Review Reports

Round 1

Reviewer 1 Report

There are no lines number so it is difficult to track with manuscript. The page 3, last paragraph, author used RCBD design with no need to block because they used pots so there are no different among pots. Also, the pots were to small to hold three plants.

Author Response

Reviewer 1.

We thank the reviewer for valuable comments.  The following changes were made to the manuscript as recommended by the reviewer.

The randomized complete block design (RCBD) is frequently used in greenhouse experiments where plants are grown in pots.  Although environmental factors are better controlled in greenhouses compared to field experiments there may still be differences in shading or temperature on a large greenhouse bench.  Recent study compared the RCBD and re-randomization in a greenhouse experiment where barley was grown in pots and the RCBD was found to outperform other experimental design of simple re-arrangements of pots.  See Hartung, J., Wagener, J., Ruser, R. et al. Blocking and re-arrangement of pots in greenhouse experiments: which approach is more effective?. Plant Methods 15, 143 (2019). https://doi.org/10.1186/s13007-019-0527-4

The pots used had 5 kg of soil but not 1.2 kg.  This mistake was corrected in the manuscript.

Reviewer 2 Report

Main comments

The authors of the manuscript took up an interesting topic of searching for solutions in the field of soil and environmental protection. The use of growth regulators in the discussed issues seems justified

The topic of the manuscript is very relevant and the manuscript is well-written.

The methodological assumptions and execution of the experiment are accurate and detailed. Clearly and precisely presented results and skillfully initiated discussion. The work is interesting and well prepared.

Comments and suggestions:

The title: GA₃ (subscript) later in the work, apart from the Abstract, it is correct. Switchgrass (Panicum virgatum L.) italic.

Abstract and other chapters: S-nitroso-N-acetylpenicillamine, gibberellic acid, control plants- these phrases with lowercase letters.

Material and Methods: 2.1. Plant Species: first line on page 3- C₄ (subscript) carbon fixation perennial grass

2.6. Plant Growth Conditions: What was the intensity of the artificial light? What fluorescent lamps were used? power in W?

2.8. Trypan Blue Staining of Roots for AMF Assessment:

Roots were stained in 0.05% Trypan blue for 15 minutes at 90˚C, and then de-stained in glycerol acidified with 2.5% HCl (acidic glycerol). redundant expression (acidic glycerol) , the information is in advance.

Author Response

Reviewer 2. 

We thank the reviewer for valuable comments.  The following changes were made to the manuscript as recommended by the reviewer.

The abbreviation GA₃ for gibberellic acid instead of GA3 was changed in the text.  The abbreviation GA (meaning Georgia) was spelled out to avoid any misunderstanding.

Lower case letters were used for chemicals, and this was changed in the text.  Two chemicals (SNAP and GSNO) contain capital letters.  Please see the following examples and references for use.  “…incorporating S-nitroso-N-acetylpenicillamine (SNAP) in a medical grade…”  ACS Appl Mater Interfaces. 2017 May 10; 9(18): 15254–15264.  Published online 2017 Apr 26. doi: 10.1021/acsami.7b01408

“…presence of S-nitrosoglutathione (GSNO) was examined…” Proc Natl Acad Sci U S A. 1996 Dec 10; 93(25): 14428–14433. doi: 10.1073/pnas.93.25.14428

We also changed “Control plants” to “control plants” in the text.

Also, we changed “Trypan blue” to “trypan blue”.

On page 4 we changed C4 changed to C₄.

Information on the artificial light was added to the text: …”cool white fluorescent lamps (10,000 Lux)

In the description on the AMF staining in roots we deleted (acidic glycol) that was mistakenly placed right after HCl.

 

Reviewer 3 Report

The issue of soil pollution by hazardous substances in the soil, their accumulation in plants is an issue that is monitored and evaluated for a long time. The use of plants for phytoremediation is still a topical issue. Of great importance is the use of plants for phytoremediation and for energy purposes, when toxic substances do not enter the food chain. This is important for food safety and quality. The manuscript is therefore interesting and addresses the issue of increasing the intake of risk elements, specifically Pb, using additives. The results are very interesting and stimulating. The manuscript is written relatively carefully, but I have a few remarks and remarks on it. In the abstract I would specify the most advantageous combination of substances and the accumulation of Pb. It may also be appropriate to supplement the values ​​obtained. The introduction is rather more general. The methodology includes a botanical description of plant material. I recommend including it in the introduction. I would add to the methodology the characteristics of the genotype, the source of seeds, etc. In the characteristics of the soil to add the content of other nutrients and pH of the soil. The results are very brief, just one paragraph. Please fill in. The discussion is rather descriptive. I also recommend evaluating my own results and previewing conclusions and statements. The use of literature is mainly older, it can be used, but to such an extent. Graphs are simple. It might be appropriate to supplement the results with correlations of measured values ​​and a variant of the experiment. 

Author Response

Reviewer 3. 

We thank the reviewer for valuable comments.  The following changes were made to the manuscript as recommended by the reviewer.

In the Abstract we outlined the most optimal treatment, and we added the values of Pb concentrations found in the plants.

The plant description was put in the Introduction.

We moved the description of the plant (switchgrass) to the Introduction. 

We also added in the text more details on the plant’s origin and genotype: “The switchgrass cultivar “Alamo” (AP13) originating from Live Oak County Texas is particularly well acclimated for use in Georgia USA [66].  Derived from the “Alamo” cultivar, a high biomass yield isolate, EG 1101 was provide by the University of Georgia and used in this study.”

We added more information on the soil such as soil pH.  We did not measure the nitrogen level in the soil since during the Pb spiking of the soil we used lead nitrate.

We elaborated more on the results and re-wrote most of the Results section.

Also, we performed a regression analysis and added to the Results section.

Reviewer 4 Report

The manuscript "Effects of exogenous application of plant growth regulators (SNP and GA3) on phytoextraction by switchgrass (Panicum virgatum L.) grown in lead (Pb) contaminated soil" by Sigurdur Greipsson, Thomas McElroy, Marina Koether and Adrianne Beavers is an important contribution to phytoremediation research. The manuscript is generally well written. However, there are major and secondary points that need to be improved and updated.

1. Latin names should be written in italics. Applies to the entire content of the manuscript.
2. In the introduction, I suggest adding information on the impact of Pb on the natural environment in order to obtain the full spectrum of this element's activity.
3. "It is estimated that 207,000 Pb contaminated sites, comprising millions of hectares, exist throughout the United States [9]". In the introduction, in addition to the data on Pb pollution in the United States, suggests adding data for the world.
4. Some information from point 2.1 should be included in the introduction, not in the methodology.
5. My major concern is the number of replicate pots. Why in the first variant, only 12 pots were selected in which a relatively small amount of soil can fit. Is it too exaggerated to infer the full range of 12 pots of 3 plants?
5. What did the plants look like on the 135th day of vegetation? Were they not lying? How high were they?
6. Why were there already 40 pots in the second part of the experiment?
7. I believe that the methodology section would be more readable if subsection 2.6 were to be placed after section 2.2.
7. The research results are described too laconically. Please extend this section or combine it with the discussion section.
8. The Conclusions section is missing from the paper. Please supplement.

Author Response

Reviewer 4. 

 We thank the reviewer for valuable comments.  The following changes were made to the manuscript as recommended by the reviewer.

All Latin names in the text were written in italics.

We added in the Introduction information on the impact of Pb on the natural environment.

We already have in the Introduction information on: “Environmental Pb contamination is a recognized global health problem…”.  We did not find a comprehensive review article on global lead soil pollution.  Although, studies on Pb soil pollution have been conduction in various countries it would not represent a global study.  There is certainly a need for such a review paper.

We moved section 2.1. Plant description into the Introduction.

The first experiment used only three replicated pots and we were convinced that we did not have to repeat the experiment with larger numbers of pots since the difference between control plants and plants receiving treatments was enormous and statistically significant (see Figures 1, 2, 3 in manuscript).  Also, since the difference between plants treated with EP vs EPS was hardly noticeable, we would only have expected to get lower standard deviations but not significant differences by using larger number of pots. 

After 135 days of growth the plants showed slight signs of toxicity expressed as yellowing of leaves (chlorosis) and at that time the longest leaf of plants was on average about 90 cm.  We added in the text the information on chlorosis. 

The second experiment was more refined and was performed after the first one.  We used 10 replicated pots for each treatment in order to lower the standard deviation. 

We moved section 2.6 right after section 2.2.

We elaborated more on the results and re-wrote most of the Results section.

We added in the text a new Conclusion section.

Round 2

Reviewer 3 Report

The authors modified the manuscript according to the comments. These comments were accepted or it was explained why some analyzes were not carried out. The level of presentation of results has significantly improved, including the addition of regression analysis. The said manuscript, once accepted, has significant potential for citation. I believe that now the quality of this manuscript is at a level where it will be possible to print it. 

Reviewer 4 Report

Ponownie widzę, że Autorzy nie stosują kursywy, pisząc nazwy łacińskie kursywą (np. w tytule rękopisu), jednak jest to uwaga techniczna, która nie wpływa na jakość rękopisu. Autorzy odpowiedzieli na komentarze i udzielili satysfakcjonujących odpowiedzi, dlatego polecam manuskrypt do publikacji.