Impact of Titanium Oxide Nanoparticles on Growth, Pigment Content, Membrane Stability, DNA Damage, and Stress-Related Gene Expression in Vicia faba under Saline Conditions
, Nabil I. Elsheery 2, Pavel Pashkovskiy 3, Vladimir Kuznetsov 3, Suleyman I. Allakhverdiev 3,4,* and Amina M. Zedan 5Round 1
Reviewer 1 Report
This work demonstrated that the capability of nTiO2 to improve plant perforformance under saline conditions, and this suggest us to apply nTiO 2 as a new strategy for improving plant growth under salt stress conditions. The experiment and data analysis are well designed. The writing is good, and there are some minor additions need to be addressed.
1. Table 1 was wrongly type set.
2. Why author choosing HSP genes for expression analysis , not the salt stress related gene.
3. The figures quality needs to be improved.
4. Is there any phenotype differences for the different treatments?
5. In Fig. 4, the total soluble proteins around 50KDa awas quiety different, especially s1 and s2 compared with control, T1,T2, how could it be. Can author have some explanation for this.
Author Response
- Table 1 was wrongly type set.
Answer: We improved Table 1.
- Why author choosing HSP genes for expression analysis, not the salt stress related gene.
Answer: HSP genes are often involved in general stress responses. They act as molecular chaperones, assisting in the proper folding of proteins and preventing protein aggregation. These functions are crucial when a plant or any organism faces stress conditions, which can lead to protein misfolding. Therefore, by studying HSP genes, researchers can obtain insights into the broader stress response of the organism. HSP genes are well studied and well characterized in many organisms. They can serve as an easily measurable indicator of a stress response, even if they are not directly related to the primary stressor (such as salt). However, we agree with the reviewer that it is essential to consider that focusing on HSP genes does not mean that salt stress-related genes are unimportant. We wanted to study whether nanoparticles can trigger the chaperone mechanism under salinity, so we chose the genes for HSPs.
- The figures quality needs to be improved.
Answer: Done.
All figures were prepared at 600 dpi, inserted in the manuscript word file and attached as separate TIFF files.
- Is there any phenotype differences for the different treatments?
Answer:
Some changes were recorded in leaf thickness, especially under salinity stress, as well as shoot and root length, but these changes were not listed in the presented data because our main interest was to investigate the physiological and molecular associated changes.
- In Fig. 4, the total soluble proteins approximately 50KDa awas quiety different, especially s1 and s2 compared with control, T1,T2, how could it be. Can author have some explanation for this.
Answer:
We added information to MS
«A protein with a size of approximately 55 kDa was specifically enriched in the control, T1, T2, S1T1 and S2T1 samples (Figure 4). We suppose that at higher NaCl concentrations (200 mM), it might induce a stronger stress response compared to control or nTiO2 concentrations. Moreover, it cannot be ruled out that the nanoparticles themselves may also influence the accumulation of this protein, which is quite intriguing. This stress can lead to changes in protein synthesis and degradation, resulting in altered levels of specific proteins.»
Reviewer 2 Report
This study estimated the “Combined and Separate Effects of TiO2 Nanoparticles and Salinity on Morphophysiological Parameters, Pigment Content, and Expression of Stress-Related Genes in Vicia faba”. I have several major concerns with the manuscript which prevent me from recommending it for publication in its current situation.
The main concern is:
Title:
(1) The MS title should be improved because “Morphophysiological Parameters” is rather general and does not give information on the specific aspects of your work.
(2) Please do not use abbreviation (TiO2) in the title.
Abstract:
(3) The abstract must be clear and concise. The experimental design and the treatments is not clearly revealed in the abstract.
I would advise the authors to re-write the abstract part focusing primarily on the foundation of the experiments they have undertaken.
(4) In L 16: Authors said that “Stressed plants showed a reduction in plant growth parameters and photosynthetic activity”.
Actually, I wonder because authors did not measure any photosynthetic activity parameters like Gas Exchange Capacity (net photosynthetic rate, stomatal conductance, and transpiration rate) and Chlorophyll Fluorescence System [the maximum quantum efficiency of PSII photochemistry (Fv/Fm), electron transport rate (ETR), actual photochemical efficiency of PSII (ΦPSII), photochemical quenching coefficient (qP), effective quantum yield of PSII photochemistry (Fv′/Fm'), and non-photochemical quenching coefficients (qN)].
Moreover, they did not measure all the plant growth parameters, only few of them.
I would advise the authors to re-write the abstract part focusing on the actually results they have obtained.
(5) Abbreviations should be defined in first mention, please revise this issue in the whole MS!
Keywords:
(6) Do not use abbreviations.
Introduction:
(7) There is no information on how Vicia faba respond to salinity.
(8) Please illustrate by more details the impact of using TiO2 Nanoparticles on the measured parameters under salt stress. Please incorporate recent references.
(9) The introduction is written chaotically. There is no hypothesis or purpose of the study.
(10) At the end of this section, Please describe the novelty of your work in comparison with previous work. What has it added that we did not know before?
Material and methods:
(11) In L 86: Authors said that “Seeds were procured from the Rice Research and Training Center (RRTC) at Kafr El Sheikh-Egypt”.
I am wondering how authors obtained Vicia faba seeds from Rice Center?.
(12) Why authors chose these specific treatments [two concentrations of nanotitanium (nTiO2) [10 ppm (T1) and 20 ppm (T2) of nTiO2]?
Moreover, how many times they added them?
(13) How much plants were taken for different analysis should be mentioned clearly.
Results:
(14) Results section needs improvement, some sentences meaning not clear.
Discussion:
(15) I think that this part should be completed. The authors make no attempt to explain the mechanism of action of salt stress as well as different levels of nanotitanium (nTiO2). Why and how salt stress increases or decreases a parameter and why and how different levels of nanotitanium (nTiO2) increases or decreases a parameter……what are the possible mechanisms, should be mentioned in the discussion part.
(16) Throughout the manuscript, there is also a lack of indication of what is innovative in this paper and what the authors have contributed to the current state of knowledge.
Conclusion:
(17) The conclusion section must be rewritten. Authors should include specific results of their research, which extend the current state of knowledge.
References
(18) References: need to be cross-checked.
Linguistic quality:
(19) English should be polished.
Moderate editing of English language required
Author Response
Title:
(1) The MS title should be improved because “Morphophysiological Parameters” is rather general and does not give information on the specific aspects of your work. Please do not use abbreviation (TiO2) in the title.
Answer: New Title: Impact of Titanium Oxide Nanoparticles on Growth, pigment content, membrane stability, DNA damage and stress-related gene expression in Vicia faba under saline conditions
Abstract:
(3) The abstract must be clear and concise. The experimental design and the treatments is not clearly revealed in the abstract.
I would advise the authors to rewrite the abstract part focusing primarily on the foundation of the experiments they have undertaken.
Answer: We improved the Abstract.
«This study investigates the effects of nanotitanium dioxide (nTiO2) on Vicia faba exposed to salinity stress. Plants were treated with either 10 or 20 ppm nTiO2 and exposed to two salinity concentrations (100 and 200 mM NaCl). Salinity led to reduced dry weight, increased electron leakage and MDA content, increased chromosomal aberrations and DNA damage and decreased transcript levels of some stress- and growth-related genes. nTiO2 treatment enhanced dry weight in unstressed plants and mitigated the salinity-damaging effect in stressed plants. nTiO2 application improved cell division, decreased chromosomal aberrations, and reduced DNA damage in plants under saline conditions. The upregulation of antioxidant genes further supports the protective role of nTiO2 against oxidative stress. Particularly significant was the nTiO2 ability to enhance the upregulation of heat shock proteins and growth-related genes. These findings underscore the potential of nTiO2 to reduce the osmotic and toxic effects of salinity-induced stress in plants.»
(4) In L 16: Authors said that “Stressed plants showed a reduction in plant growth parameters and photosynthetic activity”.
Actually, I wonder because authors did not measure any photosynthetic activity parameters like Gas Exchange Capacity (net photosynthetic rate, stomatal conductance, and transpiration rate) and Chlorophyll Fluorescence System [the maximum quantum efficiency of PSII photochemistry (Fv/Fm), electron transport rate (ETR), actual photochemical efficiency of PSII (ΦPSII), photochemical quenching coefficient (qP), effective quantum yield of PSII photochemistry (Fv′/Fm'), and nonphotochemical quenching coefficients (qN)].
Moreover, they did not measure all the plant growth parameters, only a few of them.
I would advise the authors to rewrite the abstract part focusing on the actually results they have obtained.
Answer: We are grateful to the reviewer for the remark. We improved the Abstract.
(5) Abbreviations should be defined in first mention, please revise this issue in the whole MS!
Answer: This has been done.
Keywords:
(6) Do not use abbreviations.
Answer: This has been done.
Introduction:
(7) There is no information on how Vicia faba responds to salinity.
Answer: We added information about it.
(8) Please illustrate by more details the impact of using TiO2 Nanoparticles on the measured parameters under salt stress. Please incorporate recent references.
Answer: We appreciate the reviewer's comment.
In response, we have made minor adjustments to the structure of the manuscript, and as a result, this information has been incorporated into the discussion section.
We trust that the reviewer will find our response satisfactory.
(9) The introduction is written chaotically. There is no hypothesis or purpose of the study.
Answer: We improved the introduction section.
(10) At the end of this section, Please describe the novelty of your work in comparison with previous work. What has it added that we did not know before?
Answer: We improved the introduction section.
Material and methods:
(11) In L 86: Authors said that “Seeds were procured from the Rice Research and Training Center (RRTC) at Kafr El Sheikh-Egypt”.
I am wondering how authors obtained Vicia faba seeds from Rice Center?
Answer:
It was a mistake, and it was corrected in the word file (Agriculture Research Centre (ARC), Giza, Egypt)
(12) Why authors chose these specific treatments [two concentrations of nanotitanium (nTiO2) [10 ppm (T1) and 20 ppm (T2) of nTiO2]?
Moreover, how many times they added them?
Answer: Specific treatments were chosen depending on the preexperiment and published references.
Khan, M.N. Nanotitanium dioxide (nano-TiO2) mitigates NaCl stress by enhancing antioxidative enzymes and accumulation of compatible solutes in tomato (Lycopersicon esculentum Mill.). Journal of Plant Sciences 2016, 11, 1-11. http://dio.org/10.3923/jps.2016.1.11
We added information to “2.1. Experimental conditions and treatments» section: «Thus, salt and nanoparticles were proceeding simultaneously with irrigation every three days, starting from the 10th day of vegetation; as a result, 7 treatments were made».
(13) How much plants were taken for different analysis should be mentioned clearly
Answer: We improved section 2.10. Statistisc
Each plant leaf of the middle tier fixed in liquid nitrogen was treated as a biological replicate; thus, three biological replicates were performed to determine dry weight, membrane stability, photosynthetic pigment content, comet assay, gene expression and total soluble protein (TSP). For each of these experiments, at least three parallel independent measurements were taken. The significance of differences between groups was calculated using one-way analysis of variance (ANOVA) followed by Duncan’s method using Statistical Package for Social Sciences (SPSS) software. Letters indicate significant differences between variants (p < 0.05) unless otherwise specified. Data are given as arithmetic means ± standard errors.
Results:
(14) Results section needs improvement, some sentences meaning not clear.
Answer:
We revised the Results section.
Discussion:
(15) I think that this part should be completed. The authors make no attempt to explain the mechanism of action of salt stress as well as different levels of nanotitanium (nTiO2). Why and how salt stress increases or decreases a parameter and why and how different levels of nanotitanium (nTiO2) increases or decreases a parameter……what are the possible mechanisms, should be mentioned in the discussion part.
Answer:
We added information to the Discussion section: “It is possible that at certain concentrations, nTiO2 might help plants cope with salt stress through various mechanisms, such as enhancing antioxidant activity or improving nutrient uptake. The interplay between salt stress and nTiO2 might lead to changes in the activity of enzymes such as superoxide dismutase, catalase, and peroxidase.
Nanoparticles such as nTiO2 might have the potential to bind to certain toxic ions, either mitigating or exacerbating their effects. It would be worth exploring whether nTiO2 plays any role in sodium or chloride ion dynamics within plants under salt stress.
It is also conceivable that the combined effects of salt stress and nTiO2 might be antagonistic. These antagonistic effects could be influenced by both the concentration and size of the nanoparticles. While nanoparticles may offer promise in shielding plants from the osmotic and toxic effects of salinity, their potential is not boundless. Beyond a certain threshold, the nanoparticles could become toxic themselves.»
(16) Throughout the manuscript, there is also a lack of indication of what is innovative in this paper and what the authors have contributed to the current state of knowledge.
Answer: We added this information to the MS.
The novelty of this research lies in its holistic approach, which provides an in-depth analysis of the protective effects of titanium oxide nanoparticles on V. faba plants. These insights pave the way for a more profound understanding of nanoparticle effects, which is crucial for uncovering the mechanisms of nanoparticle action in the future.
Conclusion:
(17) The conclusion section must be rewritten. Authors should include specific results of their research, which extend the current state of knowledge.
Answer: We improved the Conclusion.
«This study underscores the potential of nTiO2 to enhance plant performance under saline conditions. Cytological and comet assay analyses confirmed a mitigation in the adverse effects of salinity on chromosome abnormalities and DNA damage when cotreated with nTiO2. The nanoparticles' influence was reflected in a decline in MDA content and in dampening the effects of salinity. This attenuation was evident in the efflux of electrolytes, shifts in the concentration of photosynthetic pigments, alterations in the ratios of water to dry matter, and the clear differentiation of bands for total soluble proteins. Moreover, nTiO2 markedly modulated the expression of antioxidant enzyme-encoding genes (Fe-SOD, Cu/Zn-SOD, GR, and CAT), chaperone heat shock protein genes (HSP17.9, HSP70), and the PSII-D protein-encoding gene. This modulation is instrumental in bol-stering plant resistance to the toxic and osmotic stresses induced by salinity.”
References
(18) References: need to be cross-checked.
Answer:
Done, and it was corrected.
Linguistic quality:
(19) English should be polished.
Comments on the Quality of English Language Moderate editing of English language required
Answer:
We improved the quality of English language.
Reviewer 3 Report
In this manuscript (horticulturae-2572320) entitled ‘Combined and Separate Effects of TiO2 Nanoparticles and Salinity on Morphophysiological Parameters, Pigment Content, and Expression of Stress-Related Genes in Vicia faba’ submitted to Horticulturae, Samar Omar and colleagues employed multiple approaches to characterize the effects of TiO2 in alleviating the effects of salinity on Vicia faba L. plants. This research is interesting and complete, and I have some minor concerns to be addressed to improve the quality of this manuscript.
1. For Figure 4, a protein with size about 55KD was specifically enriched in the samples of control, T1, T2, S1T1 and S2T1, which should be carefully discussed in the revised manuscript.
2. For Figure 5 and Figure 6A, scale bar should be included in the revision. In addition, what does the arrow indicate in the Figure 5, please introduce in the legend.
3. For Figure 6, where is the summarizing title of legend? In addition, how could four cells be arranged in a line in the panel d in the Figure 6A.
4. Full names of abbreviations SOD, CAT, GR, HSP70, HSP17.9, PSII-D1 and GPRP presented in manuscript should be described at the place where they first appeared.
Moderate editing of English language required
Author Response
- For Figure 4, a protein with size approximately 55KD was specifically enriched in the samples of control, T1, T2, S1T1 and S2T1, which should be carefully discussed in the revised manuscript.
Answer: We added information to the MS: “A protein with a size of approximately 55 kDa was specifically enriched in the control, T1, T2, S1T1 and S2T1 samples (Figure 4). We suppose that at higher NaCl concentrations (200 mM), it might induce a stronger stress response compared to control or nTiO2 concentrations. Moreover, it cannot be ruled out that the nanoparticles themselves may also influence the accumulation of this protein, which is quite intriguing. This stress can lead to changes in protein synthesis and degradation, resulting in altered levels of specific proteins.”
- For Figure 5 and Figure 6A, scale bar should be included in the revision. In addition, what does the arrow indicate in Figure 5, please introduce in the legend.
Answer:
A scale bar was added to both figures.
Arrows indicate induced or lost bands. introduced in the legend
- For Figure 6, where is the summarizing title of legend? In addition, how could four cells be arranged in a line in the panel d in Figure 6A.
Answer:
A summary of the title of the legend was added.
Figure. 6. DNA genotoxicity of leaf nuclei from V. faba under different experimental treatments. [A]: Representative images of the plant cells assayed by the comet assay where a: control; b: salinity 50 mM (S1); c: salinity 100 mM (S2); d: nTiO2 10 ppm (T1); e: nTiO2 20 ppm (T2); f: salinity 100+ nTiO2 10 ppm (S1N1); g: salinity 200; nTiO2 10 ppm (S2N1); h: salinity100+ nTiO2 10 ppm (S2N1); i: salinity 200, nTiO2 20 ppm (S2N2). [B] Percentage of tailed nuclei of examined seedlings under all experimental conditions. [C] Mean tail length (µm). Slides were examined with a magnification of 40X and with an excitation filter of 174 420-490 nm. Values with different letters are significantly different at p <0.05.
It is what we got in the microscopic field, and arranged nuclei were found in different panels in the same figure i,e …. (g, I). This could be due to migration under electrophoretic conditions.
- Full names of abbreviations SOD, CAT, GR, HSP70, HSP17.9, PSII-D1 and GPRP presented in manuscript should be described at the place where they first appeared.
Answer: It is done
Comments on the Quality of English Language Moderate editing of English language required
Answer:
We improved the quality of English language.
Reviewer 4 Report
The study in the submitted article aimed to investigate the protective role of nTiO2 against the hazardous effects of salinity stress considering cytological and molecular changes. One of the objectives of the study was the assessment of changes in cell division, chromosomal aberrations, DNA damage and associated changes in the expression pattern of some stress- and growth-related genes. Egypt is a major faba bean producer, and it is mainly used due to its high protein content. This vegetable species is a sensitive crop to salinity, with a reduction of yield of up to 50% in stress conditions. Genotypes used in the experiment are salinity-sensitive, and the results of this study will enable faba bean producers to overcome the problem of growing faba beans on saline soils around the world.
The introduction explains very well why this experiment was necessary to be carried out. The hypotheses are well established. Everything is clearly described. The material and methods are described in great detail. Everything is clear and sufficient information is provided about the setting of the experiment.
The results are clearly displayed. The results have been well analyzed. In the discussion, the authors link to each segment of their experiment and supplement their findings with arguments. As the authors stated, their results are the first record of the role of NPs in stimulating the expression of GPRP and the use of the comet assay technique to assess the recovery effects of nanomaterial application on salinity stress.
The cited references are relevant to the experiment. Out of 70 cited references 35 were published within 7 last years (average within 6.8 years). Only 3 of 70 are auto citations, and they are relevant to the subject.
Specific comments:
Line 49: "comes in third place after soybeans" - please cite the source. Different information is shown in FAOstat. According to this data, soybeans are in the first place, but then there are beans, which you did not mention, chickpeas, etc.
Line 53: "the largest rhythms of consumption" - perhaps another term should be used, e.g. high level instead of rhythms.
Line 568: Please correct "upregulatregulated"..
Perhaps in the conclusion or discussion, one sentence could be written about the economic profitability of using NPs, whether it is expensive or not, and when it should be used.
Author Response
Line 49: "comes in third place after soybeans" - please cite the source. Different information is shown in FAOstat. According to these data,soybeans are in the first place, but then there are beans, which you did not mention, chickpeas, etc.
Answer: We have deleted this information.
Line 53: "the largest rhythms of consumption" - perhaps another term should be used, e.g. high level instead of rhythms.
Answer: We have deleted this information.
Line 568: Please correct "upregulatregulated"..
Answer: It is done.
Perhaps in the conclusion or discussion, one sentence could be written about the economic profitability of using NPs, whether it is expensive or not, and when it should be used.
Answer:
We added the following text: «In the broader context, the economic viability of employing NPs must be addressed, while the initial costs may be higher, the long-term benefits to plant health and yield could justify the investment, particularly in regions where salinity is a persistent challenge.»
Round 2
Reviewer 2 Report
Manuscript was improved but English language should be polished
Extensive editing of English language required
Author Response
We greatly appreciate the reviewer's meticulous attention to our manuscript. The quality of the English has been enhanced, with a native English-speaking academic reviewing the manuscript. Additionally, the text was assessed by the American Journal Experts service.
