In the Discussion section, the authors explain the superior performance of iron oxide nanoparticles by improving iron uptake by plants for metabolism. This is possible if plants are iron deficient. Wouldn't it be easier to use cheaper iron compounds than nanoparticles to supply plants with iron? What is the effectiveness of iron oxide nanoparticles over other iron-containing preparations?

12. The use of a snowflake in Figure 8 to represent cold stress is unjustified, as the manuscript used temperatures above freezing. Use a different symbol for low temperatures.

Author Response

Response to Comments of Reviewer #2:

Comment 1:

I did not find any Supplementary Materials referenced in the manuscript (lines 122 and 124).

Response 1:

Thank you for bringing this to our attention. The Supplementary Materials were inadvertently omitted from the original submission. They have now been uploaded and are appropriately cited in the revised manuscript.

Comment 2:

The Introduction and Discussion sections do not mention information on the effect of gold nanoparticles (the "standard" for nanoparticles) on wheat under low temperature conditions, including articles in the Plants.

Response 2:

We thank the reviewer for this thoughtful suggestion. While studies on gold nanoparticles in wheat under low-temperature stress provide valuable insights, our manuscript focuses specifically on the role of Fe₂O₃, ZnO, TiO₂, and CeO₂ nanoparticles in enhancing cold tolerance in rice. Therefore, to maintain the scope and focus of the manuscript, we did not include a detailed discussion of gold nanoparticles in wheat. Instead, we have expanded the Introduction and Discussion by incorporating additional literature relevant to nanoparticle-mediated cold stress tolerance in rice and closely related crop species.

Comment 3:

Add a rationale for the choice of titanium oxide nanoparticles.

Response 3:

Thank you for this valuable suggestion. We have added a clear rationale for the selection of TiO₂ nanoparticles in the Introduction section of the revised manuscript.

Comment 4:

Add a rationale for the nanoparticle concentrations used.

Response 4:

Response: Thank you for this valuable comment. We have added a clear rationale for the selected nanoparticle concentrations in the revised manuscript (Materials and Methods section 2.2).

Comment 5:

For what purpose did the authors conduct (Subsection 2.3) and present the results of meteorological measurements (Section 3.1) if the plant cultivation conditions were mentioned in the description of the experimental design (Subsection 2.2)?

Response 5:

We appreciate this important clarification question. The meteorological data (Section 2.3 and 3.1) were included to provide context for the environmental conditions during the entire growing season, particularly for the field-grown plants during the recovery and yield assessment phases. While the cold stress treatment was conducted under controlled conditions in a growth chamber (as described in Section 2.2), the plants were grown under natural field conditions before stress imposition and during the recovery period after cold treatment. We have clarified this in the revised manuscript in Section 2.3.

Comment 6:

Explain why the maximum temperatures in Figure 1a were lower than the minimum temperatures for some days.

Response 6:

We thank the reviewer for identifying this potential error. After careful verification of our data, we discovered that this was a typographical error in the figure labeling. The maximum temperatures were indeed consistently higher than minimum temperatures on all days. We have corrected Figure 1a to accurately reflect the temperature data, with maximum temperature values properly plotted above minimum temperatures.

Comment 7:

7. The subsections describing the effect of nanoparticles on rice seedlings begin with duplicate sentences. Carefully check the text for redundancy.

Response 7:

We have thoroughly reviewed the manuscript and eliminated redundant sentences. Specifically:

- Section 3.2 (Photosynthetic pigments) - removed the duplicate opening sentence

- Section 3.3 (ROS, MDA, and Proline) - revised to avoid repetition

- Section 3.4 (Antioxidant enzymes) - restructured to begin with a unique introductory statement

Each subsection now begins with a distinct, informative sentence that reflects the specific parameters being discussed.

Comment 8:

8. Is the manuscript missing information on the comparison of the responses of four varieties to nanoparticle treatment? Was the effectiveness of nanoparticles the same or different for all varieties?

Response 8:

We appreciate this important question. The cultivar-specific responses are indeed presented in our results, but we have now made them more prominent and easier to identify. We have:

1. Added a new subsection in the Results section (3.6): Cultivar-specific responses to nanoparticle treatments under cold stress that explicitly compares the four cultivars
2. Created a new table (Table S3 in Supplementary Materials) summarizing the relative effectiveness of each nanoparticle treatment for each cultivar:

 

Table S3. Relative effectiveness of each nanoparticle treatment for each cultivar

Cultivar	Most Effective NP	Least Effective NP	Overall Tolerance
LLY-7108	Fe₂O₃ > ZnO > CeO₂ > TiO₂	TiO₂	High
XZX-6	Fe₂O₃ > ZnO > CeO₂ > TiO₂	TiO₂	High
LLY-32	Fe₂O₃ > ZnO > CeO₂ > TiO₂	TiO₂	Moderate
ZJZ-17	Fe₂O₃ > ZnO > CeO₂ > TiO₂	TiO₂	Low

 

Comment 9:

A significant shortcoming of the manuscript is the lack of experimental data or discussion of the effect of the nanoparticles used by the authors on the growth, development, and yield of rice without cold stress.

Response 9:

We acknowledge this important oversight. In the revised manuscript, we have added:

Comment 10:

In Figure 2, use "µmol" (according to the SI system) as unit of measurement.

Response 10:

 We have corrected all units in Figure 2 (and throughout the manuscript) to use SI units:

- ROS: µmol g⁻¹ FW (instead of mM)

- MDA: µmol g⁻¹ FW (instead of mM) 

- Proline: µmol g⁻¹ FW (instead of mM)

We have also ensured consistency throughout the manuscript using the SI system.

 

Comment 11:

In the Discussion section, the authors explain the superior performance of iron oxide nanoparticles by improving iron uptake by plants for metabolism. This is possible if plants are iron deficient. Wouldn't it be easier to use cheaper iron compounds than nanoparticles to supply plants with iron? What is the effectiveness of iron oxide nanoparticles over other iron-containing preparations?

Response 11:

 This is an excellent and important question. We have expanded our discussion to address this point:

Added to Discussion Section 4.4: While it is true that plants could be supplied with iron through conventional iron compounds such as Fe-EDTA or ferrous sulfate, nanoparticles offer several distinct advantages beyond simple iron supplementation. First, Fe₂O₃ nanoparticles function as sustained-release iron sources, gradually releasing bioavailable Fe²⁺/Fe³⁺ over time, thereby reducing the risk of iron toxicity associated with high concentrations of soluble iron compounds. Second, nanoparticles exhibit systemic movement within plants, potentially delivering iron to specific organelles and tissues more efficiently than ionic iron. Third, the superior effectiveness of Fe₂O₃ nanoparticles compared to conventional iron fertilizers (typically 20-40% higher chlorophyll content and 25-35% higher antioxidant enzyme activities under stress) can be attributed to their unique physicochemical properties, including high surface area-to-volume ratio, controlled dissolution kinetics, and the ability to scavenge reactive oxygen species directly through their redox-active surface. Additionally, Fe₂O₃ nanoparticles can modulate stress-responsive signaling pathways independent of their iron-supplying function, as evidenced by their effects on antioxidant gene expression and hormone signaling. Thus, the benefits of Fe₂O₃ nanoparticles extend beyond simple iron nutrition, representing a multifunctional approach to stress mitigation.

Comment 12:

The use of a snowflake in Figure 8 to represent cold stress is unjustified, as the manuscript used temperatures above freezing. Use a different symbol for low temperatures.

Response 12:

We agree with this observation. The snowflake symbol was inappropriate as our cold stress conditions were above freezing (14°C/10°C). We have replaced the snowflake with a more appropriate symbol representing low temperature stress.

 

 

 

 

 

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors did the work. All my comments were taken into account and included in the manuscript.

Author Response

Comment : The authors did the work. All my comments were taken into account and included in the manuscript.

Response : Thanks for your time to reviewe our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responded to all my comments and made the necessary changes to the revised manuscript. The file provided by the authors lacks line numbers.

One comment that requires further consideration concerns the data in Figure 1 and the text describing this figure (page 9, last paragraph).

1) In Figure 1A, one of the red dots lies significantly below 17.5°C, but the text states "the lowest maximum (19.41°C) occurred on May 1 (Figure 1a)." This discrepancy between the figure and the text should be resolved.

2) The caption to Figure 1 does not match the figure: Figures 1b and 1c are labeled (c) and (b), respectively. Furthermore, as I understand the figures, "from April 8 to August 8, 2024" refers to all three figures (1a, 1b, and 1c), not just Figure 1c.

3) The axis title in Figure 1b requires clarification of the units of measurement. What units are used for Solar radiation per square meter per day?

Furthermore, the authors use different formats for writing units of measurement throughout the manuscript. For example, the following variants are used: "mg L^-1" and "U/mg." Consistently format all units of measurement in the manuscript. And "Ug/g" should be replaced with "μg/g."

Author Response

Thank you for your careful evaluation of our manuscript and for providing us with the opportunity to revise it. We appreciate the constructive comments from you and the reviewers, which have helped us improve the quality and clarity of our work. All modifications in the manuscript are highlighted red, so that any changes can be easily reviewed by editors and reviewers.

 

Comment 1.

In Figure 1A, one of the red dots lies significantly below 17.5°C, but the text states "the lowest maximum (19.41°C) occurred on May 1 (Figure 1a)." This discrepancy between the figure and the text should be resolved.

 

Response

 

We thank the reviewer for this suggestion. We have revised the results and made necessary changes.

 

Comment 2.

The caption to Figure 1 does not match the figure: Figures 1b and 1c are labeled (c) and (b), respectively. Furthermore, as I understand the figures, "from April 8 to August 8, 2024" refers to all three figures (1a, 1b, and 1c), not just Figure 1c.

Response

We thank the reviewer for this suggestion. We changed the figure caption accordingly: Daily average, minimum, and maximum temperatures (1a), rainfall intensity and monthly cumulative rainfall (1b), seasonal energy trends, and solar radiation (1c) from April 8 to August 8, 2024.

 

Comment 3.

The axis title in Figure 1b requires clarification of the units of measurement. What units are used for Solar radiation per square meter per day?

Response

We greatly appreciate this critical feedback. We use unit of MJ/m² d

 

Furthermore, the authors use different formats for writing units of measurement throughout the manuscript. For example, the following variants are used: "mg L^-1" and "U/mg." Consistently format all units of measurement in the manuscript. And "Ug/g" should be replaced with "μg/g."

Response

Thank you for this valuable suggestion. We have carefully reviewed the entire manuscript and standardized the formatting of all units of measurement to ensure consistency.

Author Response File: Author Response.docx