Membrane Proteomics to Understand Enhancement Effects of Millimeter-Wave Irradiation on Wheat Root under Flooding Stress
, Kazuna Hamada 1,†, Takashi Furuya 2, Takumi Nishiuchi 3 and Masahiko Tani 2
Round 1
Reviewer 1 Report
In this manuscript, authors aimed to understand the mechanism of flooding stress tolerance in wheat after millimeter-wave irradiation using membrane proteomics. Proteins identified using proteomic analysis were confirmed using immunoblot or polymerase-chain reaction (PCR) analyses. Basically, this manuscript is more like descriptive instead of explanatory analysis. The discrepancy between original gel pictures and main text reduced the credibility of the manuscript.
Major:
1. Table of significantly changed proteins should be included in the main text.
2. Could authors explain why there are two calnexins? In figure 2, only 70 kDa Calnexin was detected. I would assume this membrane protein sample was extracted from unirradiated and non-flooded wheat seedlings according to the manuscript. However, in figure 5, 60 kDa calnexin is the major band under the same treatment condition. Also, the identities of proteins of interest in immunoblotting analysis were only supported by antibody. I would assume an in-gel digestion followed by LC-MS/MS or MALDI-TOF would be good evidence for the protein identification/confirmation.
3. Authors proposed that “MMW irradiation of wheat seed improves root growth through cell-wall construction, unfolding-protein reduction, waste regulation, and ATP-content control in membranes”. It would be great if authors showed pictures and dry weights of root system in each treatment.
4. Figure 2 and S2. V-ATPase was labeled around 250 kDa which is inconsistence with Figure 4B. Figure S6 and Figure 5, the MW of calnexins is around 37 kDa and 60 kDa, respectively. The quality of antibodies used in the research or data interpretation is questionable to me.
Manuscript has been well written.
Author Response
Reviewer 1
In this manuscript, authors aimed to understand the mechanism of flooding stress tolerance in wheat after millimeter-wave irradiation using membrane proteomics. Proteins identified using proteomic analysis were confirmed using immunoblot or polymerase-chain reaction (PCR) analyses. Basically, this manuscript is more like descriptive instead of explanatory analysis. The discrepancy between original gel pictures and main text reduced the credibility of the manuscript.
Answer: We are sorry that we have many problems in this article. And, thank you very much for your critical comments. Based on your comments, this article has been corrected with additional experiments and re-analyses.
Major:
1.Table of significantly changed proteins should be included in the main text.
Answer: As suggested, “List of significantly changed membrane proteins in wheat-root irradiated with MMW compared with unirradiated under flooding stress.” has been included in the main text as new Table 1.
- Could authors explain why there are two calnexins? In figure 2, only 70 kDa Calnexin was detected. I would assume this membrane protein sample was extracted from unirradiated and non-flooded wheat seedlings according to the manuscript. However, in figure 5, 60 kDa calnexin is the major band under the same treatment condition. Also, the identities of proteins of interest in immunoblotting analysis were only supported by antibody. I would assume an in-gel digestion followed by LC-MS/MS or MALDI-TOF would be good evidence for the protein identification/confirmation.
Answer: We are sorry that we made a mistake for the size of calnexins. For calnexin, 60 kDa and 70 kDa are right. Figure 2 and Supplemental Figure 6 have been corrected. The explanation has been added in the section of “3.3. The Role of Endoplasmic-Reticulum Membrane in Wheat, whose Seed Were Irradiated with MMW, under Flooding Stress.” With red color. Additionally, thank you for suggesting the use of LC-MS/MS or MALDI-TOF. However, as you known, although the band on the immune blot is one, there are many proteins on the SDS-PAGE. So, it is very difficult with our technique.
- Authors proposed that “MMW irradiation of wheat seed improves root growth through cell-wall construction, unfolding-protein reduction, waste regulation, and ATP-content control in membranes”. It would be great if authors showed pictures and dry weights of root system in each treatment.
Answer: As suggested, additional experiments have been performed and the picture of root growth has been added as new Figure 8. Total-root weight has been measured with fresh weight, because dry weights were almost “zero” in the case of wheat root.
- Figure 2 and S2. V-ATPase was labeled around 250 kDa which is inconsistence with Figure 4B. Figure S6 and Figure 5, the MW of calnexins is around 37 kDa and 60 kDa, respectively. The quality of antibodies used in the research or data interpretation is questionable to me.
Answer: We are sorry that we have problems Figures and Supplemental Figures in this article. Immunoblots in Figure 2 and Supplemental Figure 2 were used proteins extracted by Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific). However, immunoblots for confirmation experiments in Figure 4B and Supplemental Figure 5 were used proteins extracted by buffer consisting of 50 mM Tris-HCl (pH 6.8), 150 mM NaCl, 1% nonided-P40, 0.5% sodium deoxycholate, 0.1% SDS, and 5% dithiothreitol. So, the native size of V-ATPase was shown in Figure 2 and Supplemental Figure 2; however, these subunits were separated by the high concentration of dithiothreitol in Figure 4B and Supplemental Figure 5. This explanation has been added in the section of results with red color.
For calnexin, 60 kDa and 70 kDa are right. Figure 2 and Supplemental Figure 6 have been corrected. Thank you very much for your comments.
Reviewer 2 Report
The manuscript titled, Membrane Proteomics to Understand Enhancement Effects of 2 Millimeter-Wave Irradiation on Wheat Root under Flooding 3 Stress, needs major revision before it could be accepted for publication.
1. The authors should write the manuscript clearly in order to convey the objectives of the research. The title talks about proteomics, and it should be reiterated in the manuscript. It is ambiguous
2. The introduction should be improved . Strengthen the aim of your study and novelty.
3. Figure 3. Make the figure stand alone. What is increase and decrease?
4. Insert Experimental Desig in the discussiion part to clearly explain your study.
5. Please grammar check and check subsections format
The format of the subsections, the texts title should not be all capitalized
Author Response
Reviewer 2
The manuscript titled, Membrane Proteomics to Understand Enhancement Effects of 2 Millimeter-Wave Irradiation on Wheat Root under Flooding 3 Stress, needs major revision before it could be accepted for publication.
Answer: Thank you very much for your comments and suggestion. Based on reviewers’ suggestion, this article has been improved with additional experiments and analyses.
- The authors should write the manuscript clearly in order to convey the objectives of the research. The title talks about proteomics, and it should be reiterated in the manuscript. It is ambiguous
Answer: Thank you very much for your suggestion. In this revised article, proteomics has been reiterated in the section of results based on suggestion.
- The introduction should be improved . Strengthen the aim of your study and novelty.
Answer: As suggested, the purpose and novelty of this study have been emphasized in the section of Introduction with red color.
- Figure 3. Make the figure stand alone. What is increase and decrease?
Answer: We are sorry for this problem. Legend of Figure 3 has been improved with more information with red color.
- Insert Experimental Desig in the discussiion part to clearly explain your study.
Answer: Experimental design was inserted in the section of results. In the section of discussion, experimental design has been clearly explained with red color.
- Please grammar check and check subsections format
Answer: English grammar has been checked by native American English speakers. Subsection format has been checked based on journal instructions.
Round 2
Reviewer 1 Report
The revised manuscript answered most of my concerns. However, the explanation of different size of V-ATPase in Figure 2 and 4 does not make sense to me. According to the manual of Mem-PER™ Plus Membrane Protein Extraction Kit, cat. 89842 "The cells are first permeabilized with a mild detergent, allowing the release of soluble cytosolic proteins, after which a second detergent solubilizes membrane proteins." I assume the native V-ATPase complex was isolated in a stronger detergent which is able to solubilize membrane proteins. Plus, for a standard SDS-PAGE analysis, protein samples will be heated up in sample buffer which contains SDS and reducing reagent. If those protocols were carried out for sample prep in this study, no matter how the membrane proteins were isolated, samples used to create figure 2 and 4 were boiled in SDS sample buffer. Under such condition, I would expect V-ATPase was denatured. I am not an expert of V-ATPase, so authors please list references to support the claim.
As for gel pictures, figure S5 and S6, the protein ladder labelings are off.
Author Response
Reviewer 1
The revised manuscript answered most of my concerns.
However, the explanation of different size of V-ATPase in Figure 2 and 4 does not make sense to me. According to the manual of Mem-PER™ Plus Membrane Protein Extraction Kit, cat. 89842 "The cells are first permeabilized with a mild detergent, allowing the release of soluble cytosolic proteins, after which a second detergent solubilizes membrane proteins." I assume the native V-ATPase complex was isolated in a stronger detergent which is able to solubilize membrane proteins. Plus, for a standard SDS-PAGE analysis, protein samples will be heated up in sample buffer which contains SDS and reducing reagent. If those protocols were carried out for sample prep in this study, no matter how the membrane proteins were isolated, samples used to create figure 2 and 4 were boiled in SDS sample buffer. Under such condition, I would expect V-ATPase was denatured. I am not an expert of V-ATPase, so authors please list references to support the claim.
Answer: Thank you for your pointing out. You are right. Although we believed that the tonoplast was purified based on proteomic results, the tonoplast is so small amount that it may not be detectable by immunoblot technique using membrane fraction. This description has been deleted because it is not possible to consider the difference in molecular weight at this stage. And also, Figure 1 and Figure S2 have been corrected. However, there is no problem with purification efficiency of the membrane fraction, because H+ATPase was significantly accumulated in the membrane fraction.
As for gel pictures, figure S5 and S6, the protein ladder labelings are off.
Answer: Carefully corrected the position of the markers in figure S5 and figure S6.
Reviewer 2 Report
The manuscript titled Membrane Proteomics… needs more improvement in writing in order to be accepted for publication.
1. Please make a paragraph on Experimental Design because the flow of the writing is vague.
2. What is Table 1? Make this more informative.
3. Add a Conclusion part.
The English is fine but writing needs improvement.
Author Response
Reviewer 2
Comments and Suggestions for Authors
The manuscript titled Membrane Proteomics… needs more improvement in writing in order to be accepted for publication.
- Please make a paragraph on Experimental Design because the flow of the writing is vague.
Answer: Thank you very much for your comments. As suggested, a paragraph of Experimental Design has been added each results section with blue color.
- What is Table 1? Make this more informative.
Answer: Thank you very much for your comments. The information has been added for Table 1 in the section “2.2. Membrane Proteomics of Wheat Irradiated with MMW” with blue color.
- Add a Conclusion part.
Answer: We are sorry that we could not understand your real meaning. “Conclusion” section was written as “5” section based on IJMS instruction. However, we decided that what you meant was to improve this section “5. Conclusion”, so we have improved it with blue color.
Comments on the Quality of English Language
The English is fine but writing needs improvement.
Answer: English has been checked by native American English speakers, again. I am sorry, if possible, could you tell us where to fix it?
Round 3
Reviewer 1 Report
The current version could be accepted depending on the editor's decision.
Reviewer 2 Report
looks good now
