Influence of Hydroxyapatite Nanoparticles on Germination and Plant Metabolism of Tomato (Solanum lycopersicum L.): Preliminary Evidence
, Antonio Filippi 1, Alessio Adamiano 2, Lorenzo Degli Esposti 2,3, Michele Iafisco 2, Alessandro Mattiello 1, Elisa Petrussa 1 and Enrico Braidot 1Round 1
Reviewer 1 Report
This paper describes some preliminary evidence on the use of hydroxyapatite nanoparticles for an agricultural application.
Title - It should be "evidence" not "evidences"
Line 94 - Change "rpm" to "g"
Was there dissolution of the particles after the centrifugation and washing? This should be measured. If there is dissolved P and C, this could impact the interpretation of the rest of findings throughout the paper.
Line 163 - The authors should also test this method using a certified reference material to check the recovery of the process.
Line 169 - It would be helpful to clarify which standards were run.
Line 234 - This should be "z-average" size, not zeta average. What does the "error" refer to. Are these standard deviation values from some number of replicate samples or is this related to the poyldispersity index? This needs to be clarified.
Line 236 - What do the authors mean by Hd variation? Was there a statistically significant difference for the 2 mg/L suspension. Also, how did the authors test if there was sedimentation?
Lines 238-241 - DLS measurements are known to be low resolution (Caputo et al., 2019, Journal of Controlled Release, 299, pages 31-42). Could an alternative method be used to test if there is agglomeration at 2 mg/L?
Figure 3 - What do the error bars correspond to? What is "n"
Could the changes in the nHA be explained by any of the stabilizers added such as the high concentration of CMC, or was this used as the control water? This is not entirely clear and should be clarified in the figure captions.
Line 333 - Correct typo.
Author Response
Agronomy (ISSN 2073-4395)
Manuscript ID: agronomy-464506
Title: Influence of Hydroxyapatite Nanoparticles on Germination and Plant Metabolism of Tomato (Solanum lycopersicum L.): preliminary evidences
Authors: Luca Marchiol * , Antonio Filippi , Alessio Adamiano , Lorenzo Degli Esposti , Michele Iafisco , Alessandro Mattiello , Elisa Petrussa , Enrico Braidot
Reviewer 1
Comments and Suggestions for Authors
This paper describes some preliminary evidence on the use of hydroxyapatite nanoparticles for an agricultural application.
Q. Title - It should be "evidence" not "evidences"
A. Done
Mat&Met
Q. Line 94 - Change "rpm" to "g"
Was there dissolution of the particles after the centrifugation and washing? This should be measured. If there is dissolved P and C, this could impact the interpretation of the rest of findings throughout the paper.
A. We thank the referee for his/her comment.
1) The centrifugation speed has been now reported in g unit. The samples were centrifuged at 3943 x g.
2) Hydroxyapatite nanoparticles were washed with ultrapure water at neutral pH. Hydroxyapatite nanoparticles separation by centrifugation and washing with water are standard procedures, as this material is well known to be stable under this condition (Ksp = 6.8 x 10-37).
Q. Line 163 - The authors should also test this method using a certified reference material to check the recovery of the process.
A. Unclear what “certified reference material” stands for in this case. In our experimental design we considered cerium bulk at the highest concentration to check an eventual CeO2 size-scale effect, assuming that this could be particularly evident at the highest concentration.
Q. Line 169 - It would be helpful to clarify which standards were run.
A. Done. A certified reference material (NIST SRM® 1573 tomato leaves) was included for quality control of analysis.
Results
Q. Line 234 - This should be "z-average" size, not zeta average. What does the "error" refer to. Are these standard deviation values from some number of replicate samples or is this related to the poyldispersity index? This needs to be clarified.
A. 1) We acknowledge the Referee for this correction and we apologize for the mistake. The term z-average has been now used all along the text.
2) The error is reported as the standard deviation of three replicated measurements. This has been made clearer in the materials and method section.
Q. Line 236 - What do the authors mean by Hd variation? Was there a statistically significant difference for the 2 mg/L suspension. Also, how did the authors test if there was sedimentation?
A. 1) The term Hd (hydrodynamic diameter) was removed to avoid confusion in the reader. What we meant to say is that we didn’t detect any statistically relevant change in the particle size distribution with time according to Student-t analysis. This has been now clarified in paragraph 2.3.8.
2) Time wise sedimentation was measured by DLS, and more in detail by checking variations of the derived count rate parameter, which is relative to the intensity of the light scattered by nanoparticles, and thus to the nanoparticle concentration. As the nanoparticles suspensions were all very stable and derived count rates didn’t change with time, this data was not reported in the text.
Q. Lines 238-241 - DLS measurements are known to be low resolution (Caputo et al., 2019, Journal of Controlled Release, 299, pages 31-42). Could an alternative method be used to test if there is agglomeration at 2 mg/L?
A. 1) Agglomeration typically increases with increasing the concentration of the nanoparticles in suspension. Since we did not observe any agglomeration for higher nanoparticle concentrations, and moreover the concentration of 2 mg ml-1 is close to the resolution limit of the instrument for hydroxyapatite (which has a low refractive index), we can reasonably ascribe the apparent agglomeration as an artifact of the instrument. This is also denoted by the high error associated to the measurement of the solution at 2 mg ml-1. In this view, the paper by Caputo et al. has been now cited in the text, highlighting the drawbacks of the DLS technique to measure highly diluted suspensions.
2) Other techniques, like nanoparticle tracking analysis or differential centrifugal sedimentation could have helped to overcome the limitations of DLS; however, this technique was not available at the time of the analysis. Moreover, we wanted to provide results that refer to a very common, widespread and interdisciplinary-known technique such as DLS.
Q. Figure 3 - What do the error bars correspond to? What is "n"
A. 1) The error bars correspond to the standard deviation of the three replicate measurements for every size point. This has been now clarified in the materials and method section and in the caption of figure 3.
2)The letter “n” before the acronym HA stands for “nano”. The acronyms have been now checked and used uniformly along the text.
Q. Could the changes in the nHA be explained by any of the stabilizers added such as the high concentration of CMC, or was this used as the control water? This is not entirely clear and should be clarified in the figure captions.
A. We carried out some preliminary observations on suspension of nHA with the purpose to define the experimental conditions. Since nHA suspensions tend to aggregate and precipitate in very short time, according to Liu and Lal (2014) we used CMC to maintain a very concentrated nHA suspension stable for at least 8 days (see image).
We did not include the equivalent concentrations of nHA without CMC in the experimental plan because this would have complicated the management of the experiment. However, we have verified that CMC does not affect the tomato germination process.
Q. Line 333 - Correct typo.
A. Done.
Reviewer 2 Report
Some improvements in the manuscript should be done before it could be suitable for publishing.
Abstract
Lane – it should be stated that nHA were stabilized with CMC
Lane 25 – since some undesirable effects were observed in seedlings exposed to the highest concentration of CMC-stabilized nHA particles, maybe this statement should be rephrased
Materials and methods
Page 3, Lane 96 – after „according to “the name of the first authors should be added before the reference number in the brackets
Page 4, lane 152 – you haven't mentioned if you performed the germination experiment on the control medium without nHA
Page 4, lane 161 – why was 1000 mg L-1 nHA concentration omitted from the exposures in hydroponic culture? Moreover, you haven’t explained why did you introduce the 2000 mg L-1 bHA treatment which was not tested in seed germination and root elongation experiment.
Page 4, lane 163-164 – how come that you used different techniques for quantification of P and Cu in nHA (ICP-OES) and in plant tissue (ICP-MS)?; Can you specify what shoot represents; stem with leaves? only stem? only leaves? Because sometimes it is shoot and sometimes it is stem in the text, and this is confusing.
Page 4, lane 168 – could you be more precise about Ca and P determination since you didn’t just want to find out if there were present in the tissue but also to measure their concentration
Page 4, lanes 170-173 – ROS abbreviation stand for Reactive oxygen species ; you didn’t resuspend the nitrogen powder but the powder obtained from plant tissues grounded with liquid nitrogen – please rephrase! Moreover, it is unclear where the results of these measurements were presented in the Results section!
Page 4, lane 174-179 – the title of this subsection should be changed to “Photosynthetic pigments quantification” since from the results section it is obvious that you didn’t measure only chlorophyll content, but also carotenoids and pheophytin!; in front of the bracket with reference number the name of the first author should be given.; what do you mean by “different plant tissues” ? – please specify what type of tissue was taken for this analysis.
Page 4, lane 182-183 – how can it be FW if it is grounded with liquid nitrogen? Maybe you weighed 100 mg of tissue and grounded? If so, please rephrase the sentence!; put the name of the first author in front of the bracket with the reference number.
Results
General comment – since you have synthesized and characterized both naked (nHA) and CMC-stabilized (CMC-nHA) particles and only CMC-nHA were used in plant exposure experiments, the abbreviation CMC-nHA should be used in all parts of the manuscript (text as well as figure and table captions) when referring to experiment with plant material.
Page 8, lane 258 – introduce the (Fig. 5) mark before the comma
Page 8, lane 264 – caption of Figure 5 – you state that different letters indicate statistically significant differences even though you don’t have different letters in your graph
Page 9, lane 273 – effects on concentration of photosynthetic pigments
Page 9, lane 278 – Table 2 – the significant difference in pheophytin concentration was obtained between 20 and 200 nHA ; could you please comment this result
Page 10, Table 3 – on which biochemical traits do you mean in the caption of this Table? Moreover, it is unclear to which results are fluorescence values related to?
Page 10, lanes 290-295 – if you presented the root results before the shoot results in the table, it would be logical to explain in the text the roots results first
Page 10, Figure 7 - you state that different letters indicate statistically significant differences even though you don’t have different letters in your graph
Page 10, lanes 306-309 – but these results are not significant!
Discussion
Page 12, lanes 371-393 – the part describing the effects of nanoparticles on germination is rather long but authors somehow failed to offer an explanation of the results that obtained in this study.
Page 13, lane 401 – it is unclear what authors mean by “photosynthetic efficiency”? they have only measure the concentration of photosynthetic pigments!
Page 13, lanes 406-407 – what about the increased pheophytin content at 200 mg L-1 concentration? Could this also be a sign of chloroplast degradation? Please comment in discussion!
Page 13, lane 411 – what do you mean by “different plant portions”?
Page 13, lane 430 – please carefully specify where Ca measurements performed in stems or shots?
Page 13, lanes 423-437 – these two paragraphs are very confusingly written; the point is that the P content was of similar values in roots and shoots, while Ca was dominant in shoots. This should be clearly stated and commented.
Conclusions
The first paragraph of the conclusion brings a very broad observation on possible application of nanomaterials in agriculture and is suitable for introduction not for conclusion; therefore it should be omitted form the conclusion section.
English language should be improved in the whole manuscript.
Author Response
Agronomy (ISSN 2073-4395)
Manuscript ID: agronomy-464506
Title: Influence of Hydroxyapatite Nanoparticles on Germination and Plant Metabolism of Tomato (Solanum lycopersicum L.): preliminary evidences
Authors: Luca Marchiol * , Antonio Filippi , Alessio Adamiano , Lorenzo Degli Esposti , Michele Iafisco , Alessandro Mattiello , Elisa Petrussa , Enrico Braidot
Reviewer 2
Some improvements in the manuscript should be done before it could be suitable for publishing.
Abstract
Q. Lane – it should be stated that nHA were stabilized with CMC.
A. Done
Q. Lane 25 – since some undesirable effects were observed in seedlings exposed to the highest concentration of CMC-stabilized nHA particles, maybe this statement should be rephrased.
A. Germination was not negatively affected by the nHA treatments. some variability of the data may suggest a negative effect at 500-1000 ppm nHA. Actually, that was only apparent. No statistically significant differences in the germination rate were detected. Instead we found a positive (statistically significant) effect of nHA on root elongation.
Materials and methods
Q. Page 3, Lane 96 – after „according to “the name of the first authors should be added before the reference number in the brackets
A. Done.
Page 4, lane 152 – you haven't mentioned if you performed the germination experiment on the control medium without nHA.
A. Done.
Q. Page 4, lane 161 – why was 1000 mg L-1 nHA concentration omitted from the exposures in hydroponic culture? Moreover, you haven’t explained why did you introduce the 2000 mg L-1 bHA treatment which was not tested in seed germination and root elongation experiment.
A. We carried out two distinct experiments, and therefore we have not used the same experimental scheme. The seeds in germination were treated only with CMC-nHA, while in hydroponics the seedlings received in addition to the treatment with CMC-nHA also the nutrient solution. As far as the bHA is concerned, often in this type of experiments it is advisable to introduce some bulk material to get feedback on the possible effects induced more than on the concentration, on the nano scale of the material used.
Page 4, lane 163-164 – how come that you used different techniques for quantification of P and Cu in nHA (ICP-OES) and in plant tissue (ICP-MS)?
A1 This paper reports the results of a collaboration between nano-chemists and plant scientists. The synthesis and characterization of nHA and the analysis on plant materials were carried out in different labs.
Can you specify what shoot represents; stem with leaves? only stem? only leaves? Because sometimes it is shoot and sometimes it is stem in the text, and this is confusing.
A.2 Since we worked with rather small plants, we have specified in MAT & MET that shoots stands for stems + leaves, also indicated as epigeal biomass.
Page 4, lane 168 – could you be more precise about Ca and P determination since you didn’t just want to find out if there were present in the tissue but also to measure their concentration.
A. The term “concentration” was added to the sentence.
Q. Page 4, lanes 170-173 – ROS abbreviation stand for Reactive oxygen species; you didn’t resuspended the nitrogen powder but the powder obtained from plant tissues grounded with liquid nitrogen – please rephrase! Moreover, it is unclear where the results of these measurements were presented in the Results section!
A. We amended the term “radical” in title of the subsection 2.3.5 as “reactive”. We rewrote the phrase “One hundred mg of nitrogen powder obtained by different plant tissues were resuspended “ as “Root and shoot samples of tomato were ground with liquid nitrogen and 100 mg of the obtained powder was resuspended…”.
Q. Page 4, lane 174-179 – the title of this subsection should be changed to “Photosynthetic pigments quantification” since from the results section it is obvious that you didn’t measure only chlorophyll content, but also carotenoids and pheophytin! in front of the bracket with reference number the name of the first author should be given.; what do you mean by “different plant tissues”? – please specify what type of tissue was taken for this analysis.
A. We changed the title of the subsection according to reviewer suggestion in “Photosynthetic pigments quantification”. According to referee suggestion, before the reference [24] we inserted Mobin and Khan and we specified “20 mg of frozen powder obtained from tomato shoot portions” instead of 20 mg of different plant tissues.
Q. Page 4, lane 182-183 – how can it be FW if it is grounded with liquid nitrogen? Maybe you weighed 100 mg of tissue and grounded? If so, please rephrase the sentence!; put the name of the first author in front of the bracket with the reference number.
A. According to reviewer suggestions we changed the sentence “Root and shoot portions were frozen with liquid nitrogen, finally ground to a fine powder and samples (100±20 mg FW) were obtained for cellular ATP measurement, following the protocol described in [25]“ as follows “Root and shoot portions were weighted (100±20 mg DW) and frozen by liquid nitrogen. A fine powder was obtained by grinding and it was used for cellular ATP measurement, according to Mattiello et al. [25].”
We also amended the related tables 2-3 (µg g-1 FW → µg g-1 DW).
Results
Q. General comment – since you have synthesized and characterized both naked (nHA) and CMC-stabilized (CMC-nHA) particles and only CMC-nHA were used in plant exposure experiments, the abbreviation CMC-nHA should be used in all parts of the manuscript (text as well as figure and table captions) when referring to experiment with plant material.
A. Done
Q. Page 8, lane 258 – introduce the (Fig. 5) mark before the comma.
A. Done.
Page 8, lane 264 – caption of Figure 5 – you state that different letters indicate statistically significant differences even though you don’t have different letters in your graph
A. Indicating, therefore, that the effect of the treatments was not statistically significant.
Q. Page 9, lane 273 – effects on concentration of photosynthetic pigments
A. The sentence has been corrected.
Q. Page 9, lane 278 – Table 2 – the significant difference in pheophytin concentration was obtained between 20 and 200 nHA; could you please comment this result.
A. At row 290 we inserted the following sentence: “Pheophytin, a known metabolite associated to Photosystem II or involved in chlorophyll turnover, exhibited a bell-shaped profile, peaking at 200 mg L-1 dose of nHA. This value showed a significant difference compared to that measured at 20 mg L-1 nHA concentration.”
Q. Page 10, Table 3 – on which biochemical traits do you mean in the caption of this Table? Moreover, it is unclear to which results are fluorescence values related to?
A. The following sentence was inserted at row 294: “The redox balance of seedling shoot and root portions was scarcely influenced by nHA treatments, as shown by ANOVA analysis (Table 3), where ROS where evaluated by means of the fluorescent probe H2DCFDA.” We inserted the following new caption for Tab. 3: “One-way ANOVA analysis performed on ROS and ATP content measured in S. lycopersicum seedlings to evaluate the effect of different concentrations of nHA and bHA.”
Q. Page 10, lanes 290-295 – if you presented the root results before the shoot results in the table, it would be logical to explain in the text the roots results, first.
A. Done.
Q. Page 10, Figure 7 - you state that different letters indicate statistically significant differences even though you don’t have different letters in your graph.
A. Similarly to previous Page 8, lane 264 this confirms that the effect of the treatments was not statistically significant.
Page 10, lanes 306-309 – but these results are not significant!
A. We did not state that they do. We have only indicated that from a quantitative point of view the lowest radical biomass value was that of the control thesis.
Discussion
Page 12, lanes 371-393 – the part describing the effects of nanoparticles on germination is rather long but authors somehow failed to offer an explanation of the results that obtained in this study.
A. As described, in our case seed germination was not affected by the treatments, then we reported a synthetic overview regarding the effects of ENMs on seed germination.
Q. Page 13, lane 401 – it is unclear what authors mean by “photosynthetic efficiency”? they have only measure the concentration of photosynthetic pigments!
A. We amended the expression “photosynthetic efficiency” with following “photosynthetic pigments”.
Q. Page 13, lanes 406-407 – what about the increased pheophytin content at 200 mg L-1 concentration? Could this also be a sign of chloroplast degradation? Please comment in discussion!
A. At line 422 we added the following comment: “In addition also pheophytin content exhibited a peak corresponding to the 200 mg L-1 dose. Although the ANOVA analysis did not show any significance for the overall treatment, the peak could represent a rearrangement of the photosynthetic machinery, possibly by chlorophyll recycling and increase of the Photosystem II presence.”
Q. Page 13, lane 411 – what do you mean by “different plant portions”?
A. See the next comment. “different plant portions” changed in “hypogeal and epigeal plant biomass”.
Q. Page 13, lane 430 – please carefully specify where Ca measurements performed in stems or shots?
A. See Ref 1 Page 4, lane 163-164.
Q. Page 13, lanes 423-437 – these two paragraphs are very confusingly written; the point is that the P content was of similar values in roots and shoots, while Ca was dominant in shoots. This should be clearly stated and commented.
A. According to reviewer suggestion, we rewrote the paragraph and we add a citation.
Regarding the amount of nutrients accumulated within the two distinct portions of the plant, P showed a marked difference in uptake with respect to Ca. Generally, P exhibits a more diffuse absorption area and a greater mobility inside the organ [59] and we suggest that, thanks to its more efficient uploading and transport inside the seedling, the content of P in the different tomato tissues was not affected by different nHa concentrations, even if compared to bHA treatment. Accordingly, the profile between the hypogeal and epigeal portion showed a similar trend (Fig. 7).
On the contrary, Ca absorption and translocation is largely localized at new root hairs or at root tip [59]. In addition, a continuous removal of Ca from roots and subsequent transfer to epigeal organs occur, without recirculation via phloem as it happens for other nutrients. This unidirectional transport of Ca via xylem is a well-known phenomenon [60] and caused Ca accumulation in the stem with an order of magnitude higher when compared to the root (Fig. 8). This process could have also masked in the shoots the effects induced by different doses of nHA, whereas a hormetic effect induced by the different treatments was detectable in the root, being this organ not the main storage site for plant Ca pools.
Conclusions
Q. The first paragraph of the conclusion brings a very broad observation on possible application of nanomaterials in agriculture and is suitable for introduction not for conclusion; therefore, it should be omitted form the conclusion section.
A. This part is definitely short. On the other hand, this study reports initial results that do not allow us to argue in more depth. Therefore, we propose to keep the text of this paragraph.
Q. English language should be improved in the whole manuscript.
A. The manuscript was revised.
