Overexpression of CsGSH2 Alleviates Propamocarb Residues and Phytotoxicity in Cucumber by Enhancing Antioxidant and Glutathione Detoxification Properties

Round 1

Reviewer 1 Report

L 37. Change “Drug” for another word as pesticide or nematicide

 

L108-112. At the final of the introduction, the objectives and hypothesis are not clear

 

L75-100. This part of the introduction is a bit heavy to read. It contains too much data about the functions and interactions between the different molecules. I recommend reducing the amount of information so that a non-specialist audience in the area can understand it.

 

Table 1. Column “Numbers” is not clear what means

 

L445-446. Indicate that it is detoxification of propamocarb hypothetical model

Author Response

Please see the attachment.Thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors,

My comments about your manuscript are:

Lane 121. 20 mL of propamocarb was applied on leaves?

Lane 136. Please, a representative chromatogram could be included in supplemental materials.

Lane 144-146. Are the methods for H2O2 and O2- correctly indicated?

Lane 153-154, Please list in supplemental materials the forward- and reverse-primers used.

Lane 161-167. What is the purpose to study the subcellular localization ofCsGSH2?

Lane 195: what is the buffer solution used?

Lane 218-219. Tukey’ test

Lane 234-239: The authors must explain the results of figure 1. How in spite of the differences in residues accumulation of propamocarb in M729 and Y3F604, there were not observed significant differences in MDA, H2O2 and O2- content?

Lane 252. Table 1. Please comment about the other gene with high expression. GST and RDR. Why were these genes not choosing for this work?

Figure 2B. How is interpreted the expression pattern of GSH2 in both cultivars? What is the correlation with the figure 1A? The authors carry out a western blot analysis? There is previous work that explain the stability of GSH2 protein to understand the accumulation pattern of residues observed in figure 1.

 

Figure 3. One description of each panel of figure 3 must be carried out.

Lane 270 : cytosolic protein

In the analysis of gene sequence, the authors identified peptide signal for cytosolic localization?

Lane 290-291. The description is not observed in figure S2.

Lane 300-301. The description of Figure 5D is not correct.

Lane 319: Fig. 6A?

Lane 339: Please, change figure 7A for 6A.

Lane 333 - 335: How were calculated the means? It is not clear.

Figure 7: Figure E is F.

Lane 355. The conversion of GSH to GSSG was enhanced.....?

Lane 367-368. No significant was observed between .....(Fig 8A-B). Please improve the description.

Discussion:

Propamocarb is a pesticide or bactericide?

Please indicate the name of genes: HMGB, MCF, ABCA.

Lane 388-390. .....the molecular mechanisms regulating propamocarb residues in cucumber by glutathione.

Lane 395. Expression and localization reveal the gene function?

Lane 429-430 ....GSH independent reactions for propamocarb detoxification,...?

Lane 442. Not Only POD and GST, also other enzymes.

Figure 9. The proposed model is not supported by the findings of the present research. The authors must include the antioxidant enzymes controlling ROS levels

The authors must discuss their results and not include a lot of previous results.

Lane 475: There was not reduction of MDA and ROS (Figure 1B,C and D)

 

With kind regards 

Author Response

Please see the attachment. Thank you.

Author Response File: Author Response.pdf

Reviewer 3 Report

Propamocarb is widely used as a pesticide/fungicide to control downy mildew. Excessive use of propamocarb causes toxicity in the plant. In this study, the authors investigated the endogenous genetic mechanism involved in propamocarb detoxification in cucumbers. The study suggests that six genes in the glutathione signaling pathway were involved in propamocarb detoxification. Among the six genes, Csa1M571280 (CsGSH2) expression was rapidly increased from 6 to 24 hours in both cucumber cultivars Y3F604 (low 106 propamocarb residues) and M729 (high propamocarb residues). Functional analysis further revealed that CsGSH2 overexpressing lines accumulate less propamocarb compared to the CsGSH2 antisense plants. In addition, the study also revealed an enhanced antioxidant system including glutathione, glutathione peroxidase, glutathione-S-transferase, glutathione reductase, glutathione peroxidase in CsGSH2-overexpressing lineages that could potentially be involved in propamocarb detoxification. The enzymatic antioxidants SOD, POD, CAT, and APX also increased in the CsGSH2-overexpressing lines. Finally, the study suggests the role of CsGSH2 in the detoxification of residual propamocarb in cucumber. The results reported here are very interesting. Study design and methodology are appropriate. English is fine. The manuscript needs revision. I suggest the authors to address the following points and improve the manuscript further.

 

 

1.        In the Materials and Methods section under subsection 2.1. Plants and Treatments, page 3 and lines 119-124, the paragraph is not clearly mentioned. The authors performed several experiments and also studied various parameters including glutathione cycle enzymes (GSH, GPX, GST, GR, etc.), antioxidant enzymes (SOD, POD, CAT, APX, DHAR, MDHAR, etc.), ROS generation, MDA salary, etc. The authors need to revise the paragraph and also properly rewrite the sentence detailing the experimental design and study endpoints.

 

2.        The significant levels a or b shown in the all graphic figures are not clear. Include the details (a or b) in 2.9. Statistical analysis section.

 

3.        Page 4; line 190-193, In the methodology section author described “histochemical staining of O2- and H2O2 was performed using nitroblue tetrazo-191 lium (NBT) and DAB, respectively, following Gong et al. (2014).” However, histochemical visualization data were missing in the main results section also in the supplementary section. Please include.

 

4.        What protocol was used to quantify MDA levels in plant tissues in this study? The authors used both methods described in subsections (2.3. Lipid peroxidation and ROS levels and 2.7. Determination of ROS and MDA levels).

 

5.        Page 13; the significant level (a, b, c or b) shown in the Table 2 are not clear. Please include details (a, b, c or b) in legend section or in the Statistical analysis section.

 

6.        Please consistent the vertical x-axis title (ex. nmol g-1) in Figure 8, also in the Figure 1

 

7.        The formats of the reference citations in the text and reference part are in two different styles. Make it consistent with the current journal style.

Author Response

Please see the attachment.Thank you.

Author Response File: Author Response.pdf