CAX1a TILLING Mutations Modify the Hormonal Balance Controlling Growth and Ion Homeostasis in Brassica rapa Plants Subjected to Salinity
, Juan Manuel Ruiz 1 and Begoña Blasco 1Round 1
Reviewer 1 Report
This manuscript present new and interesting data and the topic and scope is suitable for publication in the journal. However, the presentation and some key issues which are detailed below, such as the statistical analyses and the material and methods) should be improved before the manuscript can be considered for publication.
Specific comments:
- The English writing in the manuscript should be re-checked ad revised. There are many places where the language is awkward.
For example :
In the abstract " The hypothesis to test in this study.." should be revised.
Line 42: disbalances should be inbalances
Line 52: "The resilience of plants against stress" should be "The resilience of plants to stress"
And many more.
- Section: 2.1. Plant material and growth conditions. The description of the plant growth is lacking. Where the plants cultivated in a hydroponic solution? Was it aerated? Size and shape of the containers? Does renewed mean replace? How was the pH regulated? and more..
- Section 2.2: the experimental design is not clear. What were the 12 treatments? I can only see 8 treatments. What do you mean by" 8 plants per treatment, and 3 replications each."?
- Section 2.5: The description of the statistical analyses is insufficient and unclear. What do you mean by 'simple ANOVA'? it is not an acceptable statistical term.
- Also, Line 162: you write "n=9" ?? but you mentioned only 3 replications? or maybe 9 plants per treatment. So what is 9?? This suggests awkwardness in the statistical analysis.
- Background: The authors mention in the introduction and in the discussion several aspects of salinity damage, including Na toxicity, K and Ca deficiency, and disturbed water relations. However, they fail to mention the involvement of oxidative stress, and especially the relations between salinity-Ca-oxidative stress. This should be mentioned at list briefly. 2 Recommended manuscripts: DOI:10.1016/j.freeradbiomed.2011.03.036
https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-14-24
6. Line 40: replace " Numerous crop species" with "Most crop species".
7. Line 269: : What do you mean by incrementing?
8. Line 270: the WUE data should be presented
Author Response
REVIEWER 1
This manuscript present new and interesting data and the topic and scope is suitable for publication in the journal. However, the presentation and some key issues which are detailed below, such as the statistical analyses and the material and methods) should be improved before the manuscript can be considered for publication.
Specific comments:
Comment 1
The English writing in the manuscript should be re-checked ad revised. There are many places where the language is awkward.
For example :
In the abstract " The hypothesis to test in this study.." should be revised.
Response: Done. “The hypothesis to test in this study” was replaced by “We hypothesized that” (page 1, line 25).
Line 42: disbalances should be inbalances
Response: Done (page 2, line 45).
Line 52: "The resilience of plants against stress" should be "The resilience of plants to stress"
Response: Done (page 2, line 56).
And many more.
Response: The English writing style was thoroughly revised.
Comment 2
Section: 2.1. Plant material and growth conditions. The description of the plant growth is lacking. Were the plants cultivated in a hydroponic solution? Was it aerated? Size and shape of the containers? Does renewed mean replace? How was the pH regulated? and more..
Response: A detailed description of the plant growth conditions has been included (page 3, lines 109-117).
Comment 3
Section 2.2: the experimental design is not clear. What were the 12 treatments? I can only see 8 treatments. What do you mean by" 8 plants per treatment, and 3 replications each."?
Response: Thank you for the comment, it was a writing mistake. The experiment consisted of 8 treatments, with 3 trays of plants per treatment and 8 plants in each tray. Thus, in total, we had 24 plants per treatment. This has been clarified in the manuscript (page 3, lines 124-129).
Comment 4
Section 2.5: The description of the statistical analyses is insufficient and unclear. What do you mean by 'simple ANOVA'? it is not an acceptable statistical term.
Response: We have included some statistical details and corrected the inappropriate terms (page 4, line 155-157).
Comment 5
Also, Line 162: you write "n=9" ?? but you mentioned only 3 replications? or maybe 9 plants per treatment. So what is 9?? This suggests awkwardness in the statistical analysis.
Response: This has been clarified in section 2.2 of the manuscript; n=9 means that 9 independent replicates from each treatment were analyzed, and the mean values and standard errors were obtained from these 9 replicates (page 3, lines 124-129).
Comment 6
Background: The authors mention in the introduction and in the discussion several aspects of salinity damage, including Na toxicity, K and Ca deficiency, and disturbed water relations. However, they fail to mention the involvement of oxidative stress, and especially the relations between salinity-Ca-oxidative stress. This should be mentioned at least briefly. Recommended manuscripts: DOI:10.1016/j.freeradbiomed.2011.03.036 and https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-14-24
Response: The involvement of antioxidative metabolism in salinity tolerance of BraA.cax1a mutants was assessed in a previous study (https://doi.org/10.1016/j.envexpbot.2020.104061). Indeed, this aspect has been discussed in the manuscript concerning IAA and JA roles in ROS detoxification and lipid peroxidation. However, following reviewer’s suggestion, we have included some additional information regarding antioxidative metabolism in the introduction and in the discussion (page 2, lines 50-51; page 8, lines 279-281).
Comment 7
Line 40: replace " Numerous crop species" with "Most crop species".
Response: Done (page 2, line 43)
Comment 8
Line 269: What do you mean by incrementing?
Response: The word “incrementing” was replaced by “enhancing” (page 9, line 326)
Comment 9
Line 270: the WUE data should be presented
Response: The WUE data have not been shown in this article because they will be included in a follow-up article that will in-depth address water relations and water use efficiency of BraA.cax1a mutants.
Reviewer 2 Report
The authors are presenting an interesting paper on hormonal balance and salinity stress. The study is well designed but the manuscript needs some revisions. Some parts of the introduction do dot provide enough background information on the topic. Details are missing in the experimental design and the method. The paper could be improved by adding data on WUE and calcium fluxes.
Introduction
L72-76: The authors need to be more specific. The ameliorative effect of calcium during salinity stress varies depending on the plant species and the concentration of Ca used. Any research done in Brassica? This section on the role of Ca during salt stress is very short, it could be developed using recent literature (Manishankar et al, 2018. J of Experimental Botany 69, 4215-4226).
L78-86: This part on CAX transporters is also very short. More background information on CAX transporters and their role during salinity stress is required.
L79:80: how was CAX activity increased in Suaeda?
L84:85: unclear, explain how change in amino acids affecting the conformation of CAX could improve the function of the transporter. Has the activity of the transporter measure in the mutant?
L86: be more specific, what are these changes?
Materials and methods
L97-98, L100-101: It is difficult to visual the experimental set up as details are missing. Did the authors used pots (growth medium, pot size) or hydroponics (container size, aeration). Number of plants per container? Strength of the Hoagland solution? Add a reference or give the composition.
L108-110: there is no mention of drying the roots
L113-116: why measuring ion analysis only in leaves?
L120: which tissues? Leaves?
Results
Figures: I suggest to use white as one of the pattern for the bars on the graphs instead of 3 shades of grey (hard to see the difference between them).
Table 2: provide the abbreviations for iP, tZ,
Table 2: Check SA for BraA.cax1a-7 with NaCl (1035.42a) compare to control (317.96a) the values seems quite different but there is no significant difference
L167-168: This is not true for all mutants
Discussion
L227: Do you have any data on calcium fluxes? This information is critical.
L269: Could you show the data on WUE?
L312: be more specific, role of IAA and CK?
Author Response
REVIEWER 2
The authors are presenting an interesting paper on hormonal balance and salinity stress. The study is well designed but the manuscript needs some revisions. Some parts of the introduction do dot provide enough background information on the topic. Details are missing in the experimental design and the method. The paper could be improved by adding data on WUE and calcium fluxes.
Introduction
Comment 1
L72-76: The authors need to be more specific. The ameliorative effect of calcium during salinity stress varies depending on the plant species and the concentration of Ca used. Any research done in Brassica? This section on the role of Ca during salt stress is very short, it could be developed using recent literature (Manishankar et al, 2018. J of Experimental Botany 69, 4215-4226).
Response: Additional information about the role of Ca during salinity stress and new references regarding the ameliorative effect of Ca on Brassicaceae species were included (page 2, lines 77-80).
Comment 2
L78-86: This part on CAX transporters is also very short. More background information on CAX transporters and their role during salinity stress is required.
Response: We have included additional information about CAX transporter as the reviewer suggests (page 2, lines 86-92)
Comment 3
L79:80: how was CAX activity increased in Suaeda?
Response: In the manuscript, this sentence was confusing, and thus it has been rewritten to make it more clear (page 2, lines 89-91).
Comment 4
L84:85: unclear, explain how change in amino acids affecting the conformation of CAX could improve the function of the transporter. Has the activity of the transporter measure in the mutant?
Response: An amino acid change could affect the protein folding and therefore the CAX1 structure and its ion transport capacity. Likewise, the regulatory mechanisms of the transporter could be affected, which in turn would alter its activity. The CAX activity was not measured in the present study but it would be very interesting to do it in subsequent studies to understand the specific effect of BraA.cax1a mutations on CAX conformation and regulatory mechanisms.
Comment 4
L86: be more specific, what are these changes?
Response: The changes are explained in the material and methods, section 2.1 (page 3, lines 106-108).
Materials and methods
Comment 5
L97-98, L100-101: It is difficult to visual the experimental set up as details are missing. Did the authors used pots (growth medium, pot size) or hydroponics (container size, aeration). Number of plants per container? Strength of the Hoagland solution? Add a reference or give the composition.
Response: A detailed description of the experimental set up has been included (page 3, lines 109-117; 124-125)
Comment 6
L108-110: there is no mention of drying the roots
Response: Done (page 3, line 126)
Comment 7
L113-116: why measuring ion analysis only in leaves?
Response: The ion analysis of the root was assessed in another experiment (https://doi.org/10.1016/j.envexpbot.2020.104061). Furthermore, given that the present study has been focused on the role of leaf phytohormones, thus only the leaf ionic content has been discussed in relation to growth responses under salinity.
Comment 8
L120: which tissues? Leaves?
Response: Phytohormone concentrations were determined in leaves. This was clarified in the manuscript (page 4, line 138).
Results
Comment 9
Figures: I suggest to use white as one of the pattern for the bars on the graphs instead of 3 shades of grey (hard to see the difference between them).
Response: We have formatted the figures according to reviewer’s suggestions to make them easier to visualize (page 4, line 179; page 7; line 246).
Comment 10
Table 2: provide the abbreviations for iP, tZ,
Response: Provided in the material and methods section (page 4, lines 150-151)
Comment 11
Table 2: Check SA for BraA.cax1a-7 with NaCl (1035.42a) compare to control (317.96a) the values seems quite different but there is no significant difference
Response: The letters indicate significant differences among genotypes but not between the salinity and control treatments. Therefore, the fact that BraA.cax1a-7 subjected to salinity (1035.42a) and BraA.cax1a-7 under control conditions (317.96a) have the same letter does not indicate that there are no significant differences. The salinity effects are shown at the bottom of the table (and for this specific comparison indicate significant differences).
Comment 12
L167-168: This is not true for all mutants
Response: The sentence has been modified to more accurately explain the results (page 5, lines 208-209)
Discussion
Comment 13
L227: Do you have any data on calcium fluxes? This information is critical.
Response: We do not have data on calcium fluxes yet. In the present study, we only related the phytohormonal profile with the total concentration of Ca. In future studies, we plan to analyze the Ca concentration in each cell compartment and its fluxes to mechanistically understand the effect of BraA.cax1a mutations.
L269: Could you show the data on WUE?
Response: The WUE data have not been shown in this article because they will be included in a follow-up article that will in-depth address water relations and water use efficiency.
L312: be more specific, role of IAA and CK?
Response: The roles of IAA and CKs have been concretized (page 9, line 368).
