Characterization of Interactions between the Soybean Salt-Stress Responsive Membrane-Intrinsic Proteins GmPIP1 and GmPIP2

Round 1

Reviewer 1 Report

In this study author characterization the interactions between the soybean plasma membrane- intrinsic proteins GmPIP1s and GmPIP2s that responding to salt stress. They also did yeast hybrid, and other scientific methods were engaged in analyzing the interactions of a set of aquaporins, soybean (Glycine max) plasma membrane-intrinsic proteins (GmPIPs), in response to salt stress. The studies showed GmPIP1;5 and GmPIP1;6 formed hetero-tetramers with GmPIP2;4, GmPIP2;6, GmPIP2;8, GmPIP2;9, GmPIP2;11, and GmPIP2;13. They also detected interactions between GmPIP1;6 and GmPIP1;7, but not between GmPIP1;6 and GmPIP1;5. Furthermore, GmPIP2;9 formed homo-tetramers, and this interaction was strengthened under salt and osmotic stress. Expression analysis indicated complex and unique responses to salt stress depending on the duration of the stress. The manuscript is very well written and cover all aspect of experiments. However, for the manuscript's betterment, I would like to make few suggestions to the authors.

It would be better if the author tries to functionally characterize at least one candidate gene for natural salt stress tolerance in soybean or any other plant.

Change at

L107 were ligased using to were ligated using.

L113 two hybrid to two-hybrid.

121 Mannitol respectively to mannitol, respectively.

L122 What is OD?

L122 and then 1 ml of OD and 1 ml of the solution was poured in a cuvette to confirm the OD to Then, 1 ml of OD and 1 ml of the solution were poured into a cuvette to confirm the OD.

L142 share high similarity, we found to share a high similarity; we found.

L207 afterwards to afterward.

L237 nearly share the same section to nearly shares the same section.

L296 In this study we identified to In this study, we identified.

I found pleagerism at these lines L213-215, L158-160. Please clean it.

Author Response

Dear reviewer,

Thank you very much for your kind comments and advice.  The manuscript has been revised into a new version. All of your remarks have been concerned, and point-to-point revision was made accordingly in the new version(The track changed version has been attached). 

Best regards,

Authors     

Author Response File: Author Response.pdf

Reviewer 2 Report

Review of the manuscript entitled “Characterization of interactions between the soybean plasma 2 membrane-intrinsic proteins GmPIP1s and GmPIP2s responding to salt stress” by Liu et al. and submitted for publication to “Agronomy”.

Major comments: As described in more detail below, this manuscript suffers from many inconsistencies in its formatting, interpretation of the results, and conclusions. Below are several comments to help the reviewers updating their work. This reviewer believes that this manuscript would require a deeper and more consistent analysis to strengthen the conclusions.

1. In their abstract, the authors mentioned the “vital role of hetero- and homo-tetramers” in conferring salt tolerance to soybean plants. This reviewer believes that this conclusion is not supported by the results. To reach such a conclusion, the author should compare the salt response of mutant and wild-type plants.

2. Could the author provide consistent spacing between words especially when citing references?

3. Lines 40-43: could the author provide consistent format when naming plants and citing the bibliography [e.g., “Arabidopsis thaliana(Quigley et al. 2001)”; vs. “soybean (Glycine max; Zhang et 41 al. 2013)(Zhang et al. 2013)”; vs. “Brassica rapa (Kayum et al. 2017)”; vs. “chickpea (Cicer arietinum L.)(Deokar & Tar'an 2016)”]

4. Lines48 to 67: The authors provide a lot of information in two different paragraphs about the interaction and roles of PIP1 and 2 in transporting water. The information should be reorganized to be presented more logically. As written, the authors mix gene expression analyses and protein-protein interaction analyses.

5. Supplemental Figure 1: the authors mentioned 8 and 14 PIP1 and PIP2 proteins in soybean. Supplemental Figure 1 only highlights eight out of the 22 PIP proteins. Comparative analysis should include all the 22 PIP proteins.

6. Supplemental Figure 1: the authors created a phylogenetic tree using 10 and 14 PIP1 and PIP2 protein sequences. This was unexpected knowing that there are only 8 PIP1 proteins in soybean (see introduction).

7. Line 178: Could the authors update the following information: “(Reference to Table or Figure)”?

8. Lines 190-191: Could the author be more specific regarding the PIP pairs that have stronger interactions upon salt treatment? What were the criteria to reach this conclusion?

9. Fig 4: PIP2-11 seems to be upregulated. Besides, could the authors include the pattern of expression of GmPIP1-7? If this gene is not up-or down-regulated, what is its transcriptional response to drought stress?

10. Discussion: The authors discuss the up-regulation of numerous PIP genes in response to salt; many more than mentioned in the result section. This is a major discrepancy.

Author Response

Dear reviewer,

Thank you very much for your kind comments and advice.  The manuscript has been revised into a new version. All of your remarks have been concerned, and point-to-point revision was made accordingly in the new version(The track changed version has been attached). 

Best regards,

Authors     

Author Response File: Author Response.pdf

Reviewer 3 Report

The article by Liu and co-workers describes the protein-protein interactions and gene expression of PIPs in soybean in response to salt stress.

This is a relevant research topic regarding the negative effects of increasingly important stress in the growth and productivity of soybean plants. PIPs regulate water and solutes flow through membranes and are involved in growth and stress responses in plants. The identification of the  genes encoding PIPs in soybean was previously described and this gene family was also characterized in other species and shown to form homo- and hetero-tetramers. Soybean is a crop with a major agriculture relevance and salt stress is one of the abiotic factors that affect its productivity. In the present paper the authors studied protein interactions by Y2H and gene expression by qPCR. Data regarding sequence analysis is also presented.  

Results presented are valuable not only on a fundamental research point of view but can also be useful to improve the tolerance of crop plants to salt stress. Although the manuscript in relatively well written and describes interesting results, the methods are poorly described and some errors were detected throughout the manuscript. Manuscript organization, Legends, figure presentation and discussion should also be improved before the manuscript is acceptable for publication. My comments are as follows:   

Title: ……the soybean salt-stress responsive membrane-intrinsic proteins GmPIP1a and GMPIP2

Abstract:

Line 23: Yeast-two hybrid

Line 23: “other scientific methods” is too vague. Please specify the methods used.

Line 24: showed that ….

Line 32: improves our understanding

Keywords: gene expression

Materials and Methods:

Line 78: Seeds of soybean…..(Glycine max) from …of our lab were grown…

This information is missing: Pot size? Mr of plants per pot? Soil used? Hydroponics? Photoperiod? Humidity? Light intensity?  Growth chamber? Greenhouse?

Describe the sampling time points: 0h, 2h 12h. It is no clear if controls were collected at the beginning of the experiment of after 12h (no salt). Plant age (is described on results section but should be in M&M)

Line 95: what are the “other samples”: what is CK?

Line 97: was checked using

Line 105: “every gene repeated 3 times” is not clear. Do you mean “every construct was sequenced 3 times”?

Line 107: ligated

Line 109: How were the positive clones “prepared for the following…”

ddH20 was used for yeast….

Correct: 1ml of OD (what OD?) And 1mL of the solution (what solution?)

What program was used to design primers

All the operations were conducted following instructions (manufacturer’s?)       

Statistical analysis:

Only two replicates were used for statistical analysis? In figure 4 legend you say “3 independent experiments with each experiment performed in triplicate” It is not clear.

Please explain in M&M: how many biological samples (individual plants or pooled plants) and mg of tissue were used for RNA extractions. How many technical replicates were made in qPCR.  

Results:

Authors say that “based on phylogenetic analysis we selected some PIP for Y2H assay”. If so, authors should explain why specific PIP were chosen and based on what criteria. In the present state sequence analysis do not seem to be the most relevant result and it could be presented after the experimental results.

Figure Legends are too long and should be shortened. The details regarding yeast growth, OD, dilutions etc should be on M&M.

In fig 1 and 2 the interactions detected should be highlighted (arrow or square) to help the reader to interpret results.

Figure 4: Only one reference gene was used? (in M&M two ref genes were mentioned, please correct)

Different letters indicate….

Since authors observed and discuss similar expression profiles of some groups of genes (e.g. PIP1,5 and PIP1, 6), I suggest to re-organize the figure to help visualize this, by placing the genes with similar expression patters side by side, instead organizing them by nomenclature numbering.

Discussion:

A separate study highlighted….(Afzal et al., 2014). This is a review paper or a results paper?

Authors say that the expression patters under salt stress is partly consistent with sequence similarities and interaction patterns among Gm PIPs. This should be more developed taking into account the results obtained and sequence analysis (including duplication data).

Check the text for duplicated references (e. g. Afzal, Qiang)

Conclusions:

Was not the identification of PIP genes described in a previous paper? If so, this sentence should not be the first conclusion.        

Author Response

Dear reviewer,

Thank you very much for your kind comments and advice.  The manuscript has been revised into a new version. All of your remarks have been concerned, and point-to-point revision was made accordingly in the new version(The track changed version has been attached). 

Best regards,

Authors     

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I don't see any radical revision in this manuscript. Please send reviewer pointwise rebuttal and mark changes in the manuscript.

Author Response

Dear reviewer

Thank you very much for your comments.

Here we submit the new version of our manuscript. In the new version, the introduction, the material and method and the result have been intensively revised.

Here is the point-to-point response for the first-round comments.

L107 were ligased using to were ligated using.  Revised accordingly, please see L124.

L113 two hybrid to two-hybrid. Revised accordingly, please see L127.

121 Mannitol respectively to mannitol, respectively. Revised accordingly, please see L137.

L122 What is OD? OD value is 1.5. See L130-131

L122 and then 1 ml of OD and 1 ml of the solution was poured in a cuvette to confirm the OD to Then, 1 ml of OD and 1 ml of the solution were poured into a cuvette to confirm the OD.

This part is revised, see L130-131

L142 share high similarity, we found to share a high similarity; we found. Revised accordingly, please see L146-147.

L207 afterwards to afterward. This part is deleted in revision.

L237 nearly share the same section to nearly shares the same section. Revised accordingly, please see L263.

L296 In this study we identified to In this study, we identified. This part has been removed in revision, please see L284-291.

I found pleagerism at these lines L213-215, L158-160. Please clean it. They were cleaned off in the new version.

Best wishes

Weicong

Reviewer 2 Report

Dear Authors,

Your manuscript should be carefully edited. Several discrepancies have been identified in this new version of your manuscript (e.g., the number of regulated PIP genes does not match the list provided by the authors). Also, I strongly suggest the authors respond to the reviews through a detailed rebuttal letter rather than a general statement. This is always helpful to reviewers when reading for a second time a research manuscript. As a note, the authors did not update their introduction to clarify the information provided about the role of PIP genes and proteins.

Author Response

Dear reviewer

Thank you very much for your comments.

Here we submit the new version of our manuscript. In the new version, the introduction, the material and method and the result have been intensively revised. The revision removed the discrepancies.

Here is the point-to-point response for the first-round comments.

1. In their abstract, the authors mentioned the “vital role of hetero- and homo-tetramers” in conferring salt tolerance to soybean plants. This reviewer believes that this conclusion is not supported by the results. To reach such a conclusion, the author should compare the salt response of mutant and wild-type plants.

Answer: According to this comment, we have adjust the expression in the manuscript, to highlight our research is focusing on how the salt stress influence GmPIP (expression level and protein interaction, L32-33).

2. Could the author provide consistent spacing between words especially when citing references?

Answer: Yes, in the new version this issue has been fully addressed.

3. Lines 40-43: could the author provide consistent format when naming plants and citing the bibliography [e.g., “Arabidopsis thaliana(Quigley et al. 2001)”; vs. “soybean (Glycine max; Zhang et 41 al. 2013)(Zhang et al. 2013)”; vs. “Brassica rapa (Kayum et al. 2017)”; vs. “chickpea (Cicer arietinum L.)(Deokar & Tar'an 2016)”]

Answer: Yes, this problem has been addressed in the new version.

4. Lines48 to 67: The authors provide a lot of information in two different paragraphs about the interaction and roles of PIP1 and 2 in transporting water. The information should be reorganized to be presented more logically. As written, the authors mix gene expression analyses and protein-protein interaction analyses.

Answer: Yes, toward this issue we have revised the introduction part thoroughly.

5. Supplemental Figure 1: the authors mentioned 8 and 14 PIP1 and PIP2 proteins in soybean. Supplemental Figure 1 only highlights eight out of the 22 PIP proteins. Comparative analysis should include all the 22 PIP proteins.

Answer: In the supplemental figure, we want to demonstrate the structure of the PIPs, so here we use part of the GmPIP to showed the feature. In the new version we upload the sequences of all the GmPIPs as another supplementary file.

6. Supplemental Figure 1: the authors created a phylogenetic tree using 10 and 14 PIP1 and PIP2 protein sequences. This was unexpected knowing that there are only 8 PIP1 proteins in soybean (see introduction).

Answer: Ok, this error has been corrected.

7. Line 178: Could the authors update the following information: “(Reference to Table or Figure)”?

Answer: OK, the information has been updated.

8. Lines 190-191: Could the author be more specific regarding the PIP pairs that have stronger interactions upon salt treatment? What were the criteria to reach this conclusion?

Answer: Yes, in the new version this part has been revised accordingly, as well the figure 2 is updated with modification.

9. Fig 4: PIP2-11 seems to be upregulated. Besides, could the authors include the pattern of expression of GmPIP1-7? If this gene is not up-or down-regulated, what is its transcriptional response to drought stress?

Answer: In the new version this issue has been addressed. PIP2-11 was upregulated and described in the result part; GmPIP1-7 is downregulated under salt stress, but drought stress is not included in the qPCR analysis.

10. Discussion: The authors discuss the up-regulation of numerous PIP genes in response to salt; many more than mentioned in the result section. This is a major discrepancy.

Answer: The discrepancy has been remove in the new version, the result part was revised intensively.

Best wishes

Weicong

Reviewer 3 Report

The article by Liu and co-workers describes the protein-protein interactions and gene expression of PIPs in soybean in response to salt stress.

Although authors did some alterations according to my suggestions there are several points that were not addressed in this new version of the manuscript. The methods, namely plant growth conditions are still poorly described and some information that should be on the M&M sections is presented in figure legends.

In addition to the PDF file with track changes of the alterations, authors should provide a response file to my comments. This should include a response for every comment, whether the suggested alteration was done (indicate line), or if not, authors should justify why they decided not to make the suggested alteration. It is acceptable that some of the suggested alterations are not made but a justification is needed. 

You can find bellow the points that were not altered in this new version of the manuscript, and for which a justification is required:

Title: ……the soybean salt-stress responsive membrane-intrinsic proteins GmPIP1a and GMPIP2

Abstract:

Line 23: Yeast-two hybrid

Line 23: “other scientific methods” is too vague. Please specify the methods used.

Line 24: showed that ….

Keywords: gene expression

Materials and Methods:

Line 78: Seeds of soybean…..(Glycine max) from …of our lab were grown…

This information is missing: Pot size? Mr of plants per pot? Soil used? Hydroponics? Photoperiod? Humidity? Light intensity?  Growth chamber? Greenhouse?

Describe the sampling time points: 0h, 2h 12h. It is no clear if controls were collected at the beginning of the experiment of after 12h (no salt). Plant age (is described on results section but should be in M&M)

Line 95: what are the “other samples”: what is CK?

Line 105: “every gene repeated 3 times” is not clear. Do you mean “every construct was sequenced 3 times”?

Line 109: How were the positive clones “prepared for the following…”

ddH20 was used for yeast….

Correct: 1ml of OD (what OD?) And 1mL of the solution (what solution?)

What program was used to design primers

All the operations were conducted following instructions (manufacturer’s?)       

How many technical replicates were made in qPCR. 

Results:

Authors say that “based on phylogenetic analysis we selected some PIP for Y2H assay”. If so, authors should explain why specific PIP were chosen and based on what criteria. In the present state sequence analysis do not seem to be the most relevant result and it could be presented after the experimental results.

Figure Legends: The details regarding yeast growth, OD, dilutions etc should be on M&M.

Since authors observed and discuss similar expression profiles of some groups of genes (e.g. PIP1,5 and PIP1, 6), I suggest to re-organize the figure to help visualize this, by placing the genes with similar expression patters side by side, instead organizing them by nomenclature numbering.

Discussion:

Authors say that the expression patters under salt stress is partly consistent with sequence similarities and interaction patterns among Gm PIPs. This should be more developed taking into account the results obtained and sequence analysis (including duplication data).

Author Response

Dear reviewer

Thank you very much for your comments.

Here we submit the new version of our manuscript. In the new version, the introduction, the material and method and the result have been intensively revised. The revision removed the discrepancies.

Here is the point-to-point response for the second-round comments.

1. Title: ……the soybean salt-stress responsive membrane-intrinsic proteins GmPIP1a and GMPIP2

Answer: OK, the title has been changed

2. Abstract: Line 23: Yeast-two hybrid

Answer: OK, this part is revised accordingly

Line 23: “other scientific methods” is too vague. Please specify the methods used.

Answer: It has been revised into qPCR.

Line 24: showed that ….

Answer: OK, it is done.

Keywords: gene expression

Answer: OK, it is done.

Materials and Methods:

Line 78: Seeds of soybean…..(Glycine max) from …of our lab were grown…

This information is missing: Pot size? Mr of plants per pot? Soil used? Hydroponics? Photoperiod? Humidity? Light intensity?  Growth chamber? Greenhouse?

Answer: This information is provided in the new version.

Describe the sampling time points: 0h, 2h 12h. It is no clear if controls were collected at the beginning of the experiment of after 12h (no salt). Plant age (is described on results section but should be in M&M)

Answer: This information is presented more clear in the new version.

Line 95: what are the “other samples”: what is CK?

Answer: The sample is root, and CK is 0h.

Line 105: “every gene repeated 3 times” is not clear. Do you mean “every construct was sequenced 3 times”?

Answer: Yes, it is presented in the new version.

Line 109: How were the positive clones “prepared for the following…”

ddH20 was used for yeast….

Correct: 1ml of OD (what OD?) And 1mL of the solution (what solution?)

Answer: This part has been revised thoroughly and the OD value is 1.5 which has been added into the manuscript.

What program was used to design primers

Answer: it is primer 3.

All the operations were conducted following instructions (manufacturer’s?)       

Answer: We have provided the name and the productor of the kit.

How many technical replicates were made in qPCR. 

Answer: There are three technical replicates which has been added into the article.

Results:

Authors say that “based on phylogenetic analysis we selected some PIP for Y2H assay”. If so, authors should explain why specific PIP were chosen and based on what criteria. In the present state sequence analysis do not seem to be the most relevant result and it could be presented after the experimental results.

Answer: How we selected GmPIPs for Y2H assay is depicted in the legend of figure 2.

Figure Legends: The details regarding yeast growth, OD, dilutions etc should be on M&M.

Answer: Yes, the information has been added in the revision.

Since authors observed and discuss similar expression profiles of some groups of genes (e.g. PIP1,5 and PIP1, 6), I suggest to re-organize the figure to help visualize this, by placing the genes with similar expression patters side by side, instead organizing them by nomenclature numbering.

 Answer: The issue raised above has been addressed. Though the column charts order in figure 1 is not adjusted, new declaration has been made to emphasize the differences of the expression pattern among GmPIPs.

Discussion:

Authors say that the expression patters under salt stress is partly consistent with sequence similarities and interaction patterns among Gm PIPs. This should be more developed taking into account the results obtained and sequence analysis (including duplication data).

Answer: in the new version, the discussion concerned the mentioned part above has been revised (L285-294).

Best wishes

Weicong