Analysis of Morphological, Physiological, and Biochemical Traits of Salt Stress Tolerance in Asian Rice Cultivars at Seedling and Early Vegetative Stages

Round 1

Reviewer 1 Report (Previous Reviewer 2)

The title should change to "Analysis of Morphological, Physiological, and Biochemical Traits of Salt Stress Tolerance in Asian Rice Cultivars at Seedling and Early Vegetative Stages"

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 1)

The authors have modified the manuscript as per the comments. I have only one concern now and that is of English language. The manuscript still has several grammatical mistakes and therefore need thorough correction in the language. 

The authors have modified the manuscript as per the comments. I have only one concern now and that is of English language. The manuscript still has several grammatical mistakes and therefore need thorough correction in the language. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report (Previous Reviewer 3)

The authors have done a careful revision and the manuscript has substantially improved. Readability is much better and the hypothesis-oriented framework is clearer. From my side I can recommend acceptance. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

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

Comments

The manuscript by Alkahtani and Dwiningsih describes the screening of 116 Asian rice genotypes under salt stress condition by various morphological, biochemical and physiological parameters. The experiment is well designed and has been properly carried out. Results are properly described and discussed. However, the major concern is the English language of the manuscript. The manuscript thoroughly needs correction/improvement in English language.

Some of the minor suggestions are as below.

The introduction part contains a lot of information and some of the things are not required for this manuscript. It can be reduced in order to focus more on the work done in the manuscript.

Rewrite the sentence as “One of the constraints in rice production is soil salinity as it greatly affects rice production”

Line 14: replace word tolerant with tolerance

Line 17 and other places in manuscript: instead of writing “TCCP 266 from Philippines, IR 45427 also from Philippines” modify it as “TCCP 266 and IR 45427 from Philippines”

Line 41-42: replace word tolerance with tolerant

Line 47: replace present with presence

Line 56: replace reduce with reduced

Line 58-59: correct as “which leads to decrease in the grain yield”

Line 60: replace the sentence “In the salt-tolerant cultivar maintains…” with “The salt-tolerant cultivar maintains..”

Line 67: replace effects with affects

Line 77: correct the sentence as “Soil salinity tolerance is a quantitative trait which is controlled by…”

Line 88: delete “been”

Line 92: correct the line as “developing salt-tolerant rice”

Line 93: Reference format should be in number rather than Quin et al. Maintain the uniformity in the format.

Line 113: correct the line as “changes occur in the rice plants”

Line 119: change the line “with highly salinity tolerant” by “with high salinity tolerance”

Line 123: replace word tolerant with tolerance

Line 195: delete the word “tolerant”

Line 196-197: modify the sentence as “With the increase of Na+ concentration in the cells, water potential inside the cells is getting reduced which ultimately affect the cell membrane stability”

Line 209: delete the word “in”

Cite the reference for methods used for measurement of cell membrane stability, Na/K ratio and other biochemical/physiological parameters

The english language of the manuscript requires improvement.

Author Response

On behalf of all authors, I would like to Thank Reviewer#1 for their valuable comments:

• The manuscript by Alkahtani and Dwiningsih describes the screening of 116 Asian rice genotypes under salt stress condition by various morphological, biochemical and physiological parameters. The experiment is well designed and has been properly carried out. Results are properly described and discussed. However, the major concern is the English language of the manuscript. The manuscript thoroughly needs correction/improvement in English language. ==> Thank you for taking the time to review our manuscript. We appreciate your valuable feedback and suggestions. We would also like to express our gratitude for your specific comments, and we are trying our best to correct/improve in English language.

• The introduction part contains a lot of information and some of the things are not required for this manuscript. It can be reduced in order to focus more on the work done in the manuscript. ==> Based on your suggestions, we have already reduced several information in the Introduction section in order to focus more on our work in the manuscript (Please kindly checked our Revised Manuscript).  

• Rewrite the sentence as “One of the constraints in rice production is soil salinity as it greatly affects rice production” ==> According to your suggestions, we have already revised the sentence (Please kindly checked our Revised Manuscript).

• Line 14: replace word tolerant with tolerance ==> Based on your suggestions, we have already replaced word tolerant with tolerance in line 14 (Please kindly checked our Revised Manuscript).

• Line 17 and other places in manuscript: instead of writing “TCCP 266 from Philippines, IR 45427 also from Philippines” modify it as “TCCP 266 and IR 45427 from Philippines” ==> According to your suggestions, we have already modified the sentence in line 17 (Please kindly checked our Revised Manuscript).

• Line 41-42: replace word tolerance with tolerant ==> Based on your feedback, we have already replaced the word tolerance with tolerant in line 41-42 (Please kindly checked our Revised Manuscript become in the line 38).

• Line 47: replace present with presence ==> According to your valuable comment, we have already replaced present with presence (Please kindly checked our Revised Manuscript become in the line 43).

• Line 56: replace reduce with reduced ==> Based on your suggestions, we have already replaced reduce with reduced (Please kindly checked our Revised Manuscript become in the line 52).

• Line 58-59: correct as “which leads to decrease in the grain yield” ==>According to your suggestions, we have already corrected the sentence become “which leads to decrease in the grain yield” (Please kindly checked our Revised Manuscript become in the line 54). 

• Line 60: replace the sentence “In the salt-tolerant cultivar maintains…” with “The salt-tolerant cultivar maintains..” ==>  Based on your comments, we have already replaced the sentence “In the salt-tolerant cultivar maintains…” with “The salt-tolerant cultivar maintains..” (Please kindly checked our Revised Manuscript become in the line 56).

• Line 67: replace effects with affects ==> According to your valuable feedbacks, we have already replaced effects with affects (Please kindly checked our Revised Manuscript become in the line 62).

• Line 77: correct the sentence as “Soil salinity tolerance is a quantitative trait which is controlled by…” ==> Based on your valuable suggestion, we have already corrected the sentence as “Soil salinity tolerance is a quantitative trait which is controlled by…” (Please kindly checked our Revised Manuscript become in the line 72).  

• Line 88: delete “been” ==> According to your feedback, we have already deleted “been” (Please kindly checked our Revised Manuscript become in the line 83).

• Line 92: correct the line as “developing salt-tolerant rice” ==>Based on your suggestions, we have already corrected the line as “developing salt-tolerant rice” (Please kindly checked our Revised Manuscript become in the line 87).

• Line 93: Reference format should be in number rather than Quin et al. Maintain the uniformity in the format. ==> Thank you very much for your detail attention. Yes, we have already revised the reference format (Qin et al., 2020) become [39] (Please kindly checked our Revised Manuscript become in the line 87).

• Line 113: correct the line as “changes occur in the rice plants” ==> According to your suggestion, we have already corrected the line as “changes occur in the rice plants” (Please kindly checked our Revised Manuscript become in the line 108).

• Line 119: change the line “with highly salinity tolerant” by “with high salinity tolerance” ==> Based on your suggestion, we have already changed the line “with highly salinity tolerant” by “with high salinity tolerance” (Please kindly checked our Revised Manuscript become in the line 114).

• Line 123: replace word tolerant with tolerance ==> Based on your valuable feedback, we have already replaced word tolerant with tolerance (Please kindly checked our Revised Manuscript become in the line 118).

• Line 195: delete the word “tolerant” ==> According to your comment, we have already deleted the word “tolerant” (Please kindly checked our Revised Manuscript become in the line 189).

• Line 196-197: modify the sentence as “With the increase of Na+ concentration in the cells, water potential inside the cells is getting reduced which ultimately affect the cell membrane stability” ==> Based on your suggestions, we have already modified the sentence as “With the increase of Na+ concentration in the cells, water potential inside the cells is getting reduced which ultimately affect the cell membrane stability” (Please kindly checked our Revised Manuscript become in the line 190-191).

• Line 209: delete the word “in” ==> According to your valuable suggestions, we have already deleted the word “in” (Please kindly checked our Revised Manuscript become in the line 203).

• Cite the reference for methods used for measurement of cell membrane stability, Na/K ratio and other biochemical/physiological parameters ==> Yes, we have already added the reference for methods used for measurement of cell membrane stability, Na/K ratio and other biochemical/physiological parameters (Please kindly checked our Revised Manuscript).

Author Response File: Author Response.pdf

Reviewer 2 Report

1.     Pictures of salt tolerant and sensitive rice under salt treatment should be presented.

2.     Figure 3. Root length, root fresh weight, shoot length, and plant biomass of rice are                     different between rice cultivars under normal condition, Effect of salinity stress condition on root length (a), root fresh weight (b), shoot length (c), and plant biomass (d) of rice cultivars should be presented in relative to that of normal condition.

3.     Salinity Screening at Seedling and Early Vegetative Stage

4.     “After12 days of salt treatment, germinated 340 seedlings were transferred to a hydroponic system containing Yoshida’s medium [55] with 200 mM NaCl. Rice plants were grown in a growth-chamber at temperature 28oC/24oC for day/night with 65% humidity, and light intensity 500 µEm-2s -1 until early vegetative stage (3 leaf stage). “ Salt tolerant and sensitive rice cultivars required different days to grow to  3 leaf stage under 200 mM NaCl condition, It’s better to analyze in the same day.

5.     Why selected 200 mM NaCl?  Sensitive rice cultivars may be dead under 200 mM NaCl for two weeks.

Minor editing of English language required

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 3 Report

The work by Alkahtani and Dwiningsih advances our understating of salt tolerance and its morphological, physiological, and biochemical correlates in Asian rice. The report is well written and condensed, as well as technically appropriate. However, before being able to recommend acceptance, I invite authors to address the following amendments.

First, the introduction section should properly close (L126) with an explicit research questions, hypotheses and expected results (so far only a main goal is described). For instance, do authors hypothesize a polygenic or a Mendelian/biomodal segregation for salt tolerance in Asian rice? Are traits correlated with salt tolerance expected to exhibit antagonistic pleiotropy or conditional neutrality when comparing them in various environments?  

Second, methodological speaking authors should comment more clearly how they handled environmental variation within the study. After all, quantitative Gaussian trait segregation in Figure 1-7 can be accounted both by the polygenic model and by reaction norms triggered by environmental gradients.

Third, concerning the results, figures and tables are very well edited and insightful. Still, authors should include explicit p-values for the correlation cores in the upper half of Table 3, and not just asterisks for the significant cases.

Fourth, results must also include a second section in L241 briefly commenting on a posteriori statistical power analysis, specially taking into account that the strong influence of environmental sampling on phenotypic variance. How representative are they? Furthermore, when salt stress effects are measured and observed is a key issue to consider. Was the salt tolerance observed during the flowering phenological phase? What about terminal stress? Please expand, especially for readers coming more from a breeding side. 

Fifth, please explicitly re-visit the above sampling caveat within the discussion section (e.g., L326). Additionally, the discussion, although perceptive, should embrace broader reflections on whether salt tolerance in Asian rice could be understood as a plastic or alternative a genetic adaptive strategy to cope with osmotic imbalance. Besides, since abiotic stresses are usually pleiotropic, please also comment on potential gene functional correlates of salt tolerance with drought, heat and flooding tolerance (consider referring to Front Genet 2019 10:954), which are stresses typically correlated with salt stress in the face of hypoxia and osmotic imbalance (consider referring to PLoS One 2013 8(5):e62898). Please comment/discuss the latter list of correlations in the abiotic responses with salt tolerance in the light of ABA-mediated drought tolerance pathways such as ASR genes (consider referring to BMC Genet 2012 13:58), ERECTA-mediated drought tolerance (i.e., consider referring to Plant Sci 2016 242: 250), and DREB-based ABA-independent drought tolerance responses (consider referring to the work Theor Applied Genet 2012 125(5):1069-85). Any insights in the studied Asian rice accessions panel?

As complement to my previous comment, what about combined drought + heat treatments to study the same Asian rice panel with known salt tolerance properties? Also related to this point on pleiotropy, authors must also briefly mention whether adaptive trade-offs for salt tolerance are observed/predicted (check and comment in the light of e.g. Front Plant Sci 2018 9:128). For instance, a biomass/reproductive trade-off triggered by salt stress in Asian rice may also be evident in more subtle ways.

Sixth, although the paper provides good evidence into the genetic mechanisms for salt tolerance in Asian rice, a major question that authors should prospect in their discussion is how Asian rice improvement for salt tolerance may unlock and effectively utilize the identified trait variation (see as guidance Genes 2021 12:556). Gene editing, recurrent backcrossing and inter-specific schemes (condensed, contrasted and discussed in e.g. Front Genet 2020 11:564515, and Genes 2021 12:783, both recommended) may offer an avenue in this regard that authors should acknowledge (consider referring to Agronomy 2021 11:1978). Please envision any other recommendation (some ideas in the next paragraph).

Last but not least, the report is so far lacking a very brief closing paragraph (L327) that describes the major caveats/limitations of the present work. It is never beyond the scope of any research to explicitly acknowledge the study caveats, especially when dealing with highly variable trait profiles in a complex trait with strong GxE effects and plasticity such as salt tolerance. For instance, trait variation may exhibit antagonistic pleiotropy or conditional neutrality when comparing their presence and their effects (direction and magnitude) in contrasting environmental treatments, something to be tested in more profound trials beyond a single location). Also link to this point, a short perspectives section, just before the conclusions, would be insightful for readers to fill the identified caveats as part of future research. Specifically, what other Asian rice accessions, traits and combined abiotic stresses must future studies target? How could the identified segregant candidate genotypes (something authors should list in more detail as a main new table within the results section) source Asian rice pre-breeding and future improvement programs?

In L126 it shouldn’t read “objectives” but “objective”

Also notice that tables’ captions are disorganized. It starts by Table 3 followed by Table 1 and 2.

Author Response

On behalf of all authors, I would like to Thank Reviewer#3 for their valuable comments:

• The work by Alkahtani and Dwiningsih advances our understating of salt tolerance and its morphological, physiological, and biochemical correlates in Asian rice. The report is well written and condensed, as well as technically appropriate. However, before being able to recommend acceptance, I invite authors to address the following amendments.
• Thank you very much for your time to provide valuable feedback on our manuscript. Yes, sure we are trying our best to address your detailed suggestions.
• First, the introduction section should properly close (L126) with an explicit research questions, hypotheses and expected results (so far only a main goal is described). For instance, do authors hypothesize a polygenic or a Mendelian/biomodal segregation for salt tolerance in Asian rice? Are traits correlated with salt tolerance expected to exhibit antagonistic pleiotropy or conditional neutrality when comparing them in various environments?  Thank you very much for your detailed suggestions. In the last paragraph of the Introduction section, we tried to properly close with expected results that reflect in the line 116 – 119: “By understanding the mechanism of salinity tolerance in the rice plants based on the morphological, physiological, biochemical, and genetics effects, the development of rice varieties with high salinity tolerance by genetic engineering techniques can be accelerated [28,53].”
• Second, methodological speaking authors should comment more clearly how they handled environmental variation within the study. After all, quantitative Gaussian trait segregation in Figure 1-7 can be accounted both by the polygenic model and by reaction norms triggered by environmental gradients. Thank you very much for your valuable input. In order to handle the environmental variation within the study, we make sure the growth chamber condition that we used to grow for all the rice genotypes under controlled conditions: at temperature 28^oC/24^oC for day/night with 65% humidity, and light intensity 500 µEm^-2s^-1 until early vegetative stage (3 leaf stage). Thus, we are 100% sure that the variation values for all the parameters of all the rice genotypes because of the genetic potential of each rice genotype.
• Third, concerning the results, figures and tables are very well edited and insightful. Still, authors should include explicit p-values for the correlation cores in the upper half of Table 3, and not just asterisks for the significant cases. Thank you very much for your detailed feedback on our results. Yes, we have already revised our Table 3 title by adding the p-values (p < 0.05) in the upper half of Table 3 (Please kindly checked our Revised Manuscript).
• Fourth, results must also include a second section in L241 briefly commenting on a posteriori statistical power analysis, specially taking into account that the strong influence of environmental sampling on phenotypic variance. How representative are they? Furthermore, when salt stress effects are measured and observed is a key issue to consider. Was the salt tolerance observed during the flowering phenological phase? What about terminal stress? Please expand, especially for readers coming more from a breeding side.  Thank you very much for your valuable comments on our results. Based on Table 3, we only analyze the correlation coefficients among morphological, physiological, and biochemical parameters from 116 Asian rice cultivars under 200 mM NaCl at seedling and early vegetative stages in order to understand the salt tolerance mechanism in diverse rice genotypes.
• Fifth, please explicitly re-visit the above sampling caveat within the discussion section (e.g., L326). Additionally, the discussion, although perceptive, should embrace broader reflections on whether salt tolerance in Asian rice could be understood as a plastic or alternative a genetic adaptive strategy to cope with osmotic imbalance. Besides, since abiotic stresses are usually pleiotropic, please also comment on potential gene functional correlates of salt tolerance with drought, heat and flooding tolerance (consider referring to Front Genet 2019 10:954), which are stresses typically correlated with salt stress in the face of hypoxia and osmotic imbalance (consider referring to PLoS One 2013 8(5):e62898). Please comment/discuss the latter list of correlations in the abiotic responses with salt tolerance in the light of ABA-mediated drought tolerance pathways such as ASR genes (consider referring to BMC Genet 2012 13:58), ERECTA-mediated drought tolerance (i.e., consider referring to Plant Sci 2016 242: 250), and DREB-based ABA-independent drought tolerance responses (consider referring to the work Theor Applied Genet 2012 125(5):1069-85). Any insights in the studied Asian rice accessions panel? Thank you very much for your detailed suggestions. Yes, we have already added these suggestions in the Discussion section (last paragraph).

Since genomic locations which control the abiotic stresses tolerance is pleiotropic, the potential candidate genes correlated to salt tolerance also showed associated with heat [84], drought [85], and flooding stresses. Some of the genes that responsible to salt tolerance were directly linked to heat-responsive pathways, such as germination and seedling development (Family Glicosil-hydrolase), abscisic acid (ABA) signaling pathway (histone-like transcription factors NFYB) [86], and cell wall stability (GAE6) [84]. Salt tolerance also showed correlation to ABA-stress response (Asr) genes [86], ERECTA-mediated drought tolerance [87], and DREB-based ABA-independent drought tolerance response [88]. Each gene which correlated to salt tolerance associated with genotype x environmental association that showed allelic diversity might allow each Asian rice genotype has diverse adaptation ability under salt stress environment [89].

• As complement to my previous comment, what about combined drought + heat treatments to study the same Asian rice panel with known salt tolerance properties? Also related to this point on pleiotropy, authors must also briefly mention whether adaptive trade-offs for salt tolerance are observed/predicted (check and comment in the light of e.g. Front Plant Sci 2018 9:128). For instance, a biomass/reproductive trade-off triggered by salt stress in Asian rice may also be evident in more subtle ways. Thank you very much for your valuable feedbacks. Yes, sure we have already added this description in the Discussion section (last paragraph).

Genome-wide diversity in Asian rice cultivars showed high genetic variability that allowed them to adapt in various stress conditions, including salinity stress environment. The adaptation of Asian rice cultivars under salt stress condition are observed in morphological, physiological, and biochemical properties, such as percentage germination, root length, root fresh weight, shoot length, plant biomass, leaf rolling, chlorophyll content, Na/K ratio, cell membrane stability, proline, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and sugar content. These genome-environment association supported by Cortes and Blair [90].

• Sixth, although the paper provides good evidence into the genetic mechanisms for salt tolerance in Asian rice, a major question that authors should prospect in their discussion is how Asian rice improvement for salt tolerance may unlock and effectively utilize the identified trait variation (see as guidance Genes 2021 12:556). Gene editing, recurrent backcrossing and inter-specific schemes (condensed, contrasted and discussed in e.g. Front Genet 2020 11:564515, and Genes 2021 12:783, both recommended) may offer an avenue in this regard that authors should acknowledge (consider referring to Agronomy 2021 11:1978). Please envision any other recommendation (some ideas in the next paragraph). Thank you very much for your detailed comments. Yes, sure we have already added this explanation in the Discussion section (last paragraph).

Each gene which correlated to salt tolerance associated with genotype x environmental association that showed allelic diversity might allow each Asian rice genotype has diverse adaptation ability under salt stress environment [89]. Genome-wide association, genomic prediction, machine learning, big data approaches, and gene editing techniques (such as CRISPR-Cas9) might assist the identification and functional of genes associated with the salt tolerance in order to speed up the rice adaptability under salinity condition [91,92,93].

• Last but not least, the report is so far lacking a very brief closing paragraph (L327) that describes the major caveats/limitations of the present work. It is never beyond the scope of any research to explicitly acknowledge the study caveats, especially when dealing with highly variable trait profiles in a complex trait with strong GxE effects and plasticity such as salt tolerance. For instance, trait variation may exhibit antagonistic pleiotropy or conditional neutrality when comparing their presence and their effects (direction and magnitude) in contrasting environmental treatments, something to be tested in more profound trials beyond a single location). Also link to this point, a short perspectives section, just before the conclusions, would be insightful for readers to fill the identified caveats as part of future research. Specifically, what other Asian rice accessions, traits and combined abiotic stresses must future studies target? How could the identified segregant candidate genotypes (something authors should list in more detail as a main new table within the results section) source Asian rice pre-breeding and future improvement programs? Thank you very much for your suggestions. Yes, we have already included the closing paragraph in the Discussion section (last paragraph).

In order to validate the salt stress mechanisms in Asian rice cultivars, combination of several abiotic stress conditions, such as drought, heat, and flooding need to be completed. And also more quantity of Asian rice cultivars about 500 cultivars need to be involved.   

• In L126 it shouldn’t read “objectives” but “objective” Yes, we have already revised.
• Also notice that tables’ captions are disorganized. It starts by Table 3 followed by Table 1 and 2. Yes, we have already revised.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

no

Reviewer 3 Report

Authors have done a good job revising the current manuscript