Potential Application of Selenium and Copper Nanoparticles in Improving Growth, Quality, and Physiological Characteristics of Strawberry under Drought Stress
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear authors
In the next section you will find the comments on your manuscript, I hope they are useful to improve the document.
Introduction
In the introduction, the general objective of the work and the research hypothesis should be clearly established.
Materials and methods
Include the equipment used, example:
Z-sizer 4 (Malvern Ltd., Enigma Business Park, Grovewood Road, UK.)
Line 160, include firmness test speed (mm/s)
Line 226, change to rpm, throughout the text.
Include the number of replicates or repetitions in this section.
Results
Line 273, indicate significant differences as p≤0.05 and non-significant differences as p>0.05. homogenize throughout the document.
In the results, the increase or decrease in the response variables is mentioned in percentage, which is why it cannot be defined as significant or not, this is revealed by the ANOVA carried out. Review.
Discussion
The discussion presented is limited, there are many parameters to give greater strength to the document.
Lines 439 to 442 is introduction; this does not go here.
Some questions that will help you respond to the discussion are:
What do nanoparticulate systems have that modify the responses evaluated?
What was the synergism between Cu and Se NPs due to?
Have these NP components been evaluated in micrometric and/or non-nanostructured form?
Conclusions
The conclusions respond to the stated objective and the hypothesis.
Comments on the Quality of English LanguageNo comments
Author Response
Dear editor and reviewers：
Thank you very much for your letter and the reviewer’s comments concerning our manuscript entitled “Potential application of selenium and copper nanoparticles in improving growth, quality, and physiological characteristics of strawberry under drought stress” (Manuscript ID:3098217). Those comments are all valuable and very helpful for revising and improving our paper. We have carefully revised the manuscript according to each comment referred by the reviewers. In accordance with the requirements of the journal, we have carefully revised the paper format and corrected the grammatical errors. The revised portions were highlighted in the marked-revised manuscript. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the correction will meet with approval. Once again thank you very much.
Independent Review Report, Reviewer 1
In the next section you will find the comments on your manuscript, I hope they are useful to improve the document.
Introduction
In the introduction, the general objective of the work and the research hypothesis should be clearly established.
Response: Thanks for your positive suggestion. We are sorry about our unclear description. We have revised it. Please see the red words in Line 107-116.
We hypothesized that (i）CuNPs and SeNPs were able to increase the photosynthetic activity used for osmotic protection and enhanced plant tolerance to drought stress;（ii）Synergistic effects of CuNPs and SeNPs can alleviate drought stress-induced oxidative stress in plants. The objective of this study was to test whether application of CuNPs and SeNPs suppresses the adverse effects of drought stress on strawberry growth, photosynthetic characteristics and seeding enzymatic antioxidants of strawberry, and explored the positive effects of CuNPs and SeNPs on improving the nutritional value of strawberry fruits under drought stress. Our study will provide a theoretical basis for the study of exogenous nano-fertilizers for healthy growth of strawberry under drought stress.
Materials and methods
Include the equipment used, example:
Z-sizer 4 (Malvern Ltd., Enigma Business Park, Grovewood Road, UK.)
Response: Thanks for your positive suggestion. Done. Please see Line 187-189, 211.
Line 160, include firmness test speed (mm/s)
Response: Thanks for your positive suggestion. The firmness test speed is 1mm/s. Please see Line 189.
Line 226, change to rpm, throughout the text.
Response: Thanks for your positive suggestion. Done. We have replaced “r·min-1” into “rpm”. Please see Line 247 and 254.
Results
Line 273, indicate significant differences as p≤0.05 and non-significant differences as p>0.05. homogenize throughout the document.
Response: Thanks for your positive suggestion. Done, please see Line 301-302, 334-335, etc.
In the results, the increase or decrease in the response variables is mentioned in percentage, which is why it cannot be defined as significant or not, this is revealed by the ANOVA carried out. Review.
Response: Thanks for your positive suggestion. We couldn't agree with you more, and in expressing the difference in variables as a percentage, we deleted the expression significant or non-significant. Please see Line 305, 310, 325-328, 342-345, , etc.
Discussion
The discussion presented is limited, there are many parameters to give greater strength to the document.
Lines 439 to 442 is introduction; this does not go here.
Response: Thanks for your positive suggestion. Done, we have deleted it.
Some questions that will help you respond to the discussion are:
What do nanoparticulate systems have that modify the responses evaluated?
Response: Thanks for your positive suggestion. The mechanism of action of nanoparticles in improving the resistance of crops to drought stress mainly involves the following aspects:
(1) Antioxidant protection: Drought stress causes a large amount of reactive oxygen species (ROS) to be produced in the plant body, resulting in oxidative damage to plant cells. Nanoparticles, especially those containing antioxidant components, can provide antioxidant protection and reduce drought-induced oxidative stress.
(2) Signaling: Nanoparticles may modulate plant responses to drought stress by affecting signaling pathways in plants. For example, they may promote the synthesis and signaling of phytohormones (e.g., abscisic acid), thereby enhancing drought resistance in plants.
(3) Nutrient uptake: Nanoparticles can promote the uptake of nutrients in plants, especially trace elements. This not only helps to improve the overall health of the plant, but also helps to enhance the plant's ability to survive under drought conditions.
(4) Gene expression regulation: Nanoparticles may enhance plant tolerance to drought stress by regulating the expression of specific genes. This may involve the activation of genes encoding drought-related proteins.
In summary, nanoparticles enhance crop resistance to drought stress through multiple mechanisms, thus contributing to the improvement of crop yield and quality, especially in arid regions. However, the environmental safety of nanoparticles and their impact on the ecosystem need to be considered for practical applications.
We have added “At the sensing and signaling level, metal nanoparticles can act as signaling molecules to modulate plant responses to environmental signals. They have the ability to activate specific signaling pathways, including those involved in stress responses, nutrient uptake, and hormone signaling (Mustafa and Komatsu, 2021; Abdelkader et al., 2023). Moreover, metal nanoparticles have been shown to induce post-translational modifications of proteins such as phosphorylation, acetylation and ubiquitination, which can alter protein stability, activity and localization. These modifications are critical for regulating various cellular processes, including hormone signaling, stress responses, and plant growth regulation (Mustafa & Komatsu, 2021, Mosa et al., 2022). Therefore, the future need to understand the complex molecular interactions between metal nanoparticles and plants is crucial to increase the full potential in crop productivity and agricultural sustainability (Elemike et al., 2019) ” in discussion.
We have added it. Please see Line 584-593.
What was the synergism between Cu and Se NPs due to?
Response: Thanks for your positive suggestion.
The synergistic effect of CuNPs and SeNPs is mainly reflected in the following aspects:
(i) Antioxidant effect: nano selenium is an effective antioxidant that can scavenge free radicals in the body and protect cells from oxidative damage. Nano-copper also has some antioxidant capacity, but its antioxidant effect is mainly enhanced by synergizing with selenium, which improves the antioxidant capacity of cells and protects the health of the organism.
(ii) Growth promotion: In agriculture, nano-copper and nano-selenium can be used as plant growth promoters to enhance the disease resistance of plants, promote plant growth and increase crop yield. Nano-copper can promote photosynthesis and increase the chlorophyll content of plants, while nano-selenium can improve the antioxidant capacity of plants and protect plant cells from environmental stress. In conclusion, the synergistic effect of nano-copper and nano-selenium can produce significant effects in several areas, including antioxidant and growth promotion. The combination of the two can capitalize on their respective advantages and enhance the overall effect.
We have added it. Please see Line 594-604.
Have these NP components been evaluated in micrometric and/or non-nanostructured form?
Response: Thanks for your question. The evaluation of NP components (nanoparticle components) in micrometer and/or non-nanometer structural forms focuses on their physical, chemical and biological properties at different scales and how these properties affect their functions and applications. The main assessment methods are particle size distribution, in which we mention the diameters of 5-18 nm and 35-42 nm for SeNPs and CuNPs, respectively, from the Center for Applied Chemistry Research in Saltillo, Mexico, but did not measure the particle size distribution of selenium and copper nanoparticles by laser diffraction. In the future, we will combine scanning electron microscopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC) to evaluate the various properties of nanoparticles.
Conclusions
The conclusions respond to the stated objective and the hypothesis.
Response: Thanks for your positive suggestion. We are sorry about our unclear description. We have revised it. Please see Line 606-625.
Our study concluded that foliar spraying of CuNPs and SeNPs is an effective strategy to improve drought tolerance in strawberry. The beneficial effects of CuNPs and SeNPs on the growth performance of strawberry under different drought stress conditions can be attributed to (1) protection of photosynthetic pigments and enhancement of photosynthetic capacity, (2) activation of antioxidant system and effective maintenance of reactive oxygen species homeostasis, and (3) improvement of fruit quality by increasing the water use efficiency and enhancing the accumulation of root biomass and fruit biochemical compounds. The application of SeNPs alone was superior to that of CuNPs alone, and the combination of the two was the most effects. In summary, the application of nanomaterials provided significant protection to drought-stressed strawberry plants, highlighting the promising application of nanomaterials in agriculture.
Future in-depth research is needed in the following three areas: (1) In addition to pot plant experiments under controlled conditions, field trials could help to understand the potential applications of these advanced nanoparticles in agriculture under changing environmental conditions; (2) The potential mechanisms of nanoparticle-crop interactions need to be studied and explored in depth; (3) Toxicity evaluations of most nanomaterials have been limited to simple surface toxicity, and systematic studies of the biological effects of nanoparticles and their toxicity mechanisms are needed in the future.
Reviewer 2 Report
Comments and Suggestions for AuthorsDear author,
Thanks for your good report. My comments and questions are in the attached file.
Regards
Comments for author File: 
Comments.pdf
Author Response
Dear editor and reviewers：
Thank you very much for your letter and the reviewer’s comments concerning our manuscript entitled “Potential application of selenium and copper nanoparticles in improving growth, quality, and physiological characteristics of strawberry under drought stress” (Manuscript ID:3098217). Those comments are all valuable and very helpful for revising and improving our paper. We have carefully revised the manuscript according to each comment referred by the reviewers. In accordance with the requirements of the journal, we have carefully revised the paper format and corrected the grammatical errors. The revised portions were highlighted in the marked-revised manuscript. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the correction will meet with approval. Once again thank you very much.
Independent Review Report, Reviewer 2
In Line 42, develop ment is wrong
Response: Thanks for your positive suggestion. We have replaced “develop ment” into “development”. Please see Line 49.
Germination should not be in your literature review. Please remove it.
Response: Thanks for your positive suggestion. We have removed it.
There are some previous works about effect of Se on strawberry in drought conditions as mentioned in the references, but you did not use them in literature of introduction. Please show Gap in the last paragraph of introduction.
Response: Thanks for your question. Done. Please see Line 98-106.
Line 117, There is no information about plant material used.
Response: Thanks for your positive suggestion. We have added the information about Fen Yu No1. The strawberry cultivar was Fen Yu No. 1, cultivated by the Hangzhou 121 Institute of Agricultural Science. Fenyu No.1 is an early-ripening, disease-resistant and pink-peel strawberry cultivar (Fragaria × ananassa Duchesne) derived from a cross between Kaorino as a female parent and 2012-W-02 as a male parent. Please see Line 141-143.
Soil mixture physicochemical analysis?
Response: Thanks for your positive suggestion. We are sorry about our unclear description. The soil samples were collected at five different sites of each plot and combined into a sample mixture. We have revised it. Please see Line 156-157.
Line 135-142, How you determined and measured it that under these irrigation levels, plants are in drought stress condition?
Response: Thanks for your question. The experiment was designed for soils with different levels of drought. Soil moisture content in the pots was determined using a configuration of ML2 probes (ThetaProbe soil moisture devices, UK) and HH2 moisture meter Delta-T Devices (HH2 moisture meter Delta-T Devices, UK), with supplemental watering using drip irrigation to ensure that soil moisture was within the range set for the experiment. Please see Line 151-155.
In line 152, When plants were selected?
Response: Thanks for your question. We are sorry about our unclear description. We have added after “98 d”. Please see Line 178.
In line 155, Is there any reference for leaf area calculation?
Response: Thanks for your question. We have added reference. Dogan M, Bolat I, Karakas S, Dikilitas M, Gutiérrez-Gamboa G, Kaya O. Remediation of Cadmium Stress in Strawberry Plants Using Humic Acid and Silicon Applications. Life (Basel).. 2022 Nov 23;12(12):1962. doi: 10.3390/life12121962. Please see Line 166.
You had two factors: FC and nano materials spray. How you analyzed data one-way?
Response: Thanks for your question. We are sorry about our unclear description. The significance of differences between the treatments was analyzed by one-way, two-way analysis of variance (ANOVA) using IBM SPSS Statistical software. Please see Line 275.
Treatments should be as Table 1.
Response: Thanks for your positive suggestion. Done.
Thanks for your report. However, with only pot experiment you can not recommend these nano materials for practical application. As many factors are different in the field condition compare to a pot experiment.
Response: Thanks for your positive suggestion. We are sorry about our unclear description. We couldn't agree with you more. We have revised conclusions.
Future in-depth research is needed in the following three areas: (1) In addition to pot plant experiments under controlled conditions, field trials could help to understand the potential applications of these advanced nanoparticles in agriculture under changing environmental conditions; (2) The potential mechanisms of nanoparticle-crop interactions need to be studied and explored in depth; (3) Toxicity evaluations of most nanomaterials have been limited to simple surface toxicity, and systematic studies of the biological effects of nanoparticles and their toxicity mechanisms are needed in the future. please see Line 618-625.
Reference number 4 is the same as 62.
Response: Thanks for your positive suggestion. We are sorry about our unclear description. We have deleted reference 62.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe submitted manuscript to PLANTS-MDPI entitled “Potential application of selenium and copper nanoparticles in improving growth, quality, and physiological characteristics of strawberry under drought stress” is interesting to investigate. BUT, following are the comments that need to be addressed:
Why did the authors select to study selenium and copper nanoparticles? Are they applicable in the field conditions? Are they more economical than other plant biostimulants e.g. plant-based biostimulants which are more eco-friendly than anything else
Nanotechnology can be a threat to the environment, how do you cope with this aspect in future?
Line 16: should mention the drought levels.
How authors maintain the drought stress?
Which nanoparticles performed better for abiotic stress tolerance in strawberry?
What is the novelty in this study since extensive research has already been done on the role of nanoparticles in mitigating drought stress tolerance in different plant species?
The research gap is missing in the last paragraph of the introduction section. The research objectives are not clear too.
Line 262: Please use the roundoff values throughout the MS.
Conclusion (Line 567): Please name the enzymes and other components that played an important role in mitigating drought stress drastic effects in strawberry.
Author Response
Dear editor and reviewers：
Thank you very much for your letter and the reviewer’s comments concerning our manuscript entitled “Potential application of selenium and copper nanoparticles in improving growth, quality, and physiological characteristics of strawberry under drought stress” (Manuscript ID:3098217). Those comments are all valuable and very helpful for revising and improving our paper. We have carefully revised the manuscript according to each comment referred by the reviewers. In accordance with the requirements of the journal, we have carefully revised the paper format and corrected the grammatical errors. The revised portions were highlighted in the marked-revised manuscript. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the correction will meet with approval. Once again thank you very much.
Independent Review Report, Reviewer 1
The submitted manuscript to PLANTS-MDPI entitled “Potential application of selenium and copper nanoparticles in improving growth, quality, and physiological characteristics of strawberry under drought stress” is interesting to investigate. BUT, following are the comments that need to be addressed:
Why did the authors select to study selenium and copper nanoparticles? Are they applicable in the field conditions? Are they more economical than other plant biostimulants e.g. plant-based biostimulants which are more eco-friendly than anything else
Response: Thanks for your question.
CuNPs and SeNPs are key enzymes in the antioxidant defense system of plants, helping them to scavenge free radicals more efficiently and reduce oxidative stress. Copper is a cofactor for many enzymes and is essential for processes such as photosynthesis, respiration and nitrogen metabolism in plants. Selenium, on the other hand, is a trace element that is also important for antioxidant defense and metabolic activities in plants. CuNPs promote stomatal closure and reduce water evaporation, while SeNPs improve the osmoregulatory capacity of plants, helping them to better retain water under drought conditions. In summary, CuNPs and SeNPs contribute to the mitigation of drought stress and the improvement of plant survival and productivity through a variety of mechanisms, including the enhancement of antioxidant capacity, the promotion of growth, and the enhancement of water use efficiency in plants.
We have added it in the introduction section. Please see Line 68-75.
In recent years, metal nanoparticles have emerged as promising tools in the field of agriculture, offering new opportunities for enhancing plant growth and ensuring global food security. Nanoscale materials, such as silver, gold, copper, and zinc nanoparticles, exhibit unique physical and chemical properties that make them highly attractive for various applications in plant systems.
CuNPs and SeNPs as a new type of fertilizer additive, has the following significant advantages over traditional biostimulants:(1) Higher bioavailability: CuNPs and SeNPs are smaller and can be absorbed and utilized by plants more effectively, which helps to enhance the antioxidant capacity of plants and improve the quality of crops.(2) Wider application range: CuNPs and SeNPs is not only suitable for a wide range of crops, but can also be used at different growth stages, thus realizing the whole nutritional management. Biostimulants, usually require specific environments and conditions for optimal results. (3) Environmentally friendly: CuNPs and SeNPs has a low environmental impact during production and use, and is less likely to cause soil and water contamination. In contrast, some biostimulants may contain environmentally unfavorable ingredients that may have a negative impact on the ecosystem when used over a long period of time. (4) Enhance plant resistance: CuNPs and SeNPs can enhance plant resistance to diseases and adversities such as drought, salinity and high temperature. This helps to improve crop stability and yield and reduce losses due to environmental changes.
In summary, CuNPs and SeNPs has obvious advantages in improving crop nutrition, enhancing plant health and environmental friendliness, and is a new type of fertilizer additive worth promoting in modern agriculture.
Nanotechnology can be a threat to the environment, how do you cope with this aspect in future?
Response: Thanks for your question.
As a cutting-edge technology, nanotechnology, while bringing many benefits, does have certain environmental risks. In order to ensure the sustainable development of nanotechnology, we need to take a series of measures to deal with this aspect: (1) Strengthen research and monitoring: Continuously carry out research on the environmental behavior and ecological effects of nanomaterials, and set up a perfect monitoring system to keep abreast of the distribution, migration and transformation of nanomaterials in the environment, and to assess their long-term impacts. (2) Formulate strict standards and norms: formulate and implement standards for the production, use and disposal of nanomaterials to ensure that they meet environmental requirements in all aspects. At the same time, strengthen the safety assessment of nanoproducts to ensure that they will not cause harm to the environment and human health. (3) Promote green nanotechnology: Encourage and support the development of environmentally friendly nanomaterials and nanotechnology to reduce pollutant emissions in the production process and improve resource utilization efficiency. For example, develop degradable nanomaterials to reduce their accumulation in the environment. Through the implementation of these measures, we can effectively manage and control the environmental risks of nanotechnology to ensure that it does not pose a threat to the environment while promoting social development.
We have added “(3) Toxicity evaluations of most nanomaterials have been limited to simple surface toxicity, and systematic studies of the biological effects of nanoparticles and their toxicity mechanisms are needed in the future.” in the conclusion section. Please see Line 622-625.
Line 16: should mention the drought levels.
Response: Thanks for your positive suggestion. Done, please see Line 16. Moderate drought stress (DS, 60% FC), Severe drought stress (SS. 25%, FC).
How authors maintain the drought stress?
Response: Thanks for your question. Soil moisture content in the pots was determined using a configuration of ML2 probes (ThetaProbe soil moisture devices, UK) and HH2 moisture meter Delta-T Devices (HH2 moisture meter Delta-T Devices, UK), with supplemental watering using drip irrigation to ensure that soil moisture was within the range set for the experiment. We have added it in methods and materials section. Please see Line 151-155.
Which nanoparticles performed better for abiotic stress tolerance in strawberry?
Response: Thanks for your question. In the conclusion section, we have added “the application of SeNPs alone superior to that of CuNPs alone. Please see Line 613-614..
What is the novelty in this study since extensive research has already been done on the role of nanoparticles in mitigating drought stress tolerance in different plant species?
Response: Thanks for your question. First of all, strawberry is a herbaceous plant, mainly cultivated in facilities, which generally has a large leaf area, grows fast, and has a root system distributed in the shallow layer of the soil, which is highly susceptible to the effects of drought stress. Therefore, strawberries are more susceptible to the adverse effects of drought stress than other crops.
In addition, previous research pairs have focused on the application of nanoparticles to the soil. Foliar spraying of nanoparticles can be directly absorbed by plant leaves, avoiding adsorption and immobilization in the soil, thus accelerating the speed and efficiency of absorption. Through foliar spraying, the amount and location of nanoparticles can be more accurately controlled to avoid the waste of resources and environmental pollution caused by excessive application. Foliar spraying of nanoparticles can more directly affect the physiological processes of plants, such as promoting photosynthesis, enhancing disease resistance, etc., thus improving the yield and quality of crops. Foliar spraying is usually carried out using spraying equipment, which is simple to operate and easy to apply on a large scale. Foliar spraying of nanoparticles has obvious advantages in improving efficiency, reducing pollution, and enhancing crop performance, which makes it a nanoparticle application method worth promoting. However, fewer studies have been conducted on foliar nanoparticle application in ameliorating plant abiotic stresses.
We have added “There is a lack of research on how the combined application of SeNps and CuNPs ameliorates the adverse effects of drought stress on strawberries. Foliar spraying of nanoparticles has obvious advantages in improving efficiency, reducing pollution, and enhancing crop performance, which makes it a nanoparticle application method worth promoting. However, fewer studies have been conducted on foliar nanoparticle application in ameliorating plant abiotic stresses.” in the introduction section. Please see Line 101-106.
The research gap is missing in the last paragraph of the introduction section. The research objectives are not clear too.
Response: Thanks for your positive suggestion. Done, please see Line 107-116.
Line 262: Please use the roundoff values throughout the MS.
Response: Thanks for your positive suggestion. The MDPI Journals are required to keep two valid digits after the decimal point, so your understanding is greatly appreciated.
Conclusion (Line 567): Please name the enzymes and other components that played an important role in mitigating drought stress drastic effects in strawberry.
Response: Thanks for your positive suggestion.
Drought stress causes excessive production of reactive oxygen radicals in plants, which can damage cellular structures. Drought stress causes excessive production of reactive oxygen radicals in plants, which can damage cellular structures. SOD catalyzes the disproportionation of superoxide anion radicals to hydrogen peroxide and oxygen, thus reducing oxidative stress.CAT and POD breaks down hydrogen peroxide and reduces its toxic effects on cells. toxic effects on cells. Under drought stress, they help maintain intracellular redox balance. Like vitamin C and vitamin E, they directly scavenge free radicals and protect cells from oxidative damage. These enzymes and other components work together to help plants maintain normal physiological functions under drought stress, reduce the damage caused by drought, and improve plant survival.
We have added “SOD, POD, CAT and Vc are important in mitigating the drastic effects of drought stress in strawberry.” Please see Line 601-602.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsDear author,
Thanks for the revision file.
Line 142: Fragaria × ananassa must be italic.
Line 188: mistakes.
Reference number 30 missed in the text.
Regards
Comments on the Quality of English LanguageIt is OK.
Author Response
Line 142: Fragaria × ananassa must be italic.
Response: Thanks for your positive suggestion. Done.
Line 188: mistakes.
Response: Thanks for your positive suggestion. We have revised it. Soil Testing Equipment Professional Systems GmbH, STEP-TII Beijing Co Ltd, 10 Tianxiu Road, Beijing.
Reference number 30 missed in the text.
Response: Thanks for your positive suggestion. We have replaced “Luo et al. (2011) [31]” into Luo et al. (2011) [30]”. We have replaced “Chirinos et al. (2007) [34]” into “Chirinos et al. (2007) [31]”.
Author Response File: Author Response.pdf
