Comparative Effects of Iron Nanoparticles, Chelates, and Iron Sulfate on Biomass, Yield, and Nitrogen Assimilation in Spinach

Response to Reviewer 1 Comments

1. Summary

Dear Reviewer.

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

This study investigated the effects of foliar application of iron oxide nanoparticles (IONPs) and traditional iron sources (ferric sulfate and iron chelate) on biomass, yield, and nitrogen assimilation in spinach. By systematically comparing the effects of different iron sources and concentrations, the study clearly demonstrated for the first time the advantages of IONPs in optimizing nitrogen metabolism in spinach. The findings have certain practical application value. However, I feel there are a number of issues which the authors really need to address before accepted for publication.

Comments 1: My main concern is that the experiments use seedlings grown in iron defficient media as control to compared the effects of different source of iron. The objective of this study appears to be identifying the most suitable iron source for spinach growth. However, if under normal growth conditions (with adequate iron supply) spinach already achieves good growth and yield, is there still a need to search for alternative iron sources? I think the experimental design should have been based on the nutrient solution formulas commonly used in current production (i.e., with normal iron supply), and then assess whether different iron sources and their concentrations can further increase yield beyond that baseline. This would allow for the selection of the most appropriate iron source and optimal foliar iron concentration. Therefore, I think it inappropriate to use an iron-deficient condition as the control for comparing different treatments. A proper control with normal iron supply should have been included. This is a fundamental basic control that really needs to be performed if the authors are to draw any realistic conclusions from their data.

Response 1: We sincerely thank the reviewer for this valuable observation regarding the need to include a control treatment with adequate iron supply in the nutrient solution. It is true that in our experiment, iron was not added to the nutrient solution, as our aim was to directly and comparatively assess the effects of different iron sources and concentrations applied via foliar spray.

We acknowledge that this decision may limit the extrapolation of the results to commercial production systems where iron is routinely supplied through fertigation. However, the primary goal of this study was to isolate and evaluate the physiological efficiency of each iron source (FeSO₄, Fe-EDDHA, and IONPs) under controlled partial iron-deficiency conditions, thereby minimizing the confounding influence of root uptake pathways.

This approach has been employed in previous studies investigating the foliar absorption efficiency of micronutrients, particularly in the case of novel formulations such as nanoparticles (e.g., Raliya et al., 2015; Dimkpa & Bindraban, 2016). Moreover, induced deficiency conditions enhance the plant system’s sensitivity to treatments, allowing subtle physiological differences to be detected that might otherwise remain hidden under optimal nutrition.

We genuinely appreciate your suggestion to include, in future trials, an additional control with standard iron supply in the nutrient solution (e.g., using Fe-EDTA or Fe-EDDHA). This would allow for a more comprehensive comparison of the added value of foliar iron applications against a standard agronomic reference. We will certainly consider this valuable recommendation in the design of future experiments.

Comments 2: Typical phenotypic pictures of plants under different treatments should be provided to visually demonstrate the effects of the various fertilization treatments. Images offer a clearer and more intuitive representation of the results.

Response 2. We appreciate the reviewer’s suggestion regarding the inclusion of representative phenotypic images. We fully agree that visual documentation can greatly enhance the interpretation and clarity of experimental results. While this study focused primarily on physiological and biochemical parameters, future research will consider the systematic inclusion of photographic records under standardized conditions to complement quantitative findings. This approach will enable a more comprehensive presentation of plant responses to different iron fertilization treatments.

Comments 3: I am not sure what the difference is between Figure 4 and Figure 5—one represents biomass and the other yield. But isn’t plant biomass essentially the same as yield? What exactly distinguishes the two? Is it just the difference between dry weight and fresh weight of biomass? If that’s the case, I suggest unifying the terminology and referring to them consistently as either "biomass" or "yield." Additionally, I recommend that the authors separate plant biomass into aboveground and root biomass in the presentation. This would allow readers to assess which iron source has a better effect based on root development as well.

Response 3.  We thank the reviewer for this important observation and the opportunity to clarify our terminology. In our study, the term “biomass” refers to the total dry weight of the plant, including both leaves and roots, while “yield” specifically refers to the fresh weight of the leaves, which represent the edible portion and are the primary economic product in spinach cultivation.

We acknowledge that the distinction may not have been sufficiently clear in the initial version of the manuscript. Therefore, we have revised the Methods section (Section 2.5.1, Lines 188-195) to explicitly define both variables and highlight the difference in units (g D.W. plant⁻¹ for biomass vs. g F.W. plant⁻¹ for yield).

Regarding the suggestion to present aboveground and root biomass separately, we respectfully note that the main objective of this study was to assess the overall physiological response of the plant to different iron sources, with emphasis on nitrogen assimilation and yield performance. For this reason, we focused on total dry biomass as a growth indicator.

However, we agree that root vs. shoot partitioning is a valuable parameter and we will consider including it in future studies aimed at root-specific physiological responses. We have added a sentence in the Discussion acknowledging this as a potential area for further investigation.

Comments 4: I suggest removing Figure 6, as the SPAD values shown in this figure essentially represent the same indicator as the chlorophyll content presented in Table 2. Since the directly measured chlorophyll content provides more accurate results, Figure 6 can be omitted to save space, especially given the limited length of the manuscript.

Response 4: We appreciate the reviewer’s thoughtful suggestion. However, we respectfully prefer to retain Figure 6, as it provides complementary, not redundant, information to that of Table 2. While both variables are related to chlorophyll status, they differ significantly in methodology, resolution, and application:

• Table 2 reports values of chlorophyll a, b, total chlorophyll, and carotenoids determined via destructive biochemical extraction, offering detailed pigment concentrations.
• Figure 6, in contrast, presents SPAD index values obtained from non-destructive, in vivo leaf readings, which are widely used in agronomic studies as a rapid diagnostic tool for leaf greenness and nitrogen status.

SPAD measurements are particularly useful in field applications because they enable real-time monitoring of plant health without the need for destructive sampling. They also reflect integrated physiological responses, including chlorophyll density, leaf thickness, and nitrogen availability. As highlighted in our introduction, one of the study’s aims is to explore practical indicators of nitrogen assimilation and iron-induced physiological changes under different Fe sources, which justifies the inclusion of SPAD as a relevant variable.

Moreover, retaining SPAD data supports our hypothesis regarding the physiological efficiency of iron nanoparticles and their potential use in precision agriculture. For these reasons, we believe that Figure 6 adds scientific value and enhances the applicability of our findings. We kindly ask to retain it in the final version.

Comments 5: I suggest that the authors supplement the study by measuring photosynthetic gas exchange parameters and chlorophyll fluorescence-related indicators. This would help further explain the differences in biomass among plants under different treatments from the perspectives of photosynthetic efficiency and light energy utilization. Such additions would make the results richer, more comprehensive, and more nuanced, as relying solely on chlorophyll pigment content provides somewhat limited evidence.

Response 5: We sincerely appreciate this valuable suggestion. We fully agree that photosynthetic gas exchange and chlorophyll fluorescence measurements would enrich the interpretation of plant physiological performance and offer deeper insights into energy use efficiency under different iron sources.

However, due to technical and logistical constraints during the experimental stage, it was not possible to include these measurements in the present study. Instead, we focused on non-destructive SPAD readings and chlorophyll pigment quantification as reliable indicators of photosynthetic capacity and nutritional status. These variables are commonly used in agronomic and physiological research as proxies to assess chlorophyll content, nitrogen assimilation, and plant vitality.

In addition, biomass accumulation, yield, and nitrogen-related biochemical markers (soluble proteins and free amino acids) were evaluated to integrate the physiological response to iron treatments. We acknowledge the value of including photosynthetic efficiency parameters in future studies and will consider expanding the methodological scope accordingly.

Comments 6: The authors conducted an iron biofortification experiment, but the results lack data on nutrient element contents in plant leaves and roots, especially iron content. This omission makes the study incomplete. When comparing the effects of iron biofortification, measuring the iron content in the plants is essential.

Response 6: We appreciate the reviewer’s insightful comment. However, we would like to clarify that the objective of this study was not to conduct a biofortification trial aimed at increasing the edible iron content in plant tissues. Instead, our primary aim was to evaluate the physiological role of different iron sources (IONPs, Fe-EDDHA, and Fe₂(SO₄)₃) on nitrogen assimilation and associated metabolic responses in spinach.

Our focus was specifically on nitrogen-related parameters—including free amino acids, soluble proteins, SPAD index, and photosynthetic pigments—as indicators of nitrogen metabolism efficiency. These variables were chosen to elucidate how iron availability, depending on the source, may influence the enzymatic pathways involved in nitrogen assimilation.

While we recognize the value of determining tissue iron content, particularly in biofortification studies, it falls outside the scope of the present work. Future studies may incorporate iron quantification to further correlate nutrient uptake with physiological responses.

Comments 7: The authors need to clarify in the discussion that although IONPs100 treatment significantly increased biomass (+49.8%) and yield (+40%)—the highest among all treatments—the photosynthetic pigment content under this treatment actually decreased, and nitrogen metabolism enzyme activity was even significantly inhibited. These observations require further detailed explanation in the discussion.

Response 7. We thank the reviewer for this important observation. As suggested, we have expanded the discussion to address the discrepancy between the improved biomass/yield under the IONPs100 treatment and the observed reductions in photosynthetic pigments and nitrate reductase (NR) activity.

We hypothesize that the high dose of IONPs (100 ppm) may have exceeded the optimal concentration range for certain metabolic processes, leading to subtle oxidative stress or nutrient imbalances that impaired chlorophyll biosynthesis and NR enzyme activity, despite an overall increase in biomass. This apparent contradiction suggests a differential sensitivity of physiological pathways to iron levels: while structural growth may continue under slightly stressful conditions, enzymatic and pigment pathways may be more tightly regulated and vulnerable to excess Fe accumulation.

We have added a new paragraph discussing this trade-off in Section 3.5 (Lines 435–451) and included references highlighting dose-dependent effects of nanoparticles on metabolic activity and photosynthetic pigment stability (e.g., Feng et al., 2022; Tombuloglu et al., 2024). These results support the idea that IONPs50 may represent the most physiologically efficient treatment, while IONPs100, though effective in biomass accumulation, may not sustain optimal metabolic balance.

Response to Reviewer 2 Comments

1. Summary

Dear Reviewer.

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

The following are the review comments on this article:

Comments 1: Line 18-20, the authors did not specify what the control is.

Response 1: Thank you for your comment. We agree that the original version of the summary did not explicitly define the control treatment used as a reference for comparison. In response, we have added "0 ppm" (line 18) where application rates are specified to clarify that all percentage increases in physiological parameters refer to comparisons with the untreated control (0 ppm), i.e., plants that did not receive foliar iron application. This adjustment improves transparency and facilitates accurate interpretation of the results.

Comments 2: Line 21-22, SPAD values reflect chlorophyll content to some extent. It would be better to choose one of them.

Response 2: Thank you for your observation. While SPAD values are indeed correlated with chlorophyll content, we respectfully consider that they provide complementary rather than redundant information. SPAD is a non-destructive, rapid, and integrative measurement of leaf greenness and nitrogen status, whereas the quantification of photosynthetic pigments (chlorophyll a, b, total, and carotenoids) is obtained through destructive biochemical extraction.

Both methods were included intentionally: SPAD allows in vivo assessment under field-like conditions, while pigment analysis provides detailed quantification. Including both strengthens the interpretation of physiological responses and aligns with our aim of evaluating nitrogen metabolism under different iron sources.

For this reason, we have kept both measurements in the study.

Comments 3: Line 23, expressions like "up to 50%" and "47% more" are inappropriate. Specific numerical values should be used directly.

Response 3: We thank the reviewer for this valuable observation. While we understand the importance of presenting precise data, we chose to retain percentage-based expressions in the abstract to improve clarity and facilitate quick understanding of treatment effects, especially for readers outside the immediate research field.

However, we have revised the phrasing to ensure accuracy and specificity. The expressions now read as "biomass (up to 50%), yield (40%), and nitrate reductase activity (246%) compared to the untreated control." This format balances scientific rigor with readability and maintains consistency with how treatment effects are discussed in the main text.

Comments 4: Line 24-25 The authors did not measure photosynthetic parameters such as net photosynthesis. It is not reasonable for the authors to directly reflect photosynthesis through chlorophyll content.

Response 4: We thank the reviewer for this important clarification. We agree that chlorophyll content does not directly measure photosynthetic performance, and we would like to clarify that our manuscript does not claim to assess net photosynthesis or use pigment data as a direct substitute for gas exchange parameters.

In this study, the determination of chlorophyll a, b, and carotenoids was used as an indirect indicator of leaf physiological status, specifically to assess the impact of different iron sources on nitrogen metabolism, which includes chlorophyll biosynthesis pathways. The pigments were evaluated as part of the plant’s integrated response to iron-mediated nitrogen assimilation, not as a proxy for photosynthetic rate.

Given the specific focus of the research—nitrogen assimilation and related biochemical markers (nitrate reductase activity, soluble protein, and amino acids)—we opted not to include photosynthetic gas exchange parameters. We will revise the sentence in the manuscript to avoid any misinterpretation.

Comments 5: Line 36-38 is unreasonable. Nitrogen is important, but there is no need to elaborate on it in the first paragraph.

Response 5: We thank the reviewer for this observation. We agree that a detailed discussion of the biochemical functions of nitrogen in the first paragraph could detract attention from the broader context. In response, we have restructured the introductory paragraph to emphasize the global challenges facing agricultural systems and the need for improved nutrient management, without overemphasizing nitrogen at this early stage.

The revised paragraph introduces nitrogen (N) and iron (Fe) as essential strategic nutrients for maintaining crop productivity, laying the groundwork for a more in-depth analysis of their physiological roles in later sections. This change improves the scientific focus, better fits the scope of the manuscript and improves the logical coherence of the introduction.

"Increasing demands on agricultural systems, driven by climate change, population growth and the need to intensify food production, have highlighted the urgency of adopting more sustainable and efficient nutrient management strategies. Among these, optimizing the use of key elements such as nitrogen (N) and iron (Fe) is essential to support crop yields and ensure long-term productivity"

We believe this revision improves the consistency and clarity of the introduction, in line with the reviewer's recommendation.

Comments 6: Mentioning N multiple times in the early part is not appropriate. It is suggested to rewrite it. First, the differences among the three types of Fe should be introduced, and then the importance of nitrogen uptake can be discussed.

Response 6: Thank you for this valuable recommendation. In response, we have restructured the introduction to first present the rationale for comparing three iron sources—ferric sulfate, iron chelate (Fe-EDDHA), and iron oxide nanoparticles (IONPs)—highlighting their differences.

We then introduce the relevance of nitrogen assimilation and its regulation by iron-dependent enzymes. This reorganization improves the flow of the introduction and reinforces the justification of the study's objectives. We appreciate this suggestion, which has contributed to improving the scientific clarity of our manuscript.

Comments 7: Line 60-61, regarding the importance of metal nanoparticles, it is recommended to cite this article (https://doi.org/10.1016/j.crope.2025.03.004).

Response 7: We thank the reviewer for this helpful suggestion. As recommended, we have now cited the article by Li et al. (2025) in the corresponding section (Line 61) to strengthen the discussion on the physiological benefits of metal nanoparticles in crops. This reference adds valuable evidence on the role of ZnO nanoparticles in improving biomass accumulation, chlorophyll content, and stress resilience, supporting the relevance of nanomaterials in nutrient efficiency studies.

Comments 8: In terms of the characterization of IONPs, only basic information such as shape, size, and purity is provided. Detailed descriptions of properties such as crystal structure, specific surface area, and surface charge are lacking. It is suggested to supplement these details.

Response 8: We thank the reviewer for this insightful suggestion. In response, we have substantially expanded the nanoparticle characterization section in the manuscript. The Fe₂O₃ nanoparticles used in our study were produced via wet chemical synthesis and identified as the maghemite (γ-Fe₂O₃) polymorph, confirmed through X-ray diffraction (XRD) analysis. Morphological and elemental composition were verified through scanning and transmission electron microscopy (SEM, TEM) and EDX, respectively, revealing uniform particle distribution, sub-50 nm size, and high purity (99.7%).

While specific surface area (BET) and zeta potential were not reported by the supplier and could not be independently measured during this study, the crystalline phase, morphology, and elemental purity were rigorously assessed and met the requirements for foliar application. We have updated the manuscript accordingly to include this additional information.

Comments 9: Why IONPs can more effectively promote nitrogen assimilation and photosynthesis in spinach within a certain concentration range, but have the opposite effect at high concentrations, has not been discussed in detail. Are there different absorption, transport, or metabolic pathways compared with traditional iron sources?

Response 9: We appreciate your valuable comment. We have added a new paragraph at the end of Section 3.5 (lines 446-462) to address the differential behavior of iron oxide nanoparticles (IONPs) in comparison to conventional iron sources. This paragraph discusses how, at moderate concentrations (e.g., 50 ppm), IONPs can act as a slow-release source of Fe³⁺, enhancing nitrogen assimilation by maintaining optimal levels of bioavailable iron without inducing toxicity. Their nanometer size facilitates foliar penetration through cuticular pores and stomata, and their subsequent apoplastic translocation differs from the classical ionic uptake pathway followed by fully soluble sources such as Fe-EDDHA and Fe₂(SO₄)₃. However, at higher concentrations (100 ppm), excessive nanoparticle internalization can disrupt redox homeostasis and cause oxidative stress, which could inhibit enzymatic processes such as nitrate reduction and degrade photosynthetic pigments. The paragraph highlights the need to better understand the relationship between nanoparticles and plants and the delicate balance between beneficial and toxic thresholds in nano-based fertilization strategies.

Comments 10: The intrinsic connections and comprehensive analysis of some experimental results are insufficient. For example, the correlation analysis between biomass, yield of spinach and indicators such as photosynthetic pigments and nitrate reductase activity is relatively simple. The quantitative relationships among these indicators and how they jointly affect the growth and yield formation of spinach have not been thoroughly explored.

Response 10: We are deeply grateful for this comment, which allowed us to reflect on the integrated interpretation of the results. Although the main objective of this study was not to establish predictive statistical relationships between biomass, yield, and physiological variables, we did observe consistent patterns that support a physiologically coherent interpretation.

We have expanded the discussion in Section 3.5 by integrating observations on how variations in photosynthetic pigments and nitrate reductase activity correlate. For example, treatments such as IONPs50 not only showed elevated levels of NRmax activity and chlorophyll content but also exhibited the highest increases in yield, suggesting a possible functional relationship between greater photosynthetic efficiency and nitrogen assimilation capacity. In contrast, higher concentrations, such as IONPs100, although increasing biomass, were associated with enzymatic inhibition and pigment reduction, indicating a metabolic imbalance that may limit resource utilization.

We acknowledge that future research could benefit from more robust multivariate models (such as multiple regression analysis) to more precisely quantify these interactions. However, in this study, we opted for an interpretative approach that emphasizes the physiological behavior of each treatment under controlled conditions.

Response to Reviewer 3 Comments

1. Summary

Dear Reviewer.

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

Response to Reviewer 4 Comments

1. Summary

Dear Reviewer.

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors