Morpho-Physiological Characterization of Diverse Rice Genotypes for Seedling Stage High- and Low-Temperature Tolerance
, Akanksha Seghal 1, Salah Jumaa 1,2, Raju Bheemanahalli 1, Naqeebullah Kakar 1, Edilberto D. Redoña 3, Chathurika Wijewardana 1, Firas Ahmed Alsajri 1,2, Daryl Chastain 3, Wei Gao 4, Shasthree Taduri 1 and Ajaz A. Lone 1Round 1
Reviewer 1 Report
It is very well designed study and authors have taken significant efforts to present their research findings concisely. However there is still some more work needs to be done to accept this manuscript. Please see the comments in the attached file.
Review Comments:
It is very well designed study and authors have taken significant efforts to present their research findings concisely. However, there is still some more work needs to be done to accepts this manuscript. Please see the below comments.
Abstract: Currently there are 275 words in the abstract which is significantly higher than the allowed word count (200 words maximum). Please revise the abstract and make the required corrections.
Line 24: “below-ground traits” I think it would be better if you just say root traits
Introduction: Very well written authors have provided sufficient information on the background and supplemented with relevant literature.
Materials and methods: Overall experiment is designed well but it would have been better if authors would have repeated this experiment two times in the same setting so that the experimental variation could have been properly understood.
Line 183: “number of tips (RT), number of forks (RF), number of crossings (RC)” please replace “RT” as the number of root tips, “RF” as the number of root forks and “RC” as the number of root crossings. These are the standard definitions and I think it would be in reader’s interest if they are presented this way.
Results and Discussion:
Line 229: Rewrite this sentence, the cohesiveness is missing in the later part of this sentence particularly linking first part of the sentence with the later part here “variations and that directly impacts rice productivity”
Line 232: “continue” should be “continues” please correct it accordingly
Line 239: (Figure 1A;) should be formatted according to the journal format
Line 314: “And” I would not start a sentence with And rather I would start it by saying “These are the useful…..”
Lines 249, 291, 301, 328, 343 and ???: Authors are interchangeably presenting low temperature (LT) regime either as 22/14 oC or 21/14 oC. Please be consistent throught the manuscript as per the defined LT regime.
Lines 342-343: “tips, the number of forks, the number of crossings,” please refer back to comment for Line 183.
Line 380: “22-14 °C,” presentation style is different than similar LT regime presented elsewhere in the manuscript.
Line 382: “IR65600-81-5-2-3 (an” please check if there is extra space
Line 385-386: “red- 385 cued” correct the spelling
Line 395: “25th to 75th” please check if use of superscript is warranted
Line 471-472: “Our results suggest the existence of different levels of heat tolerance between vegetative and reproductive in rice.” Something is missing in the later part of the sentence, please revise it accordingly.
Conclusion:
Line 533-535: “By screening a diverse set of rice genotypes, encompassing both the indica and japonica major rice subspecies, we were able to identify genotypes, including released varieties and experimental breeding lines still under development, that are tolerant of extreme temperatures.”. It’s too speculative to call and report heat and cold tolerant genotypes just based on one experiment. As a breeder, I will repeat this experiment one more time to confidently declare the identified genotypes as cold or heat tolerant if they show similar performance in the repeated experiment.
Line 547: “climes” should be “climates” please correct the same.
Comments for author File: 
Comments.pdf
Author Response
Reviewer 1:
Abstract: Currently there are 275 words in the abstract which is significantly higher than the allowed word count (200 words maximum). Please revise the abstract and make the required corrections.
Response: We have reduced the abstract word count to 200. Thank you for bringing this mistake to our notice.
Line 24: “below-ground traits” I think it would be better if you just say root traits
Response: Considering the reviewer's comment, the word “below-ground traits” has now been replaced with “roots” throughout the text. All our edits have been track changed.
Line 183: “number of tips (RT), number of forks (RF), number of crossings (RC)” please replace “RT” as the number of root tips, “RF” as the number of root forks and “RC” as the number of root crossings. These are the standard definitions and I think it would be in reader’s interest if they are presented this way.
Response: Thanks for pointing this inaccuracy. Necessary changes have now been made in the reviewed version. We have also provided abbreviations and their full discerptions at the end of the manuscript.
Line 229: Rewrite this sentence, the cohesiveness is missing in the later part of this sentence particularly linking first part of the sentence with the later part here “variations and that directly impacts rice productivity”
Response: The sentence edited for clarity. Following is the revised sentence “It is now clear that the emphasis on stress tolerance in plants has to be addressed from the vegetative to reproductive stages because of their greater sensitivity to environmental variations.”
Line 232: “continue” should be “continues” please correct it accordingly
Response: Thank you. “continue” replaced with “continues”.
Line 239: (Figure 1A;) should be formatted according to the journal format
Response: We have looked recently published articles and formatted accordingly.
Line 314: “And” I would not start a sentence with And rather I would start it by saying “These are the useful…..”
Response: Suggestion adopted.
Lines 249, 291, 301, 328, 343 and ???: Authors are interchangeably presenting low temperature (LT) regime either as 22/14 oC or 21/14 oC. Please be consistent throught the manuscript as per the defined LT regime.
Response: We thank reviewer for pointing this typo error. The text has now been edited for consistency.
Lines 342-343: “tips, the number of forks, the number of crossings,” please refer back to comment for Line 183.
Response: Suggestion accepted. Necessary changes have now been made in the reviewed version.
Line 380: “22-14 °C,” presentation style is different than similar LT regime presented elsewhere in the manuscript.
Response: Changed. 22-14 °C replaced with 22/14 °C
Line 382: “IR65600-81-5-2-3 (an” please check if there is extra space
Response: Corrected.
Line 385-386: “red- 385 cued” correct the spelling
Response: Corrected. “red-cued” has been changed to “reduced.”
Line 395: “25th to 75th” please check if use of superscript is warranted
Response: Changed made as required.
Line 471-472: “Our results suggest the existence of different levels of heat tolerance between vegetative and reproductive in rice.” Something is missing in the later part of the sentence, please revise it accordingly.
Response: Thank you. Considering the reviewer suggestion sentence has been edited for clarity.
Corrected sentence- “These observations reveal the existence of different high-temperature stress tolerance mechanisms between vegetative and reproductive in rice”.
Line 533-535: “By screening a diverse set of rice genotypes, encompassing both the indica and japonica major rice subspecies, we were able to identify genotypes, including released varieties and experimental breeding lines still under development, that are tolerant of extreme temperatures.”. It’s too speculative to call and report heat and cold tolerant genotypes just based on one experiment. As a breeder, I will repeat this experiment one more time to confidently declare the identified genotypes as cold or heat tolerant if they show similar performance in the repeated experiment.
Response: Thanks for the comment. Knowing the limitation of our study; in the conclusion, we advised validating the performance of selected genotypes under field conditions before recommending them to the producers and breeders to accomplish the maximum benefit.
Reviewer 2 Report
The manuscript article by Reddy et al. entitled 'Morpho-Physiological Characterization of Diverse Rice Genotypes for Seedling Stage High- and Low-Temperature Tolerance' is very scientifically sound research work based on the breeding phenotype and physiology aided characterization of a set of rice lines for their heat and cold stress tolerance in seedling stage level.
The manuscript is written very well and easy to understand language. The author pointed out the major strategy work on the seedling stage because it is very much prone to environmental stresses and important for biomass and yield production.
I have some specific questions/comments to the authors for possible corrections:
Line 43-44: Please update the information till date (2020-21).
Line 149: It would be 'day/night respectively'.
Line 159: Please use any one either 'content' or 'index' and be consistent of this throughout the entire text in the manuscript. Also, how does the author justified the SPAD values with real chlorophyll values? Do they perform and bulk chlorophyll estimation and regression with the SPAD value to get an equation for the samples?
Line 169: Please change 'Phenology and Growth' to 'Phenotyping of agronomical traits' because the listed traits are not phenology of the crop.
Please mention the references for equation 1, 2, 3 and 4. If those are new, please justify their usage.
Line 200-201: Please write in the full name of the abbreviated terminologies.
Line 210: Please mention which package of R they used for ANOVA and PCA?
Figure 6B: Please describe why R2 values of physiological traits are very low (around zero)?
Figure 7: Please increase the font size as those are very hard to read.
Author Response
Reviewer 2:
Line 43-44: Please update the information till date (2020-21).
Response: We would love to keep the information up-to-date; however, we did not find consistent and valid information.
Line 159: Please use any one either 'content' or 'index' and be consistent of this throughout the entire text in the manuscript. Also, how does the author justified the SPAD values with real chlorophyll values? Do they perform and bulk chlorophyll estimation and regression with the SPAD value to get an equation for the samples?
Response: Thank you. We have now retained the “chlorophyll index” in the revised manuscript.
SPAD chlorophyll meter is a portable device designed to capture variation in leaf nitrogen level as a function of chlorophyll content nondestructively. The SPAD reading concept is a time-tested and well-accepted proxy for tedious “laboratory chlorophyll estimation.” Knowing the stronger association (up to 95%) between SPAD reading and the chlorophyll pigment at various growth stages across different stresses, we did not find a reason to perform wet-lab extraction.
Line 169: Please change 'Phenology and Growth' to 'Phenotyping of agronomical traits' because the listed traits are not phenology of the crop.
Response: Suggestion adopted.
Please mention the references for equation 1, 2, 3 and 4. If those are new, please justify their usage.
Response: Relevant references added.
Line 200-201: Please write in the full name of the abbreviated terminologies.
Response: We have provided a separate section of abbreviations and their discerptions at the end of the manuscript
Line 210: Please mention which package of R they used for ANOVA and PCA?
Response: Statistical analyses were carried out using RStudio 3.6.1 (https://rstudio.com/). An ANOVA was performed for each of the traits to estimate the significance of genetic variability among rice genotypes under treatments using the library ("agricolae"). The principal component analysis was performed using XLSTAT.
Figure 6B: Please describe why R2 values of physiological traits are very low (around zero)?
Response: This is a million-dollar question. It requires a series of follow up system biology experiments to underpin the reasons for the weak correction.
In response to changes in temperature, plant cell quickly perceives the stress signal, which alters physiological processes by triggering specific pathways at a biochemical and molecular levels to combat stress injury. For example, depending on the intensity of stress and duration of stress, cell damage occurs within minutes due to the excess production of reactive oxygen species. These events change the properties or activity of cells, which primarily lead to the weak physiological adaptations followed by morphological of the plant under stress condition. We did not speculate much about such results in the paper due to a lack of data from our study.
However, the following sentence has been included in the text. “High temperature alters cell properties or activities such as enzyme kinetics, protein binding, and membrane fluidity due to the excess production of reactive oxygen species [59]. Above stress-induced changes in the cell physical properties partially explain the reason for weaker relationship between CHTRI and physiological traits”.
Figure 7: Please increase the font size as those are very hard to read.
Response: We did consider increasing the font size; however, genotypes overplay each other, and it became much harder to read as we increase the font size.
