Effects of Sulfate on the Physiology, Biochemistry, and Activity of Group 1 Sulfate Transporters in Seedlings of Brassica pekinensis
, Jigna Tank 5,* and Ashish Patel 3,*
Round 1
Reviewer 1 Report
The paper is written very well. Data compiled correctly. Very interesting and innovative topic. The remarks that came to my mind and that should be introduced are: clearly specify the goals of the work / research hypotheses, because there are none. Conclusions should be rewritten so that they fully reflect the results of the research. Change the keywords so that they do not repeat those in the title. Replace old (more than 10 years old) literature with newer ones.
Author Response
The paper is written very well. Data compiled correctly. Very interesting and innovative topic.
We would like to thank the esteemed reviewer for insightful remarks and excellent ideas, which have really aided us in improving our manuscript. The changes that were made are listed below:
1. The remarks that came to my mind and that should be introduced are: clearly specify the goals of the work / research hypotheses, because there are none.
Answer- The goal of the study has made clear in the L- 100-105 in introduction section of the manuscript as suggested by respected reviewer.
2. Conclusions should be rewritten so that they fully reflect the results of the research.
Answer- Conclusion has been updated in L-636-647 as per the suggestion of the respected reviewer.
3. Change the keywords so that they do not repeat those in the title.
Answer- Changes were made in keywords as per the suggestion of the respected reviewer.
4. Replace old (more than 10 years old) literature with newer ones.
Answer- The authors have replaced the old references with the recent ones in the revised manuscript, as per the suggestion of the respected reviewer.
Reviewer 2 Report
Dear Authors,
Your manuscript is original, current and has a great scientific importance. All segments of the paper are very well written, content-wise, and technically successfully arranged. The results are presented clearly, tabularly and graphically, concisely, with extensive discussion. The list of references is adequate and fully corresponds to the research.
I have no objections to the paper.
Best regards
Author Response
Dear Authors,
Your manuscript is original, current and has a great scientific importance. All segments of the paper are very well written, content-wise, and technically successfully arranged. The results are presented clearly, tabularly and graphically, concisely, with extensive discussion. The list of references is adequate and fully corresponds to the research.
I have no objections to the paper.
Best regards
Author Response: The Authors would like to express their gratitude to the highly respected reviewer for providing insightful feedback that will significantly boost the morale of the authors.
Reviewer 3 Report
Effects of Sulphate on Physiological, Biochemical, and Activity of Group Sulphate Transporter in Seedlings of Brassica Pekinensis
The main problem is the performance of these experiments, they are not clear and please add flowchart to explain:
The main treatments, main measurements, and how old are seedlings in each period of each experiment?
1- Seeds were germinated in vermiculite, ten-day-old seedlings were transferred to 30-liter containers (hydroponics), then applying sulfate treatments? Plants were harvested 3 h after the onset of the light period….?
2- This is enough time to uptake sulphate by roots and shoots? Only 3 hours?
I think this time is not enough? How can the authors guarantee the complete and equilibrium of this uptake?
You can imagine the biomass values in table (2):
0.08 ± 0.00a, 0.08 ± 0.00a, 0.09 ± 0.01b 0.11 ± 0.00,…….
The values are very small, how can recommend these results of these values?
3- And why for the Fumigation experiment, plants were transferred after 14 days to 13-L stainless steel containers????
4- And again not clear:
“Atmospheric H2S and pedospheric S nutrition, exposed to 0, 0.1, 0.2, and 0.3 μl l -1 H2S for six days.”
5- Why different period for many experiments?
6- Again, not clear:
“Upon harvest, plants were removed from the nutrient solution, and then roots were rinsed three times in ice-cold, demineralised water for 20 seconds each. Roots and shoots were harvested separately and weighed.”
Which harvest, and when for different experiments?
7- Not clear again, which anions, please be very clear:
“2.4. Anions: Frozen plant material was homogenised in demineralised water…” these are mainly sulphate and nitrate?
All figures must be in color form to be easy to differ by readers?
Major revision
Moderate editing of English language required
Author Response
Comments and Suggestions for Authors
Effects of Sulphate on Physiological, Biochemical, and Activity of Group Sulphate Transporter in Seedlings of Brassica Pekinensis
- The main problem is the performance of these experiments, they are not clear and please add flowchart to explain: The main treatments, main measurements, and how old are seedlings in each period of each experiment?
Answer- As per suggestion of respected reviewer, the authors have added the flowchart of all treatments and measurements in materials and methods section as Figure 1.
- Seeds were germinated in vermiculite, ten-day-old seedlings were transferred to 30-liter containers (hydroponics), then applying sulfate treatments? Plants were harvested 3 h after the onset of the light period….? This is enough time to uptake sulphate by roots and shoots? Only 3 hours? I think this time is not enough? How can the authors guarantee the complete and equilibrium of this uptake?
Answer- The authors have mentioned in the methodology section that they germinated Brassica pekinensis seeds in vermiculate for 10 days, then transferred them into a 25% Hoagland solution with sulfate (MgSO4) and without sulfate (MgSO4 replaced by Mgcl2) for 4 days (to gain enough weight before transferring them into the fumigation cabinet). Followed by transferring seedlings into a fumigation cabinet for 6 days and then exposing them to different concentrations of H2S. In which, transfer of seedlings into 30-liter containers (hydroponics) in a climate-controlled room from vermiculate is then harvested for analysis of all sulfur metabolites and gene expression at 0 days, 1 day, 2 days, 3 days, and 4 days from the climate-controlled room, and then after 6 days from the fumigation cabinet. So, as mentioned in the methodology section of the manuscript, plants were harvested 3 h after the onset of the light period, meaning 9 seedlings of each treatment from a total of 60 seedlings (with sulphur and without sulphur) were harvested for all 0-day analyses after 3 h of the onset of the light period. Also, please see the attached publication for more detailed information.
Zuidersma, E.I., Ausma, T., Stuiver, C.E.E., Prajapati, D.H., Hawkesford, M.J. and De Kok, L.J., 2020. Molybdate toxicity in Chinese cabbage is not the direct consequence of changes in sulphur metabolism. Plant Biology, 22(2), pp.331-336.
- You can imagine the biomass values in table (2): 0.08 ± 0.00a, 0.08 ± 0.00a, 0.09 ± 0.01b 0.11 ± 0.00,…….The values are very small, how can recommend these results of these values?
Answer- Actually, the authors wanted to compare (after 10 days in vermiculate) 0 day (in 25% Hoagland solution) biomass with overdays (0-4 days) and after 6 days in fumigation cabinet. The growth was very low at starting days of treatments but we had to take them for comparison with later days treatments on seedlings.
- And why for the Fumigation experiment, plants were transferred after 14 days to 13-L stainless steel containers????
Answer- Seedlings were very small in size after 10 days from vermiculate, so, for gaining enough weight and size (so the authors have observed the impact of hydrogen sulphide treatments on plants). Further, the authors have transferred them into climate-controlled room for 4 days without H2S gas treatment which was followed by transferring them into a 13-L stainless steel containers. In addition to this, it was impossible to accommodate containers in the fumigation cabinet of size more than 13 L.
- And again not clear: “Atmospheric H2S and pedospheric S nutrition, exposed to 0, 0.1, 0.2, and 0.3 μl l -1 H2S for six days.”
Answer- In fumigation cabinets seedlings (which are in 25% Hoagland solution with sulfate (MgSO4) and without sulfate (MgSO4 replaced by MgCl2) were exposed to 0, 0.1, 0.2, and 0.3 μl l -1 H2S, which are in pedospheric sulfur medium already (25% Hoagland solution with sulfate (MgSO4) and without sulfate (MgSO4 replaced by MgCl2).
- Why different period for many experiments?
Answer- Germinated Brassica pekinensis seeds in vermiculate for 10 days, then transferred into 25% Hoagland solution with sulfate (MgSO4) and without sulfate (MgSO4 replaced by MgCl2) for 4 days for gain enough weight and size before transferring into fumigation cabinet and then transferred seedlings for 6 days in fumigation cabinet and then exposed by different concentration of H2S.
- Again, not clear: “Upon harvest, plants were removed from the nutrient solution, and then roots were rinsed three times in ice-cold, demineralised water for 20 seconds each. Roots and shoots were harvested separately and weighed.” Which harvest, and when for different experiments?
Answer- To remove ions and other particles attached to the root, roots were rinsed three times by demineralised water in ice cold condition. Furthermore, the authors have performed all the analysis of roots and shoots separately. Further, roots and shoots were harvested for overdays (0-4 days from 25% Hoagland solution in climate-controlled room) and after 6 days in fumigation cabinets. Also, please see the attached publication for more detailed information.
Zuidersma, E.I., Ausma, T., Stuiver, C.E.E., Prajapati, D.H., Hawkesford, M.J. and De Kok, L.J., 2020. Molybdate toxicity in Chinese cabbage is not the direct consequence of changes in sulphur metabolism. Plant Biology, 22(2), pp.331-336.
- Not clear again, which anions, please be very clear: “2.4. Anions: Frozen plant material was homogenised in demineralised water…” these are mainly sulphate and nitrate?
Answer- Thank you for this valuable comment and suggestion. The anions were mainly sulfate and nitrate.
- All figures must be in color form to be easy to differ by readers?
Answer- Thank you for this valuable comment and suggestion. As per the suggestion of respected reviewer, the said figures (Figure 2 and Figure 3) are shown into colour form in the revised manuscript.
Major revision
Comments on the Quality of English Language
- Moderate editing of English language required
Answer- English language has been thoroughly revised throughout the manuscript.
Reviewer 4 Report
In the manuscript titled, 'Effects of Sulphate on Physiological, Biochemical, and Activity of Group 1 Sulphate Transporter in Seedlings of Brassica pekinensis' the authors present results of a study performed to asses the impact of atmospheric hydrogen sulfide on the expression of high affinity sulfate transporters as well as additional growth and biochemical parameters. The authors designed an appropriate set of experiments that first establish the response of these parameters under sufficient sulfate and deficient sulfate conditions. Next, the authors reevaluated these parameters when atmospheric H2S was added under sulfate sufficient and deficient conditions. The major observation from these studies was that the expression of high affinity group 1 sulfate transporters, which are responsible for sulfate uptake in the roots, are not regulated by H2S supplied in the atmosphere.
While I agree with the main conclusions of the paper, some of the data are not consistent with previous observations. Specifically, previous reports using H2S as the sole sulfur source have shown up to a 5-fold increase in shoot thiols (https://www.frontiersin.org/articles/10.3389/fpls.2019.00743/full), and others have shown that shoot-to-root thiol signaling does impact the expression of high affinity sulfate transporters (https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2012.04924.x). These discrepancies should be addressed in the discussion and differences between these studies and the present study should be highlighted
English language is acceptable. Certain words, such as 'remarkable', are not as precise as possible (and used too often), but could easily be addressed.
Author Response
In the manuscript titled, 'Effects of Sulphate on Physiological, Biochemical, and Activity of Group 1 Sulphate Transporter in Seedlings of Brassica pekinensis' the authors present results of a study performed to assess the impact of atmospheric hydrogen sulfide on the expression of high affinity sulfate transporters as well as additional growth and biochemical parameters. The authors designed an appropriate set of experiments that first establish the response of these parameters under sufficient sulfate and deficient sulfate conditions. Next, the authors reevaluated these parameters when atmospheric H2S was added under sulfate sufficient and deficient conditions. The major observation from these studies was that the expression of high affinity group 1 sulfate transporters, which are responsible for sulfate uptake in the roots, are not regulated by H2S supplied in the atmosphere.
We would like to thank the esteemed reviewer for insightful remarks and excellent ideas, which have really aided us in improving our manuscript. The changes that were made are listed below:
- While I agree with the main conclusions of the paper, some of the data are not consistent with previous observations. Specifically, previous reports using H2S as the sole sulfur source have shown up to a 5-fold increase in shoot thiols (https://www.frontiersin.org/articles/10.3389/fpls.2019.00743/full).
Answer- The authors have taken information and enriched the content of our manuscript from the suggested publication (https://www.frontiersin.org/articles/10.3389/fpls.2019.00743/full) L- 589 to 596.
- Others have shown that shoot-to-root thiol signaling does impact the expression of high affinity sulfate transporters (https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2012.04924.x). These discrepancies should be addressed in the discussion and differences between these studies and the present study should be highlighted.
Answer- The authors have cited your suggested publication (https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2012.04924.x) in the manuscript. In which it shows that shoot-to-root thiol signaling does impact the expression of high affinity sulfate transporters. The authors have compared this with present study data and added contents in discussion part of the manuscript in L- 581 to 583.
Comments on the Quality of English Language
- English language is acceptable. Certain words, such as 'remarkable', are not as precise as possible (and used too often), but could easily be addressed.
Answer- The Authors have rectified as per your suggestions in the manuscript.
Round 2
Reviewer 3 Report
thanks for corrections
accepted in this form
Moderate editing of English language required
