Round 1

Reviewer 1 Report (Previous Reviewer 2)

Reviewer’s Recommendation: Major Revision

Reviewer’s comments to Authors

Authors should revise the manuscript carefully in light of below comments...............

1.   Grammatical errors are present, please revise the whole manuscript to remove any possible grammatical and typos errors.

2.   Error in sentence formation, please revise the whole manuscript to avoid the use of long sentences and confusing sentences/paragraphs.

3.   Please maintain uniformity while in-text citation and referencing in the entire manuscript.

4.   The reference does not meet the format requirements of the Journal so please check the references as per the authors guideline of the Journal.

5.   It is advised to check and avoid too many self-cited papers. Authors are advised to cite maximum two self-papers.

6.   The beginning of a new paragraph should be after some space, check in complete manuscript.

7.      Throughout the whole manuscript the plant names should be in italic format.

8.      This paper lacks final revision by the author as many general repetitions, typos, grammatical, sentence formation errors were found in the manuscript. It is not possible to mention all such errors. Thus revise the manuscript accordingly.

Abstract:

Authors should revise the manuscript carefully in light of below comments...............

1. An abstract must be fully self-contained and make sense by itself, without further reference to outside sources or to the actual paper. It is important to provide the relevance or importance of your work and the main outcomes. Please revise the abstract accordingly.
2. The abstract is not clear and the objective of the paper is not clearly validated from the abstract.
3. The future perspective of the experiment should be mentioned in the abstract.
4. The abstract should appropriately over the contents of the manuscript.
5. In the keywords, it is strongly advisable to use suitable words that can aid in finding out the manuscript in current registers or indexes. Strictly avoid the use of title words in the keywords.

6.      A graphical abstract is recommended for better perception of the present study.

7.      A novelty statement is also encouraged to be added in the manuscript for bringing out the uniqueness of your study and its importance.

8.      Please ensure that “Highlights” of the present work should be clearly mentioned in the manuscript.

Introduction:

Authors should revise the manuscript carefully in light of below comments...............

1.   The literature from past work done in the same field missing to strengthen the introduction section. The need and importance of the present work should be clearly written in the introduction section.

2.   The new aspects and innovations of this manuscript should be clearly and briefly described in this section.

3.   The present state of knowledge in the subject should be described in introduction.

4.   The literature should be sufficiently critical, current, and internationally evaluated.

Materials and Methods:

Authors should revise the manuscript carefully in light of below comments...............

1.         Please try to merge the different sub-sections of the methodology as an individual mention for each component seems a bit unscientific method.

2.         The size of manuscript seems to be large. It should be crisp and appropriate. Please revise it.

3.         The text presented across the manuscript should be simple so that the scientist/workers in other disciplines will understand. Please revise it.

4.         Introduction, Result, and Discussion sections are poorly cited with the references and required to update and validation with previous studies. The relevant papers listed below may be considered to enhance the scientific quality of manuscript significantly.

·         Kumar, D. et al. (2023). Titanium dioxide nanoparticles potentially regulate the mechanism (s) for photosynthetic attributes, genotoxicity, antioxidants defense machinery, and phytochelatins synthesis in relation to hexavalent chromium toxicity in Helianthus annuus L. Journal of Hazardous Materials, 454, p.131418.

·         Agnihotri, A. et al. (2018). Counteractive mechanism (s) of salicylic acid in response to lead toxicity in Brassica juncea (L.) Czern. cv. Varuna. Planta, 248, pp.49-68.

·         Singh, D. et al. (2017). Interactive effects of EDTA and oxalic acid on chromium uptake, translocation and photosynthetic attributes in Indian mustard (Brassica juncea L. var. Varuna). Current Science, pp.2034-2042.

 

Results and Discussion:

Authors should revise the manuscript carefully in light of below comments...............

1. The results and discussion section needs to be elaborated more. The results should be clearly described in light of available knowledge and hypothesis and must be strongly validated with previous reports in the related subject area.
2. The non-significant results was not clearly validated from the previous papers.
3. Please carefully check, verify, and correct the results of the present experiments from the tables/figures/graphs provided in the manuscript.
4. The discussion does not describe the results with proper facts and even does not validate the result with appropriate references. Please enrich it significantly.
5. The discussion did not provide a specific reasons for the results. The provided explanation should be strengthen significantly.
6. The strong hypothesis, scientific facts, and validation of previous reports are entirely missing. Please revise it. 

Conclusion:

Authors should revise the manuscript carefully in light of below comments...............

1. The conclusion section failed to enlighten the spirit of the present finding or work so required to revise it accordingly.
2. In the conclusion section the authors have only mentioned the data but major finding is missing from the conclusion part. Need to revise and incorporate this important concern of reviewer.
3. The conclusion section seems like abstract so there is a need to revise the conclusion part accordingly.

Figures and Tables:

Authors should revise the manuscript carefully in light of below comments...............

·         Please provide the clear figures and tables.

·         The authors should write the descriptive, elaborated legends for the figures and the tables.

·         Please remove the redundancy from the legends of the figures and tables.

·         The legends of the figures and tables are not crisp and not completely bringing out the sense of the figures and tables. Rewrite it accordingly.

·         The placement of tables and figures in the manuscript should be done appropriately, which is missing in this manuscript. Please revise it.

·         The figures are overlapping the legends, the editing needs to be done.

·         The proper explanation of statistical analysis and its importance for describing the results should be mentioned.

·         There should not be monotony in representation of the results for instance all should not be represented in bar graph form vise-versa.

 

Author Response

Comment 1: Grammatical errors are present, please revise the whole manuscript to remove any possible grammatical and typos errors.

Response: Thank you very much for your careful review of our manuscript. We greatly appreciate your critique and advice, and hope that the corrections will meet your approval. The English language has been polished by the standard language editing in MDPI, which was marked in red in the revised manuscript. Once again, thank you very much for the comments and suggestions. Thank you most sincerely!

 

Comment 2:  Error in sentence formation, please revise the whole manuscript to avoid the use of long sentences and confusing sentences/paragraphs.

 Response: Thank you for the constructive suggestion. We have used the editing services by MDPI to improve the English of our manuscript. 

 

Comment 3: Please maintain uniformity while in-text citation and referencing in the entire manuscript.

Response: Thank you most sincerely! We have checked the formatting of the complete manuscript.

 

Comment 4: The reference does not meet the format requirements of the Journal so please check the references as per the authors guideline of the Journal.

Response: Thank you most sincerely! We have checked the formatting of the complete manuscript.

 

Comment 5:  It is advised to check and avoid too many self-cited papers. Authors are advised to cite maximum two self-papers.

 Response: We sincerely thank the reviewer for the careful reviews! According to the previous research about the effects of bicarbonate concentration on the plant. We have reduced the number of self-cited articles. Thank you most sincerely!

 

Comment 6:  The beginning of a new paragraph should be after some space, check in complete manuscript.

Response: Thank you most sincerely! We have checked the formatting of the complete manuscript.

 

Comment 7: Throughout the whole manuscript the plant names should be in italic format.

Response: Thank you. We have revised the plant names of the complete manuscript. Thank you most sincerely!

 

Comment 8: This paper lacks final revision by the author as many general repetitions, typos, grammatical, sentence formation errors were found in the manuscript. It is not possible to mention all such errors. Thus revise the manuscript accordingly.

 Response: Thank you for the constructive suggestion. We have used the editing services by MDPI to improve the English of our manuscript.

Abstract:

Comment 9: An abstract must be fully self-contained and make sense by itself, without further reference to outside sources or to the actual paper. It is important to provide the relevance or importance of your work and the main outcomes. Please revise the abstract accordingly.

 Response: Thank you for the constructive suggestion. The abstract has been added and revised as "Karst ecological stresses are harmful to plant growth, especially high bicarbonate, drought, high pH, etc. In this study, it was investigated the effects of 0, 2.0, 7.0 and 12.0 mmol L^-1 sodium bicarbonate on the biomass, electrophysiological information, intracellular water metabolism, nutrient transport, photosynthesis and chlorophyll fluorescence of Coix lacryma-jobi L.. The results show that 2.0 mmol L^-1 sodium bicarbonate effectively improved the biomass formation of Coix lacryma-jobi L., and notably increased its intrinsic capacitance (IC) and decreased its intrinsic resistance (IR), intrinsic impedance (IZ), intrinsic capacitive reactance (IXc) and intrinsic inductive reactance (IXL), as well as reliably enhanced its intracellular water metabolism, nutrient transport and photosynthesis capacities. However, Sodium bicarbonate of 7.0 and 12.0 mmol L^-1 exhibited notably inhibiting effects on the photosynthetic rate, stomatal conductance, transpiration rate and dry weight, whereas they could not significantly change intracellular water metabolism and nutrient transport capacities of Coix lacryma-jobi L.. This study highlights that appropriate bicarbonate could enhance the intracellular water metabolism, nutrient transport, photosynthesis and growth of Coix lacryma-jobi L., and which could be rapidly obtained by plant’s electrophysiological information. Importantly, in the future study of plant physiology, plant’s electrophysiological information can be used as an ingeniously technology for capturing the metabolism information of the dissolved inorganic carbon from atmosphere and soil in plants." Thank you most sincerely!

 

Comment 10: The abstract is not clear and the objective of the paper is not clearly validated from the abstract.

 Response: Thank you very much for your careful review of our manuscript. We have revised the abstract accordingly. Thank you most sincerely!

 

Comment 11: The future perspective of the experiment should be mentioned in the abstract.

 Response: Thank you very much for your careful review of our manuscript. We have revised the abstract accordingly. "... Importantly, in the future study of plant physiology, plant electrophysiological measurement can be used as an method for studying the metabolism of dissolved inorganic carbon from the atmosphere and soil in plants.

 

Comment 12: The abstract should appropriately over the contents of the manuscript.

 Response: Thank you very much for your careful review of our manuscript. The corresponding sentence has been changed in the revised manuscript. 

 

Comment 13: In the keywords, it is strongly advisable to use suitable words that can aid in finding out the manuscript in current registers or indexes. Strictly avoid the use of title words in the keywords.

 Response: Thank you very much for your careful review of our manuscript. The keywords have been changed in the revised manuscript. Thank you most sincerely!

 

Comment 14:  A graphical abstract is recommended for better perception of the present study.

Response: Thank you very much for your careful review of our manuscript. We have made the graphical abstract in the revised manuscript. Please line 32.

 

Comment 15:  A novelty statement is also encouraged to be added in the manuscript for bringing out the uniqueness of your study and its importance.

 Response: Thank you very much for your careful review of our manuscript. We have added it in the revised abstract. Please see line 26.

Comment 16:  Please ensure that “Highlights” of the present work should be clearly mentioned in the manuscript.

 Response: We have added it in the revised abstract. electrical signal technology can perform multi-parameter detection (WTR/IWAC/NAC, etc.), which is significantly superior to photosynthesis indicators. Thank you most sincerely!

 

Introduction:

Comment 17: The literature from past work done in the same field missing to strengthen the introduction section. The need and importance of the present work should be clearly written in the introduction section.

Response: Thank you for the constructive suggestion. The corresponding parts have been added in the revised manuscript. Thank you most sincerely!

 

Comment 18: The new aspects and innovations of this manuscript should be clearly and briefly described in this section.

Response: Thank you for the constructive suggestion. The corresponding parts have been added in the revised manuscript.

 

Comment 19: The present state of knowledge in the subject should be described in introduction.

Response: Thank you for the constructive suggestion. The corresponding parts have been added in the revised manuscript. Thank you most sincerely!

 

Comment 20: The literature should be sufficiently critical, current, and internationally evaluated.

Response: Thank you for the constructive suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely!

 

Materials and Methods:

Comment 21: Please try to merge the different sub-sections of the methodology as an individual mention for each component seems a bit unscientific method.

Response: Thank you for the constructive suggestion. It is a good suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely!

 

Comment 22: The size of manuscript seems to be large. It should be crisp and appropriate. Please revise it.

Response: Thank you for the constructive suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely!

 

Comment 23: The text presented across the manuscript should be simple so that the scientist/workers in other disciplines will understand. Please revise it.

Response: Thank you for the constructive suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely!

 

Comment 24: Introduction, Result, and Discussion sections are poorly cited with the references and required to update and validation with previous studies. The relevant papers listed below may be considered to enhance the scientific quality of manuscript significantly.

• Kumar, D. et al. (2023). Titanium dioxide nanoparticles potentially regulate the mechanism (s) for photosynthetic attributes, genotoxicity, antioxidants defense machinery, and phytochelatins synthesis in relation to hexavalent chromium toxicity in Helianthus annuus L. Journal of Hazardous Materials, 454, p.131418.
• Agnihotri, A. et al. (2018). Counteractive mechanism (s) of salicylic acid in response to lead toxicity in Brassica juncea (L.) Czern. cv. Varuna. Planta, 248, pp.49-68.
• Singh, D. et al. (2017). Interactive effects of EDTA and oxalic acid on chromium uptake, translocation and photosynthetic attributes in Indian mustard (Brassica juncea L. var. Varuna). Current Science, pp.2034-2042.

 Response: Thanks very much for the reviewer's careful reviews and good comment on our manuscript! It is a good suggestion. We have cited the relevant paper in the revised article (Kumar, D. et al. 2023；Agnihotri, A. et al. 2018；Singh, D. et al. 2017). In the future work, we will give priority to these references. Thank you most sincerely!

Results and Discussion:

Comment 25: The results and discussion section needs to be elaborated more. The results should be clearly described in light of available knowledge and hypothesis and must be strongly validated with previous reports in the related subject area.

Response: Thank you for the constructive suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely!

 

Comment 26: The non-significant results was not clearly validated from the previous papers.

Response: Thank you for the constructive suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely! 

 

Comment 27: Please carefully check, verify, and correct the results of the present experiments from the tables/figures/graphs provided in the manuscript.

Response: Thank you for the constructive suggestion. We have checked the results. Thank you most sincerely!

 

Comment 28: The discussion does not describe the results with proper facts and even does not validate the result with appropriate references. Please enrich it significantly.

Response: Thank you for the constructive suggestion. It is a good suggestion. We have enriched the discussion. Thank you most sincerely!

 

Comment 29: The discussion did not provide a specific reasons for the results. The provided explanation should be strengthen significantly.

Response: Thank you for the constructive suggestion. It is a good suggestion. We have enriched the discussion. Thank you most sincerely!

 

Comment 30: The strong hypothesis, scientific facts, and validation of previous reports are entirely missing. Please revise it. 

Response: Thank you for the constructive suggestion. It is a good suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely!

 

Conclusion:

Comment 31: The conclusion section failed to enlighten the spirit of the present finding or work so required to revise it accordingly.

Response: Thank you for the constructive suggestion. It is a good suggestion. We have revised it. Thank you most sincerely!

 

 

Comment 32: In the conclusion section the authors have only mentioned the data but major finding is missing from the conclusion part. Need to revise and incorporate this important concern of reviewer.

Response: Thank you for the constructive suggestion. It is a good suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely!

 

Comment 33: The conclusion section seems like abstract so there is a need to revise the conclusion part accordingly.

Response: Thanks very much for the reviewer's careful reviews and good comment on our manuscript! It is a good suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely!

 

Figures and Tables:

• Please provide the clear figures and tables.
• The authors should write the descriptive, elaborated legends for the figures and the tables.
• Please remove the redundancy from the legends of the figures and tables.
• The legends of the figures and tables are not crisp and not completely bringing out the sense of the figures and tables. Rewrite it accordingly.
• The placement of tables and figures in the manuscript should be done appropriately, which is missing in this manuscript. Please revise it.
• The figures are overlapping the legends, the editing needs to be done.
• The proper explanation of statistical analysis and its importance for describing the results should be mentioned.
• There should not be monotony in representation of the results for instance all should not be represented in bar graph form vise-versa.

Response: Thank you for the constructive suggestion. The corresponding parts have been changed in the revised manuscript. Thank you most sincerely!

Reviewer 2 Report (New Reviewer)

This study estimated the “Appropriate bicarbonate enhances the intracellular water metabolism, nutrient transport and photosynthesis capacities of Coix lacryma-jobi L.”. There are some comments that should be taken into account by authors.

The main concern is:

Title:

(1) Could be improved.

“Appropriate sodium bicarbonate concentration enhances the intracellular water metabolism, nutrient transport and photosynthesis capacities of Coix lacryma-jobi L.”

Introduction:

(2) Abbreviations should be defined in first mention, please revise this issue in the whole ms!

(3) Authors should expand the state of the art by adding new references. There is a lack of information about what is new.

(4) Please illustrate by more details the impact of using sodium bicarbonate on the measured parameters.

(5) At the end of this section, Please describe the novelty of your work in comparison with previous work. What has it added that we did not know before?

Discussion:

(6) I think that this part should be completed. The authors make no attempt to explain the reasons for the difference in plant response to different levels of sodium bicarbonate.  

The mechanism of action of different levels of sodium bicarbonate is missing in all parameters. Why and how these levels increases or decreases a parameter ……what are the possible mechanisms, should be mentioned in the discussion part.

(7) Throughout the manuscript, there is also a lack of indication of what is innovative in this paper and what the authors have contributed to the current state of knowledge.

Conclusion:

(8) Add the significance and future prospect of the study.

References

(9) References: Please use the newest one.

Linguistic quality:

(10) English should be polished. 

Author Response

Title:

Comment 1: Could be improved.

“Appropriate sodium bicarbonate concentration enhances the intracellular water metabolism, nutrient transport and photosynthesis capacities of Coix lacryma-jobi L.”

Response: Thank you very much for your careful review of our manuscript. We greatly appreciate your critique and advice, and hope that the corrections will meet your approval. We have revised the title. Thank you most sincerely!

 

Introduction:

Comment 2: Abbreviations should be defined in first mention, please revise this issue in the whole ms!

Response: Thank you for the constructive suggestion. We have revised it in the whole revised manuscript. Thank you most sincerely!

 

Comment 3: Authors should expand the state of the art by adding new references. There is a lack of information about what is new.

Response: Thanks very much for the reviewer's careful reviews and good comment on our manuscript! It is a good suggestion. The corresponding parts have been changed in the revised manuscript. The total number of the references is 57, only three references are before 2000 and most of the references were published after 2010. Thank you most sincerely!

 

Comment 4: Please illustrate by more details the impact of using sodium bicarbonate on the measured parameters.

Response: Thank you for the constructive suggestion. The corresponding parts have been added in the revised manuscript. Thank you most sincerely!

 

Comment 5: At the end of this section, Please describe the novelty of your work in comparison with previous work. What has it added that we did not know before?

Response: Thank you for the constructive suggestion. The corresponding parts have been changed in the revised manuscript. This study highlights that appropriate bicarbonate could enhance the intracellular water metabolism, nutrient transport, photosynthesis and growth of Coix lacryma-jobi L.

 

Discussion:

Comment 6: I think that this part should be completed. The authors make no attempt to explain the reasons for the difference in plant response to different levels of sodium bicarbonate.  

The mechanism of action of different levels of sodium bicarbonate is missing in all parameters. Why and how these levels increases or decreases a parameter ……what are the possible mechanisms, should be mentioned in the discussion part.

Response: Thanks very much for the reviewer's careful reviews and good comment on our manuscript! The corresponding parts have been added as “In this work, the results exhibit that 2.0 mmol L^-1 sodium bicarbonate effectively enhanced the photosyntheic rate and Φp of Coix lacryma-jobi L., and notably promoted its IWHC, WTR (or NTR), UAF, NAC and NTC, and then reliably improved its intracellular water metabolism and nutrient transport, thereby observably enhanced its growth and biomass formation. Meanwhile, 12.0 mmol L^-1 sodium bicarbonate exhibited notably inhibiting effects on the chlorphyll content, photosynthetic rate, stomatal conductance, transpiration rate and biomass of Coix lacryma-job L. ” in the revised manuscript. Thank you most sincerely!

 

Comment 7: Throughout the manuscript, there is also a lack of indication of what is innovative in this paper and what the authors have contributed to the current state of knowledge.

Response: Thank you for the constructive suggestion. The corresponding parts have been added in the introduction and discussion of the revised manuscript.

 

Conclusion:

Comment 8: Add the significance and future prospect of the study.

Response: Thank you for the constructive suggestion. The corresponding parts have been added in the revised manuscript.

 

References

Comment 9: References: Please use the newest one.

Response: Thank you for the constructive suggestion. We have cited the latest references in the revised manuscript. Thank you most sincerely!

 

Linguistic quality:

Comment 10: English should be polished. 

Response: Thank you very much for your careful review of our manuscript. We greatly appreciate your critique and advice, and hope that the corrections will meet your approval. The English language has been polished by the standard language editing in MDPI, which was marked in red in the revised manuscript. Once again, thank you very much for the comments and suggestions. Thank you most sincerely!

Round 2

Reviewer 1 Report (Previous Reviewer 2)

I looked at the revised manuscript and it is excellently revised and perfect. Please go ahead for publication.

Author Response

Thank you very much for your careful review of our manuscript. Thank you most sincerely for your approval!

Reviewer 2 Report (New Reviewer)

I wonder the authors did not responded to all the critical issues indicated. In my opinion, the manuscript does not improved and is not ready for publication.

The authors should follow abbreviation rule – first define a term, and then use the abbreviated form. e.g. L 35: HCO₃^-

 

The Introduction and Discussion must be improved – the authors did not present any possible mechanisms of sodium bicarbonate on plant growth and photosynthesis, please refer to other literatures on sodium bicarbonate.

For example,

Rapid and Positive Effect of Bicarbonate Addition on Growth and Photosynthetic Efficiency of the Green Microalgae Chlorella Sorokiniana (Chlorophyta, Trebouxiophyceae). Appl. Sci. 2020, 10(13), 4515; https://doi.org/10.3390/app10134515

 

Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta). Photosynth Res 2016 Jun;128(3):259-70. doi: 10.1007/s11120-016-0240-3.

 

 At the end of Introduction section, authors did not describe the novelty of their work in comparison with previous work. What has it added that we did not know before? 

 

The Discussion did not improved. The mechanism of action of different levels of sodium bicarbonate is missing in all parameters. Why and how these levels increases or decreases a parameter ……what are the possible mechanisms, should be mentioned in the discussion part.

Comments for author File: Comments.docx

Moderate editing of English language required

Author Response

Comment 1: I wonder the authors did not respond to all the critical issues indicated. In my opinion, the manuscript does not improve and is not ready for publication.

 Response: Thank you very much for your careful review of our manuscript. We greatly appreciate your critique and advice, and hope that the corrections will meet your approval. Once again, Thank you most sincerely! We have improved the introduction and discussion. Please see responses of comment 3, 4 and comment 5.

Comment 2: The authors should follow abbreviation rule – first define a term, and then use the abbreviated form. e.g. L 35: HCO₃^- 

Response: Thank you for the constructive suggestion. We have revised it in the whole revised manuscript. Please forgive me for my carelessness. Thank you most sincerely!

HCO₃^-: Bicarbonate

d :      specific effective thickness

This has been revised in the revised article. Please see line 35 and line 466.

 

Comment 3: The Introduction and Discussion must be improved – the authors did not present any possible mechanisms of sodium bicarbonate on plant growth and photosynthesis, please refer to other literatures on sodium bicarbonate.

For example,

1. Rapid and Positive Effect of Bicarbonate Addition on Growth and Photosynthetic Efficiency of the Green Microalgae Chlorella Sorokiniana (Chlorophyta, Trebouxiophyceae). Appl. Sci. 2020, 10(13), 4515; https://doi.org/10.3390/app10134515

2. Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta). Photosynth Res 2016 Jun;128(3):259-70. doi: 10.1007/s11120-016-0240-3.

 

Response: Thanks very much for the reviewer's careful reviews and good comment on our manuscript! It is a good suggestion. We have cited the two relevant papers in the revised article (Zhou et al. 2020；Giovanna et al 2016). In the future work, we will give priority to these references. Thank you most sincerely!

1. “Zhouet al. reported that 3~4 mmol L^-1 sodium bicarbonate concentration added to the cultures was conducive to growth and the improvement of carbonic anhydrase activity in macroalgae [22]”. Please see line 61.

It was revised as “Some reports have shown that HCO₃^- not only provides short-term carbon and water sources for plants under drought stress, but also promotes stomatal opening and restores photosynthesis [16-20]. Meanwhile, HCO₃^- could promote carbon and nitrogen metabolism by regulating the activities of key enzyme involved in carbon and nitrogen metabolism, and by participating in the regulation of complex physiological processes including carbon assimilation and nitrogen reduction in plants [15, 21]. Zhou et al. reported that 3~4 mmol L^-1 sodium bicarbonate concentration added to the cultures was conducive to growth and the improvement of carbonic anhydrase activity in macroalgae [22]. Additionally, HCO₃^- could also change the activity of glycolysis and pentose phosphate pathways by the distribution of their substrates, so as to enhance the stress resistance of plants and obtain survival opportunities [8]. Wu et al. systematically summarized the karst adaptive mechanisms of plants in terms of morphology, ecology, photosynthetic capacity, utilization of inorganic nutrients, carbonic anhydrase activity, biodiversity, calcium regulation root organic acid exudation, etc. [1].”

2. “Giovanna et al. reported that added HCO₃^-is beneficial for plant growth and improving photosynthetic efficiency [59]”. Please see line 406.

Comment 4: At the end of Introduction section, authors did not describe the novelty of their work in comparison with previous work. What has it added that we did not know before? 

Response: In this study, we used both the traditional photosynthesis indicators and the new plants’ electrophysiological parameters. plants’ electrophysiological parameters can perform multi-parameter detection (WTR, IWHC,NAC, etc.), which is significantly superior to photosynthesis indicators. Compared with the traditional photosynthesis measurement, plant electrophysiological information can faster and more effective reflect the physiological response of plants to inorganic carbon. Thank you most sincerely!

Comment 5: The Discussion did not improved. The mechanism of action of different levels of sodium bicarbonate is missing in all parameters. Why and how these levels increases or decreases a parameter ……what are the possible mechanisms, should be mentioned in the discussion part.

Response: Bicarbonate can have contrasting effects on plant growth and development depending on its concentration. At the appropriate concentrations of the bicarbonate condition, the positive effect is greater than the negative effect, such as: conducive to carbon assimilation; stimulate stomatal opening and improves drought resistance etc. In contrast, at the excessive high concentrations of the bicarbonate condition, it can bring anionic toxicity; physiological drought; stomata closure; reducing metabolite synthesis and transport etc. It has reported that high concentrations of HCO₃^- can reduce the uptake of Fe in plants by limiting the expression of Fe acquisition genes, leading to Fe deficiency [12],

The electrical parameters can be responded to bicarbonate stress rapidly. The stronger the growth of plants, the higher IC, IWHC, WTR, NAC， and the lower its IR, IZ, IXc and IXL value.

Our previous works have shown that in karst environments, plants can alternately use bicarbonate from soil and atmospheric carbon dioxide, and when plants encounter some adversity, the stomatal conductance decreases, carbonic anhydrase activity increases, and the use of bicarbonate also increases, improving the intercellular water status, nutrient transport, photosynthetic capacity, which in turn increases the use of atmospheric carbon dioxide by plants [1,15-16, 20].

 

Comment 6: Moderate editing of English language required

Response: We have used the editing services by MDPI to improve the English of our manuscript.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Review:  Appropriate bicarbonate enhances the intracellular water metabolism, nutrient transport and photosynthesis capacities of Coix lacryma-jobi L

 

General comments

The topic is interesting due to karstic areas covering many hectares of arable land producing a decrease in the plant yield and consequently an economic impact. As the authors mentioned, studies in plant performance on karstic conditions are quite difficult due to the complexity of the stress which has multifactorial parts like (high pH, high bicarbonate, drought, and high). Several studies are focused on A thaliana, the model plant in dicotyledonous, which help to understand the general mechanism of non-tolerant to karstic conditions species.  Studies in crops that can be naturalized to mild karstic conditions, like the present study, may elucidate the different physiological mechanisms.

The general comment is positive with some little comments that can help the authors to have a final and better version of the present manuscript. Some sentences are not clear, perhaps it is a translation problem. As I’m not an English native, I detect some sentence constructions that are not clear. Congrats to the authors, keep doing science.

 

Introduction: In general, the introduction is clear and contains the main information to follow the story

Line 36 à Is not well defined the difference between calcareous and karstic differences.

Line 43 to 59 àThe Introduction is focused on the benefits of bicarbonate. Also, bicarbonate at higher concentrations produces a decrease in plant yield that is not well introduced.

 

Material and methods: I missed some important information in the part of material and methods and statistical analysis. Here, I add point by point

Line 103  à“All other reagents were analytical grade” In this sentence, I understood that there are other chemicals that you were using and are not here?

Line 105à” Three-leaf seedlings with growth uniformly of Coix lacryma-jobi L. ‘Yizhu ’ were used as experimental materials”. What are technical replicates? What are biological replicates?

Line 112 à How old were the plants?

Line 115à “Based on the preliminary experimental results and the sodium bicarbonate concentration (1.2~3.3 mmol 116 L-1) in the field planting soils of Coix lacryma-jobi L. Which previous experiment? Where is the citation? Where is this data? The authors mentioned several times these data. In my opinion, this is quite important and can improve massively the quality of the paper.

Line 125-127à  “Generally, the salt stress occurred when the concentration of sodium was in the 125 range of 25 -50 mM [41]. Hence, the Coix lacryma-jobi L. seedlings did not suffer from the salt stress because the concentration of sodium bicarbonate was far below 25 mM in this study”. In my opinion, this sentence is not appropriate here, you can add it in the introduction. Also, can be placed in the discussion.

Line 208 à Statistical analysis you should add something like that: The normal distribution of data was confirmed by Levene’s test. Treatment effects were analyzed by one-way ANOVA. Post-hoc analyses for the significance of differences were realized using the Tukey test.

 

Results

Line 215 à Figure 1, Figure 2 and Figure 3 add brackets or capital to the graphic letter to make it more aesthetic. Otherwise is confusing with statistical letter

Line 215à “The concentrations of the HCO3- increased with the increasing sodium bicarbonate added”. I do not understand the meaning of the sentence. For me, this has no sense.

Figure Captation change “Small letters indicate significant differences at 5 % level (p < 0.05)” to  Letters indicate significant differences between treatments (n= 3; p < 0.05 according to Tukey’s HSD).

 

Conclusions

Line 216 and 218à It is very bizarre to indicate a sentence using numbers. Please, reformulate the sentence.

Line 222 to 224à “The results presented here reveal that the appropriate concentration of sodium bicarbonate significantly enhanced the growth of Coix lacryma-jobi L., while 7.0~12.0 mmol L-1 sodium bicarbonate exhibited an inhibiting effect on Coix lacryma-job L. growth”.  To me, this is not a result.  This sentence should be placed in the discussion or conclusion part.

Line 227 à Figure 1. Effects of sodium bicarbonate on the growth of Coix lacryma-jobi L. Values indicate the 227 mean ± SD of three replicates. Small letters indicate significant differences at a 5 % level (p < 0.05).

 

Discussion

Line 312 à Change HCO^-₃ to Bicarbonate. It is not appropriate to initiate a sentence with numbers or chemical nomenclature.

Line 313 à “Bicarbonate has both positive and negative effects on plant growth and development” This statement is true for all the plant species or only for the plant of this study?

Line313 à I suggest changing the sentence “Bicarbonate has both positive and negative effects on plant growth and development” to “Bicarbonate can have contrasted effect on the plant growth and development depending on the concentration”

Line 316-318 à Do not explain again the results, remove significantly (p < 0.05)

Line 318à I suggest changing “non-added sodium bicarbonate” to “control conditions”

Line 320 à” We also found that the sodium bicarbonate concentration in the field planting soils of Coix lacryma-jobi L. in Guizhou planting areas was about 1.2~3.3 mmol L-1.” This information is really interesting. Where did you find this, it is an experiment that your lab performed, at another lab? Where is this data? Where is the citation?

 

Line 327 à  Rao and Wu citation. In the way that the sentence is written seems that the author found a similar response in the same plant. Perhaps is better to say the authors Rao and Wu found similar results than yours in other plant species (Camptotheca acuminate seedlings)

Author Response

General comments

The topic is interesting due to karstic areas covering many hectares of arable land producing a decrease in the plant yield and consequently an economic impact. As the authors mentioned, studies in plant performance on karstic conditions are quite difficult due to the complexity of the stress which has multifactorial parts like (high pH, high bicarbonate, drought, and high). Several studies are focused on A thaliana, the model plant in dicotyledonous, which help to understand the general mechanism of non-tolerant to karstic conditions species.  Studies in crops that can be naturalized to mild karstic conditions, like the present study, may elucidate the different physiological mechanisms.

1. The general comment is positive with some little comments that can help the authors to have a final and better version of the present manuscript. Some sentences are not clear, perhaps it is a translation problem. As I’m not an English native, I detect some sentence constructions that are not clear. Congrats to the authors, keep doing science.

 Response: Thanks very much for the reviewer's careful reviews on our manuscript! We appreciate for reviewer’s warm work earnestly, and hope that the correction will meet with approval. Once again, thank you very much for the comments and suggestions.

The corresponding has been changed in the revised article

Introduction: In general, the introduction is clear and contains the main information to follow the story

Comment 1：Line 36    Is not well defined the difference between calcareous and karstic differences.

Response: Calcification is a typical characteristic of karst soils. Calcareous soils are identified by the presence of calcium carbonate in the parent material and an accumulation of lime. Calcareous soils generally have more than 15% of CaCO₃ in various forms (Taalab et al., 2019). HCO₃^- concentration in calcareous soils was usually high. Calcareous is with high content of calcium carbonate. contain high quantity of calcite. Low nutrient availability and biological activity are the main challenges in calcareous soils. 

Karst environments are regions where sparingly soluble rocks outcrop and efficient acid hydrolysis creates spectacular dissolution landforms. The release of CO₂ from karst waters to the atmosphere causes precipitation of calcium-carbonate deposits. Karst is the whole of the soluble rock areas which contain the calcium carbonate, calcium magnesium carbonate, and so on. High HCO₃^- concentration is usually observed in Karst soils.

Corresponding Reference:

Taalab A S, Ageeb G W, Siam H S, and Safaa A. Some Characteristics of Calcareous soils. A review [J]. Middle East J, 2019, 8(1): 96-105.

 

Comment 2：Line 43 to 59     The Introduction is focused on the benefits of bicarbonate. Also, bicarbonate at higher concentrations produces a decrease in plant yield that is not well introduced.

 Response: The high concentration HCO₃^- can reduce the uptake of Fe in plants by limiting the expression of Fe acquisition genes, leading to Fe deficiency (García et al, 2014; Ding et al, 2020). The Fe deficiency strongly limits the biosynthesis of chlorophyll (Kallala et al, 2018), which will result in a significant decrease for photosynthesis.

It has been added in the revised article

Corresponding Reference

García M J, García‐Mateo M J, Lucena C, Romera FJ, Rojas CL, Alcántara E, Pérez-Vicente R. Hypoxia and bicarbonate could limit the expression of iron acquisition genes in Strategy I plants by affecting ethylene synthesis and signaling in different ways[J]. Physiologia Plantarum, 2014, 150(1): 95-106. DOI: 10.1111/ppl.12076.

Ding W, Clode P L, Lambers H. Effects of pH and bicarbonate on the nutrient status and growth of three Lupinus species[J]. Plant and Soil, 2020, 447: 9-28. DOI: 10.1007/s11104-019-03980-8.

Kallala N, M’sehli W, Jelali K, Kais Z, Mhadhbi H. Inoculation with efficient nitrogen fixing and indoleacetic acid producing bacterial microsymbiont enhance tolerance of the model legume Medicago truncatula to iron deficiency[J]. BioMed research international, 2018, 9134716. DOI: 10.1155/2018/9134716.

 

Comment 3：Material and methods: I missed some important information in the part of material and methods and statistical analysis. Here, I add point by point

Line 103    “All other reagents were analytical grade” In this sentence, I understood that there are other chemicals that you were using and are not here?

Response: Thanks for the careful reviews. Please excuse our slip of the pen. All  reagents were analytical grade. This has been revised  in the revised article. Please see line 109.

 

Comment 4：Line 105   Three-leaf seedlings with growth uniformly of Coix lacryma-jobi L. ‘Yizhu ’ were used as experimental materials”. What are technical replicates? What are biological replicates?

Response: It was set three replicates, and each replicate contained nine plants as the biological replicates. One plant was randomly selected from the five plants on the two diagonals. At last, just three plants were used for investigation.

 

Comment 5：Line 112    How old were the plants?

Response: The plants seeding was 14 days before treatment. And the whole treatment cycle was also 14 days.

 

Comment 6：Line 115    “ Based on the preliminary experimental results and the sodium bicarbonate concentration (1.2~3.3 mmol L^-1) in the field planting soils of Coix lacryma-jobi L. Which previous experiment? Where is the citation? Where is this data? The authors mentioned several times these data. In my opinion, this is quite important and can improve massively the quality of the paper.

Response: We sincerely thank the reviewer for the careful reviews! According to the previous research of our research group. Our team has recently published many articles about the effects of bicarbonate concentration on the field planting soils. Such as:

Luo, L.; Wu, Y.; Li, H.; Xing, D.; Zhou, Y.; Xia, A. Drought Induced Dynamic Traits of Soil Water and Inorganic Carbon in Different Karst Habitats. Water 2022, 14, 3837. DOI: 10.3390/w14233837 

Yao, K.; Wu, Y. Rhizospheric bicarbonate improves glucose metabolism and stress tolerance of Broussonetia papyrifera L. seedlings under simulated drought stress. Russian Journal of Plant Physiology. 2021, 68(1), 126-135. DOI: 10.1134/S1021443721010209.

Wu, Y. Is bicarbonate directly used as substrate to participate in photosynthetic oxygen evolution. Acta Geochimica 2021, 40, 650–658.

Wu, Y. Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality. Acta Geochimica 2023, 42, 77–88.

 

Comment 7：Line 125-127   “Generally, the salt stress occurred when the concentration of sodium was in the 125 range of 25 -50 mM [41]. Hence, the Coix lacryma-jobi L. seedlings did not suffer from the salt stress because the concentration of sodium bicarbonate was far below 25 mM in this study”. In my opinion, this sentence is not appropriate here, you can add it in the introduction. Also, can be placed in the discussion.

Response: Thanks for constructive suggestion. It has been placed to the discussion in the revised article. Please see line 348.

 

Comment 8：Line 208    Statistical analysis you should add something like that: The normal distribution of data was confirmed by Levene’s test. Treatment effects were analyzed by one-way ANOVA. Post-hoc analyses for the significance of differences were realized using the Tukey test.

 Response: Thanks for constructive suggestion. The data are shown as the mean ±SD. The data were subjected to analysis of variance (ANOVA). The means of the different groups were compared via Tukey’s test (p < 0.05). Small letters indicate significant differences between treatments (n= 3; p < 0.05). The corresponding statement has been changed in the revised article. Please see line 217.

 

Results

Comment 9：Line 215     Figure 1, Figure 2 and Figure 3 add brackets or capital to the graphic letter to make it more aesthetic. Otherwise is confusing with statistical letter

Response: We sincerely thank the reviewer for the careful reviews! All the Figures has added brackets. The corresponding figures have been changed in the revised article.

 

Comment 10：Line 215    “The concentrations of the HCO₃^- increased with the increasing sodium bicarbonate added”. I do not understand the meaning of the sentence. For me, this has no sense.

Response:  We have deleted it in the revised article. Please see line 225.

 

Comment 11：Figure Captation change “Small letters indicate significant differences at 5 % level (p < 0.05)” to Letters indicate significant differences between treatments (n= 3; p < 0.05 according to Tukey’s HSD).

 Response: The data are shown as the mean ±SD. The data were subjected to analysis of variance(ANOVA). The means of the different groups were compared via Tukey’s test (p < 0.05). Small letters indicate significant differences between treatments (n= 3; p < 0.05). The corresponding statements have been changed all in the revised article. respectively.

 

Conclusions

Comment 12：Line 216 and 218    It is very bizarre to indicate a sentence using numbers. Please, reformulate the sentence.

Response: Thanks, we have corrected it as ‘Sodium bicarbonate of 2.0 mmol L^-1 significantly (p < 0.05) promoted plant height, shoot part fresh weight and plant fresh weight compared with non added sodium bicarbonate (control conditions)’. And so on in the whole revised article.

 

Comment 13：Line 222 to 224   “The results presented here reveal that the appropriate concentration of sodium bicarbonate significantly enhanced the growth of Coix lacryma-jobi L., while 7.0~12.0 mmol L^-1 sodium bicarbonate exhibited an inhibiting effect on Coix lacryma-job L. growth”.  To me, this is not a result.  This sentence should be placed in the discussion or conclusion part.

 Response: Thanks for your suggestion. We have deleted this sentence in the result part. And you can see it in the discussion part. Please see Line328.

 

Comment 14：Line 227   Figure 1. Effects of sodium bicarbonate on the growth of Coix lacryma-jobi L. Values indicate the  mean ± SD of three replicates. Small letters indicate significant differences at a 5 % level (p < 0.05).

 Response: Thanks for your suggestion. We have changed it. Values indicate the mean ± SD of three replicates. Small letters indicate significant differences between treatments (n= 3; p < 0.05 according to Tukey’s HSD).

 

Discussion

Comment 15：Line 312   Change HCO₃^- to Bicarbonate. It is not appropriate to initiate a sentence with numbers or chemical nomenclature.

 Response: Thanks for your suggestion. We have changed it in the revised article.

 

Comment 16：Line 313    “Bicarbonate has both positive and negative effects on plant growth and development” This statement is true for all the plant species or only for the plant of this study?

Response: It is a good question. This statement is true for all the plant species in the existing knowledge.

 

Comment 17：Line313   I suggest changing the sentence “Bicarbonate has both positive and negative effects on plant growth and development” to “Bicarbonate can have contrasted effect on the plant growth and development depending on the concentration”

Response: Thanks for your constructive suggestion. The corresponding sentence has been changed in the revised article. Please see line 343.  

 

Comment 18：Line 316-318   Do not explain again the results, remove significantly (p < 0.05)

Response: Thanks for your careful reviews. The corresponding statement has been changed in the revised article. We have deleted it in the result part. And it was only reserved in the discussion part.

 

Comment 19：Line 318   I suggest changing “non-added sodium bicarbonate” to “control conditions”

Response: Thanks for the careful reviews. The corresponding statement has been changed in the revised article.

 

2. Line 320   ” We also found that the sodium bicarbonate concentration in the field planting soils of Coix lacryma-jobi  in Guizhou planting areas was about 1.2~3.3 mmol L-1.” This information is really interesting. Where did you find this, it is an experiment that your lab performed, at another lab? Where is this data? Where is the citation?

Response: We sincerely thank the reviewer for the careful reviews! According to the previous research of our research group. Our team has recently published many articles about the effects of bicarbonate concentration on the field planting soils. Such as:

Luo, L.; Wu, Y.; Li, H.; Xing, D.; Zhou, Y.; Xia, A. Drought Induced Dynamic Traits of Soil Water and Inorganic Carbon in Different Karst Habitats. Water 2022, 14, 3837. DOI: 10.3390/w14233837 

Yao, K.; Wu, Y. Rhizospheric bicarbonate improves glucose metabolism and stress tolerance of Broussonetia papyrifera L. seedlings under simulated drought stress. Russian Journal of Plant Physiology. 2021, 68(1), 126-135. DOI: 10.1134/S1021443721010209.

Wu, Y. Is bicarbonate directly used as substrate to participate in photosynthetic oxygen evolution. Acta Geochimica 2021, 40, 650–658.

Wu, Y. Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality. Acta Geochimica 2023, 42, 77–88.

 

3. Line 327    Rao and Wu citation. In the way that the sentence is written seems that the author found a similar response in the same plant. Perhaps is better to say the authors Rao and Wu found similar results than yours in other plant species (Camptotheca acuminateseedlings)

Response: Thanks for the careful reviews. The corresponding statement has been changed in the revised article. Please see line 362.

Reviewer 2 Report

Paper Title- Appropriate bicarbonate enhances the intracellular water metabolism, nutrient transport and photosynthesis capacities of Coix lacryma-jobi L.

Ms ID/ Ref no.:

Journal Name: Agronomy

Reviewer Recommendation: Major Revision

 

Reviewer’s Comments to Authors:

1.      Grammatical errors are present, please revise the whole manuscript to remove any possible grammatical and typos errors.

2.      Error in sentence formation, please revise the whole manuscript to avoid the use of long sentences. Paraphrasing requires at several places in the Ms.

3.      Please check the formatting and editing according to the Journal’s requirements.

4.      The Ms. requires a rigorous update on the References as large number of old references have been cited in the Ms. Please see and follow the Reviewer’s observation and suggestion in the reference section comment.

Abstract

1.      Line 15-20, page 1; please avoid the usage of large sentences.

2.      The abstract fails to answer whether the cultivation in karst areas is harmful or helpful, i.e., the bicarbonate stress should be given at field level to obtain an increase in the studied parameters.

Introduction

1.      The references used to describe the introduction section includes a few old papers. The references can be therefore, updated accordingly.

2.      Overall, the introduction is well written covering almost all the aspects of the study. However, the areas that have been reported to contain high concentration of HCO₃^- can also be added under this section.

Materials and Methods

1.      Line 103-105, page 3; the methodology and the conditions adopted for seedling growth is missing.

2.      Line 116, page 3; please give the reference to the preliminary experiment is published previously.

3.      Line 125-128, page 3; it is good to mention the salt stress range but please specify the observed salt concentration as well.

4.      Line 211, page 5; please specify the post-hoc test if used for determining the significance as indicated by different letters- a,b,c

References

Introduction, Result, and Discussion sections poorly cited with the references and strongly recommended to update and validation with previous studies. Therefore, the relevant papers listed below should be considered and cited appropriately in the Introduction, Result, and Discussion sections of this Ms. which will certainly upgrade and enhance the Ms. quality significantly. Omitting any of these papers will certainly compromise the scientific quality of this manuscript.

·         Kumar D., et al., (2023). Titanium dioxide nanoparticles potentially regulate the mechanism (s) for photosynthetic attributes, genotoxicity, antioxidants defense machinery, and phytochelatins synthesis in relation to hexavalent chromium toxicity in Helianthus annuus L. Journal of Hazardous Materials. https://doi.org/10.1016/j.jhazmat.2023.131418

·         Gupta, P., et al., (2022). 24-Epibrassinolide Regulates Functional Components of Nitric Oxide Signalling and Antioxidant Defense Pathways to Alleviate Salinity Stress in Brassica juncea L. cv. Varuna. Journal of Plant Growth Regulation, 1-16.

Results

1.      Line 213, page 5; please check and correct the 3.1 heading.

2.      Line 215, page 5; how was the bicarbonate concentration determined and its result to validate the statement

3.      Line 247-257, page 6; with the currently reported results will it be fair to conclude 2.0 mmol L^-1 is optimum concentration under the given factors.

4.      Line 289, page 7; please check and correct “owned an inhibiting”

5.      Line 297-298, page 8; please check and correct “The Fv/Fm of Coix lacryma-jobi L. was no significant (p < 0.05) difference”

Discussion

1. Line 312-332, page 8; the major focus of this part seems to be result only rather than the discussion of the obtained results.
2. The discussion for the electrical parameters a 7, and 12.0 mmol L^-1 should be improved.
3.  The discussion across some of the paragraphs focuses firstly on discussing the results of other authors which is then followed by the findings of the current study.

Conclusion

1.      Line 412, page 10; please check and correct the “Conclusions”

Figures and Tables

1.      The legends of table need to be modified including the full forms of all the abbreviations used across the table.

2.      The statement mentioned as (a) below both the tables seems to be an inappropriate way of writing the information. It can be modified such that it becomes a part of the legend.

 

Author Response

Reviewer’s Comments to Authors:

1. Grammatical errors are present, please revise the whole manuscript to remove any possible grammatical and typos errors.
2. Error in sentence formation, please revise the whole manuscript to avoid the use of long sentences. Paraphrasing requires at several places in the Ms.
3. Please check the formatting and editing according to the Journal’s requirements.
4. The Ms. requires a rigorous update on the References as large number of old references have been cited in the Ms. Please see and follow the Reviewer’s observation and suggestion in the reference section comment.

 

Abstract

Comment 1：Line 15-20, page 1; please avoid the usage of large sentences.

Response: We sincerely thank the reviewer for the careful reviews! The large sentences has been rewritten.

 

 

Comment 2： The abstract fails to answer whether the cultivation in karst areas is harmful or helpful, i.e., the bicarbonate stress should be given at field level to obtain an increase in the studied parameters.

Response: Plants grown in karst areas often suffer from bicarbonate stress. Karst ecological stresses is harmful to plant growth, especially high bicarbonate, drought, high pH, etc.

 

 

Introduction

Comment 3：The references used to describe the introduction section includes a few old papers. The references can be therefore, updated accordingly.

Response: We sincerely thank the reviewer for the careful reviews! We have added five new references. Such as:

 

1. Gupta, Praveen，Seth, Chandra Shekhar. 24-Epibrassinolide Regulates Functional Components of Nitric Oxide Signalling and Antioxidant Defense Pathways to Alleviate Salinity Stress in Brassica junceaL. cv. Varuna. Journal of Plant Growth Regulation, 2022, 1-16. DOI: 10.1007/s00344-022-10884-y
2. 2. Luo, L.; Wu, Y.; Li, H.; Xing, D.; Zhou, Y.; Xia, A. Drought Induced Dynamic Traits of Soil Water and Inorganic Carbon in Different Karst Habitats. Water 2022, 14, 3837. DOI: 10.3390/w14233837
3. 3. García M J, García‐Mateo M J, Lucena C, Romera FJ, Rojas CL, Alcántara E, Pérez-Vicente R. Hypoxia and bicarbonate could limit the expression of iron acquisition genes in Strategy I plants by affecting ethylene synthesis and signaling in different ways[J]. Physiologia Plantarum, 2014, 150(1): 95-106. DOI: 10.1111/ppl.12076.

4.Ding W, Clode P L, Lambers H. Effects of pH and bicarbonate on the nutrient status and growth of three Lupinus species[J]. Plant and Soil, 2020, 447: 9-28. DOI: 10.1007/s11104-019-03980-8.

5.Kallala N, M’sehli W, Jelali K, Kais Z, Mhadhbi H. Inoculation with efficient nitrogen fixing and indoleacetic acid producing bacterial microsymbiont enhance tolerance of the model legume Medicago truncatula to iron deficiency[J]. BioMed research international, 2018, 9134716. DOI: 10.1155/2018/9134716.

 

Comment 3：Overall, the introduction is well written covering almost all the aspects of the study. However, the areas that have been reported to contain high concentration of HCO₃^- can also be added under this section.

Response: We sincerely thank the reviewer for the careful reviews! This is a good suggestion. We have added this part. Please see line 48-line 52.

 

Materials and Methods

Comment 4：Line 103-105, page 3; the methodology and the conditions adopted for seedling growth is missing.

Response: Thanks for your suggestion. The corresponding statement has been added in the revised manuscript. Please see line 112and line 126-132 in the revised article.

 

Comment 5：Line 116, page 3; please give the reference to the preliminary experiment is published previously.

Response: We sincerely thank the reviewer for the careful reviews! According to the previous research of our research group. Our team has recently published many articles about the effect of bicarbonate concentration to the field planting soils. Such as:

Luo, L.; Wu, Y.; Li, H.; Xing, D.; Zhou, Y.; Xia, A. Drought Induced Dynamic Traits of Soil Water and Inorganic Carbon in Different Karst Habitats. Water 2022, 14, 3837. DOI: 10.3390/w14233837

Yao, K.; Wu, Y. Rhizospheric bicarbonate improves glucose metabolism and stress tolerance of Broussonetia papyrifera L. seedlings under simulated drought stress. Russian Journal of Plant Physiology. 2021, 68(1), 126-135. DOI: 10.1134/S1021443721010209.

Wu, Y. Is bicarbonate directly used as substrate to participate in photosynthetic oxygen evolution. Acta Geochimica 2021, 40, 650–658.

Wu, Y. Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality. Acta Geochimica 2023, 42, 77–88.

 

Comment 6：Line 125-128, page 3; it is good to mention the salt stress range but please specify the observed salt concentration as well.

Response: According to the previous research of our research group. we observed the maximum bicarbonate concentration was up to 3.99 mm/L in karst soil.

In this experiment, the sodium did not reach the condition of salt stress. It is indicated that the sodium salt stress is 25-50 mM (Haro et al, 1993). However, in our experiments, the highest sodium was no more than 12 mM, which has not yet reached the salt-stressed condition (far below 25mM). Therefore, we regarded the bicarbonate instead of sodium affected the growth of plants.

The sodium did not reach the condition of salt stress. Neither Na⁺ or K⁺ reached toxic concentrations (Lu, 2009; Zhang,2018).

1.Lu, S., Zhang, S., Xu, X., Korpelainen, H. , & Li, C.. Effect of increased alkalinity on Na+ and K+ contents, lipid peroxidation and antioxidative enzymes in two populations of Populus cathayana, Biologia Plantarum. 2009. 53 (3): 597-600.

2.Hui-hui Zhang, Nan Xu, Xuye Wu, Junrui Wang, Songliang Ma, Xin Li & Guangyu Sun. Effects of four types of sodium salt stress on plant growth and photosynthetic apparatus in sorghum leaves, Journal of Plant Interactions, 2018. 13:1, 506-513, DOI:10.1080/17429145.2018.1526978

3.Haro R; Banuelos M. A.; Quintero F. J.; Rubio F.; Rodriguez-Navarro A.. Genetic basis of sodium exclusion and sodium tolerance in yeast, A model for plants, Physiologia Plantarum. 1993, 89: 868－874.

 

Comment 7：Line 211, page 5; please specify the post-hoc test if used for determining the significance as indicated by different letters- a,b,c

Response: The data were subjected to analysis of variance (ANOVA). The means of the different groups were compared via Tukey’s test (p < 0.05). Small letters indicate significant differences between treatments (n= 3; p < 0.05).

 

References

Comment 8：Introduction, Result, and Discussion sections poorly cited with the references and strongly recommended to update and validation with previous studies. Therefore, the relevant papers listed below should be considered and cited appropriately in the Introduction, Result, and Discussion sections of this Ms. which will certainly upgrade and enhance the Ms. quality significantly. Omitting any of these papers will certainly compromise the scientific quality of this manuscript.

•  
• (Gupta, Praveen，Seth, Chandra Shekhar)    Gupta, P., et al., (2022). 24-Epibrassinolide Regulates Functional Components of Nitric Oxide Signalling and Antioxidant Defense Pathways to Alleviate Salinity Stress in Brassica juncea L. cv. Varuna. Journal of Plant Growth Regulation, 1-16. DOI: 10.1007/s00344-022-10884-y

Response: Thanks very much for the reviewer's careful reviews and good comment on our manuscript! It is a good suggestion. We have cited the relevant paper in the revised article. The total number of the references is 56, only three references are before 2000 and most of the references were published after 2010.

 

Results

Comment 9：Line 213, page 5;   please check and correct the 3.1 heading.

Response: Thanks very much for the reviewer's careful reviews. We have revised the head 3.1.

Comment 10：Line 215, page 5;   how was the bicarbonate concentration determined and its result to validate the statement.

Response: Four treatments were designed with 0, 2.0, 7.0 and 12.0 mmol L^-1 sodium bicarbonate, respectively. The concentration of the bicarbonate dissolution in the medium were observed by titration. But, it has no sense to the whole article. We have deleted it in the revised article.

 

Comment 11：  Line 247-257, page 6; with the currently reported results will it be fair to conclude 2.0 mmol L^-1 is optimum concentration under the given factors.

Response: From this study, the optimum concentration is 2.0 mmol L^-1 bicarbonate. It enhanced the photosynthesis of Coix lacryma-jobi L. and promoted its growth.

 

Comment 12：Line 289, page 7; please check and correct “owned an inhibiting”         

Response: We have changed the sentence as “bicarbonate of 7.0~12.0 mmol L^-1 inhibit the growth of Coix lacryma-job L.”. Please see line 314.

 

Comment 13：Line 297-298, page 8; please check and correct “The Fv/Fm of Coix lacryma-jobi L. was no significant (p < 0.05) difference”

Response: Yes, the Fv/Fm of Coix lacryma-jobi L. was no significant (p < 0.05) difference in 0, 2.0, 7.0 and 12.0 mmol L^-1 sodium bicarbonate treatments.

 

Discussion

Comment 14：Line 312-332, page 8; the major focus of this part seems to be result only rather than the discussion of the obtained results.

Response: Thanks very much for the reviewer's careful reviews. We have deleted this part.

 

Comment 15：The discussion for the electrical parameters at 7, and 12.0 mmol L^-1 should be improved.  363-364

 

Response: Thanks very much for the reviewer's careful reviews. In this work, 7.0 and 12.0 mmol L^-1 sodium bicarbonate owned inhibiting effects on the chlorophyll content, photosynthetic rate, stomatal conductance and transpiration rate of Coix lacryma-job L. The intensification of osmotic stress and the decrease of transpiration rate caused by high HCO₃^- concentration might be the mainly reasons for the decrease of photosynthetic capacity of Coix lacryma-job L.

 

Comment 16： The discussion across some of the paragraphs focuses firstly on discussing the results of other authors which is then followed by the findings of the current study.

Response: Thanks very much for the reviewer's careful reviews. The corresponding has been changed in the revised article.

 

Conclusions

Comment 17： Line 412, page 10; please check and correct the “Conclusions”

Response: OK. The conclusions has been changed in the revised article.

Figures and Tables

Comment 18：The legends of table need to be modified including the full forms of all the abbreviations used across the table.

Response: Thanks for your suggestion. The corresponding statement has been changed in the revised manuscript. We have made a table of the abbreviations in the revised article.

 

Abbreviations

C       capacitance

Z       impedance

R        resistance

F         force

Xc      capacitive reactance

XL      inductive reactance

IC       intrinsic capacitance

IR       intrinsic resistance

IZ        intrinsic impedance

IXc      intrinsic capacitive reactance

IXL      intrinsic inductive reactance

IWHC     ntracellular water-holding capacity

IWUE      intracellular water use efficiency

IWHT       intracellular water-holding time

WTR        water transfer rate

UNF        nutrient flux per unit area

NAF     flow of nutrient

NTR      nutrient transfer rate

NTC      nutrient transport capacity

NAC       nutrient active transport capacity

 

Comment 19：The statement mentioned as (a) below both the tables seems to be an inappropriate way of writing the information. It can be modified such that it becomes a part of the legend.

Response: Thanks for your suggestion. The corresponding statement has been changed in the revised article.

Reviewer 3 Report

 

L386: The authors are making unsubstantial claims about the involvement of HCO3 in electron transfer in the chloroplast reaction centers; they state: “ This positive effects probably were related to HCO3 utilized by Coix lacryma-jobi L. from the soils, which was tightly [closely] in the reaction center of PSII, which played an important role in the transfer of electrons and improved the photosynthetic rate of plants” . To the best of my knowledge, bicarbonate is not involved in electron transport in the chloroplast or in the mitochondrion.

A good part of the results reported in the manuscript are speculative. The bulk of what has been reported in the manuscript relates to the plant’s electrophysiological activities including charge separation, electron movement, proton and dielectric transport, the main processes responsible for energy production chloroplasts and mitochondria and nutrient transport.  These inferences were made on electrical measurements made on whole crushed leaves (clamped) rather than at the membrane level or on phloem exudates or vacuolar fluids. The method used does not distinguish between apoplastic water and dissolved salts, osmotica in the vacuole and phloem sap (symplast); the method does not take into account the all-important compartmentation of ions in plant cells and tissues.

Actually, the method was only recently reported by authors from the same group of researchers who did the present study:

Zhang, C.; Wu, Y.; Su, Y.; Xing, D.; Dai, Y.; Wu, Y.; Fang, L. A plant’s electrical parameters indicate its physiological state: A 519 study of intracellular water metabolism. Plants. 2020, 9, 1256. DOI: 10.3390/plants9101256. 520 40.

Zhang, C.; Wu, Y.; Su, Y.; Li, H.; Fang, L.; Xing, D. Plant’s electrophysiological information manifests the composition and 521 nutrient transport characteristics of membrane proteins. Plant Signaling & Behavior. 2021, 16(7), e1918867. DOI: 522 10.1080/15592324.2021.1918867.

Some other notes that need to be addressed:

7 mM NaHCO3 in not stressful by the authors own statement in the introduction.

7 mM NaHCO3 had no effect on chlorophyl content (SPAD) contrary to what has been stated.

The higher concentrations of NaHCO3 did not affect Pn despite a marked reduction in stomata conductance; why? If the authors imply that the reduction in mesophyll  CO2 was compensated by CO2 release from HCO3 breakdown, they should show it. They should also measure internal CO2 conc.

The authors used vague terms to describe physiological phenomena without defining what those terme mean. For instance, water metabolism; are they referring to water activity, water potential or osmotic potential? In what part of the plant or tissue? “appropriate supply of biocarbonate” ? appropriate in relation to what? Besides, you measured 2 and 7 mM; how can we know if 3 or 4 mM is not more appropriate?

The manuscript needs to be revised for English language:

L40, 41, 61, 74, 97 : frequent  use of “etc” and “so on”;

L70 you mean “rural areas”?

L84 you mean theoritically ?

L99, 105, 113, 117 re-phrase

L106 “unit of roots” you mean root gs?

L108 incomplete sentence;

L129-136 how FW was measured? On subsamples?

L147 you mean self-made capacitor?

No need to use the scientific name of the plant with  the authority in all the text.

 

 

Author Response

Comment 1：L386: The authors are making unsubstantial claims about the involvement of HCO₃ in electron transfer in the chloroplast reaction centers; they state: “ This positive effects probably were related to HCO₃ utilized by Coix lacryma-jobi L. from the soils, which was tightly [closely] in the reaction center of PSII, which played an important role in the transfer of electrons and improved the photosynthetic rate of plants” . To the best of my knowledge, bicarbonate is not involved in electron transport in the chloroplast or in the mitochondrion.

Response: Some reports have shown that HCO₃^- not only provided short-term carbon and water sources for plants under drought stress, but also promoted stomatal opening and restored photosynthesis [16-20]. Meanwhile, HCO₃^- could promote plant’s carbon and nitrogen metabolism by regulating the key enzyme activities of carbon and nitrogen metabolism, and participate in the regulation of complex physiological processes including carbon assimilation and nitrogen reduction in plants [15, 21]. Additionally, HCO₃^- could also change the distribution of glycolysis and pentose phosphate pathways by affecting glucose metabolism disproportionation, so as to enhance the stress resistance of plants and obtain survival opportunities [8].

Our previous works have shown that in karst environments, plants can alternately use bicarbonate from soil and atmospheric carbon dioxide, and when plants encounter some adversity, the stomatal conductance decreases, carbonic anhydrase activity increases, and the use of bicarbonate also increases, improving the intercellular water status, which in turn increases the use of atmospheric carbon dioxide by plants [1,15-16, 20].

Meantime, bicarbonate can stimulate plants release oxygen, which called bicarbonate effect [55]. Recently, Wu even proposed that bicarbonate may be directly used as substrate to participate in photosynthetic O₂ evolution, resulting in the chemical equilibria, HCO₃^-+H⁺®1/2O₂+2e^-+2H⁺+CO₂, which provides with electrons, and concentrates CO₂ into Calvin cycle for photosynthetic carbon assimilation [51, 53, 54].

 

Corresponding reference:

51. Wu, Y. Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality. Acta Geochim. 2023, 42, 77–88. DOI: 10.1007/s11631-022-00580-9.

53 Wu, Y. Is bicarbonate directly used as substrate to participate in photosynthetic oxygen evolution. Acta Geochim. 2021, 40(4):650–658. DOI: 10.1007/s11631-021-00484-0.

54.Wu, Y. Bicarbonate use and carbon dioxide concentrating mechanisms in photosynthetic organisms. Acta Geochim. 2021, 40(5):846–853. DOI: 10.1007/s11631-021-00488-w.

55.Warburg, O.; Krippahl, G. Hill-Reaktionen. Z Naturforsch. 1958, 13b: 509-514

 

Comment 2：A good part of the results reported in the manuscript are speculative. The bulk of what has been reported in the manuscript relates to the plant’s electrophysiological activities including charge separation, electron movement, proton and dielectric transport, the main processes responsible for energy production chloroplasts and mitochondria and nutrient transport.  These inferences were made on electrical measurements made on whole crushed leaves (clamped) rather than at the membrane level or on phloem exudates or vacuolar fluids. The method used does not distinguish between apoplastic water and dissolved salts, osmotica in the vacuole and phloem sap (symplast); the method does not take into account the all-important compartmentation of ions in plant cells and tissues.

Actually, the method was only recently reported by authors from the same group of researchers who did the present study:

Zhang, C.; Wu, Y.; Su, Y.; Xing, D.; Dai, Y.; Wu, Y.; Fang, L. A plant’s electrical parameters indicate its physiological state: A 519 study of intracellular water metabolism. Plants. 2020, 9, 1256. DOI: 10.3390/plants9101256. 520 40.

Zhang, C.; Wu, Y.; Su, Y.; Li, H.; Fang, L.; Xing, D. Plant’s electrophysiological information manifests the composition and 521 nutrient transport characteristics of membrane proteins. Plant Signaling & Behavior. 2021, 16(7), e1918867. DOI: 522 10.1080/15592324.2021.1918867.

Some other notes that need to be addressed:

Response: We sincerely thank the reviewer for the careful reviews! First of all, based on the theory of plant electrophysiology, our team has recently published many articles on plant water, nutrition, metabolism and other aspects (Frontiers in Plant Science, 13: 845628. Scientia Horticulturae, 276 (2021) 109763. Scientia Horticulturae, 2019,256,108642. Agronomy 2022, 12, 2758. Plants 2022, 11, 2313. Plants 2020, 9, 1256. Horticulturae2023, 9, 218. Horticulturae 2022, 8, 1082. Plant Signaling & Behavior, 2022, 17:1, 2073420. Horticulturae 2022, 8, 182. Horticulturae 2022, 8, 185. Plants 2021, 10, 1772. Horticulturae 2021, 7（8）, 206. Plant Signaling & Behavior, 2022, e1950899. Horticulturae 2021, 7, 137. Plant Signaling & Behavior, 2021, e1918867. Journal of Plant Interactions,2019, 14(1), 610-616.). Our many results have also verified the correctness of the plant electrophysiology theory that we established and have broad prospects in the evaluation of plant physiology. Secondly, most plant mesophyll cells can be divided into long cylindrical (or prismatic) palisade tissue cells and irregularly spherical sponge tissue cells. In order to simplify the scientific problem, mesophyll cells are uniformly regarded as spherical in this study. The mesophyll cells of all tested plants were regarded as spherical, thus the obtained results were consistent and comparable. The leaf thickness in plants is highly variable as dependent mainly on plant species, the water state of leaf and maybe also on mineral nutrition. Additionally, Beilby (1979) and Philip (2003) reported that the Nernst equation can be used to quantitatively describe the potential or concentration of ions, ion groups and electric dipoles inside and outside of the cell membrane. Same as them, the Nernst equation was same used to quantitatively describe the potential of ions, ion groups and electric dipoles inside and outside of the cell membrane in this study. The concentration of ions, ion groups and electric dipoles in mesophyll cells is most strongly related to electrical parameters (IZ, IXc and IC). And then, every mesophyll cell can be regarded as a capacitor, many aligned mesophyll cells are connected by plasmodesmata to form integrated leaf capacitor. Thus, plasmodesmata are equivalent to conductor. The capacitance and resistance of plasmodesmata in plants relative to mesophyll cells is very low and negligible. Thus, we humbly believe that the reference of our previous works (Plants. 2020, 9, 1256. and Plant Signaling & Behavior. 2021, 16(7), e1918867.) in this article is correct, and we sincerely hope to get your recognition and understanding. Thank you most sincerely!

 

Comment 3： 7 mM NaHCO₃ is not stressful by the authors own statement in the introduction.

Response: According to the previous research of our research group. The average of the bicarbonate concentration is 2.0 mmol/L. We observed the maximum bicarbonate concentration was up to 3.99 mmol/L in karst soils. In this study, we design the 7.0 mmol/L for identify the difference. In some cases, it is a slightly larger optimal concentration,

 

Comment 4：7 mM NaHCO₃ had no effect on chlorophyl content (SPAD) contrary to what has been stated.

Response:  7 mM NaHCO₃ had no effect on chlorophyl content (SPAD) contrary to the treatment with non added sodium bicarbonate (control conditions).

 

Comment 5：The higher concentrations of NaHCO₃ did not affect Pn despite a marked reduction in stomata conductance; why? If the authors imply that the reduction in mesophyll  CO₂ was compensated by CO₂ release from HCO₃ breakdown, they should show it. They should also measure internal CO₂ conc.

Response: HCO₃^- not only provided short-term carbon and water sources for plants under drought stress, but also promoted stomatal opening and restored photosynthesis [10-14]. Meanwhile, HCO₃^- could promote plant’s carbon and nitrogen metabolism by regulating the key enzyme activities of carbon and nitrogen metabolism, and participate in the regulation of complex physiological processes including carbon assimilation and nitrogen reduction in plants [15-16]. Additionally, HCO₃^- could also change the distribution of glycolysis and pentose phosphate pathways by affecting glucose metabolism disproportionation, so as to enhance the stress resistance of plants and obtain survival opportunities [17]. Our previous works have shown that in karst environments, plants can alternately use bicarbonate from soil and atmospheric carbon dioxide, and when plants encounter some adversity, the stomatal conductance decreases, carbonic anhydrase activity increases, and the use of bicarbonate also increases, improving the intercellular water status, which in turn increases the use of atmospheric carbon dioxide by plants [1,15-16, 20].

 

Comment 6：The authors used vague terms to describe physiological phenomena without defining what those terms mean. For instance, water metabolism; are they referring to water activity, water potential or osmotic potential? In what part of the plant or tissue? “appropriate supply of biocarbonate” ? appropriate in relation to what? Besides, you measured 2 and 7 mM; how can we know if 3 or 4 mM is not more appropriate?

Response:  We sincerely thank the reviewer for the careful reviews!  We had changed responding part. The leaf of seedlings was detected by LCR.

These suggestions are quite reasonable. We will consider it in the future study.

 

Comment 7：The manuscript needs to be revised for English language:

L40, 41, 61, 74, 97 : frequent  use of “etc” and “so on”;

Response:  Thanks for the suggestions. The corresponding has been changed in the revised manuscript.

 

Comment 8：L70 you mean “rural areas”?

Response: Yes.

 

Comment9：L84 you mean theoritically ?

Response: Yes, based on the theory of plant electrophysiology, our team has recently published many articles on plant water, nutrition metabolism and other aspects.

 

Comment 10：L99, 105, 113, 117 re-phrase

Response: Thanks for your suggestion. The corresponding has been changed in the revised article.

 

Comment 11：L106 “unit of roots” you mean root gs?

Response: It means the weight of the root.

 

Comment 12：L108 incomplete sentence;

Response: we have revised as: Hoagland nutrient solution contained

 

Comment 13：L129-136 how FW was measured? On subsamples?

Response: No, the weight was determined at the end of the harvest.

 

Comment 14：L147  you mean self-made capacitor?

Response: Yes. It was designed and produced by our team. It was based on the theory of plant electrophysiology, our team has recently published many articles on plant water, nutrition metabolism and other aspects.

 

Comment 15：No need to use the scientific name of the plant with  the authority in all the text.

 Response: OK. We have modified it in the revised article.