A Na+/H+ Antiporter Gene from Rosa multiflora (RmNHX2) Functions in Salt Tolerance via Modulating ROS Levels and Ion Homeostasis
Round 1
Reviewer 1 Report
The manuscript by Luo et al., titled "A Na+ /H+ Antiporter gene from Rosa multiflora (RmNHX2) functions 1 in salt tolerance via modulating ROS levels and ion homeosta- 2 sis" provides positive results in demonstrate that 22 RmNHX2 enhances plant salt tolerance by maintaining proper ion homeostasis, as well as by accel- 23 erating ROS scavenging.
Authors should improve the grammar, spelling, punctuation, and overall English of the manuscript. The abbreviations should be explained in full during the first mention.
Describes 11 putative 12 RmNHXs in Rosa multiflora according to the genome-wide analysis. The RmNHX genes were classi- 13 fied into 3 classes. Overall, the manuscript is clear and easy to follow. The introduction is clear and well written. The design is simple and coherent with the objectives. It would be useful to include a more detailed section regarding the treatment of stress. The results read well and present an accurate reflection of the data. ANOVA (salt treatment) of such data is appropriate. Authors should pay more attention to the reference section. Many formatting mistakes have been observed. The tables and figures are very clear and easy to interpret.
Authors should pay more attention to the reference section. Many formatting mistakes have been observed.
Author Response
Response to reviewer 1
We appreciate the comments and suggestions very much, which are valuable in improving the quality of our manuscript. We have substantially revised our manuscript according to the comments and suggestions.
1.Authors should improve the grammar, spelling, punctuation, and overall English of the manuscript.
We apologize for the mistake of our writing. We have carefully revised the English of the manuscript.
2 The abbreviations should be explained in full during the first mention.
The abbreviations had been explained in full during the first mention.
3 It would be useful to include a more detailed section regarding the treatment of stress.
Thank you for this suggestion. The stress treatment of transgenic tobacco and VIGS lines had beed added in the 4.9.
4 Authors should pay more attention to the reference section. Many formatting mistakes have been observed.
We apologize for this mistake. The formatting reference had been revised.
Author Response File: 
Author Response.doc
Reviewer 2 Report
A Na+/H+Antiporter gene from Rosa multiflora (rmnhx2) functions
in salt tolerance via modulating ROS levels and ion homeostasis
The manuscript presents interesting results about the role of a Na+/H+Antiporter isolated from Rosa multiflora and whose functional characterization in tobacco plants demonstrated an association with Na and K regulation and Redox homeostasis during high NaCl exposures. The used approaches are proper for this aim and the study is relevant for understanding as the control at gene expression level of Na+/H+ antiporter influences other tolerance mechanism in a woody ornamental plant. However, the improvement of discussion section is needed. Other published results should be included, mainly to answer (1) what is the connection between the control of ionic homeostasis and the control of the redox state of the plant, (2) whether the expression of a vacuolar transporter for Na is increased (it was not shown in which part of the plant it is being expressed) because the plant does not exhibit a Na+ exclusion mechanism, characterized by Na+ retention in the roots. Na+ and K+ data show the activation of barriers that prevent the entry or exclusion of sodium across the plasmatic membrane. It should be explained.
Other points:
How to explain that highly similar genes NHX1, NHX2 and NHX3 in relation to conserved motif and cis-acting regulatory element show different expression levels?
Figure 3C and D should be improved. Please improve the description of figure 3D.
The percentual increase (177-179, 209-210 and others) should be improved.
I suggested that the authors include the data of control plants in the figures.
Lane 202: 30-60%. Please, revise the values.
Why the authors grown the plants with different salt level (200 and 300 mM NaCl)?
Lane 237-239, 269-270, 294-296: The phrases should be removed of results section.
Lane 279-280: Please, the comment should be improved.
Lane 306-308: For this comment, the authors should carry out correlation analysis. I suggest change the comment.
Lane 309: leaves in the figure.
Lane 325: It seems that all Rosa multiflora NHX genes are responsive to salt stress.
In the discussion include the figures (figure 1, 2 or…)
Lane 329-330: Please, verify this information about FvNHX2. In phylogenetic tree, RmNHX2 is like other gene.
Lane 333-338; 347-350: Please, improve the phrase.
Lane 360-362: The phrase must be reinforced with other works.
The conclusion (495-503) should be improved.
Author Response
Response to reviewer 2
We appreciate the comments and suggestions very much, which are valuable in improving the quality of our manuscript. We have substantially revised our manuscript according to the comments and suggestions.
1 The manuscript presents interesting results about the role of a Na+/H+Antiporter isolated from Rosa multiflora and whose functional characterization in tobacco plants demonstrated an association with Na and K regulation and Redox homeostasis during high NaCl exposures. The used approaches are proper for this aim and the study is relevant for understanding as the control at gene expression level of Na+/H+ antiporter influences other tolerance mechanism in a woody ornamental plant. However, the improvement of discussion section is needed. Other published results should be included, mainly to answer (1) what is the connection between the control of ionic homeostasis and the control of the redox state of the plant,
The presence of high concentrations of salt in plant cells disturbs the kinetic steady state of Na+ transport. The adverse effects of accumulation of these two ions at high concentrations in apoplast give rise to hyperosmotic osmotic stress, ionic toxicity, and nutritional imbalance. As a result, high salinity restricts uptake of water, leading to intracellular water loss, altering the osmotic potential of the salt-exposed cells, which may eventually lead to the crop yield decline
(2) whether the expression of a vacuolar transporter for Na is increased (it was not shown in which part of the plant it is being expressed) because the plant does not exhibit a Na+ exclusion mechanism, characterized by Na+ retention in the roots. Na+ and K+ data show the activation of barriers that prevent the entry or exclusion of sodium across the plasmatic membrane. It should be explained.
Plant sodium/proton exchangers (NHX) are membrane proteins that transport protons (H+) across a membrane in exchange for Na+ or K+. Plant NHX antiporters are major players in maintaining ion homeostasis and pH regulation. Increasing evidence indicates that cellular injury derived from salt stress is predominantly due to the accumulation of Na+ to a level that plant cells are unable to deal with. The cellular K+ level is also a key factor influencing salt stress tolerance. External Na+ can inhibit uptake of K+, resulting in K+ deficiency in the cytosol and an increase in the Na/K ratio. This, again, leads to inactivation of enzymes that are important for physiological processes.
Other points:
How to explain that highly similar genes NHX1, NHX2 and NHX3 in relation to conserved motif and cis-acting regulatory element show different expression levels?
The 2.0 kb promoter region of the RmNHXs was adopted to identify the cis-acting elements using PlantCARE database. The promoter region of RmNHXs had various cis-acting elements (e.g., CAAT-box, CCAAT-box, and TATA-box). Moreover, plant hormone-responsive and stress-responsive elements were also identified in the promoter region of RmNHXs, including salicylic acid responsive elements (TCA-elements), auxin responsive elements (TGA-elements), MeJA-responsive elements (CGTCA-motif, TGACG-motif), ABA responsive elements (ABREs), gibberellinresponsive elements (TATC-box), low-temperature responsive elements (LTR), defense and stress responsiveness elements (TC-rich repeats). It should be noticed that there were different number of hormone and stress response motif in the promoter region of RmNHXs. The gene structure of 11 RmNHXs showed diverse intron-exon patterns with the number of introns ranging from 7 to 24. On the other hand, the longest intron of RmNHX2 is 2.9 kb. As a result, the genome sequences of RmNHX2 reached 7.8 kb
Figure 3C and D should be improved. Please improve the description of figure 3D.
The description of figure 3D had been revised to “To determine putative subcellular location of RmNHX2, its open reading frame (ORF) without stop codon was inserted into pHBT-GFP-NOS vector as driven by CaMV 35S, generating a fusion protein RmNHX2-GFP. In addition, the tonoplast marker protein VAC-RK was fused to C-terminal RFP. The mentioned plasmids were co-transformed into Arabidopsis protoplast cells. As demonstrated from transient expression assays, the green fluorescence of RmNHX2-GFP perfectly overlapped with tonoplast marker fluorescent signals (Figure 3D). The mentioned results suggested that RmNHX2 was localized at the tonoplast.”
The percentual increase (177-179, 209-210 and others) should be improved.
Done. It had been improved.
I suggested that the authors include the data of control plants in the figures.
There was no morphological difference between WTs and the transgenic line in the normal condition. We next compared the salt tolerance of these overexpression plants grown under salt treatment.
Lane 202: 30-60%. Please, revise the values.
The values had been revised.
Why the authors grown the plants with different salt level (200 and 300 mM NaCl)?
Tobacco and wild rose showed different tolerance to NaCl treatment.
Lane 237-239, 269-270, 294-296: The phrases should be removed of results section.
The phrases had been removed of results section.
Lane 279-280: Please, the comment should be improved.
“As the antioxidant enzymes is involved in the regulation of ROS scavenging, we analyzed the catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) enzyme activities” had been improved.
Lane 306-308: For this comment, the authors should carry out correlation analysis. I suggest change the comment.
“As ionic homeostasis is a major factor causing salt stress, therefore, we examined the accumulation of Na+ and K+ in the leaves and roots.” had been improved.
Lane 309: leaves in the figure.
It had been revised to leaves in the figure.
Lane 325: It seems that all Rosa multiflora NHX genes are responsive to salt stress.
Line 325 had been revised to “In this study, RmNHX2 was induced to the greatest degree by salt treatment, implying that RmNHX2 may play a critical role in regulation of salt response”
Lane 329-330: Please, verify this information about FvNHX2. In phylogenetic tree, RmNHX2 is like other gene.
Lane 329-330 had been revised to “The phylogenetic analysis indicated that RmNHX2 pertained to the vacuolar Na+/H+ antiporter proteins and displayed closer associations with FvH4 (6g04140.1).”
Lane 333-338; 347-350: Please, improve the phrase.
The phrase had been improved.
Lane 360-362: The phrase must be reinforced with other works.
“Increasing evidence demonstrated that a high antioxidant capacity to scavenge the ROS is linked to increased tolerance to environmental stresses [34]. This study demonstrated that RmNHX2 functions in salt tolerance through a more powerful ROS-scavenging capacity.” Had been added in the line 376.
The conclusion (495-503) should be improved.
The conclusion had been revised to “In this study, we confirmed 11 RmNHXs proteins in Rosa multiflora through the genome-wide analysis. RmNHX2 was drastically and progressively elevated by the salt stress. RmNHX2 was localized at the tonoplast. RmNHX2 overexpression in tobacco led to the enhanced salt tolerance, while down-regulation of RmNHX2 in Rosa multiflora increased salt susceptibility. Endogenous Na+ concentration in the transgenic tobacco lines was reduced in the leaves and roots, leading to the lowered Na/K ratios. RmNHX2-mediated salt tolerance might be at least in part via the antioxidant system and ion homeostasis (Figure. 9). Accordingly, RmNHX2, as an important candidate gene, had a great potential for molecular breeding of salt-tolerant woody ornamental plants.”
Author Response File: Author Response.doc
Reviewer 3 Report
In present study, the authors, Luo et al. have analyzed the sodium ion transporters from Rosa multiflora. Abiotic stresses specifically salt stress is growing concern in global climate change and increasing temperatures. These results successfully revealed that 22 RmNHX2 enhances plant salt tolerance by maintaining proper ion homeostasis, as well as by improving the ROS scavenging potential. Moreover, the authors have identified 11 putative RmNHXs in Rosa multiflora based on to the genome-wide study of Rose. The study is significant improvement on current knowledge of antiporters in Rose family. However, prior to consideration for publication I recommend certain improvements the manuscript:
For figures legends: change wording to ‘before or after salt treatment’ instead of ‘before or after salt’
In Figure 5F: Clearly label each microcentrifuge tube.
Figure 3D: The size of scale bar is not visible.
Provide figures, specifically the drawing and microscopic image with higher resolution.
Authors need to provide catalog numbers of the chemicals used in the study.
Include the growth conditions, like humidity, soil media, light.
Authors need to improve and enrich the introduction on abiotic stress and ROS analysis based recent literature, for this I suggest a review on salt stress: https://doi.org/10.1016/j.xplc.2022.100417 and literature of antioxidants: https://doi.org/10.3390/antiox11081630 ; these resources would be useful for improving the quality of discussion or introduction.
The conclusions should be expanded and include future implications of present study on abiotic stress management in Rose and other garden plants species.
Which test was applied to calculate the statistical significance? Please provide this information. It seems ANOVA was used, why not student t-test?
The study could be considered for publication after revision of present manuscript.
Author Response
Response to reviewer 3
In present study, the authors, Luo et al. have analyzed the sodium ion transporters from Rosa multiflora. Abiotic stresses specifically salt stress is growing concern in global climate change and increasing temperatures. These results successfully revealed that 22 RmNHX2 enhances plant salt tolerance by maintaining proper ion homeostasis, as well as by improving the ROS scavenging potential. Moreover, the authors have identified 11 putative RmNHXs in Rosa multiflora based on to the genome-wide study of Rose. The study is significant improvement on current knowledge of antiporters in Rose family. However, prior to consideration for publication I recommend certain improvements the manuscript:
We appreciate the comments and suggestions very much, which are valuable in improving the quality of our manuscript. We have substantially revised our manuscript according to the comments and suggestions.
For figures legends: change wording to ‘before or after salt treatment’ instead of ‘before or after salt’
In the figures legends, ‘before or after salt’ had been revised to ‘before or after salt treatment’
In Figure 5F: Clearly label each microcentrifuge tube.
Each microcentrifuge tube had been labeled.
Figure 3D: The size of scale bar is not visible.
The size of scale bar had been added in the Figure 3D.
Provide figures, specifically the drawing and microscopic image with higher resolution.
The figures with higher resolution had been provided.
Authors need to provide catalog numbers of the chemicals used in the study.
The catalog numbers of the chemicals had been provided.
Include the growth conditions, like humidity, soil media, light.
The growth conditions had been added.
Authors need to improve and enrich the introduction on abiotic stress and ROS analysis based recent literature, for this I suggest a review on salt stress: https://doi.org/10.1016/j.xplc.2022.100417 and literature of antioxidants: https://doi.org/10.3390/antiox11081630 ; these resources would be useful for improving the quality of discussion or introduction.
“Abiotic stresses also led to the accumulation of ROS, including hydrogen peroxide (H2O2) and hydroxyl radicals, O2- (superoxide radicals), in plant cells [10]. ROS scavenging enzymes is responsible for protection against oxidative damages on cell membranes [10]. Furthermore, given that, it will be of high significance for exploiting the favorable genes to increase the salt tolerance of plants” had been added in the introduction.
The conclusions should be expanded and include future implications of present study on abiotic stress management in Rose and other garden plants species.
The conclusion had been revised to “In this study, we confirmed 11 RmNHXs proteins in Rosa multiflora through the genome-wide analysis. RmNHX2 was drastically and progressively elevated by the salt stress. RmNHX2 was localized at the tonoplast. RmNHX2 overexpression in tobacco led to the enhanced salt tolerance, while down-regulation of RmNHX2 in Rosa multiflora increased salt susceptibility. Endogenous Na+ concentration in the transgenic tobacco lines was reduced in the leaves and roots, leading to the lowered Na/K ratios. RmNHX2-mediated salt tolerance might be at least in part via the antioxidant system and ion homeostasis (Figure. 9). Accordingly, RmNHX2, as an important candidate gene, had a great potential for molecular breeding of salt-tolerant woody ornamental plants.”
Which test was applied to calculate the statistical significance? Please provide this information. It seems ANOVA was used, why not student t-test?
“ANOVA was used to compare statistical difference based on Fisher’s least significant difference test, statistical differences were determined at the significance level of P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).” Had been added in the line 515.
Author Response File: Author Response.doc
Round 2
Reviewer 2 Report
Dear authors,
The main points of the manuscript were unsatisfactorily answered. The authors must explain why RmNHX2, a vacuolar Na+/H+ antiporter, did positively influence on redox state of plant tissues through increased activity of antioxidative enzymes. Moreover, whether the expression of RmNHX2 is increased (it was not shown in which part of the plant it is being expressed) why the plant does not exhibit a Na+ exclusion mechanism, characterized by Na+ retention in the roots (high Na+ accumulated in roots and low Na+ accumulated in aerial tissues). Na+ and K+ data show reduced levels at both tissues, suggesting that the activation of barriers that prevent the entry or extrusion of sodium across the plasmatic membrane could be involved. It should be explained.
With kind regards.
Author Response
ROS levels has been well accepted that in biological systems ROS accumulation is related to physiological perturbation and ROS levels can reflect the degree of damage to cellular components. Increased activity of antioxidative enzymes led to the lower ROS accumulation. RmNHX2 might not directly influenced the antioxidative enzymes. So, RmNHX2 functions in salt tolerance by, at least partially, the activation of the antioxidant system. The overexpressing RmNHX2 could increase the efflux of Na+ from the cell and compartmentalizing Na+ into vacuoles from the cytosol, to prevent superfluous accumulation of Na+ in cytosol, so that plant cells can sustain the ion homoestasis. In the overexpression RmNHX2 lines, the Na+ level in the root is lower than in the leaves. However, in the silencing RmNHX2 lines, the Na+ level in the root is higher than in the leaves.
Reviewer 3 Report
Authors have revised the manuscript satisfactorily.
Author Response
We thank you very much for giving us an opportunity to revise our manuscript.
Round 3
Reviewer 2 Report
Dear authors,
Line 368-369:
This study demonstrated that the Rm NHX2 (a vacuolar Na+/H+ antiporter) regulate the Na+ and K+ homeostasis, which promove optimal conditions in cytosol and organelles to function antioxidative enzymes.
With kind regards
Author Response
We appreciate the comments and suggestions very much, which are valuable in improving the quality of our manuscript. We have substantially revised our manuscript according to the comments and suggestions.
Line 368-369: “This study demonstrated that RmNHX2 functions in salt tolerance through a more powerful ROS-scavenging capacity.” has been revised to This study demonstrated that the Rm NHX2 (a vacuolar Na+/H+ antiporter) regulate the Na+ and K+ homeostasis, which promove optimal conditions in cytosol and organelles to function antioxidative enzymes.
