Responsible Mechanisms for the Restriction of Heavy Metal Toxicity in Plants via the Co-Foliar Spraying of Nanoparticles

Round 1

Reviewer 1 Report

The Ms by Emamverdian et al. is dealing with an investigation aimed at evaluating the role of two types of nanoparticles (NPs) in alleviating the deleterious effects of As in bamboo plants by spraying leaves with iron oxide (IONPs) and selenium (Se-NPs) nanoparticles. Beside growth traits and As content, a wide array of biochemical and physiological parameters involved in plant response to metal stress was measured, allowing to display a portrait on how the supply with NPs of As-treated plants reduces the harmful effects associated to the exposure of plants to this metalloid. The experimental design is well structured and the analysis of the different investigated parameters appropriately performed. Results can be of certain interest and can expand the knowledge on the matter. I have some concerns that authors should tackle prior the paper being acceptable for the publication.

L26 I suggest to mention the amount of NPs sprayed on leaves throughout the experimental trial

L34 Not clear if the combination of NPs means that 60 mg/L of each NP was used or the total amount of NPs was 60 mg/L as resulting from 30 mg/L of each NP. Please explain it better also in materials and methods section

L128 Please check and correct the number of the table. Moreover, it is not clear which samples were taken for biochemistry measurements (I suppose leaf samples) and how they are maintained before being analysed. Please refer to which samples were analised for each measured parameter (i.e. antiox. enzyme activity, hydrogen peroxide etc.)

L142 Better find another way to abbreviate these parameters (TISH/R) as in this way it seems a ratio of shoot to root

L182 Please check about PAO activity as it was not shown in the result section

L194-196 Authors state that they performed a 2-way ANOVA followed by a Tukey’s test and in my opinion the statistical approach is correct. Anyway, the results of the statistical test expressed by the letters of significance are not convincing to me as the criterion followed to assign the letters sounds not clear, or the assignment of the letters of significance was not appropriately performed. For example, in Figure 1A, there is a value marked with “ef” without any other value marked with “e”; the same for “dg” in Figure 1B. So, not clear how the homogeneous groups were identified. Again, in Figure 2 (A side) it is not clear to me why values in  As10 are marked by “bc” and higher values in SeNPs+As20 are marked by “cde” while in-between you can find values in IONPs+As40 marked with “de”. So, which is the interval of the homogenous group corresponding to “c”? Other cases can be found in B side of the Figure 2. Really, also Figures 3, 4, 5, 6, and 7 showed a similar situation to be revised. Moreover, an excessive data overlapping occurs in all the figures, making extremely difficult to evaluate the real differences among theses and therefore with the obvious risk of missing the main information brought by the different analyses performed in the experimental work. So, I suggest to apply a different statistical test, as for example Duncan’s test, to avoid excessive data overlapping and make a clearer overview of the statistical differences among theses.

 L238 In table 2 authors reported a statistical evaluation of control plants in which the values are obviously corresponding to zero or 1 depending on the parameter. This approach is without sense to me as no data interval can occur and therefore no mean and standard deviation of the mean can be calculated, making not possible to subject these data to statistical assessment. Therefore, a new statistical evaluation should be made without data referring to control plants

L269 These data are reported in figure 4 instead of erroneously mentioned in the text as figure 3 and viceversa.

L410 Authors mentioned how in literature is reported that NPs can stimulate growth and absorbance of nutrients. However, in their work a beneficial effect at multiple level in plants treated with NPs was reported, especially for that regards the oxidative status of plants. In this regard, authors should better discuss which can be the role of NPs in ameliorating this status respect to control, as it seems that control plants have undergone a certain degree of  oxidative stress that the treatment with NPs was able to alleviate

L418 I think authors mean “adsorb”

L420 It should be better explained how the foliar spray of NPs can reduce the uptake and translocation of As, i.e. which mechanism can be activated in plants able to reduce the absorption of As at root level

L421 I think authors mean “absorption”

L424-427 I wonder if the differences in As tolerance in the leaves (measured through various parameters) can be related to the slight reduction of As content occurring in plants, i.e approx. 23 ppm to 18 ppm in plants treated with 40 ppm As,  in control and NPs treated plants respectively

L509 No evidence about “infiltration” of leaves with NPs was highlighted in this work

L522 The role of polyamine seems overestimated in this so peremptory statement given the overall results presented. As authors discussed, the higher tolerance of plants to As is probably due to the capability of NPs to reduce the As accumulation in the different organs so lowering the oxidative stress effects associated to the metalloid presence. Anyway, the mechanism through which it occurs remains unclear and a hypothesis about that could be made.

The Ms would benefit from a thorough revision of the text to amend punctuation and typing errors.

Minor editing of English language required. Punctuation should be revised.

Author Response

Response to Reviewer Comments 1#

Dear Reviewer

Thank you for your valuable comments and thank you for your precious time spent reviewing this paper. The manuscript (ID: agronomy-2415817) was carefully reviewed, and all the sections and parts were modified according to the comments. Moreover, linguistically, the manuscript was edited by native expert in English. The authors hope that the manuscript in the revised form meets the expectations of the reviewer and can express the content of our research to the community with more clarity.

Comments and Suggestions for Authors

The Ms by Emamverdian et al. is dealing with an investigation aimed at evaluating the role of two types of nanoparticles (NPs) in alleviating the deleterious effects of As in bamboo plants by spraying leaves with iron oxide (IONPs) and selenium (Se-NPs) nanoparticles. Beside growth traits and As content, a wide array of biochemical and physiological parameters involved in plant response to metal stress was measured, allowing to display a portrait on how the supply with NPs of As-treated plants reduces the harmful effects associated to the exposure of plants to this metalloid. The experimental design is well structured and the analysis of the different investigated parameters appropriately performed. Results can be of certain interest and can expand the knowledge on the matter. I have some concerns that authors should tackle prior the paper being acceptable for the publication.

L26 I suggest to mention the amount of NPs sprayed on leaves throughout the experimental trial

It has been mention in the text

Bamboo plants exposed to four levels of As (0, 10, 20, and 40 mg L^-1) were foliar-sprayed by 60 mg L^-1 Se-NPs and 60 mg L^-1 IONPs alone and in combination.

L34 Not clear if the combination of NPs means that 60 mg/L of each NP was used or the total amount of NPs was 60 mg/L as resulting from 30 mg/L of each NP. Please explain it better also in materials and methods section

It has been corrected.

In sum, Se-NPs and IONPs improved bamboo endurance, yet the most effective approach for increasing bamboo's ability to recover from As toxicity was the concurrent use of 60 mg L^-1 Se-NPs and 60 mg L^-1 IONPs.

In materials and methods section

60 mg L^-1 of Se-NPs and 60 mg L^-1 IONPs alone and in combined form was sprayed as treatments twice, 20 days (30 mg L^-1 Se-NPs, 30 mg L^-1 IONPs ) and 40 days (30 mg L^-1 Se-NPs, 30 mg L^-1 IONPs) after the trial started.

L128 Please check and correct the number of the table. Moreover, it is not clear which samples were taken for biochemistry measurements (I suppose leaf samples) and how they are maintained before being analysed. Please refer to which samples were analised for each measured parameter (i.e. antiox. enzyme activity, hydrogen peroxide etc.)

The number of the table has been corrected. Yes, that s right .it was leaf samples. The samples were maintained in the refrigerator. The leaf samples has used for parameter (i.e. antiox. enzyme activity, hydrogen peroxide etc.). It has corrected in the text.

L142 Better find another way to abbreviate these parameters (TISH/R) as in this way it seems a ratio of shoot to root

It has been corrected in the text

L182 Please check about PAO activity as it was not shown in the result section

polyamines activities (POA) are including The putrescine (Put) and spermidine (Spd)

So it has changed to ‘’The putrescine (Put) and spermidine (Spd) as two polyamines activities (POA) varied significantly across treatments (p<0.001)’’.

L194-196 Authors state that they performed a 2-way ANOVA followed by a Tukey’s test and in my opinion the statistical approach is correct. Anyway, the results of the statistical test expressed by the letters of significance are not convincing to me as the criterion followed to assign the letters sounds not clear, or the assignment of the letters of significance was not appropriately performed. For example, in Figure 1A, there is a value marked with “ef” without any other value marked with “e”; the same for “dg” in Figure 1B. So, not clear how the homogeneous groups were identified. Again, in Figure 2 (A side) it is not clear to me why values in  As10 are marked by “bc” and higher values in SeNPs+As20 are marked by “cde” while in-between you can find values in IONPs+As40 marked with “de”. So, which is the interval of the homogenous group corresponding to “c”? Other cases can be found in B side of the Figure 2. Really, also Figures 3, 4, 5, 6, and 7 showed a similar situation to be revised. Moreover, an excessive data overlapping occurs in all the figures, making extremely difficult to evaluate the real differences among theses and therefore with the obvious risk of missing the main information brought by the different analyses performed in the experimental work. So, I suggest to apply a different statistical test, as for example Duncan’s test, to avoid excessive data overlapping and make a clearer overview of the statistical differences among theses.

The Duncan test has been used for statistical differences, and all figures and tables have been corrected accordingly.

 L238 In table 2 authors reported a statistical evaluation of control plants in which the values are obviously corresponding to zero or 1 depending on the parameter. This approach is without sense to me as no data interval can occur and therefore no mean and standard deviation of the mean can be calculated, making not possible to subject these data to statistical assessment. Therefore, a new statistical evaluation should be made without data referring to control plants

The new statistical evaluation without data referring to control plants has been added to the table according to the Duncan test.

L269 These data are reported in figure 4 instead of erroneously mentioned in the text as figure 3 and viceversa.

It has been corrected in the text.

L410 Authors mentioned how in literature is reported that NPs can stimulate growth and absorbance of nutrients. However, in their work a beneficial effect at multiple level in plants treated with NPs was reported, especially for that regards the oxidative status of plants. In this regard, authors should better discuss which can be the role of NPs in ameliorating this status respect to control, as it seems that control plants have undergone a certain degree of oxidative stress that the treatment with NPs was able to alleviate

Thank you for your suggestion. We have added in the discussion parts

In this study, our result has reported beneficial effects at multiple levels in plants treated with NPs.

Our result show while different levels of As increase oxidative stress in plant with increasing ROS compounds (H₂O₂ and O₂^•−), MDA content and electrolyte leakage that the addition of NPs reduce oxidative stress case by over generation of ROS in plant under As levels in compare with control treatment.

L418 I think authors mean “adsorb”

It has corrected.

L420 It should be better explained how the foliar spray of NPs can reduce the uptake and translocation of As, i.e. which mechanism can be activated in plants able to reduce the absorption of As at root level.

It has been added to the discussion part.

This can be related to the high surface area of nanoparticles' potential to adsorb metal ions (Mobasherpour et al., 2011). So, it has been reported that van der Waals forces are involved in the physical adsorption of metal ions onto the adsorbent (Burakovet al., 2018). 

L421 I think authors mean “absorption”

It has corrected

Absorption

L424-427 I wonder if the differences in As tolerance in the leaves (measured through various parameters) can be related to the slight reduction of As content occurring in plants, i.e approx. 23 ppm to 18 ppm in plants treated with 40 ppm As, in control and NPs treated plants respectively

Multiple mechanisms are involved, in increasing plant tolerance under arsenic treated with NPs. reduction of As content can be one of them. The antioxidant defense mechanisms are the main defense mechanisms that can increase As tolerance in the plant organs.

L509 No evidence about “infiltration” of leaves with NPs was highlighted in this work

It has been corrected in the text

We proved that nanoparticles (Se-NPs and IO-NPs) may be effective foliar spray agents

L522 The role of polyamine seems overestimated in this so peremptory statement given the overall results presented. As authors discussed, the higher tolerance of plants to As is probably due to the capability of NPs to reduce the As accumulation in the different organs so lowering the oxidative stress effects associated to the metalloid presence. Anyway, the mechanism through which it occurs remains unclear and a hypothesis about that could be made.

We concluded that Se-NPs and IONPs, both alone and in combination, can elevate bamboo resilience to As Which we hypothesis that NPs by raising polyamine content and limiting As uptake and translocation can be increased bamboo tolerance under As toxicity. However, need more study to understand the involved mechanism.

Reviewer 2 Report

The topic of the article covers an important field of knowledge that may be of interest to a large number of scientists, the discussion of the results is clear and satisfactorily interpreted in terms of working hypotheses. Previous studies are also mentioned, and a discussion of their perspectives is well developed. The text is well structured, easy to read and understand.

Several minor remarks are:

Please explain, which is the reason why the authors chose to apply the exact NP concentration of 60 mg L^-1?

Line 135. Delete the acronym TF from the title

Line 142 Tolerance index is TI. I suggest using in the formula the same acronym.

Author Response

Response to Reviewer Comments 2#

Dear Reviewer

Thank you for your valuable comments and thank you for your precious time spent reviewing this paper. The manuscript (ID: agronomy-2415817) was carefully reviewed, and all the sections and parts were modified according to the comments. Moreover, linguistically, the manuscript was edited by native expert in English. The authors hope that the manuscript in the revised form meets the expectations of the reviewer and can express the content of our research to the community with more clarity.

Comments and Suggestions for Authors

The topic of the article covers an important field of knowledge that may be of interest to a large number of scientists, the discussion of the results is clear and satisfactorily interpreted in terms of working hypotheses. Previous studies are also mentioned, and a discussion of their perspectives is well developed. The text is well structured, easy to read and understand.

Several minor remarks are:

Please explain, which is the reason why the authors chose to apply the exact NP concentration of 60 mg L-1?

The concentrations of NPs were chosen according to the preliminary studies conducted by other researchers. So 60 mg/l was selected according to high and low levels within the tolerance range of the bamboo species.

Line 135. Delete the acronym TF from the title

It has been deleted.

Line 142 Tolerance index is TI. I suggest using in the formula the same acronym.

It has been corrected in the text.

TI of Shoot = (shoot dry weight of As) / (Shoot dry weight of control)                                                                                      

TI of Root = (root dry weight of As) / (root dry weight of control)                

Reviewer 3 Report

The reviewer greatly thanks the Editor for having allowed reviewing this manuscript. The paper agronomy-2415817 entitled "Responsible mechanisms for restriction of heavy metals' toxicity in plants' by Co-foliar spraying of nanoparticles" is attractive and has information relevant to the audience. The paper is generally well-written and structured. However, some revisions must be conducted before its acceptance. Here are some general comments:

1.     The authors should include a short introduction to the paper in the first two sentences of the abstract.

2.     The authors should include the main findings (values) of their research in the abstract.

3.     The authors should explain the paper's novelty in the introduction before stating the work's objectives.

4.     The lab instruments (names, selling company's name, city and country) used for lab analyses should be provided.

5.     Lines 271-274, include, in the discussion section, the scientific explanation and reason behind the highest antioxidant activity.

6.     Some texts overlap with existing published ones (lines 283-286, 306-310, etc.); rewrite the phrases to avoid such an unethical issue.

7.     There are some grammatical errors and punctuation that must be fixed and polished.

There are some grammatical errors and punctuation that must be fixed and polished.

Author Response

Response to Reviewer Comments 3#

Dear Reviewer

Thank you for your valuable comments and thank you for your precious time spent reviewing this paper. The manuscript (ID: agronomy-2415817) was carefully reviewed, and all the sections and parts were modified according to the comments. Moreover, linguistically, the manuscript was edited by native expert in English. The authors hope that the manuscript in the revised form meets the expectations of the reviewer and can express the content of our research to the community with more clarity.

Comments and Suggestions for Authors

The reviewer greatly thanks the Editor for having allowed reviewing this manuscript. The paper agronomy-2415817 entitled "Responsible mechanisms for restriction of heavy metals' toxicity in plants' by Co-foliar spraying of nanoparticles" is attractive and has information relevant to the audience. The paper is generally well-written and structured. However, some revisions must be conducted before its acceptance. Here are some general comments:

1. The authors should include a short introduction to the paper in the first two sentences of the abstract.

Bamboo is particularly significant across the world because the shoots are high in calories and nutritional fiber while being low in cholesterol however recent researches proved that bamboo shoots contain a substantial quantity of heavy metals, including Arsenic (As). Therefore, we explored whether the co-application of Iron Oxide (IONPs) and Selenium nanoparticles (Se-NPs) would attenuate As toxicity in bamboo plants (Pleioblastus pygmaeus). A greenhouse experiment has been performed to investigate plant response to arsenic toxicity.

2. The authors should include the main findings (values) of their research in the abstract.

Abstract has been modified as requested and main findings were introduced in the revised version.

3. The authors should explain the paper's novelty in the introduction before stating the work's objectives.

It has been corrected in the text.

To the best of our knowledge, this is the first research using foliar-applied Se-NPs and IONPs either alone or in combination to decrease As toxicity in bamboo plants, even though the utilization of Fe and Se in the amelioration of HMs stress has previously been documented.  Thus, in the current work, we aimed to investigate the impact of Se-NPs and IONPs in single and combination forms in bamboo plants under As toxicity, emphasizing involved mechanisms in As detoxification. We suggest that IONPs and Se-NPs can increase bamboo tolerance through antioxidant machinery and polyamine activity, decrease bioavailability, and, subsequently, As toxicity.

4. The lab instruments (names, selling company's name, city and country) used for lab analyses should be provided.

Names, selling company's name, city, and country used for lab analysis have been provided in the Material and Method part.

5. Lines 271-274, include, in the discussion section, the scientific explanation and reason behind the highest antioxidant activity.

This can be related to the reduction of HM accumulation in tissues by NPs which can reduce oxidative stress and resulting in increased antioxidant capacity in plant under stress. With increasing plant nutrient ions' availability, we also suggest that NPs may provide some essential co-factors involved in antioxidant molecule synthesis which can help improve antioxidant activity. The increasing antioxidant capacity by NPs was reported by several studies (100, 101).

6. Some texts overlap with existing published ones (lines 283-286, 306-310, etc.); rewrite the phrases to avoid such an unethical issue.

Indicated paragraphs were rephrased. Please see in the revised version.

7. There are some grammatical errors and punctuation that must be fixed and polished.

Needed edits were done accordingly.

Comments on the Quality of English Language

There are some grammatical errors and punctuation that must be fixed and polished.

Grammar has been double checked and revisions made accordingly.

Round 2

Reviewer 1 Report

The revised version of the Ms by Emamverdian et al. addresses most of the remarks made on the original submission. Notably, authors completely revised the statistical assessment of data improving the quality of the presentation and discussion about them. Anyway, in my opinion some issues remain not well tackled and authors should better deal with them. With regret, I should underline that authors did not follow the general rule for answering to a referee’s comment, i.e. indicate where in the text (page, line) they addressed the remark made by the referee, so to help the referee to properly evaluate the authors’ response.

A list of issues not well tackled is following, referred to the document with the responses of the authors:

Abstract

L 39. Not clear what the term “respectively” refers to.

L26 I suggest to mention the amount of NPs sprayed on leaves throughout the experimental trial

It has been mention in the text

Unfortunately, authors did not deal with the amount on NPs sprayed on leaves. They reported data expressed as concentration but such data are not sufficient to appreciate how many NPs reached the leaves as no volume of spraying was reported so to calculate the amount of NPs supplied to plants.

 L182 Please check about PAO activity as it was not shown in the result section polyamines activities (POA) are including The putrescine (Put) and spermidine (Spd)

So it has changed to ‘’The putrescine (Put) and spermidine (Spd) as two polyamines activities (POA) varied significantly across treatments (p<0.001)’’.

Authors should absolutely correct this part, that they approached very badly. PAO is the acronym of Poliamine Oxidase, the enzyme catabolysing polyamines, so any reference to PAO in this work is not correct and, therefore, misleading. The acronym POA (never heard about it) makes no sense to me, as polyamines are usually expressed as concentrations, and referring to them as  an “activity” is wrong and, again, misleading. So, please delete any reference to PAO or POA in this work. Further, not clear why authors did not evaluate also the spermine content, usually presented together with spermidine, that was proven to have higher metal chelation properties given the higher number of amino groups in the molecule. Lastly, authors should consider that, chemically, putrescine is a diamine not a polyamine.

L420 It should be better explained how the foliar spray of NPs can reduce the uptake and translocation of As, i.e. which mechanism can be activated in plants able to reduce the absorption of As at root level.

It has been added to the discussion part.

This can be related to the high surface area of nanoparticles' potential to adsorb metal ions (Mobasherpour et al., 2011). So, it has been reported that van der Waals forces are involved in the physical adsorption of metal ions onto the adsorbent (Burakovet al., 2018). 

Authors did not tackle properly this issue, as they did not explain (or formulate a hypothesis about it) how the spraying of leaves with NPs may result in a lower As uptake from soil by roots. In fact, if a role for NPs in reducing the movement of As, once absorbed, from roots to the upper parts of plants is discussed, mentioning possible explanations for this mechanism, the effect of the spraying leaves with NPs on the lowering the As uptake by roots is not considered, even if it represents an important finding obtained in this experimental trial.  

L522 The role of polyamine seems overestimated in this so peremptory statement given the overall results presented. As authors discussed, the higher tolerance of plants to As is probably due to the capability of NPs to reduce the As accumulation in the different organs so lowering the oxidative stress effects associated to the metalloid presence. Anyway, the mechanism through which it occurs remains unclear and a hypothesis about that could be made.

We concluded that Se-NPs and IONPs, both alone and in combination, can elevate bamboo resilience to As Which we hypothesis that NPs by raising polyamine content and limiting As uptake and translocation can be increased bamboo tolerance under As toxicity. However, need more study to understand the involved mechanism.

As underlined in my previous comment, authors did not formulate any hypothesis about the mechanism “limiting As uptake” and therefore they should not deal with it as a concluding remark. Anyway, I am still thinking that, given the information provided, the role of polyamines in limiting the damaging effects of As is overestimated.

Minor editing of English language required

Author Response

Response to Reviewer Comments 1#

Dear Reviewer

Thank you for your valuable comments and thank you for your precious time spent reviewing this paper. The manuscript (ID: agronomy-2415817) was carefully reviewed, and all the sections and parts were modified according to the comments. Moreover, linguistically, the manuscript was edited by native expert in English (Professor James Barker). The authors hope that the manuscript in the revised form meets the expectations of the reviewer and can express the content of our research to the community with more clarity.

The revised version of the Ms by Emamverdian et al. addresses most of the remarks made on the original submission. Notably, authors completely revised the statistical assessment of data improving the quality of the presentation and discussion about them. Anyway, in my opinion some issues remain not well tackled and authors should better deal with them. With regret, I should underline that authors did not follow the general rule for answering to a referee's comment, i.e. indicate where in the text (page, line) they addressed the remark made by the referee, so to help the referee to properly evaluate the authors' response.

A list of issues not well tackled is following, referred to the document with the responses of the authors:

Abstract

Question # 1

L 39(35-36). Not clear what the term "respectively" refers to.

Response # 1

To better understand for readers, we corrected data (as percentage) in abstract

Please check Line 28-37

Regarding the reviewer question respectively refers to Table 2 data which shows the tolerance ındex (TI) detected in the shoots and roots. Please see the revised version of the sentence in the abstract in L38-39: " Co-application of Se-NPs + IONPs with 10, and 20 mg L^-1 As raised TI by 14%, and 9%, in the shoot and by 18%, and 14%, in the root, respectively ".

Question # 2

L26 I suggest to mention the amount of NPs sprayed on leaves throughout the experimental trial

Unfortunately, authors did not deal with the amount on NPs sprayed on leaves. They reported data expressed as concentration but such data are not sufficient to appreciate how many NPs reached the leaves as no volume of spraying was reported so to calculate the amount of NPs supplied to plants.

Response # 2

As suggested, this query (L26) was mentioned in the text.

Thank you for this comment. I hope we can clarify the amount of NPs sprayed with the following explanation: Se-NPs (30 mg) were solvated in water (500 mL) and sprayed onto the leaves of each bamboo plant after 20 from the trial’s start. This was repeated after 40 days. (total 60 mg L^-1 Se-NPs). It was similar for IONPs too. For co-application treatment, the first spray of 15 mg of Se-NPs + 15 mg IONPs was solvated in 500 mL water and then sprayed onto each bamboo plant’s leaves 20 days from the trial’s start This was repeated after 40 days (total 60 mg L^-1 Se-NPs+ IONPs ).

Please check L 138-148 in the revised version of the manuscript.

Question # 3

L182 Please check about PAO activity as it was not shown in the result section polyamines activities (POA) are including The putrescine (Put) and spermidine (Spd)

As requested, the PAO activity was explained as : 'The putrescine (Put) and spermidine (Spd) as two polyamines activities (POA) varied significantly across treatments (p<0.001)" in the revised version.

Authors should absolutely correct this part, that they approached very badly. PAO is the acronym of Poliamine Oxidase, the enzyme catabolysing polyamines, so any reference to PAO in this work is not correct and, therefore, misleading. The acronym POA (never heard about it) makes no sense to me, as polyamines are usually expressed as concentrations, and referring to them as  an "activity" is wrong and, again, misleading. So, please delete any reference to PAO or POA in this work. Further, not clear why authors did not evaluate also the spermine content, usually presented together with spermidine, that was proven to have higher metal chelation properties given the higher # of amino groups in the molecule. Lastly, authors should consider that, chemically, putrescine is a diamine not a polyamine.

Response # 3

Thank you for the suggestion. We have deleted any reference to POA, which was an obvious mistake. We corrected this issue in the text by directly mentioning putrescine (Put) and spermidine (Spd).

Please check the revised version of Lines 220, 221, 222, 400, 401, 408, 410, 558,559,560,594,595.

On the other hand, we also thank you for your valuable suggestion, we thought that the spermidine (Spd) measurement could achieve a similar result of spermine content, so we didn't further measure spermine content. According to your comments, we will definitely consider measuring spermidine with spermine in future studies.

As the reviewer requested, putrescine has been considered one diamine in the text. Please see L 560.

Question # 4

L420 It should be better explained how the foliar spray of NPs can reduce the uptake and translocation of As, i.e. which mechanism can be activated in plants able to reduce the absorption of As at root level.

Thank you. This notion was introduced in the discussion part.

The reduction in root As level can be related to the high surface area of nanoparticles' potential to adsorb metal ions (Mobasherpour et al., 2011). It was also reported that the van der Waals forces are involved in the physical adsorption of metal ions onto the adsorbent (Burakovet al., 2018). 

Authors did not tackle properly this issue, as they did not explain (or formulate a hypothesis about it) how the spraying of leaves with NPs may result in a lower As uptake from soil by roots. In fact, if a role for NPs in reducing the movement of As, once absorbed, from roots to the upper parts of plants is discussed, mentioning possible explanations for this mechanism, the effect of the spraying leaves with NPs on the lowering the As uptake by roots is not considered, even if it represents an important finding obtained in this experimental trial. 

Response # 4

Line 472-483

We hypothesize that the nanoparticles, after penetrating the plant through the stomata of the bamboo leaves, are transferred to the bamboo rhizome junction and root surface and inhibit the arsenic uptake by bamboo roots which Kerui Guo has reported a similar mechanism on spraying nano‐materials on cucumber leaves (Guo et al., 2019). So the reduction of arsenic uptake by nanoparticles in roots can be related to the high surface area of nanoparticles' potential to adsorb metal ions (Mobasherpour et al., 2011). The van der Waals forces are also reported here to be involved in the physical adsorption of metal ions onto the adsorbent (Burakovet al., 2018). Therefore,  the nanoparticles reduce the translocation of arsenic to aerial parts by limiting arsenic uptake via roots. It was also reported that the nanoparticles, by modifying gene expression, and rising antioxidant capacity in plants, limit metal translocation from root to shoot (Rizwan et al., 2021).

Guo K, Hu A, Wang K, Wang L, Fu D, Hao Y, Wang Y, Ali A, Adeel M, Rui Y, Tan W. Effects of spraying nano-materials on the absorption of metal(loid)s in cucumber. IET Nanobiotechnol. 2019 Sep;13(7):712-719. doi: 10.1049/iet-nbt.2019.0060. PMID: 31573540; PMCID: PMC8676231.

Rizwan, M.; Ali, S.; Zia ur Rehman, M.; Riaz, M.; Adrees, M.; Hussain, A.; Zahir, Z.A.; Rinklebe, J. Effects of nanoparticles on trace element uptake and toxicity in plants: A review. Ecotoxicol. Environ. Saf. 2021, 221, 112437.

Question # 5

L522 The role of polyamine seems overestimated in this so peremptory statement given the overall results presented. As authors discussed, the higher tolerance of plants to As is probably due to the capability of NPs to reduce the As accumulation in the different organs so lowering the oxidative stress effects associated to the metalloid presence. Anyway, the mechanism through which it occurs remains unclear and a hypothesis about that could be made.

Based on our hypothesis, we concluded that Se-NPs and IONPs, both alone and in combination, can elevate bamboo resilience to As,  which we thought that NPs could increase bamboo tolerance against As toxicity by raising polyamine content and limiting As uptake and translocation. However, more study is needed to understand the whole involved mechanisms better.

As underlined in my previous comment, authors did not formulate any hypothesis about the mechanism "limiting As uptake" and therefore they should not deal with it as a concluding remark. Anyway, I am still thinking that, given the information provided, the role of polyamines in limiting the damaging effects of As is overestimated.

Response # 5

Since we previously stated our theory regarding the mechanism limiting As uptake and included it in the manuscript, we believe it may be one of the processes improving bamboo tolerance to stress. As suggested by the reviewer, we removed the reference to polyamines' role in the adverse effects of As in the conclusion

 Please check Line 600-605

Minor editing of English language required

The manuscript has been re-checked by a native English speaker among authors (Professor James Barker).