Calcium Route in the Plant and Blossom-End Rot Incidence

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

i)               Reviewer 1

Reviewer’s Evaluation

Response and Revisions

Authors’ Responses: Thank you. We agree with the reviewer’s overall comments on the manuscript.

3. Point-by-point response to Comments and Suggestions for Authors

Reviewer 1

a) The article is a review on calcium, an important element for plants. However, it is necessary to consider aspects that differentiate this papers from others in the same line. Therefore, I suggest discussing the proposed objective based on calcium signaling in plant metabolism.

Review the entire text and avoid too many repetitions.

Line 7 – Substitute macro-mineral essential by macronutrient

Lines 12 and 13 – Change - Soil available Ca2+ enters the root apoplast with the water flow to Water and calcium are absorbed in completely different ways.

Lines 35-56 - Avoid repetitions

Authors’ Responses: Thank you for your suggestions. We elaborated on calcium signaling more in the revised version. We also removed repetitions from the entire text. Substitute ‘macro-mineral with ‘macronutrient.’

Please see the revised highlighted manuscript.

b) Figure 1- Improve the figure. Instead of dots mention the concentration of ​​calcium distribution.

Authors’ Responses: Thank you for the suggestion. We updated the figure as follows.

c) Lines 91-93 - Avoid repetitions

Lines 146-156 - Which signaling? The figure below shows the signaling that is not in the text. Show this idea in the next paragraph.

Lines 175-177 - It will improve understanding of how specific stress signals modulate Ca2+ homeostasis to orchestrate adaptive responses [76]. Was this idea mentioned again to clarify the problem presented in the objective?

Authors’ Responses: Thank you for pointing this out. We detailed more about signaling in the revised manuscript, particularly under recent advances. We also include a figure on it as follows.

Please see the revised manuscript for more detail.

d)

Lines 246-256- The apoplast has a lot of calcium but the mineral does not enter with water. The absorption is different.

Line 261 - Foliar application of Ca2+ @ 0.9% increases. What does @ mean?

Line 290 - Ca2+ @100 ppm. Why not mg L^-1 ?

Lines 304-320 - Review calcium entry into the endodermis.

Lines 370-377 - Avoid repetitions.

Lines 442-444- After having described the signs of deficiency in the text, what is the contribution of table 1?

Lines 445-460 - What is the influence of pH on calcium availability? Avoid repetitions.

Lines 469-479 - When does inadequate Ca2+ compartmentalization occur (Figure 4)?

Authors’ Responses: Thank you for pointing them out. We replaced ‘@’ and ‘ppm’ with ‘at’ and ‘mg L^-1’. The influence of pH on calcium availability has been described. We also described the entry of calcium into the epidermis and inadequate calcium compartmentalization. We deleted the repetitions from the entire manuscript.

Please see the highlighted revised manuscript.

e) Lines 543-590- Leading the discussion based on calcium signaling may be a better contribution to the review. Review about calcium signaling and calcium in fruits:

70.           Naz, M., Afzal, M. R., Raza, M. A., Pandey, S., Qi, S., Dai, Z., & Du, D. (2024). Calcium (Ca2+) signaling in plants: A plant stress 750 perspective. South African Journal of Botany, 169, 464-485. https://doi.org/10.1016/j.sajb.2024.04.047

71.           Pirayesh, N., Giridhar, M., Khedher, A. B., Vothknecht, U. C., & Chigri, F. (2021). Organellar calcium signaling in plants: An 754 update. biochimica et biophysica Acta (bbA)-molecular Cell Research, 1868(4), 118948. 755 https://doi.org/10.1016/j.bbamcr.2021.118948

72.           Hocking, B., Tyerman, S. D., Burton, R. A., & Gilliham, M. (2016). Fruit calcium: transport and physiology. Frontiers in plant 992 science, 7, 569. https://doi.org/10.3389/fpls.2016.00569

         Song, W., Yi, J., Kurniadinata, O. F., Wang, H., & Huang, X. (2018). Linking fruit ca               uptake capacity to fruit growth and pedicel anatomy, a cross-species study.                       Frontiers in Plant Science, 9. https://doi.org/10.3389/fpls.2018.00575

Authors’ Responses: We reviewed calcium signaling in fruits following the suggested references. Please see the updated manuscript.

f)

Quality of English Language

( ) The English could be improved to more clearly express the research.
(x) The English is fine and does not require any improvement.

Authors’ Responses: Thank you.

4. Additional clarifications

Authors’ Responses: Thanks a lot for the detailed review.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer 2

Reviewer’s Evaluation

Response and Revisions

Authors’ Responses: Thank you so much. We agree with the reviewer’s overall comments on the manuscript.

2. Point-by-point response to Comments and Suggestions for Authors

Reviewer 2

a)              Quality of English Language

( ) The English could be improved to more clearly express the research.
(x) The English is fine and does not require any improvement.

Authors’ Responses: Thank you.

b)             Major Comments: Although not a novel topic, the writeup and language in the manuscript are adequate. However, the article lacks in-depth discussion, failing to point out research gaps and future directions systematically.

I will encourage authors to add a section on “recent advances and future directions” to discuss topics such as novel calcium delivery systems using nanotechnology, CRISPR-mediated gene manipulation of calcium transporters, and imaging and biosensors for calcium dynamics.

Indeed, climate change is affecting almost every aspect of nutrient uptake and homeostasis; therefore, authors should briefly discuss the effects of climate change on Ca²⁺ nutrition and the balance of reactive oxygen species (BER).

The MS also contain several plagiarized sentences and paragraphs from onlone sources. Authors must paraphrase these areas.

Authors’ Responses: The research gap and future directions have been included. Though research gap was explained at the end of Introduction, future directions were discussed under a new subheading - 11. Recent advances and future directions. Please see the revised manuscript.

We also included a topic on Climate change and calcium availability, and explain it with a figure as follows.

For more details, please see the revised manuscript.

The plagiarized sentences are from the first author’s PhD thesis, excluding the thesis the similarity was 11% for the submitted manuscript.

c)

Minor comments

Please write “BER” in the keywords section

Line 31: “is found within plants in higher quantities than any other divalent inorganic……………………”

Please insert citation

Line 34: I suggest authors use “tissues” instead of “body” and replace “essentiality” with a more fitting word.

Figure 1: Please consider writing “Ca2+” withing the Figure, instead of in the legend.

Line 57: Please insert citation and estimated values of BER economic losses reported

Line 91 “[15] “ remove this citation, “Ca2+ performs numerous plant functions” is a common statement to be cited.

Authors’ Responses: We followed all the suggestions and included citations as suggested. We removed the citation [15] from the text.

For more details, please see the revised manuscript.

d)

Lines 99: Please insert citation at the of this statement “……structural ones, and lignin (secondary cell wall).” Authors may consider citing this ref here https://doi.org/10.3390/jof9010052

Lines 99-100: “Cell wall Ca2+ mainly………..” Please insert citation here

Line 115: Citation is required after “…degradation, and cell death”

Lines 152-153: “A slight increase in Ca2+…………………enzyme” This sentence is ambiguous and isolated from the previous one. Please rephrase.

Line 181 an many parts: “[Ca2+]” why in brackets?

Authors’ Responses: We followed all the suggestions and included citations as suggested. Please see the revised manuscript.

“[Ca2+] indicates calcium concentration.

e)

Authors should include a figure summarizing the current understanding of genetic regulation of calcium uptake, translocation in plants, and assimilation.

The conclusion should be enriched to include the prospect for future research, pointing to research gaps within the topic being reviewed.

calcium uptake and translocation in plants

Authors’ Responses: We modified the conclusion as follows:

Calcium is an essential element for plant growth, development, and productivity. While most soils worldwide are not typically deficient in Ca²⁺, calcium-deficiency disorders in crops are prevalent and lead to substantial yield losses in several horticultural crops. Among the Ca²⁺-deficiency disorders, BER is widely prevalent worldwide. The complex route of Ca²⁺ from soil to the appropriate cellular compartments, such as cytosol, depends on multiple factors, e.g., soil (moisture availability, the competition of Ca²⁺  with other cations, pH, anoxia, and salinity), plant [genotypes, growth habit (dwarf, tall), xylem network, root and shoot growth, and yield], and environment [temperature (air and root zone), relative humidity, vapor pressure deficit, and transpiration] that renders it difficult to control BER. Moreover, the cellular Ca²⁺ compartmentalization, particularly in the vacuoles, depletes cytosolic Ca²⁺ levels and may disintegrate the plasma membrane, leading to BER development despite having high Ca²⁺ content in the blossom end of the fruit. Therefore, possible ways of minimizing and controlling BER include i) an integrated approach that ameliorates soil, plant, and environmental factors towards supplying sufficient Ca²⁺ into the cells; ii) appropriate cultural and physiological management of crops; iii) a favorable environment; and iv) BER-resistant and Ca²⁺-efficient cultivars. Other approaches, such as agronomic, physiological, breeding, and molecular methods, may also contribute to minimizing BER. Although calcium is well studied, the molecular identity and regulation of many calcium channels, pumps, and sensors remain uncharacterized. The relation between calcium as a nutrient and secondary messenger is also poorly understood. Therefore, future investigations should explore how calcium signatures encode specific responses and how the complex interactions between calcium signaling and nutritional or hormonal networks interplay.

The calcium uptake, translocation in plants and assimilation have been discussed.

3. Additional clarifications

Authors’ Responses: Thanks a lot for the detailed review.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer 3

Reviewer’s Evaluation

Response and Revisions

Authors’ Responses: Thank you so much. We agree with the reviewer’s overall comments on the manuscript.

Introduction

Line 35 “development [5].  It plays roles in”

Please remove the space before It

Line 42 [11-12].  The Ca²⁺ content of

Please remove the extra space before The

Authors’ Responses: Thank you for pointing this out. We removed the extra spaces as indicated. Please see the highlights in the revised manuscript.

Figure 1.

Please redraw this Figure and incorporate the actual parts of the cell and show the concentration of Calcium. This cartoon diagram is really not acceptable.

Authors’ Responses: Thank you for the suggestion. We updated the figure as follows. For more details, please see the revised manuscript.

2.              Function of calcium in plants

Line 127

environment.  Other cations

Please check the extra space here

Line 142

Moreover,  Ca²⁺ is required

Please check the extra space after the moreover

·                Calcium channels

Line 225 “allowing Ca2+ to enter the cytosol.”

Please check Ca2+ symbol and correct it.

Please check the type error through out the text and correct it where it is required.

Authors’ Responses: Thank you. We edited the manuscript following your suggestions. Please see the highlights in the revised manuscript.

Comments:

Point 1: The English language must be improved where it is required.

Authors’ Responses: Thank you. We improved the English languages.

3. Additional clarifications

Authors’ Responses: Thank you for your valuable comments.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer 3

Reviewer’s Evaluation

Response and Revisions

Authors’ Responses: Thank you so much. We agree with the reviewer’s overall comments on the manuscript.

Introduction

Line 35 “development [5].  It plays roles in”

Please remove the space before It

Line 42 [11-12].  The Ca²⁺ content of

Please remove the extra space before The

Authors’ Responses: Thank you for pointing this out. We removed the extra spaces as indicated. Please see the highlights in the revised manuscript.

Figure 1.

Please redraw this Figure and incorporate the actual parts of the cell and show the concentration of Calcium. This cartoon diagram is really not acceptable.

Authors’ Responses: Thank you for the suggestion. We updated the figure as follows. For more details, please see the revised manuscript.

2.              Function of calcium in plants

Line 127

environment.  Other cations

Please check the extra space here

Line 142

Moreover,  Ca²⁺ is required

Please check the extra space after the moreover

·                Calcium channels

Line 225 “allowing Ca2+ to enter the cytosol.”

Please check Ca2+ symbol and correct it.

Please check the type error through out the text and correct it where it is required.

Authors’ Responses: Thank you. We edited the manuscript following your suggestions. Please see the highlights in the revised manuscript.

Comments:

Point 1: The English language must be improved where it is required.

Authors’ Responses: Thank you. We improved the English languages.

3. Additional clarifications

Authors’ Responses: Thank you for your valuable comments.