Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigates the antioxidative protective effects of foliar-applied zinc oxide nanoparticles (ZnO NPs) on Coffea arabica seedlings under acidic soil stress. The authors systematically evaluate the impact of different ZnO NP concentrations, revealing a clear dose-dependent response. The findings offer valuable insights for the cultivation and production of Coffea arabica. Furthermore, the study suggests a potential novel strategy for future research on pest and disease resistance, namely by modulating reactive oxygen species (ROS) levels using nanoparticles to enhance stress tolerance. The manuscript is generally well-structured; however, the following points should be addressed to further improve its clarity and impact:

1. Phenotypic Documentation: The authors are strongly encouraged to provide representative phenotypic photographs of the Coffea arabica seedlings. Visual evidence showing the phenotypic differences among the various ZnO NP treatment groups would greatly strengthen the presentation of the results and provide direct support for the physiological and biochemical data.

2. Figure Diversity: The graphical representation of data could be enhanced. Currently, the figures predominantly consist of bar graphs, which limits visual variety and readability. Incorporating other graph types (e.g., line graphs for dose-response trends, representative images from assays, or schematic diagrams) where appropriate would improve the manuscript's overall appeal and effectiveness in communicating the findings.

3. Dose-Response Mechanism and Application: The results demonstrate significant differences in the physiological and oxidative balance regulation of Coffea arabica across the tested ZnO NP concentrations. A more in-depth discussion is warranted to elucidate the potential mechanisms behind these divergent effects, particularly the contrasting outcomes between moderate (25-50 mg L⁻¹) and high (100 mg L⁻¹) doses. Additionally, the authors should discuss the implications of their findings for practical application, specifically commenting on the optimal concentration range for real-world production scenarios based on their identified "safe and physiologically beneficial window."

Comments on the Quality of English Language

Good

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article investigates morphophysiological, biochemical, and mineral nutrient parameters of Coffea arabica L. seedlings grown in acidic soil and sprayed with ZnO-NPs at 10, 25, 50 and 100 mg L⁻¹. Valuable and interesting data has been obtained in this study, but their interpretation requires significant revision.

The title does not correspond to the content and conclusion of the article, since no reduction in the values ​​of oxidative stress markers after plant treatment was detected.  In addition, this study pays great attention to photosynthetic parameters and mineral nutrition status. There is no strong evidence in this work that ZnO foliar nanoparticles protect C. arabica seedlings against oxidative stress induced by soil acidity. The title of the article needs to be changed. Period at the end of the article title should not be put.

Lines 17-19. The abstract says that ZnO NP doses (25–50 mg L⁻¹) reduce hydrogen peroxide and lipid peroxidation in roots and leaves, and, in contrast, 100 mg L⁻¹ induced oxidative damage, increased H₂O₂ and MDA accumulation. This statement contradicts results (section 2.6, figure 5).

Line 84.  It's better to replace “SPAD index” with “relative chlorophyll content”.

A suggestion for the results description: it is better to provide exact p-values ​​in the presence of significant differences instead of p < 0.05.

Latin (C. arabica) is in italics in some places, not in others (e.g. line 109). To achieve consistency, highlight the Latin names of species in italics.

Lines 111-114. It is claimed that “The positive control (optimal pH) presented the highest values for the main macronutrients: K (15,354.94 ± 963.14 μg g⁻¹), Ca (14,822.83 ± 829.93 μg g⁻¹), and Mg (2,599.46 ± 113.82 μg g⁻¹), whereas the acidic control showed notable reductions in K (13,863.24 ± 819.12 μg g⁻¹) and Mg (3,356.89 ± 251.87 μg g⁻¹).” But the value of Mg at optimal pH (2,599.46) is lower than acidic control showed (3,356.89). It is necessary to correct the contradiction.

Lines 127-129. It is stated that “Under optimal pH, the positive control recorded the highest Fe (541.74 ± 69.91 μg g⁻¹) and Mg (3,334.63 ± 104.20 μg g⁻¹) contents, whereas acidic soil significantly reduced K (6,170.85 ± 273.35 μg g⁻¹) and Fe (294.46 ± 10.66 μg g⁻¹).”  Please rephrase the sentence, since a decrease in content when grown on acidic soil was noted for K, Mg, Fe, as shown in the Table 2.

The names of the section s 2.4. Pigmentos fotosintéticos, 2.6. Peroxidación lipídica, 2.7. Peróxido de hidrógeno , should be translated to English

Line 174. The reference to Figure 4 should be moved to line 178, where you are writing about roots.

Lines 191-198. It is stated that “…the acidic soil control showed a higher concentration, evidencing an increase in oxidative damage under acidic conditions.”; “…Foliar applications of 10 and 25 mg L⁻¹ ZnO NPs slightly reduced MDA content…”; “…higher doses (50 and 100 mg L⁻¹) resulted in increased lipid peroxidation (5.48 ± 0.44 and 5.41 ± 0.95 nmol g⁻¹ FW, respectively), suggesting a pro-oxidant effect at elevated nanomaterial concentrations.” But, in fact, as it shown at the Figure 5a, there is no significant differences between acidic soil and other variants. Significant differences are shown only between optimal soil and 50 mg L⁻¹ ZnO NPs. Section 2.6 (lines 191-198) should be completely rewritten. Please, be more accurate with the interpretation of the results of statistical hypotheses testing.

Figure 6a. Is it really no significant differences between variants of optimal soil and 100 mg L⁻¹ ZnO NPs?

line 319. The statement "The foliar increase in MDA at 50–100 mg L⁻¹” contradicts the data shown in the Figure 5a, where significant differences are shown only between optimal soil and 50 mg L⁻¹ ZnO NPs.

Lines 514-515. The statement  “In contrast, the highest dose (100 mg L⁻¹) increased MDA and H₂O₂ levels, indicating oxidative stress  and a loss of photosynthetic functionality” contradicts the data shown in the Figures 5a and 6a.

There is no reference to Supplementary Materials (Table S1. Physical and chemical properties of the soils used in the experiment) in the manuscript.  The reference to Supplementary Materials should be added to section 4.1. Plant Material and Experimental Conditions.

Comments on the Quality of English Language

The names of the sections 2.4. Pigmentos fotosintéticos, 2.6. Peroxidación lipídica, 2.7. Peróxido de hidrógeno should be translated to English.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors!

The cited references are mostly recent publications (within the last 5 years) and relevant. The reference does not include an excessive number of self-citations. The manuscript is scientifically sound and the experimental design is appropriate to test the hypothesis. The article’s results are reproducible based on the details given in the methods section. The figures and tables are appropriate, they properly show the data, easy to interpret and understand. The statistical analysis is sufficient, the conclusions are consistent with the evidence and arguments presented.

Introduction. If possible, add more information about different physiological responses (photosynthesis, redox metabolism) of plants under acidity of soil.

The manuscript is interesting, relevant for the field and presented in a well-structured manner, but it is not clear. My recommendations:

L 113. You should check the sentence “…Mg (2,599.46 ± 113.82 µg g⁻¹), whereas the acidic control showed notable reductions in K (13,863.24 ± 819.12 µg g⁻¹) and Mg (3,356.89 ± 251.87 µg g⁻¹)…”. I did`n see the reduction.

L 139-140. Rephrase the sentence: “The evaluation of photosynthetic pigments revealed responses to the foliar application of ZnO NPs in plants grown under acidic soil conditions”. Probably to “The evaluation of photosynthetic pigments revealed advantageous responses of leaves of plants grown under acidic soil conditions due to the foliar application of ZnO NPs”.

L 168-169, 222-223. Rephrase the sentence: “Effect of ZnO NPs on C. arabica seedlings under acid stress on photosynthetic pigment contents” to “Effect of ZnO NPs on photosynthetic pigment contents of C. arabica seedlings under acid stress”, “Effect of ZnO NPs on C. arabica seedlings under acid stress on H₂O₂ content in (a) leaves and (b) roots” to “Effect of ZnO NPs on H₂O₂ content in (a) leaves and (b) roots of C. arabica seedlings under acid stress”.

L 210-212. Rephrase the sentences: ”In leaves (Figure 6a), significant differences were detected among treatments. The optimal pH control presented 507.4 ± 1.7 nmol g⁻¹ FW, whereas the acidic soil showed a slightly lower value (468.9 ± 7.8 nmol g⁻¹ FW). ” to “The plants grown under optimal pH of soil (control) reached 507.4 ± 1.7 nmol g⁻¹ FW of hydrogen peroxide content, whereas the acidic soil showed a slightly lower value of H2O2 (468.9 ± 7.8 nmol g⁻¹ FW).”

L 285-286. You should add the word ZnO NPs to the sentence: “Accordingly, the reduction observed during 10 mg L⁻¹ ZnO NPs application could be…” In general, you should check and add the name of investigated parameters near the concentration throughout the manuscript.

L 239-240. You should check Mg or Mn you were discussing about.

L 486. You should decipher the abbreviations W and V.

Comments on the Quality of English Language

L 138. You should write Pigmentos fotosintéticos (line 138), Peroxidación lipídica (line 188), Peróxido de hidrógeno (line 207) in English.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Coffee is a major economic crop worldwide, while soil acidity is a major abiotic stressor that limits its growth, development, yield, and quality. Previously, nanoparticles was used in agriculture and agronomy to alleviate abiotic stress tolerance. The manuscript preliminarily reported that the protective effect of ZnO nanoparticles on Coffea arabica seedlings under soil acidity conditions. It is important to further understand the possible mechanisms of ZnO nanoparticles-alleviated acid stress in plants. However, I think that the following comments should be considered during the revision.

(1) TITLE: According to the core content of the manuscript, the Title should be revised, it was not only the oxidative stress. If this Title, antioxidant system including antioxidant enzymes and non-enzymatic antioxidants should be supplemented. Also, According to the figures 5, 6, peroxidative product MDA and H2O2 had no significant accumulation compared with the acidic soil.

(2) ABSTRACT: Firstly, the full names of the abbreviations, such as H2O2, MDA, should be appeared first. Secondly, the implication of this findings should be clearly stated.

(3) KEYWORDS: According to the core content and the Title of the manuscript, oxidative stress should be added in the KEYWORDS section.

(4) INTRODUCTION: According to the core content and the Title of the manuscript, the possible mechanisms of soil acidity-induced oxidative stress should be supplemented in the INTRODUCTION section.

(5) RESULTS: g_s should be supplemented in the y-axis of the figure 1. Also, the subtitle " 2.3. Elemental Analysis (Leaves and Roots) ", including 2.4, 2.6, 2.7, and 2.8, seems to be the METHODS, it should be changed to result subtitle.

(6) DISCUSSION: In general, the first paragraph of the Discussion section, the main findings should be clearly summarized, including research gap, and then separately discussed. Also, according to the core content of the manuscript, the Discussion section should be properly shortened.

(7) METHODS: Similar to the result section, the subtitles "4.2, 4.3, 4.4, 4.6, 4.7, 4.8, " seem to be the RESULTS, they should be changed to method subtitle, such as "determination of .......".

(8) CONCLUSION: The first sentence is incomplete, please check it! Also, according to the figures 5, 6, peroxidative product MDA and H2O2 had no significant accumulation compared with the acidic soil. In addition, implication and future research should be supplemented.

Comments on the Quality of English Language

above

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The new title of the article reflects its content much more accurately.  However, the use of the word "modulation" is not very appropriate in this context, I recommend replacing it with the word "effect".

Lines 24-25 The statement “Elemental analysis revealed enhanced Ca, Fe, K, and P contents and lower Na at intermediate doses, suggesting improved ion transport and membrane stability” contradicts results (table 1). For example, for K there is not significant differences except values for optimal soil and 100 mg L⁻¹ ZnO NPs. Also, the sentence needs to be more specific, is it talking about leaves or roots?

The manuscript contains many inconsistencies between the text descriptions of the results and the data presented in tables and figures. Here are some of them:

Line 118 (caption to figure 2). It is claimed that “Different letters indicate significant differences according to the Tukey HSD test (p = 0.055).” This statement contradicts the caption to figure 1 of the first version of manuscript (Different letters indicate significant differences according to Tukey’s HSD test (p ≤ 0.05). The value p = 0.055 is greater than significance level 0.05. If it's not a typo, then you cannot claim there are any significant differences in this case.  In that case, the statement “Moderate ZnO NP doses (25–50 mg L⁻¹) improved stomatal conductance and partially restored net photosynthesis” (lines 19-20; 110-111) is not statistically confirmed. 

Lines 144-145. It is claimed that “Ca also increased to 10,902.36 ± 570.84 μg g⁻¹, evidencing a stabilizing effect on cationic uptake.” But in table 1 there is no difference between the values 9136.7 ± 91.03 bc and 10902.36 ± 570.84 c.

Lines 138-141. It is claimed that “The soil with optimal pH presented the highest values for the main macronutrients: K (15 354.94 ± 963.14 μg g⁻¹), Ca (14 822.83 ± 829.93 μg g⁻¹) and Mg (2599.46 ± 113.82  μg g⁻¹), while the plantations established in acidic soil showed reductions of 9.72% in K  and 38.36% in Ca compared to the soil with optimal pH”. But the value of Mg (Table 1) at optimal pH (2,599.46) is lower than that of the acidic control (3,356.89). It is necessary to correct the contradiction.

Authors should carefully check that the description and interpretation of results corresponds to the data presented in tables and figures. Please, be more accurate with the interpretation of the results of statistical hypotheses testing. In accordance with this, it is also necessary to recheck and correct the conclusion and abstract.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript has been improved according to the comments, so I think it should be considered to accept for publication.

Comments on the Quality of English Language

no

Author Response

We sincerely appreciate the reviewer's positive feedback, which helped improve the quality of the manuscript. We also appreciate the recommendation to accept the manuscript for publication.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Authors should carefully check that the description and interpretation of results corresponds to the data presented in tables and figures. In accordance with this, it is also necessary to check and correct the conclusion and abstract.

Line 26. It is claimed that “Leaf elemental analysis showed increased Ca and P concentrations at 50 mg L⁻¹ ZnO NPs, while K remained statistically similar across treatments and Na decreased only at the highest doses (50–100 mg L⁻¹).” Authors claim that as shown in Table 1, the Ca concentration under the 50 mg L⁻¹ ZnO NP treatment (10,902.36 ± 570.84 μg g⁻¹; group “c”) was significantly higher than that of the acidic soil and the 25 mg L⁻¹ treatments (“bc" and "b", respectively). Why do you claim that the values ​​marked with “bc" and "c" differ significantly, since they both contain the same letter? If we follow this logic, for example, the meanings for  K in table 1,  13863.24 ± 819.12 ab (Acidic soil) and 12047.39 ± 467.54 b (ZnO 10 mg L⁻¹ )(marked with “ab" and "b", respectively) also should differ from each other or the meanings. The presence of at least one identical letter in a pair of values ​​means that there is no difference between them.

Table 1 and Table 2 – A note needs to be added about what the letters above the given values ​​mean. Do they indicate significant differences?

Line 27 It is claimed that “Na decreased only at the highest doses (50–100 mg L⁻¹ ZnO NPs)”. Here it is necessary to clarify what the Na decreased in relation to - judging by Table 1, there was a reduction at the highest doses relative to optimal soil and 10-25 mg L⁻¹ ZnO NPs, but no differences were found relative to acidic soil.

Lines 158-159 Under optimal pH conditions, the plants presented the highest concentrations of Fe 158 (541.74 ± 69.91 μg g⁻¹)

Lines 158-159 It is claimed that ”Under optimal pH conditions, the plants presented the highest concentrations of Fe (541.74 ± 69.91 μg g⁻¹)” but the values 578.56 ± 40.52 and 590.75 ± 70.19 are not lower than 541.74.

The statement in lines 25-26 “Leaf elemental analysis showed increased Ca and P concentrations at 50 mg L⁻¹, while K remained statistically similar across treatments and Na decreased only at the highest doses (50–100 mg L⁻¹ ZnO NPs)” contradicts the conclusion in lines 540-542 “Low to moderate  doses (10–25 mg L⁻¹ ZnO NPs) maintained intermediate stomatal conductance and net  photosynthesis values and contributed to improved nutrient balance in leaves and roots  (higher Ca, Fe, P, and K contents)”.

Author Response

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Author Response File: Author Response.pdf