Review Reports

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 aim of the work was to verify the effects brought about by foliar treatment of iodine in the form of nanoparticulate iodine in comparison with potassium iodide in lettuce with the aim of demonstrating that the structural properties of this soil conditioner were able to improve nitrogen metabolism in the plant to the benefit of a physiological improvement in product quality and yield.

Objective and purpose are the only clear aspects of the manuscript, and the experimental design, adopted and also described in detail, is in line with these points. Everything else, however, needs to be revised, starting with the abstract and the introduction, which are unclear especially with regard to the motivation behind the study.

The choice, then, of describing Results and Discussion together in the various sub-sections devoted to the different classes of parameters analysed prevents an overall and critical comparison of the various effects taken into consideration that could lead to a general conclusion of the study carried out. Moreover, in many places the interpretations of the results are not in line with the data presented.

It should be emphasised that most of the references cited in support of the arguments given in the Introduction and the interpretations in Results and Discussion are either not inherent to the themes for which they are given or are misinterpreted.

In particular

In Abstract:

Comments 1:  A sentence explaining the choice of using just INPs and KI should be included.

Response 1: We thank the reviewer for pointing out the need to clarify the rationale behind the selection of iodine forms. In response, we have added a sentence in the abstract that explicitly states the purpose of comparing a conventional ionic source (KI) with a nanoparticulate formulation (INPs), which are increasingly relevant in plant nutrition research. This contrast helps to evaluate not just iodine delivery but also the potential physiological implications of nanostructuring.

Comments 2:  It should be better developed in the results part: the advantageous effect of using INPs over KI is not clearly explained and, in any case, conclusions are drawn from a misinterpretation of the results, as will be pointed out later, and therefore not true.

Response 2: We appreciate the reviewer’s observation regarding the interpretation of results. In the revised abstract, we have rephrased our conclusions to present a more balanced perspective. We now highlight that INPs and KI elicited distinct physiological effects, and neither treatment consistently outperformed the other across all measured variables. The conclusion emphasizes the differentiated responses and potential relevance of iodine formulations in metabolic modulation, without overstating superiority. This revision ensures that the conclusions remain firmly grounded in the observed data and accurately reflect the complex outcomes.

In Introduction:

Comments 3:  Support, justify and link the sentence at Line 46-47 better than the previous sentence.

Response 3: Thank you for this valuable observation. In the revised version, we restructured the paragraph to improve the logical flow and strengthened the transition between the role of nitrogen and the possible influence of iodine. Specifically, we now emphasize how nitrogen is essential for key metabolic processes and then introduce iodine as a micronutrient with emerging relevance in nitrogen metabolism, supported by recent studies (Blasco et al., 2008; Medrano-Macías et al., 2016) [5,6].

Comments 4:  The contents of the articles (reference 6,8,9) at line 51 do not seem to be relevant to the sentence for which they are quoted. This is especially with reference to the role of iodine. Add already at this point more relevant articles testifying to the role of Iodine as an influential enzyme cofactor(?) in the metabolism and utilization efficiency of nitrogen.

Response 4: We appreciate your careful evaluation of our references. In response, we removed the previous references (6, 8, 9) and replaced them with more appropriate and up-to-date citations that explicitly support the role of iodine in modulating nitrogen metabolism and nitrate reductase activity, such as Blasco et al. (2011) and Kiferle et al. (2022) [7,8]. These studies provide direct evidence of iodine's influence on nitrate reductase activity and related metabolic processes in plants.

Comments 5: The references 13,14,15 at line 53 are not applicable. In reference 13, urea enriched with iodine is used as a fertilizer but in the form of Iodate in soil, and the effect of decreasing the volatilization of urea nitrogen is attributed to the reduced activity of the urease enzyme in soil. The study described in ref.14, on the other hand, apart from the fact that it is very dated, is actually a study linked to the subject of biofortification and presents contradictory results on the use of Iodine, which would lead to a decrease in N uptake in the plant studied and at the same time to an increase in NUE, unlike iodate, which would improve all nitrogen-related parameters. Reference 15 is completely irrelevant.

Response 5: Thank you for this clarification. We have carefully reviewed and removed references 13, 14, and 15 from the original version. Instead, we incorporated more relevant and recent studies focusing specifically on the physiological effects of iodine forms in plants. The revised references better align with the context of nitrogen assimilation and nanoparticle efficacy, such as Shalaby (2025) [16] and recent reviews on nanomaterials and foliar nutrition (Rico et al., 2011; Servin et al., 2015) [10,11].

Comments 6: The sentence at lines 55-57 is inaccurate. In reference 12 cited by the way, the possibility of using the iodate ion as an alternative substrate (and not ‘alternative cofactor’) of the enzyme nitrate reductase linked to a possible mechanism of absorption of Iodine as Iodide and thus linked to the Biofortification process is emphasised. In reference 12 there is no link between Iodine Biofortification and direct ameliorative effect for N assimilation in plants: this statement should be better reinforced.

Response 6: Thank you for highlighting this conceptual inaccuracy. We have corrected the statement in the revised manuscript by clarifying that iodate may act as an alternative substrate of nitrate reductase, not a cofactor, as proposed by Blasco et al. (2011). We also removed any misleading implications regarding a direct and confirmed role in nitrogen assimilation enhancement, and instead focused on the observed physiological outcomes reported in relevant studies.

Comments 7: Reference 14 (line 61), as already mentioned, is outdated (almost 15 years ago) and should be replaced with studies leading to the same statements, which are not exact, as reported by the authors, if one is to rely on this study alone.

Response 7: We acknowledge this recommendation and have excluded the outdated reference. It has been replaced with more recent and contextually appropriate literature, such as Shalaby (2025) [16], which reports on the effects of iodine foliar applications on antioxidant status and nitrate content in lettuce under stress conditions. This directly supports the relevance of iodine in modulating nitrogen-related parameters.

Comments 8: In reference 10 at line 64 (a very recent and accurate review of studies on Nanomaterials- based Biofortification), there is no mention of any article concerning the use of iodine nanoparticles and thus on the possible positive effects in particular for N assimilation.

Response 8: Thank you for this observation. We have adjusted the narrative accordingly. In the revised version, we explicitly state that the current literature on iodine nanoparticles is limited, and that their potential role in nitrogen metabolism remains underexplored. This revision strengthens the rationale for our study as a novel contribution in this emerging area, as supported by recent studies on the physiological impacts of nanomaterials (Tripathi et al., 2017; Rai et al., 2021) [13,14].

Comments 9: In the final analysis, in view of my comments on the bibliography cited, it is not clear how the arguments given in the introduction support and justify the study carried out and the expectations set out at lines 82-88.

Response 9: We appreciate your concern and have restructured the entire introduction to provide a clearer and more cohesive justification for the study. The revised version highlights: (1) the global challenge of nitrogen use efficiency; (2) the physiological importance of nitrogen; (3) the potential of iodine and its forms (particularly INPs) to modulate nitrogen-related processes; and (4) the existing gap in comparative studies between INPs and conventional iodine sources. These revisions now provide a stronger and more coherent foundation for the hypothesis and objectives of the study.

In Materials and Methods

Comments 10: What is the composition of the ‘..modified Hoagland nutrient...’?

Response 10: We appreciate the reviewer’s observation. The composition of the modified Hoagland nutrient solution has now been explicitly included in the revised manuscript (Lines 101–104). This detailed formulation follows the protocol proposed by Sánchez et al. (2004), adapted to meet the physiological requirements of Lactuca sativa during the experiment.

Comments 11: Reference 23 cited at line 105 is not only very old (2002) but also does not seem to be relevant.

Response 11: Thank you for your suggestion. The original reference [23] has been replaced with a more recent and relevant source (Sánchez et al., 2004) [18], which provides the specific formulation of the nutrient solution used in this study.

Comments 12: Please better clarify the sentence at lines 111-114. Add a description of the method used for ("wet chemical method") for the synthesis of the nanoparticles with suitable reference, and for their characterisation (including information about the instrumentation used).

Some fundamental data on the structural characteristics of the nanoparticles used, such as size distribution and surface energy, are missing. In addition, considering that the images in Figures 1 and 2 are shown, their content and significance, which is illustrated rather summarily in the text, must be adequately described.

Response 12: We thank the reviewer for their insightful comment. The section has been revised to improve clarity and provide additional detail on the synthesis and characterization of the iodine nanoparticles. As now indicated in the manuscript, the INPs were synthesized by a specialized nanomaterials company (Investigación y Desarrollo de Nanomateriales S.A. de C.V.) via a wet chemical reduction process from potassium iodate. This information was obtained from the product’s technical sheet provided by the manufacturer.

Additionally, the morphological and structural characteristics of the nanoparticles are now described, including the size range (80–150 nm), purity (97.3%), and methods used for characterization (SEM and EDS), as well as references to Figures 1 and 2. These additions address the reviewer’s request for greater specificity regarding the synthesis protocol and the physicochemical properties of the INPs.

Comments 13: It is incorrect to name section 2.3 “Nanoparticles preparation”.

Response 13: We appreciate the reviewer’s comment. The section title “Nanoparticles preparation” has been revised to “Preparation of iodine nanoparticle suspensions” to accurately reflect the procedure used in this study. As the nanoparticles were not synthesized in our laboratory but obtained as a commercial powder, this section now properly describes the preparation of working suspensions from the stock material.

Comments 14: At lines 144-145 better detail how the foliar application of soil improvers and the treatment plan (how many applications? At what times?).

Response 14: We thank the reviewer for the helpful suggestion. The manuscript has been revised to include specific details about the foliar application protocol. We now indicate that all treatments were applied using a manual pressurized sprayer at a volume of approximately 50 mL per plant, and that applications were carried out on days 45, 52, 59, and 66 after sowing (DAS), under controlled environmental conditions.

Comments 15: In 2.6.2 Extraction and assay of NR (E.C. 1.6.1) ? Enzyme entry has changed should be EC 1.7.7.1

Response 15: We appreciate the reviewer’s suggestion. However, the enzyme activity measured in our study corresponds to the NADH-dependent nitrate reductase, using a colorimetric assay based on the quantification of nitrite produced in the presence of NADH as electron donor. Therefore, the correct enzyme classification is EC 1.7.1.1, which reflects the NADH: nitrate oxidoreductase commonly used in plant physiological assays. The manuscript has been reviewed to ensure this classification is accurately and consistently stated.

Comments 16: At line 170 ‘root and leaf portions’ equal to ? amount?

Response 16: We thank the reviewer for the observation. The amount of tissue used for each extraction has now been specified as 100 mg of fresh root or leaf tissue.

Comments 17: At line 172 ‘DTT ’? Enter the full name of the reagent first and then the abbreviation in brackets. The same applies for ‘PVPP’.

Response 17: As suggested, the full names of dithiothreitol (DTT) and polyvinylpolypyrrolidone (PVPP) have been written out in full upon first mention, followed by their abbreviations in parentheses.

Comments 18: For all reagents used and entered in chapters 2.6.2, 2.6.3, 2.6.4 the brand name and the degree of purity must be specified.

Response 18: We appreciate this recommendation. The brand name and purity level of each reagent used in Sections 2.6.2, 2.6.3, and 2.6.4 have now been included in the revised manuscript to ensure clarity and reproducibility.

Comments 19: At line 174 insert model and brand name of the centrifuge used

Response 19: The brand and model of the centrifuge used for extractions (Hermle Z326K, Hermle Labortechnik GmbH, Germany) has now been specified in the methods section, as requested.

Comments 20: At line 177 insert name of main author before reference number 28.

Response 20: The name of the main author, Kaiser, has now been inserted before reference [28] to enhance clarity.

Comments 21: Two different articles are cited regarding the NR assay. But does the method described refer to reference 28 or 29?

Response 21: Thank you for pointing this out. We clarify that the NR assay followed the biochemical procedure described by Kaiser and Lewis (1984) [28], while the colorimetric quantification step using diazotization with sulphanilamide and naphthyl-ethylenediamine was based on Hageman and Hucklesby (1971) [29]. This distinction is now clearly indicated in the revised manuscript.

Comments 22: Specify the meaning and the difference between Chl a and Chl b with asterisk and without asterisk in equations 1,2,3.

Response 22: We thank the reviewer for the observation. The manuscript has been updated to clarify that Chl a* and Chl b* refer to intermediate values calculated from absorbance, while Chl a and Chl b are the final concentrations expressed in μg cm⁻² FW after volume and area adjustment.

Comments 23: At line 201 ‘A volume of 0,5 g’ is incorrect: replace with An amount.

Response 23: Thank you for the suggestion. The phrase “A volume of 0.5 g” has been corrected to “An amount of 0.5 g,” which accurately reflects the measurement of fresh leaf tissue used in the extraction.

In Results and Discussion

Comments 24: Chapter 3.1 needs profound changes:

Reference 35 at line 262 does not seem relevant

The sentence at line 265-267 is not accurate: no statistically significant difference is observed for treatments with KI

At line 268-269 it reads "The notable increase in biomass observed with INPs can be attributed to improved efficiency in the absorption and utilisation of nitrogen, an essential element for the formation of plant structures."  The subsequent sentences (Lines 270-276), with their supporting citations (ref. 36 and ref. 37) which should support the previous assumption, are not convincing.

Reference 36 is not relevant to the assumption to which it refers, whereas in reference 37 it only speaks of an improved effect on the antioxidant properties of lettuce brought about by an increased production of antioxidant compounds at certain levels of the iodine ion.

Furthermore, once again there is mention of the possibility of the iodate ion acting as a possible cofactor of the enzyme nitrate reductase. This is inaccurate, as previously commented on reference 12 (lines 55-57): the possibility of iodate ion being used as an alternative substrate and not as an “alternative cofactor” of the nitrate reductase enzyme. And then in the current work the experimentation is done with KI and with iodine.

The interpretation of the content of reference 39, which, by the way, being a review, also reports conflicting results from different studies, is also somewhat controversial, especially when iodine is attributed the role of ".......regulator of nitrogen metabolism."

Response 24: We appreciate the reviewer’s detailed feedback. In response, we have significantly revised section 3.1 to eliminate unsupported claims and replace non-pertinent references. Reference [35] has been substituted with a recent study (Riyazuddin et al., 2022 [35]) that clearly supports the bio-stimulatory role of iodine. We have clarified that potassium iodide (KI) treatments did not produce statistically significant increases in biomass, and therefore, no misleading conclusions were drawn. Additionally, we removed the inaccurate claim regarding iodate as an “alternative cofactor” for nitrate reductase. Instead, the revised text refers more generally to the modulation of nitrogen metabolism enzymes. Prior references [36] and [37] have been replaced with appropriate sources discussing nanoparticle uptake and nutrient assimilation (e.g., Servin et al., Siddiqi & Husen, Blasco et al.). The misinterpretation of Sularz et al. was also addressed by removing that citation entirely.

Comments 25: At line 302-304 it is stated among other things that ".....does not negatively affect reproductive

processes or the filling of harvest structures, even at relatively high concentrations"

On the other hand, it is well documented that Iodine can have negative effects on plant physiology depending on the amount added, and the toxic dose is cultivar dependent. The authors should also discuss this point.

Response 25: Thank you for the observation. We have now included a detailed discussion of the potential phytotoxic effects of iodine at high doses or in sensitive genotypes, supported by relevant literature (Blasco et al., 2008; Voogt et al., 2010 [5,34]). This clarifies the dual (biphasic) nature of iodine responses and acknowledges the threshold-dependent variability of physiological tolerance, which strengthens the interpretation of our results.

Comments 26: References 41 and 42 are not at all relevant and therefore do not support the statements made at lines 302-312.

Response 26: We agree with the reviewer that references [41] and [42] were not appropriate for supporting the claims in the yield section. These references have been removed and replaced by recent studies on nanoparticle-based delivery systems that enhance nitrogen use efficiency and stabilize physiological responses under field conditions (Mejías et al., 2021 [36]; Kumar et al., 2022 [37]; Saurabh et al., 2024 [38]). The revised section now reflects a more evidence-based interpretation.

Comments 27: Reference 44 is also inappropriate and its content has nothing to do with the sentence it refers to. The same applies to reference 45 where drought stress is mentioned and nanoparticles are never mentioned, and to reference 46 where there is no mention of Iodine nanoparticles, so the statements made at lines 330-343 must be scientifically better corroborated in light of the results reported.

Response 27: Thank you for your observation regarding references 44–46 in the original manuscript. These citations have now been removed and replaced with updated and directly relevant literature.

Specifically, we have included recent studies demonstrating:

The positive role of iodide (I⁻) in foliar absorption, systemic translocation, and the activation of nitrate reductase (Humphrey et al., 2019; Weng et al., 2008; Gonzali et al., 2019).

The inhibitory effect of iodine nanoparticles (INPs) on NR activity due to nanoparticle-induced oxidative stress and the compartmentalization of iodine away from the cytosol (Rastogi et al., 2019; Ma et al., 2010).

These references support the physiological interpretation of our results and ensure the scientific validity of the statements made. The revised text has been corrected accordingly to reflect this improved foundation.

Comments 28: All comments at line 362-372 are based on statistically insignificant differences and therefore conclusions such as those assumed cannot be drawn from them.

Response 28: Thank you for your valuable observation. We agree that conclusions based solely on statistically insignificant differences must be interpreted cautiously. In response, we have revised this section to clearly differentiate between statistically supported findings and observed physiological trends.

The revised paragraph emphasizes that no statistically significant differences were found (p > 0.05), and it now interprets the minor variations observed in treatments with iodine nanoparticles (INPs) as potential physiological adjustments, not definitive effects. The text avoids asserting any conclusive impact and instead frames the discussion in the context of previously reported mechanisms of nanoparticle interactions, supported by literature, while acknowledging the exploratory nature of these findings.

Comments 29: In 3.5 reference 51 is not appropriate and therefore does not support the assumption for which it is referenced: in the article cited the study is not carried out with nanoparticulate Iodine and then what is stated is not based on an established trend but only on a statistically valid value for a single of the 6 different treatments carried out. In fact, only the value of soluble amino acids obtained with the treatment with INPs 40 is statistically different from the control, and this alone could be debated.

Response 29: Thank you for your insightful comment. We acknowledge that the original reference [51] was not appropriate to support the assumption regarding nanoparticulate iodine. In the revised version, we have removed this reference and replaced it with more suitable literature that supports the potential physiological effects of nanomaterials on amino acid accumulation, such as Rico et al. (2011), Siddiqi & Husen (2017), and Selim et al. (2022). Moreover, the paragraph was rewritten to clearly state that only the INPs 40 μM treatment showed a statistically significant increase in soluble amino acids compared to the control, and we now avoid any overgeneralization across all INPs treatments.

We have also refined the discussion to focus on possible physiological mechanisms linked to the observed response at 40 μM, such as glutamate pathway activation and nitrogen assimilation enhancement, without extrapolating beyond the supported results. These changes aim to improve the scientific accuracy and transparency of our findings.

Comments 30: The above comments also apply to Chapter 3.6. One cannot extrapolate hypotheses and conclusions based on data without statistical validation. The soluble protein values obtained for the KI treatments show no significant difference to the control. The fluctuating trend in the values for the INPs treatments, on the other hand, compared to the control shows a certain influence and this alone should be discussed by the authors. Again, however, only the difference with INPs 160 is statistically significant. Therefore, what was reported at line 423-428 regarding an alleged effect due to KI and its interpretation cannot be considered valid.

Response 30: Thank you for your observation. We have revised the paragraph to ensure that no conclusions are extrapolated without statistical support. The new version specifies that only the treatment with INPs at 160 μM showed a statistically significant reduction in soluble protein content compared to the control, while all KI treatments, although showing slight upward trends, did not present significant differences. The text now refrains from attributing any confirmed physiological effect to KI treatments.

Comments 31: The content of reference 54 (moreover, the year of publication is 2018 and not 2011 as erroneously reported) also does not seem directly connected with the assumption to which it refers. The same applies to reference 43 at line 433, cited in connection with a possible interference by INPs '......with the ribosomal machinery or induce protein recycling mechanisms (autophagy), thereby reducing the net concentration of soluble proteins [43]. ". In the review cited above, work is reported on significant results obtained using other types of nano-fertilisers, e.g. nano-ZnO, nano-Zn-chitosan, nanomaterials made up of amorphous pyrogenic SiO2, nano CeO, nano-TiO.  INPs fertiliser is never mentioned.

Response 31: We acknowledge your remark and have removed the previously cited references [43] and [54], which were not directly related to iodine nanoparticles. Instead, we included Smoleń et al. (2014) as a valid reference regarding the impact of iodine form and dose on nitrogen metabolism and leaf protein content. We also referred to studies on metallic nanoparticles (e.g., ZnO, CeO₂) to illustrate that high concentrations can negatively impact protein expression, while clearly stating that such effects for INPs had not been previously documented. This revision better supports our hypothesis while maintaining scientific rigor and clarity.

Comments 32: The sentences at lines 455-461 are imprecise and unclear. How can the recorded correlation between amino acids amouns and yeld and biomass accumulation confirm that ‘....iodine nanoparticles (INPs) treatments favour a more efficient nitrogen assimilation pathway’, when there is no statistical difference between treatments and control for the latter two parameters?

Subsequent interpretations are also questionable in light of the results obtained and should be reviewed.

Response 32: We appreciate the reviewer’s observation regarding the interpretation of the correlation results. In response, we have revised the paragraph to clarify that correlations do not imply causality and do not replace statistical comparisons between treatments. Rather than concluding that INPs directly enhanced nitrogen assimilation, we now describe the observed significant correlations between soluble amino acids and productivity parameters (biomass and yield) as potential functional indicators of internal nitrogen economy. These associations are discussed in the context of existing literature on nanofertilizers, which report similar physiological trends without immediate biomass increases. The revised text also avoids generalizations and frames these relationships as hypotheses to be further explored. We believe the new version adequately addresses the concern and improves the scientific rigor of our interpretation.

Comments 33: Therefore, in the light of my previous comments, it is natural that the Conclusions, being based on questionably interpreted results, are themselves questionable.

Response 33: We appreciate the reviewer’s critical assessment regarding the interpretation of our results and its impact on the conclusions. In response, we have carefully revised the conclusion section to avoid any overstatements and to reflect the nuanced and preliminary nature of our findings. The updated conclusions now distinguish between the physiological effects of iodine in its ionic (KI) and nanoparticulate (INP) forms, acknowledging that these effects are dose-dependent and potentially mediated by redox balance and intracellular distribution.

Rather than making definitive claims about productivity improvements, the revised version emphasizes metabolic adjustments (e.g., amino acid accumulation, nitrate reductase activity) and suggests a functional role for iodine under specific conditions. We also explicitly recognize the complementary nature of responses between treatments and frame our findings as preliminary evidence that may inform future precision fertilization strategies based on physiological markers.

We trust this revised conclusion aligns more closely with the data and addresses your concern appropriately.

Response to Reviewer 1 Round 1 Comments

1. Summary

Dear Reviewer.

We sincerely thank you for your positive assessment of our revised manuscript and for recommending its acceptance. We greatly appreciate the time and effort you dedicated to reviewing our work, as your comments and suggestions were essential to improving the quality, clarity, and scientific rigor of the study.

Response to Reviewer 2  Round 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 second version of the manuscript has been extensively modified by the authors. Nevertheless, only the chapter on Materials and Methods, in which the requested improvements have been substantially applied, has been significantly improved. On the other hand, the assumptions underlying the study remain unclear, confusing, and unsupported by scientific evidence.

In the Introduction, but also in the Results and Discussion sections, the statements, interpretations, and supporting arguments continue to be invalid in many places because, although many citations have been changed, most of them continue to be inappropriate in relation to the arguments for which they are cited, or their content is even misinterpreted and forcibly adapted to the authors' argument.

The description of the results has certainly improved. All my comments on the statistical significance of the data have been accepted but, unfortunately, in many places their interpretations remain mere assumptions, precisely because of what has been stated above.

Below I detail the points under discussion:

In Introduction:

As pointed out by the authors, the narrative of the introduction has been completely revised. Here are the comments on the changes:

Comments 1:  Reference 2 is very old and reflects a situation that was valid in the previous decade. The statement in lines 34-36 needs to be supported with recent references.

Response 1: Thank you for the insightful comment. We have replaced the previous 2014 reference with a current peer-reviewed study from 2023 (You et al., 2023) that clearly demonstrates the persistently low NUE across global croplands and quantifies how it can be improved through optimized nutrient, crop, and soil management. The sentence has been revised to accurately reflect these findings and situate our study within the most current scientific context.

Comments 2:  References 7 and 8 (line 48) are not appropriate: both articles report the results of soil (not foliar) application of iodate (not iodide), as erroneously reported in line 45.

Response 2: We appreciate the reviewer’s observation and fully agree with the concern. As suggested, we have removed the previous references that reported soil application of iodate, which were not aligned with the claim about foliar potassium iodide (KI) application.

In their place, we have incorporated two appropriate and recent studies:

(1) Lawson et al. (2015), who demonstrated that foliar KI application in lettuce increases biomass, pigment accumulation, and iodine content, and

(2) Silva-Marrufo and Tinoco (2020), who showed that foliar sprays of KI in hydroponic strawberries enhanced chlorophyll, vitamin C, phenolic compounds, and overall plant growth.

Comments 3:  References 10-12 also do not support the argument made in lines 51-55: these are reviews that address the issue from a nanotoxicity perspective rather than highlighting the beneficial effects on plants. Finally, none of the three mention nanostructured iodine-based soil improvers.

Response 3: We fully agree with this observation. The original references were general reviews on nanomaterials and did not specifically report beneficial effects of INPs in agricultural contexts. We have therefore removed those citations and rephrased the paragraph to clarify that, while INPs have not yet been tested in crops, their development is scientifically grounded in the broader context of nanofertilizers. Our study introduces the concept of INPs in agriculture for the first time, and this novelty is now clearly stated and properly framed in the revised version.

Comments 4:  The sentence at lines 55-58 is too generic and needs to be better supported by published results. Reference 13 cited is not adequate as it reports beneficial effects determined by the use of an organic and mineral nanofertilizer (STYMJOD) composed mainly of 57% amino acids and organic compounds, organic nitrogen, numerous inorganic micronutrients, and only a small percentage of iodine. Therefore, the effects reported in the study cannot be attributed primarily to the presence of iodine, as hypothesized in lines 57-58.

Response 4: We acknowledge the limitation of Reference 13. As noted, the product evaluated contains multiple active components and does not allow attribution of effects specifically to iodine. Accordingly, we have removed that reference and rephrased the sentence to avoid unsupported generalizations. The revised paragraph now focuses on the scientific rationale and novelty of our approach using INPs, without relying on studies that do not isolate iodine as the active component.

Comments 5: In lines 65-66, it is stated that “Some studies have shown that iodine nanoparticles applied foliar can improve metabolic efficiency by stimulating nitrogen-associated pathways.” The references added do not validate this statement. Reference 14 reports the results of a study carried out with iodide and/or selenate in combination with each other without silicon nanoparticles (there is no mention of the use of iodine nanoparticles). In reference 15, iodine (in the form of iodide and/or iodate salt and not nanoparticles) is also studied as a complex with nanoparticulate chitosan. Finally, in references 16 and 17, the effect of foliar applications of iodate salts is studied, and there is no mention of nanoparticulate iodine.

Response 5: We appreciate the reviewer’s careful attention to the cited references. We agree that none of the studies directly involve iodine nanoparticles (INPs), and that the original sentence overstated the evidence. To address this, we have revised the paragraph to clarify that INPs have not yet been applied in crops, and that the cited studies refer to either conventional iodine salts or iodine-containing nano-complexes. The new text avoids unjustified generalizations while still presenting a valid rationale for investigating INPs based on prior experimental outcomes using iodine-related treatments.

In Results and Discussion

Comments 6: The sentences at lines 288-298 are too generic and not sufficiently connected to each other: the whole section needs to be better contextualized in light of the two articles cited (which, moreover, never mention nanoparticulated iodine) and the results related to biomass presented in this Section. Above all, the rationale behind the conclusions drawn must be clarified, especially with regard to the potential interaction of iodine nanoparticles with specific metabolic pathways and, in particular, with enzymatic patterns involving key enzymes involved in nitrogen metabolism.

Response 6: Thank you for this observation. We have thoroughly revised and restructured the section to improve the coherence and connection between ideas, ensuring that all conclusions are logically derived from the experimental results. We have clarified that, although previous studies report beneficial effects of conventional iodine forms on biomass, none to date have investigated iodine nanoparticles in lettuce. Thus, we present this finding as a novel contribution. Speculative statements regarding enzymatic pathways have been removed or rephrased cautiously, indicating that further research is needed to elucidate potential physiological mechanisms.

Comments 7: The conclusion in lines 306-308 is too hasty, considering that it is based on the only result discussed so far.

Response 7: Thank you for this remark. The previous concluding sentence has been removed. We now provide a measured interpretation focused solely on biomass results, avoiding broader conclusions at this stage. A more integrative interpretation will be provided in the general discussion after all variables are analyzed.

Comments 8: In any case, reference 6 cited in support is a review that addresses various aspects of the topic and presents the results of several studies, some of which are contradictory. It would be better to mention the work cited in the review that is most relevant to the statement.

Response 8: Thank you for pointing this out. We have revised the citation of reference 6, Medrano-Macías et al. (2021), to specify the original study cited within the review, which reported increased biomass in lettuce following potassium iodate and nitrogen application. This avoids citing the review as general support and focuses on a relevant primary result.

Comments 9: Finally, the interpretations reported in lines 313-318 should also be included only in the conclusions after discussing and interpreting all the results and not based solely on experimental biomass data.

Response 9: We appreciate your comment. The broader interpretations previously included at the end of the section have been removed and relocated to the conclusion section. The current version presents only a focused interpretation based on the observed biomass data, without making generalized claims about biostimulant mechanisms or physiological effects.

Comments 10: Summarizing what was stated in lines 331-334, no statistically significant effect was recorded in any of the treatments in terms of yield, whether KI or NPI was used at all concentrations. Slight differences are observed in both cases, but they are dose-dependent, and there is no real positive trend as stated in line 450. Therefore, the only conclusion that can be drawn from this data is the exclusion of toxicity effects at the experimental concentrations of both species administered (which would seem to contradict what was stated in the previous sections regarding biomass). Everything reported in lines 334-347 is merely speculation not supported by the results presented in this section. Once again, the notes reported in the references cited (5,6,36-38) contradict and do not corroborate the results of this study. Furthermore, studies that are not relevant to the present study are reported: articles 36-38 discuss nanofertilisers composed of mixtures of macro- and micronutrients and their effects compared to conventional fertilisers, but there is no mention of iodine as either KI or NPI.

Furthermore, what does ‘....do not limit the translocation of assimilates...’ mean in line 335?

Response 10: Thank you for your detailed observation. We have carefully revised the entire section on yield to address the lack of statistical significance in our results. The revised paragraph now explicitly states that no significant differences were found between treatments (p > 0.05), and we have removed any speculative claims regarding performance trends. We also acknowledge the absence of dose-dependency in the numerical variations observed.

In addition, we replaced the previous references (36–38), which referred to general nanofertilizer literature, with more specific and relevant studies focused on iodine fertilization in lettuce, such as Lawson et al. (2015) and Duborská et al. (2020). These works clearly indicate that iodine fertilization does not consistently affect yield but may still play a role in nutritional enhancement.

We also eliminated the ambiguous phrase “do not limit the translocation of assimilates” and instead provided a more accurate explanation based on possible differential allocation of biomass in the short growth cycle of lettuce.

We believe the revised section now provides a more cautious and scientifically consistent interpretation of our findings. Thank you for pointing out these important issues.

Comments 11: Even the references chosen to replace the previous ones (current references 41, 42, 43) seem to be of little relevance to the results reported in this section:

Reference 41 essentially studies the mechanisms of iodine uptake and translocation depending on its different forms (such as iodide and iodate) and never hypothesizes an effect “which could favor processes associated with nitrate reduction and nitrate reductase activation” as stated in lines 369-370. Furthermore, one of the conclusions of the study cited states that “... Spinach leaves can absorb iodine via foliar fertilization, but translocation is severely limited,” contrary to what is reported in lines 368-369.The same topic is addressed in ref. 42, and again, nowhere does it state that “iodide supply promotes nitrogen accumulation and stimulates key enzymes in nitrogen metabolism, including NR...” (lines 371-372).The topic of ref. 43 essentially concerns biofortification strategies aimed at improving the iodine nutritional status of a population without going into the specifics of the effects of its application on plants.Therefore, the interpretations of these results in lines 366-375 are not supported by the scientific articles cited and remain only scientifically unsupported hypotheses.

Response 11:  We sincerely thank the reviewer for the valuable comments regarding the interpretation of NR activity and the adequacy of supporting references.

In the revised version, we carefully addressed the concerns by:

1. Removing the previous references [41–43] that were not directly relevant to nitrate reductase or foliar iodine application, as pointed out.
2. Replacing them with three appropriate and scientifically grounded studies that directly support our experimental findings:
• Silva-Marrufo et al. [8] demonstrated that foliar application of potassium iodide (KI) significantly increased nitrogen concentration in strawberry plants under hydroponic conditions.
• Shalaby et al. [18] reported that foliar KI application in lettuce reduced nitrate levels while increasing amino acid content, suggesting improved nitrogen assimilation.
• Smoleń and Sady [40] showed that iodide application via foliar spraying improved nitrogen metabolism efficiency and nitrate accumulation in spinach.

These references validate the observed enhancement of NR activity following moderate foliar application of KI in our experiment and reflect the form-dependent behavior of iodine in nitrogen metabolism.

1. Regarding iodine nanoparticles (INPs), we reformulated the interpretation to emphasize that the inhibitory effect is a plausible hypothesis, based on known mechanisms of nanoparticle-induced oxidative stress. We cite Rastogi et al. [41], a comprehensive review demonstrating how metal and metal oxide nanoparticles can generate reactive oxygen species (ROS), disrupting the redox balance and affecting redox-sensitive enzymes like NR. This is presented not as a definitive claim, but as a physiologically reasonable explanation consistent with our observations.

We believe these changes align the discussion with robust scientific evidence and improve the clarity and credibility of our interpretation, as requested.

Comments 12: The reference 10 cited in support of the sentence at lines 414-421 does not seem to be relevant: the article cited is actually a review that considers studies essentially focused on the uptake by plants of different types of nanoparticles (there is no mention of studies that consider nanoparticulated iodine) from the environment, their translocation, accumulation, and phytotoxic effects. The article is very old and therefore considers studies carried out up to 2011, and it would be preferable to replace it with more recent work. However, it would be more appropriate to mention the work cited in the article that is most relevant to the statement.

The same can be said for reference 31.

In their response to comment 28, the authors state that in the new version they have modified the comments in Section 3.4 so that “The text avoids asserting any conclusive impact and instead frames the discussion in the context of previously reported mechanisms of nanoparticle interactions, supported by literature, while acknowledging the exploratory nature of these findings.” Unfortunately, the references chosen to support this are not appropriate, as previously clarified.

Response 12: Thank you for your insightful comments. In response to your concern about references 10 and 31 in the previous version, we have carefully revised Section 3.4 to replace them with updated and relevant primary research. Specifically, we now cite:

Silva-Marrufo et al. (2020), who reported enhanced pigment levels and nitrogen metabolism following KI application in strawberries;

Shalaby (2023), who documented improved photosynthetic stability in lettuce after iodide treatment;

Smoleń & Sady (2012), who demonstrated positive effects of foliar iodide on chlorophyll levels in spinach.

Regarding iodine nanoparticles (INPs), we avoided overinterpreting the results and emphasized that the physiological trends observed may relate to indirect effects, such as ROS generation or subcellular sequestration, rather than direct phytotoxicity. The discussion now acknowledges the exploratory nature of these findings and avoids unsupported conclusions. We trust this new version better reflects current scientific evidence and addresses your valuable feedback.

Comments 13: At lines 453-460, the discussion focuses on the comparison between the results obtained for the soluble amino acids parameter, discussed in this section, and those for soluble proteins, which have not yet been commented on or shown. The results of the latter parameter are discussed in the following section, and perhaps this comment should be included there. However, in the end, the whole thing is unclear as it is reported: it is not clear whether what is highlighted is attributed to a phytotoxic effect (there is mention of stress) or leads to biostimulation.

I think the clarification at lines 461-468 is unnecessary.

The sentence at lines 472-473 is too generic and absolutist:  considering that the previous sentence referred to nanoparticles, the authors here assert a general effect of biostimulation in plants' nitrogen metabolism determined solely by the addition of nanoparticles, whatever their nature.

Response 13: We appreciate the reviewer’s careful observation. In the revised version, we have removed the premature comparison between soluble amino acids and soluble proteins to maintain clarity and avoid anticipatory interpretation. This comparison is now addressed in the appropriate section (3.6), where soluble protein results are discussed in detail.

Additionally, we have revised the discussion regarding biostimulation by nanoparticles to avoid overgeneralization. The revised text now explicitly refers to specific studies involving metal-based nanoparticles (e.g., Zn, Fe, Se) under defined conditions, and highlights that similar behavior is suggested but not yet demonstrated for iodine nanoparticles. This cautious interpretation aligns with the current evidence and avoids unsubstantiated general statements.

Furthermore, the paragraph about nitrogen sufficiency and the use of the modified Hoagland solution has been streamlined into a single sentence to reduce redundancy, as suggested.

Comments 14: In this section, rather than the previous one, the results relating to soluble proteins and those relating to the analysis of soluble amino acids should be commented on and interpreted jointly. However, this should be done bearing in mind the statistical significance of the data, namely:

The amino acid analysis shows no significant difference compared to the control for the treatments with KI; only for the treatment with INPs 40 does the data deviate significantly from the control.

Even in the case of soluble proteins, the values obtained with different doses of KI show no effect; in the case of treatments with NPIs, however, the only negative significance is found with NPIs at the maximum dose (for amino acids, unlike at the lowest dose), while the overall trend is fluctuating (beyond the statistical significance of the data): first lower than the control at the minimum dose (INPs 40), an increase at the intermediate dose (INPs 80), and finally a significant decrease at the maximum dose. 

Response 14: We appreciate your comment on the need to discuss and interpret the results of soluble proteins and soluble amino acids together, taking into account the statistical significance of the data. In the revised manuscript, we have kept the results for soluble proteins and soluble amino acids in separate subsections for clarity, but we have now incorporated a comparative interpretation within the soluble proteins section that explicitly links both variables. Specifically:

In the case of soluble amino acids, a significant increase was only observed with INPs at 40 µM, while KI treatments showed no differences compared to the control.

As for soluble proteins, KI also showed no significant differences at any concentration, and the only significant negative effect was recorded with INPs at 160 µM.

We now analyze the dose-response trend of INPs, describing a possible biosynthetic effect at low concentrations and a possible catabolic or inhibitory effect at high concentrations, relating it to nitrogen redistribution or phenolic compound biosynthesis.

Recent references have been added to support this dose-dependent pattern, in line with observations from nanoparticle-based biofortification studies.

Therefore, although the two parameters are still presented in separate subsections for structural reasons, their physiological relationship and the statistical trends you have indicated are now discussed together, as you suggested.

Comments 15: First of all, as repeatedly pointed out, even the references included do not support the statements for which they are cited. For example, at line 539, ref. 48 reports a study focusing on the toxic effects of Ag nanoparticles and the possible defense strategies activated in tomatoes characterized by a decrease in soluble proteins, an increase in amino acids, and the activity of antioxidant enzymes (there is no mention whatsoever of an effect of “...more efficiency mobilization or nitrogen storage capacity...” " (lines 537-538). The topic of ref. 51, which discusses the use of nanoparticulate chitosan in sustainable agriculture and its effectiveness under high CO2 levels, is also completely out of context.

The statements in lines 540-543 are unclear and incorrect: in section 3.5, the correlation shown takes into account the data for the individual parameters of all seven different treatments. Therefore, it is not possible to associate some of the analysis results specifically with treatments using nanoparticulate iodine rather than KI. Needless to say, the content of reference 54 is unrelated to the context to which it refers.

Similarly, at lines 552-558, the negative correlations highlighted are erroneously attributed solely to treatments using nanoparticles.

Response 15: We thank the reviewer for the valuable comments and for pointing out the inconsistencies in the interpretation and the lack of correspondence between some references and the statements made. In the revised version of Section 3.7, we have:

1. Replaced out-of-context references (former refs. 48, 51, 54, 55) with peer-reviewed studies that directly address correlations between amino acids, nitrogen metabolism, and plant productivity under various nutritional regimes, ensuring that each citation fully supports the statement it accompanies.
2. Reformulated the text to avoid attributing correlation results to specific treatments (e.g., INPs vs. KI), as the correlation matrix was generated using combined data from all treatments.
3. Clarified the interpretation of positive and negative correlations, framing them as general physiological patterns rather than treatment-specific effects, and ensuring consistency with the scope and limitations of the correlation analysis.
4. Integrated all conclusions into the main text to make the section more concise and focused, as per the reviewer’s guidance.

We believe these changes address the concerns raised and improve the accuracy, clarity, and scientific rigor of this section.