Water-Soluble Carbon Nanoparticles Improve Seed Germination and Post-Germination Growth of Lettuce under Salinity Stress

Round 1

Reviewer 1 Report

The paper subject is quite studied. There are many scientific findings on the influence of carbon nanoparticles on seed germination, but the experiment is very well thought out and elaborated so that the results presented could be important for the scientific community. 

Twenty-seven lettuce varieties have been used in this experiment. Authors show that water soluble carbon nanoparticles can significantly promote seed germination without affecting seedling growth. This study indicate that water soluble carbon nanoparticles can significantly improve lettuce seed germination under salinity stress, which provide fundamental evidences on the potential of nanoparticles in agricultural application to improve crop yield and quality under stressful conditions.

The paper is well written.

The plagiarism report was 3% for the paper without references.

I recommend accept the paper in present form.

Author Response

Reviewer 1

 

The paper subject is quite studied. There are many scientific findings on the influence of carbon nanoparticles on seed germination, but the experiment is very well thought out and elaborated so that the results presented could be important for the scientific community. 

Twenty-seven lettuce varieties have been used in this experiment. Authors show that water soluble carbon nanoparticles can significantly promote seed germination without affecting seedling growth. This study indicate that water soluble carbon nanoparticles can significantly improve lettuce seed germination under salinity stress, which provide fundamental evidences on the potential of nanoparticles in agricultural application to improve crop yield and quality under stressful conditions.

The paper is well written.

The plagiarism report was 3% for the paper without references.

I recommend accept the paper in present form.

 

To Reviewer 1:

Response:

We appreciate the reviewer’s positive comment on our research findings. I agree with this reviewer’s comment on some studies of the application of nanoparticles to different plant species, however, with the rapid advancement of techniques for manufacturing nanomaterials, some unique characteristics of these nanoparticles could exhibit distinct effects on promoting plant growth. Therefore, examining the beneficial effect of these novel nanoparticles on plant growth may promote their utilization in promoting crop production and reducing the management cost.  Again, I thank this reviewer for recognizing the merit of our work.

 

Reviewer 2 Report

The manuscript ‘Water-Soluble Carbon Nanoparticles Improve Lettuce Seed Germination under Salinity Stress’ by Hanna Baz, Mathew Creech, Jianjun Chen, Haijun Gong, Kent Bradford and Heqiang Huo, is a well-written study containing clear results with both scientific value and above all practical importance. The authors examined the possibility of improving the germination and growth of lettuce (Lactuca sativa) seedlings under conditions of salt stress using carbon nanoparticles (nanocarbon). Salt stress is one of the most common types of environmental stress in crop plants, and thus all attempts to develop effective and inexpensive methods to eliminate its harmful effects are of agricultural importance. The choice of lettuce as an experimental organism is also appropriate, due to the large acreage of this crop around the world. Lettuce is grown in the ground and under cover, so that in many countries it is commercially available throughout the year. Moreover, it is a popular vegetable among consumers due to its nutritional, sensory and dietary value. The experimental design and well-chosen classical methods for determining the biological properties of seeds provided valuable information about the protective potential of carbon nanoparticles against salt and heat stress in various lettuce varieties.

 

Specific comments:

 

In addition to the process of lettuce seed germination, the authors also analysed the biometric and biochemical parameters of seedlings, so I suggest including this aspect of the research in the title. In addition, it seems to me that the title should take into account the fact that the study investigated not only the effect of carbon nanoparticles on tolerance to salinity, but also on heat tolerance.

 

Line 81 - State the composition of Plant Preservative Mixture, or just the name and content of the disinfectant compound.

Line 93 - Is the Vulpes Corp nanocarbon preparation used in this study a commercial product, and if so, what is its use? What is the shape of the carbon nanoparticles? This information is very important, because the morphology of nanoparticles has a decisive effect on their bioavailability and potential biological effects.

In section 2.4. ‘Thermoinhibition Tests of CNP-Treated Seeds’, please state after what time seed germination was counted.

Line 118 - In the Methods section, the authors state that they determined the length of the stems, but this parameter is not included in the results presented in Table 2.

Line 266 – ‘In addition, CNP pretreatment also promoted leaf elongation and number of Little Gem seeds’. Table 2 does not show the number of seeds!

Line 300 – In my opinion, the sentence is missing the words ‘ion toxicity’.

 

Why the authors used the Porra et al. (1989) method for determination of chlorophyll content and not the classic method according to Arnon (1949) or Lichtenthalar and Wellbourn (1983)?

Improve and complete the method for determination of chlorophylls content:

- please write in detail how extracts for chlorophyll determination were prepared

(were the discs cut from the leaves?, was the plant material ground ?, were the whole 21 days old seedlings used for making the extracts or only the leaf rosette?)

-it seems to me that the authors should correct the volume of solvent (800 µL?), which they used to prepare the extract of nine 21 days old seedlings!

-why did the authors use the equation for the determinations of chlorophylls in N, N'-dimethylformamide (DMF) instead in methanol, since they used methanol to extract the chlorophylls?

In which units the chlorophyll concentrations are expressed in Table 3?

Present all charts in the same graphic form (chart 4 is different from the others)

It seems to me that the statistical significance of results was not marked on some charts.

In the Discussion, where the authors analyse their own results, they cite examples in which carbon nanoparticles induced opposite biological effects (stimulation or inhibition of root growth) in various plant species or varieties. In my opinion, the authors should clarify whether the cited authors used the same concentrations of the same nanoamaterial and applied it in the same manner, so that the physiological effect can be recognized as dependent on the plant species alone. Previous research results indicate that the nature of the effect of nanomaterials also depends on other factors, such as their concentration, size, physicochemical properties, degree of dispersion in the dispersion medium, or type of coating substance. Please consider this in the discussion.

What do the authors think may be the reason that carbon nanoparticles have stronger protective properties against salt stress than against heat stress?

Author Response

 

To Reviewer 2:

The manuscript ‘Water-Soluble Carbon Nanoparticles Improve Lettuce Seed Germination under Salinity Stress’ by Hanna Baz, Mathew Creech, Jianjun Chen, Haijun Gong, Kent Bradford and Heqiang Huo, is a well-written study containing clear results with both scientific value and above all practical importance. The authors examined the possibility of improving the germination and growth of lettuce (Lactuca sativa) seedlings under conditions of salt stress using carbon nanoparticles (nanocarbon). Salt stress is one of the most common types of environmental stress in crop plants, and thus all attempts to develop effective and inexpensive methods to eliminate its harmful effects are of agricultural importance. The choice of lettuce as an experimental organism is also appropriate, due to the large acreage of this crop around the world. Lettuce is grown in the ground and under cover, so that in many countries it is commercially available throughout the year. Moreover, it is a popular vegetable among consumers due to its nutritional, sensory and dietary value. The experimental design and well-chosen classical methods for determining the biological properties of seeds provided valuable information about the protective potential of carbon nanoparticles against salt and heat stress in various lettuce varieties.

 Response:

We appreciate reviewer’s contribution to providing insightful and scientific-merit-based comments on our manuscript.

Specific comments:

Q:  In addition to the process of lettuce seed germination, the authors also analyzed the biometric and biochemical parameters of seedlings, so I suggest including this aspect of the research in the title. In addition, it seems to me that the title should take into account the fact that the study investigated not only the effect of carbon nanoparticles on tolerance to salinity, but also on heat tolerance.

A: Based on reviewer’s suggestion, we have modified our title to “Water-Soluble Carbon Nanoparticles Improve Seed Germination and Post-Germination Growth of Lettuce under Salinity Stress”

Q: Line 81 - State the composition of Plant Preservative Mixture, or just the name and content of the disinfectant compound.

A: The active ingredients of PPM are 5-chloro-2-methyl- 3(2H)-isothiazolone and 2-methyl-3(2H)- isothiazolone. More information could be found at this website https://www.plantcelltechnology.com/plant-preservative-mixture-ppm-100-ml/

Information has been added into the main text, too.

Q: Line 93 - Is the Vulpes Corp nanocarbon preparation used in this study a commercial product, and if so, what is its use? What is the shape of the carbon nanoparticles? This information is very important, because the morphology of nanoparticles has a decisive effect on their bioavailability and potential biological effects.

A: The Vulpes Corp is a startup company located at St Luis, Missouri, USA. The commercial product of this nanoparticle is named as “Bifunctional Plant Growth Promoter”. The shape of this carbon nanoparticle is spherical (see Figure R1 below), and the features of these particles are listed in Table R1 and Figure R2. These tables and figures were not granted to us to be included in the present manuscript. Therefore, please DO NOT disseminate the following information without the permission of Vulpes Corp and Authors of this manuscript.

 

Table R1. Properties of water-soluble carbon nanoparticles.

	Data	Analytical method or instrument
Name	Bifunctional plant promoter	/
Batch number	A1	/
C	60.39 wt%	Vario MAX cube
H	0.95 wt%	Vario MAX cube
O	37.39 wt%	Vario MAX cube
N	1.27 wt%	Vario MAX cube
Source	Black Diamond Material Science	/
Particle size range	Z-Average 124.7nm*	Zetasizer Nano-ZS
Zeta potential	-52.5mV	Zetasizer Nano-ZS
Specific surface area	13.489m²/g	AUTOSORB-IQ2-MP
Porosity	4.0%	AUTOSORB-IQ2-MP
Content of impurities	No obvious impurity	HITACHI Z-5000 AA
SEM image	Photograph**	HITACHI SU-8010

Figure R1 SEM image of carbon nanoparticles

 

Figure R2: size distribution by number.

 

Q: In section 2.4. ‘Thermoinhibition Tests of CNP-Treated Seeds’, please state after what time seed germination was counted.

A: The seed germination data were collected 9 days after imbibition, and this information has been added into the method section accordingly.

Q: Line 118 - In the Methods section, the authors state that they determined the length of the stems, but this parameter is not included in the results presented in Table 2.

A: We are sorry for this confusion caused by this typo. Here it should be “lengths of leaves”. The two longest leaves were measured for each seedling. The data about leaf length are listed in the Table 2.  We have fixed this typo.

Q: Line 266 – ‘In addition, CNP pretreatment also promoted leaf elongation and number of Little Gem seeds’. Table 2 does not show the number of seeds!

A: We thank this reviewer very much for your careful reading. We rewrite this sentence to make this statement more accurate and clear.

“In addition, CNP pretreatment also promoted leaf elongation and increased leaf number in Little Gem”

Q: Line 300 – In my opinion, the sentence is missing the words ‘ion toxicity’.

A: Revised.  

Q: Why the authors used the Porra et al. (1989) method for determination of chlorophyll content and not the classic method according to Arnon (1949) or Lichtenthalar and Wellbourn (1983)?

A: Several methods have been developed after the one described by Arnon in the middle of last century (Arnon, 1949). Although Arnon’s method have been highly cited and used, the imprecision of this method was fully depicted and discussed by other authors; newer and more precise spectrometric determinations have later been established and published (Jeffrey & Humphrey, 1975; Lichtenthaler, 1987; Porra et al., 1989). Porra et al . (1989, 2002) noticed that the use of Arnon's method leads to substantial and important errors. Thus, the use of Arnon's procedure could lead to flawed conclusions regarding many different plant functional and evolutionary aspects. A detailed discussion could be found in the comments of Eseban et al (2018).  

Another major reason for using Methanol instead of DMF and Acetone is that methanol is compatible with the polystyrene plates for BioTek spectrometer reader, while DMF and Acetone can quickly melt the plate, resulting in the misleading O.D. readings.

References

Arnon DI. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 24: 1–15.

Esteban R, Garcia-Plazaola JI, Hernandez A. and Fernandez-Marin B. 2018. On the recalcitrant use of Arnon’s method for chlorophyll determination. New Phytologist 217:474-476.

Jeffrey SW, Humphrey GF. 1975. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochemie und Physiologie der Pflanzen 167: 191–194.

Lichtenthaler HK. 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology 148: 350–382.

Porra RJ. 2002. The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynthesis Research 73: 149–156.

Porra RJ, Thompson WA, Kriedemann PE. 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta (BBA) – Bioenergetics 975: 384–394

Q: Improve and complete the method for determination of chlorophylls content:

- please write in detail how extracts for chlorophyll determination were prepared

(were the discs cut from the leaves?, was the plant material ground ?, were the whole 21 days old seedlings used for making the extracts or only the leaf rosette?)

A: To determine chlorophyll content, a single leaf from each 21-day-old seedling was weighed and incubated in 800uL of the methanol with 1% HCl solution in a 1.5mL microcentrifuge tube for 24 hours in the dark at 4°C. 200 µl of extraction for each sample was used for chlorophyll determination with a Synergy H1 microplate reader (BioTek Instruments, USA) at the O.D. of 652 and 665 nm with the path length correction. Nine seedlings were applied to each sample per treatment.

Q: -it seems to me that the authors should correct the volume of solvent (800 µL?), which they used to prepare the extract of nine 21 days old seedlings!

A: It is 21 days old seedling, the seedling grew very slow under 150 mM NaCl stress. As shown in the Figure R3, Paris Island seeds were treated with or without CNP, and germinated seeds were placed on ½ MS medium containing 150mM NaCl. The seedlings did not grow fast under salt stress in our tissue culture room.  (Figure R3). Single leaf could be placed into the 1.5ml centrifuge tube.

Figure R3: Growth of Paris Island control (left) and CNP-treated (right) for 21 days on the ½ MS media containing 150mM NaCl.

-why did the authors use the equation for the determinations of chlorophylls in N, N'-dimethylformamide (DMF) instead in methanol, since they used methanol to extract the chlorophylls?

A: I really appreciate this review’s critical and helpful comment. I also apologize for this wrong formula that copied from a combined protocol, which describing the method for DMF, acetone, and methanol methods. We have carefully examined the original readings of Biotek at different O.D. including 647, 664, 652 and 665 nm,  and we used the following equations for recalculating the content of Chla, Chlb and total Chla+Chlb: Chla = (16.29*[OD 665])-(8.54*[OD 652]), Chlb = (30.66*[OD 652])-(13.58*[OD 665]), and Total Chla+Chlb = (22.12*[OD 652] )+(2.71*[OD 665]). The description of this data in Table 3 were also modified in the main text. To double confirm the reliability of our data, we have repeat this experiments with newly grown seedlings, data in the Table 3 has been updated. That is the reason that it took us more than one month to submit our revised manuscript.

Q: In which units the chlorophyll concentrations are expressed in Table 3?

A: We have convert our data to µg/g FW (Fresh Weight)

Q: Present all charts in the same graphic form (chart 4 is different from the others)

A: We have modified the graphic form.

Q: It seems to me that the statistical significance of results was not marked on some charts.

A: We have rechecked all statistical analysis and added labels on Figure 4 and Figure 5.

Q: In the Discussion, where the authors analyse their own results, they cite examples in which carbon nanoparticles induced opposite biological effects (stimulation or inhibition of root growth) in various plant species or varieties. In my opinion, the authors should clarify whether the cited authors used the same concentrations of the same nanoamaterial and applied it in the same manner, so that the physiological effect can be recognized as dependent on the plant species alone. Previous research results indicate that the nature of the effect of nanomaterials also depends on other factors, such as their concentration, size, physicochemical properties, degree of dispersion in the dispersion medium, or type of coating substance. Please consider this in the discussion.

A: Thank you very much for your suggestions, we have modified our discuss and added one sentence-statement as follows
“Additionally, the conflicting results from the same species may be caused by the ways of NP applications (e.g. as supplements of culture media or direct addition into soil or foliar spray etc.) and distinct physicochemical properties of different nanoparticles (e.g. application concentration, particle size, and degree of dispersion etc.) [8,42].”

Q: What do the authors think may be the reason that carbon nanoparticles have stronger protective properties against salt stress than against heat stress?

A: I thank this author for this in-depth question. The research interest in my lab focuses on understanding the effect of abiotic stress (in particular, temperature) on horticultural crop growth. Although this argument is still presumptive, yet the preliminary observation from our tests showed that application of this soluble nanoparticles may greatly alleviate the deleterious effect from drought stress. We also admitted that this preliminary data should be consolidated by minimizing environmental variables such as light etc. Based on this observation, we argue that this CNP may help lettuce combat the high temperature as well since lettuce is quite sensitive to high temperature.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have followed my suggestions and recommendations and cleared up all my doubts regarding their work. I am impressed by their commitment to improving the manuscript. In its current form, I believe it meets all the criteria for publication in the journal Agronomy Basel. As I stated earlier, the research presented by the authors is valuable both for its contribution to scientific knowledge and its practical applications. I wish the authors continued success in producing great scientific ideas and publishing their research results.