Genome-Wide Characterization of Shi-Related Sequence Gene Family and Its Roles in Response to Zn²⁺ Stress in Cucumber

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The title of the manuscript is: Genome-Wide characterization of SRS genes family and its roles in responses to Zn metal stress in Cucumber

 Line 62 and 63: …”In 62 lotus japonicas” Is it Lotus japonicus? and must be in italics,

Line 79: …”is highly is limited in cucumber..” Should It be is highly limited?

I suggest that the authors should emphasize the yield reduction due to the abiotic and biotic stress for example. We know that due to the climate change the crop yield is decreased. In the country or region the authors come from, is cucumber an important crop for economic activity?

Why do the authors mention the importance of Zinc finger proteins in the response of abiotic stress such as drought stress but how they relate the Zinc stress in Cucumber?

Materials and Methods

Line 108 and 109: Please review the sentence.

Do the authors compare with another Cucumis and not also other species such as Arabidopsis, maize?

Results

The footnote of Table 2 could be modified and be more specific.

The authors mentioned about the analyses of the exon-intron pattern that were the same in the 8 genes of Cucumber. However, this part needs more explanation about intron and exon length, GC content for example. I suggest that this information should be added in the manuscript. In Figure 3, the authors mentioned exon-intron structures but not well explained in the Results section.

Do the authors could explain better about the results of  gene structure?

Line 244: Please review it and check the spelling of the words.

I suggest the authors should review the sentences, because they used the word “mental” instead of metal? Please verify all of them.

I also suggest that the quality of Fig 5 must improve.

Line 281 and 282: Please review the sentence.

I suggest that the authors could not use “elevated“ expression, maybe could use higher or great?

I also suggest that the part of Expression pattern of SRS genes could be written in more detail. From the 8 genes which one has the greatest expression in roots, leaves or stems? Not only mention gene by gene.

In general, I suggest to re-written this section because the authors have information that did not show and explain in Discussion section.

Discussion

In this section the authors mentioned about results that they did not mention before. For example, talking about no tandem duplication, IXHG domain, etc. That I did not find in Results section.

Line 349: ….”Interestingly, some genes showed significant changes in their response against Zn2+ treatments in different plants…” Please, could the authors specify what genes they are referring?

Finally, I suggest that this section could improve and explain in more detail the comparison of other studies.

Comments on the Quality of English Language

In general terms, I suggest the authors must edit the English language with an Editing English services.                                                           

There are parts in the manuscript that the authors mentioned mental stress. Could it be metal stress?

Author Response

The title of the manuscript is: Genome-Wide characterization of SRS genes family and its roles in responses to Zn metal stress in Cucumber

 Line 62 and 63: …”In 62 lotus japonicas” Is it Lotus japonicus? and must be in italics,

Response:

We have corrected it in the manuscript.

Line 79: …”is highly is limited in cucumber..” Should It be is highly limited?

Response:

We have corrected it in the manuscript.

I suggest that the authors should emphasize the yield reduction due to the abiotic and biotic stress for example. We know that due to the climate change the crop yield is decreased. In the country or region the authors come from, is cucumber an important crop for economic activity?

Response:

Thank you for your suggestions. The crop yield is important for almost all crops, and abiotic and biotic stress can certainly cause yield reduction in cucumber. However, in this study, we mainly focused on the genome-wide characterization of SRS family and their possible role in Zn²⁺ stress response. Thus, we do not focused on the crop yields in this study.

Why do the authors mention the importance of Zinc finger proteins in the response of abiotic stress such as drought stress but how they relate the Zinc stress in Cucumber?

Response:

Thank you for your comments. SRS proteins have the conserved Zinc finger domain. Thus, we mentioned it in the manuscript, it has no relation to the Zinc stress. However, Zn²⁺ could bind to the conserved domain of SRS proteins.

Materials and Methods

Line 108 and 109: Please review the sentence .

Response:

We have corrected it in the manuscript (L107-108).

Do the authors compare with another Cucumis and not also other species such as Arabidopsis, maize?

Response:

Thank you for your suggestions. We did not compare SRS protein sequences in different varieties of cucumbers. We constructed phylogenetic trees to study the phylogenetic relationships of SRS proteins in the process of species evolution.

Results

The footnote of Table 2 could be modified and be more specific.

Response:

Thank you for your suggestions. It has been modified in the manuscript.

The authors mentioned about the analyses of the exon-intron pattern that were the same in the 8 genes of Cucumber. However, this part needs more explanation about intron and exon length, GC content for example. I suggest that this information should be added in the manuscript. In Figure 3, the authors mentioned exon-intron structures but not well explained in the Results section.

Response:

Thank you for your comments. We have found the exon-intron pattern does not make much sense, and we have corrected it in the manuscript.

Do the authors could explain better about the results of  gene structure?

Response:

Thank you for your suggestions. We found gene structure does not make much sense and there may be written mistakes in the manuscript. We have made a major revision on the protein structure analysis in the manuscript.

Line 244: Please review it and check the spelling of the words.

Response:

We have corrected it in the manuscript.

I suggest the authors should review the sentences, because they used the word “mental” instead of metal? Please verify all of them.

Response:

We have corrected it in the manuscript.

I also suggest that the quality of Fig 5 must improve.

Response:

Thank you for your suggestions. We have improved it in the manuscript.

Line 281 and 282: Please review the sentence.

Response:

We have corrected it in the manuscript.

I suggest that the authors could not use “elevated“ expression, maybe could use higher or great?

Response:

Thank you for your comments. We have corrected it in the manuscript.

I also suggest that the part of Expression pattern of SRS genes could be written in more detail. From the 8 genes which one has the greatest expression in roots, leaves or stems? Not only mention gene by gene.

In general, I suggest to re-written this section because the authors have information that did not show and explain in Discussion section.

Response:

Thank you for your comments. We focused on the expression of each SRS gene under Zn²⁺ stress to see whether they response to the stress. Thus, we can identify the genes that may function in the metal stress response. And we have revised the section in Discussion.

Discussion

In this section the authors mentioned about results that they did not mention before. For example, talking about no tandem duplication, IXHG domain, etc. That I did not find in Results section.

Response:

We noticed the mistakes and have corrected it in the manuscript.

Line 349: ….”Interestingly, some genes showed significant changes in their response against Zn2+ treatments in different plants…” Please, could the authors specify what genes they are referring?

Finally, I suggest that this section could improve and explain in more detail the comparison of other studies.

Response:

Thank you for your comments. We have corrected it in the manuscript (L354-355).

Reviewer 2 Report

Comments and Suggestions for Authors

 

Comments/suggestions:

- Perhaps change the structure of the introduction and put the second paragraph first.

- lines 79: “… response is highly is limited in cucumber”. Erase second is

- line 227: emphasize in the text in which genes they appear, in particular environmental stress related elements (LTR and ERE)

- line 244: „heay mentals pose a threat…“. Do you mean heavy metals ?

- lines 247-249, move to M&M

- line 255 „And the expression of CsSRS3 was not…“. Start the sentence differently

- line 361 „… may function in mental stress tolerance in cucumber“????? What mental stress in a cucumber. This is the second time you have used the term mental

- Expand the conclusion in accordance with the results obtained

-  For this topic, the references cited must be predominantly from the last 5 years. Include references from this period in the Introduction and Discussion chapters, as currently only 7 (17.5%) of the 40 papers cited have been published within the last 5 years.

Comments for author File: Comments.pdf

Author Response

Comments/suggestions:

- Perhaps change the structure of the introduction and put the second paragraph first.

Response:

Thank you for your suggestions. We have re-written this part.

- lines 79: “… response is highly is limited in cucumber”. Erase second is

Response:

We have corrected it in the manuscript.

- line 227: emphasize in the text in which genes they appear, in particular environmental stress related elements (LTR and ERE)

Response:

Thank you for your suggestions. We have corrected it in the manuscript (L234-235).

- line 244: „heay mentals pose a threat…“. Do you mean heavy metals ?

Response:

We have corrected it in the manuscript.

- lines 247-249, move to M&M

Response:

We have corrected it in the manuscript (L150-152).

- line 255 „And the expression of CsSRS3 was not…“. Start the sentence differently

Response:

We have corrected it in the manuscript (L262-263).

- line 361 „… may function in mental stress tolerance in cucumber“????? What mental stress in a cucumber. This is the second time you have used the term mental

Response:

We have corrected it in the manuscript.

- Expand the conclusion in accordance with the results obtained

Response:

We have corrected it in the manuscript (L363-370).

-  For this topic, the references cited must be predominantly from the last 5 years. Include references from this period in the Introduction and Discussion chapters, as currently only 7 (17.5%) of the 40 papers cited have been published within the last 5 years.

Response:

Thank you for your suggestions. We have corrected it in the manuscript.

 

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

 

In this study, the authors identify potential SRS genes in cucumber genome, provide description of the genes/proteins and their phylogenetic relationships, then characterize their expression behaviors and potential molecular function through heterologous expression of two CsSRSs in E.coli. The descriptive part (Figs. 1~4) of the study is very confusing because there are many inconsistencies among different parts of the manuscript and figures. The experimental part (Figs. 5~6) also suffers major issues such as insufficient explanation of the design and lack of proper control, making it difficult for a reader to understand the results properly. Also, this manuscript requires extensive English editing.

Specific comments:

Fig. 1: This panel is not significant enough to be a main figure. Gene locus IDs in Table 1 are sufficient to understand which chromosome they belong to. Fig. 1 does not seem to add anything relevant to the rest of the manuscript. I suggest removing it or moving it to supplement.

The grouping (I~III) is confusing and not consistent throughout the manuscript. l.321-323 does not seem to make sense - according to the phylogenetic analysis in Fig. 2, Group II has only two proteins from cucumber. Group III contains both monocots (Zm, Os) and dicots (At, Sl). l.192 states 35 SRSs, while the next sentence l.193 states 37. Fig. 2 seems to have 35 entries, not 37 as claimed in the legend of Fig. 2 (the species abbreviations need to be clarified in the legend, too).

What are the defining characteristics of the groups I~III? CsSRS8 and CsSRS4 have substitutions in the conserved IGGH domain but that does not seem to have a big effect on the grouping in Fig. 2.

L.183: “except for only the RING domain found in CsSRS8 and CsSRS4 (Fig. S1)”: the sentence is not straightforward and should be rephrased.

l.199-201: It sounds very speculative as it is not clear how the intron size differences are tied to functions of the cucumber SRS genes.

It also claims that “all genes have the same number of motifs” but the other sentence implies CsSRS8 and CsSRS4 do not have IGGH.

Fig. 2~4 can be reorganized to improve clarity of the logic. Fig. 3 says “structure analysis of cucumber SRS genes” but only 3b refers to the gene structure. 3a can be a part of Fig. 2, Fig. 3c can be a part of Fig. S1 where protein motifs are discussed, and Fig. 3b can be combined with Fig. 4 where the gene structure including cis-regulatory elements are examined.

l.243-: From the generic descriptives of the CsSRS genes (Figs 1~4), including cis-regulatory element analysis suggesting that the genes can be involved in many different physiological pathways, it suddenly shifts focus on Zn-tolerance. The transition needs to be explained better. This can be a part of the reorganization mentioned above.

L.246-247: what do you mean by “CsSRS1 cannot be cloned”? If you simply failed to clone the gene, please state as it is – “we could not clone CsSRS1”. If you identify reasons that make the cloning fundamentally impossible, please explain.

The SRS gene expression analysis (Fig. 5), It is not clear what are being compared to generate the “relative” values and evaluate the statistical significance, making it impossible to understand the results. Also, Student's t-test is designed for comparison of two groups and not appropriate for evaluating “the overall significance of the results” involving multiple groups (l.150-151). The number of asterisks in the figure and the legend do not match. The Y axis needs to be clarified as well (is it log2?)

Fig. 6 lacks a control panel where the same dilution is spotted on media without ZnSO4. As such, it cannot be excluded that the observed difference is simply due to difference in the concentration of the starting culture. Also, I’m not sure about the relevance of this experiment to the function of CsSRSs in cucumber. It simply shows that CsSRS overexpression may confer Zn tolerance in E. coli when overexpressed, but nothing about how CsSRSs would function in cucumber. Do CsSRSs contribute to differential expression of Zn stress responsive genes in E.coli?  Do bacterial promoter elements targeted by CsSRSs share the same TF binding elements with plants?

 

Comments on the Quality of English Language

The manuscript needs an extensive and thorough English editing. Below are only a few examples.

 

l.163: “isolated” should be “identified”.

l. 229: “The possible mediated biological process prediction suggested…” this doesn’t make sense and needs correction.

l.232-233: “it is reasonable to predicted that…” this needs correction.

l.244: “Heay mental” à “Heavy metal”

l.250: “it was showed that SRS genes demonstrated…” this needs correction.

l.259, l.355, l.361: mental stress à metal stress

Author Response

In this study, the authors identify potential SRS genes in cucumber genome, provide description of the genes/proteins and their phylogenetic relationships, then characterize their expression behaviors and potential molecular function through heterologous expression of two CsSRSs in E.coli. The descriptive part (Figs. 1~4) of the study is very confusing because there are many inconsistencies among different parts of the manuscript and figures. The experimental part (Figs. 5~6) also suffers major issues such as insufficient explanation of the design and lack of proper control, making it difficult for a reader to understand the results properly. Also, this manuscript requires extensive English editing.

Response:

Thank you for your suggestions and comments, which are very helpful for improving our manuscript. The manuscript has been revised extensively, including the language editing.

Specific comments:

Fig. 1: This panel is not significant enough to be a main figure. Gene locus IDs in Table 1 are sufficient to understand which chromosome they belong to. Fig. 1 does not seem to add anything relevant to the rest of the manuscript. I suggest removing it or moving it to supplement.

Response:

Thank you for your suggestions. Fig. 1 shows clearly and concisely the exact position they are at and the chromosome they belong to, which may be better for readers to understand. And the similar figure used as the main figure has been found in other study, such as research in beans (doi: 10.1007/s10528-021-10108-0).

The grouping (I~III) is confusing and not consistent throughout the manuscript. l.321-323 does not seem to make sense - according to the phylogenetic analysis in Fig. 2, Group II has only two proteins from cucumber. Group III contains both monocots (Zm, Os) and dicots (At, Sl). l.192 states 35 SRSs, while the next sentence l.193 states 37. Fig. 2 seems to have 35 entries, not 37 as claimed in the legend of Fig. 2 (the species abbreviations need to be clarified in the legend, too).

What are the defining characteristics of the groups I~III? CsSRS8 and CsSRS4 have substitutions in the conserved IGGH domain but that does not seem to have a big effect on the grouping in Fig. 2.

Response:

Thank you for your comments. We noticed the mistakes and conducted the protein structure analysis of 8 SRS proteins, and re-constructed the tree with 38 protein sequences. The results showed that the phylogenetic relationships are consistent with the protein structure (L192-211).

L.183: “except for only the RING domain found in CsSRS8 and CsSRS4 (Fig. S1)”: the sentence is not straightforward and should be rephrased.

Response:

We have corrected it in the manuscript (L172-173).

l.199-201: It sounds very speculative as it is not clear how the intron size differences are tied to functions of the cucumber SRS genes.

Response:

We noticed the mistakes and have deleted it in the manuscript.

It also claims that “all genes have the same number of motifs” but the other sentence implies CsSRS8 and CsSRS4 do not have IGGH.

Response:

We noticed the writing mistakes and have corrected it in the manuscript.

Fig. 2~4 can be reorganized to improve clarity of the logic. Fig. 3 says “structure analysis of cucumber SRS genes” but only 3b refers to the gene structure. 3a can be a part of Fig. 2, Fig. 3c can be a part of Fig. S1 where protein motifs are discussed, and Fig. 3b can be combined with Fig. 4 where the gene structure including cis-regulatory elements are examined.

Response:

Thank you for your comments. We redo the figures and re-write the manuscript to make it more logic.

l.243-: From the generic descriptives of the CsSRS genes (Figs 1~4), including cis-regulatory element analysis suggesting that the genes can be involved in many different physiological pathways, it suddenly shifts focus on Zn-tolerance. The transition needs to be explained better. This can be a part of the reorganization mentioned above.

Response:

Thank you for your comments. We have revised it in the manuscript.

L.246-247: what do you mean by “CsSRS1 cannot be cloned”? If you simply failed to clone the gene, please state as it is – “we could not clone CsSRS1”. If you identify reasons that make the cloning fundamentally impossible, please explain.

Response:

We have re-written and explained it in the manuscript. We tried many times using different primers and DNA polymerases, and the sequence of putative CsSRS1 cannot be cloned by PCR. In addition, the expression of CsSRS1 in all samples is almost 35-42 cycles like that of negative control. Thus, CsSRS1 was not included in Zn²⁺ stress response study.

The SRS gene expression analysis (Fig. 5), It is not clear what are being compared to generate the “relative” values and evaluate the statistical significance, making it impossible to understand the results. Also, Student's t-test is designed for comparison of two groups and not appropriate for evaluating “the overall significance of the results” involving multiple groups (l.150-151). The number of asterisks in the figure and the legend do not match. The Y axis needs to be clarified as well (is it log2?)

Response:

Thank you for your comments. Generally, we conducted qRT-PCR to see the expression trend of a gene but not the absolute qPCR to figure outthe exact number of mRNA that are available in tissues. Thus, the expression of a gene may be used as control that compared by all others in the 2^-ΔΔCT method. This is very common in other studies (10.1038/s41467-023-38726-9 and 10.1126/science.adk8838). We used the multiple comparison in this study, and we noticed the writing mistakes and corrected it in the manuscript. The writing mistakes in the legends has been corrected as well.

Fig. 6 lacks a control panel where the same dilution is spotted on media without ZnSO4. As such, it cannot be excluded that the observed difference is simply due to difference in the concentration of the starting culture. Also, I’m not sure about the relevance of this experiment to the function of CsSRSs in cucumber. It simply shows that CsSRS overexpression may confer Zn tolerance in E. coli when overexpressed, but nothing about how CsSRSs would function in cucumber. Do CsSRSs contribute to differential expression of Zn stress responsive genes in E.coli?  Do bacterial promoter elements targeted by CsSRSs share the same TF binding elements with plants?

 Response:

Thank you for your comments. We have added the control panel in Fig. 6. In this experiment, we adjust the concentration of E. coli cells to the same OD₆₀₀ before dilution, so it is not an issue in this study.

Zinc (Zn) plays a structural and/or catalytic role in many biological processe. As for the molecular mechanisms of how CsSRSs enhanced Zn²⁺ stress in E. coli, we do not know. We inspect that Zn²⁺ may bind to the CsSRSs to enhance the tolerance of cells in an unknown mechanism. Although it is not within the scope of this study, it worths more in-depth research in the future.

The manuscript needs an extensive and thorough English editing. Below are only a few examples.

 Response:

Thank you for your comments. We have corrected it in the manuscript.

l.163: “isolated” should be “identified”.

Response:

We have corrected it in the manuscript.

1. 229: “The possible mediated biological process prediction suggested…” this doesn’t make sense and needs correction.

Response:

We have corrected it in the manuscript.

l.232-233: “it is reasonable to predicted that…” this needs correction.

Response:

We have corrected it in the manuscript.

l.244: “Heay mental” à “Heavy metal”

Response:

We have corrected it in the manuscript.

l.250: “it was showed that SRS genes demonstrated…” this needs correction.

Response:

We have corrected it in the manuscript.

l.259, l.355, l.361: mental stress à metal stress

Response:

We have corrected it in the manuscript.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The revision has improved the manuscript by addressing some of the concerns, but there are a few that have not been fully addressed.

> And the similar figure used as the main figure has been found in other study, such as research in beans (doi: 10.1007/s10528-021-10108-0).

In the reference presented, the chromosome distribution is discussed in comparison with other species, providing a meaning to the particular figure. The brief description of Fig 1 in this manuscript seems to lack the merit to display “uneven distribution” of cucumber SRS genes. My suggestion is to elaborate the implication of the figure more, like the reference does.

>revised Fig.2 and Fig. 3

The revisions have improved Figs 2-3 and the writing. Throughout the manuscript, please replace “protein structure analysis” with “protein motif analysis” for accuracy. A “structure analysis” implies 3D conformation of protein folding which is not applicable here.

> Fig. 5

Fig. 5 is still difficult to understand because the analysis procedure is unclear. The previous comment asked for clarification on what served as a control to the treatment in the 2^-ΔΔCT comparison. You described actin as the internal reference gene, but the 2^-ΔΔCT method also requires “control sample vs treatment sample”. The control sample should be clarified in the method or in the legend. For example, in the reference provided (10.1038/s41467-023-38726-9, Fig 5 d-f), it is clear that the wild type serves as the control (1.0) while the expression was normalized by the rice Ubiquitin gene. In this manuscript’s Fig. 5, was the control 0-hour treatment? Why are some fold-change values so low (0.2 or less) even for 0 mmol/L samples? Also, it is not clear how each sample received p-value (for asterisks) in a two-ANOVA test. If a post-hoc method is used after ANOVA to do so, please clarify it too. The error bars need to be explained as well.

> Fig. 6-related

The added control improved the figure. It is clear that heterologous overexpression of CsSRS5 appears to confer Zn tolerance in E.coli, which is bacteria. However, it is still not clear how this result “indicated their roles in Zn2+ stress response as well (l.355)” for plants, including cucumbers, where gene expression regulation by transcription factors would differ from bacteria. An acknowledgement of the gap (i.e. bacteria vs plants) in the discussion would clarify the limitation and future directions.

Author Response

Dear Editor and Reviewers:

Thank you for your comments on our manuscript entitled " Genome-Wide characterization of SRS genes family and its roles in response to Zn²⁺ metal stress in Cucumber " (horticulturae-3202291). Those comments were very helpful for improving our manuscript. We believed we have answered all reviewers’ comments. The main corrections in the paper and the responds to reviewers’ comments are as follows:

 

> And the similar figure used as the main figure has been found in other study, such as research in beans (doi: 10.1007/s10528-021-10108-0).

In the reference presented, the chromosome distribution is discussed in comparison with other species, providing a meaning to the particular figure. The brief description of Fig 1 in this manuscript seems to lack the merit to display “uneven distribution” of cucumber SRS genes. My suggestion is to elaborate the implication of the figure more, like the reference does.

 

Response:

Thank you for your suggestions. We had noticed the writing mistake “uneven distribution” in the first edition of manuscript, which has been corrected in the revised manuscript submitted before. We have added comparison in Discussion (L318 -320) as suggested.

 

>revised Fig.2 and Fig. 3

The revisions have improved Figs 2-3 and the writing. Throughout the manuscript, please replace “protein structure analysis” with “protein motif analysis” for accuracy. A “structure analysis” implies 3D conformation of protein folding which is not applicable here.

Response:

Thank you for your suggestions. We have corrected it in the manuscript.

 

 

 

> Fig. 5

Fig. 5 is still difficult to understand because the analysis procedure is unclear. The previous comment asked for clarification on what served as a control to the treatment in the 2^-ΔΔCT comparison. You described actin as the internal reference gene, but the 2^-ΔΔCT method also requires “control sample vs treatment sample”. The control sample should be clarified in the method or in the legend. For example, in the reference provided (10.1038/s41467-023-38726-9, Fig 5 d-f), it is clear that the wild type serves as the control (1.0) while the expression was normalized by the rice Ubiquitin gene. In this manuscript’s Fig. 5, was the control 0-hour treatment? Why are some fold-change values so low (0.2 or less) even for 0 mmol/L samples? Also, it is not clear how each sample received p-value (for asterisks) in a two-ANOVA test. If a post-hoc method is used after ANOVA to do so, please clarify it too. The error bars need to be explained as well.

 

Response:

Thank you for your comments. We are sorry for not clarifying it clearly in the manuscript. In our research, three technology and biology repetitions were used in qRT-PCR study, so all the samples cannot be included in one 96-cells plate. Thus, an “inter-run” with the same sample and sample gene was running repeatedly in each plate, the Ct value of which was used to count as control (1) in each plate when doing the math. Therefore, you may see values of the 0 mmol/L samples were not one, but it does not change or influence actual folds between any two samples in each bar chart. As for the p-value, we did take a two-ANOVA test, but we failed to open to see the calculating detail both “ordinary two-way ANOVA” and “Dunnett’s multiple comparisons test” were used to do the p-value (see picture below).

The value of “0 mmol treatment” at each time point is used as control for p-value (for asterisks) calculation in each bar chart. As for the error bars is the SD among the values of three biological repeats. We noticed that we did not put these informations in the manuscript and some asterisks were forgotten to add, and we have corrected it in the figure and add the details in the manuscript as required.

 

 

 

> Fig. 6-related

The added control improved the figure. It is clear that heterologous overexpression of CsSRS5 appears to confer Zn tolerance in E.coli, which is bacteria. However, it is still not clear how this result “indicated their roles in Zn2+ stress response as well (l.355)” for plants, including cucumbers, where gene expression regulation by transcription factors would differ from bacteria. An acknowledgement of the gap (i.e. bacteria vs plants) in the discussion would clarify the limitation and future directions.

 

Response:

Thank you for your suggestions. We have added it in the manuscript as required (L359-363).

 

Author Response File: Author Response.pdf