Agronomic and Physiological Indices for Reproductive Stage Heat Stress Tolerance in Green Super Rice

Round 1

Reviewer 1 Report

The authors investigated the effect of heat stress on agronomic and physiological parameters in 22 green super rice lines and 4 local rice cultivars. Given the global warming attracts increasing concerns, and the present study is on the rice performance under heat stress, I think the results should be of value for assessing responses of green super rice to future warming. However, the manuscript has some issues need be addressed.

Major concern: At the rice flowering stage, the authors maintained temperature (40-45 °C) as heat stress via covering the plants with polythene sheets. I wonder how to maintain temperature within 40-45 °C by polythene sheets？Were 40-45 °C in both day and night? Or different temperatures between day and night? The precise description of temperature fluctuation in the study is important for interpreting the results. To my knowledge, 40-45 °C heat duration can significantly affect the rice pollen germination, pollen tube growth and fertilization. The 40-45 °C heat impact on rice plants in previous reports showed much worse than the present results. Were negative effects of high temperatures partially offset by lower temperatures at night? Please make an explanation for your real temperature fluctuation in the study.

Questions and suggestions.

What are the genetic background of green super rice (GSR) in the study? Where did the seeds of 22 GSR lines come from?

Pollen Fertility Test.  At the rice flowering stage, the pollen has been developed. The pollen fertility is not pollen vitality. Pollen fertility already formed during flowering whenever encounter high temperature or not. Only high temperature during pollen mother cell differentiation and development has significant effects on pollen fertility.

Statistical significance should be added in Figure 2, Figure 3 and Figure 5.

Figure 5.C lacks mean ± SE.

Discussion. The findings were actually based on a short period of high temperature treatment at the flowering stage, however, high temperature often last longer, not only last in a short period of time, but also in a growing season, a whole lifespan, even multigenerations. On the other hand, plants may be acclimated by lone-term high temperature, and accumulate more mutations after multigenerational exposure to high temperatures (Genome Biol 2021, 22, 160). Thus, plants may also make changes in response to long-term warming. This should be discussed in a scenario of lone-term warmer future.

 

Author Response

The authors investigated the effect of heat stress on agronomic and physiological parameters in 22 green super rice lines and 4 local rice cultivars. Given the global warming attracts increasing concerns, and the present study is on the rice performance under heat stress, I think the results should be of value for assessing responses of green super rice to future warming. However, the manuscript has some issues need be addressed.

Dear Reviewer, Thank you for your careful reading of our manuscript and your helpful comments and questions. We have carefully revised this manuscript according to yours comments. A point-by-point reply to these comments is below:

Major concern: At the rice flowering stage, the authors maintained temperature (40-45 °C) as heat stress via covering the plants with polythene sheets. I wonder how to maintain temperature within 40-45 °C by polythene sheets？Were 40-45 °C in both day and night? Or different temperatures between day and night? The precise description of temperature fluctuation in the study is important for interpreting the results. To my knowledge, 40-45 °C heat duration can significantly affect the rice pollen germination, pollen tube growth and fertilization. The 40-45 °C heat impact on rice plants in previous reports showed much worse than the present results. Were negative effects of high temperatures partially offset by lower temperatures at night? Please make an explanation for your real temperature fluctuation in the study.

Response: Thank you for highlighting this important point. In this study, heat stress (40-45°C) was applied by covering the plants with polythene sheets during the day time from 10:00 am to 03:00 pm. After that, polythene sheets were removed to reduce the temperature to normal (25-35°C). This was done to make the experiment realistic. As you mentioned, this is quite possible that negative effects of high temperatures during day were partially offset by lower temperatures at evening/night. We have mentioned this in the section “2.1 Experimental site and design”.

Questions and suggestions.

What are the genetic background of green super rice (GSR) in the study? Where did the seeds of 22 GSR lines come from?

Response: GSR was developed as a combined effort of international scientists from International Rice Research Institute (IRRI), Philippines and Chinese Academy of Agricultural Sciences (CAAS), China. Based on the information on cloned green genes and loci, large-scale cross and backcross breeding was conducted to generate IL populations and lines abundant in green traits with wild rice, core germplasm, and specific local varieties as the donors. Further details about GSR background and development can be found in Yu et al. 2020, Theoretical and Applied Genetics, 133, 1427–1442.

Recently, 552 GSR advanced lines from CAAS, China were introduced at National Institute for Genomics and Advanced Biotechnology (NIGAB), National Agriculture Research Council (NARC), Islamabad (Pakistan), to develop rice cultivars that retain sustainable yield even under unfavorable environmental conditions. Out of these 552, 22 GSR lines on the bases of preliminary screening were selected and evaluated under different stress conditions. Secondly, these lines were already evaluated under drought stress (Ahmad et al., 2022) and salt stress (Amanat et al., 2022) conditions. So, we selected these GSR lines to check the effect of heat stress in this study.

Pollen Fertility Test.  At the rice flowering stage, the pollen has been developed. The pollen fertility is not pollen vitality. Pollen fertility already formed during flowering whenever encounter high temperature or not. Only high temperature during pollen mother cell differentiation and development has significant effects on pollen fertility.

Response: Yes, we agreed with the reviewer that at the flowering stage, pollen has been developed. But in this study, we applied heat stress at pre-anthesis stage and termed as “flowering stage”. Normally, pollens are in the developmental process at this stage, but possibility of variation in pollen development times among GSR and checks could not be ruled out.

Statistical significance should be added in Figure 2, Figure 3 and Figure 5.

Response: Thank you for pointing this. Suggestion is incorporated, please see the updated figures 2, 3, and 5.

Figure 5.C lacks mean ± SE.

Response: Thank you for pointing this. Suggestion is incorporated, please see the updated figures 5C.

Discussion. The findings were actually based on a short period of high temperature treatment at the flowering stage, however, high temperature often last longer, not only last in a short period of time, but also in a growing season, a whole lifespan, even multigenerations. On the other hand, plants may be acclimated by lone-term high temperature, and accumulate more mutations after multigenerational exposure to high temperatures (Genome Biol 2021, 22, 160). Thus, plants may also make changes in response to long-term warming. This should be discussed in a scenario of lone-term warmer future.

Response: Thank you for nice suggestion. Suggestion is incorporated, please see the discussion.

Reviewer 2 Report

Dear Authors

I would like to make comment the interesting trial naming “Agronomic and Physiological Indices for Reproductive Stage Heat Stress Tolerance in Green Super Rice“

 

There were included some interesting results and providing effective scientific information. However, it will need to improve several points for data arrangement and discussion. I would like to provide several comments for improvement as follows.

 

1)    The data for yield components, such as, heading date (growing stages among rice accessions at the heat treatment), culm length and panicle length instead of plant high, Numbers of panicle per plant instead of tillers per plant (impossible to count the numbers of tiller at harvest time, only panicle No.), weight of whole plant instead of straw yield per plant, Harvest index (Panicle weight/ weight of whole plant), numbers of spikelet per plant, percentage of fertility spikelet per plant, and 1000-grainn weight, should be shown as the genetic variations among rice accessions, and should be used for the characterization for heat stress tolerance. Specially, the degrees of fertility spikelet are the most important trait in this study.

2)    Four control varieties were used in this study, but there were not explained the degrees for heat stress tolerance in the “Introduction” and Material and methods”. The information should be written in the manuscript.

3)    There was a few information for Green Super Rice (GSR) varieties in the “Introduction”. The information, such as special characters and breeding process and so on, will be added in the “Introduction”

4)    The data of trait should be shown as relative values which were calculated traits’ values under the heat stress condition against normal condition.

5)    Based on the data of grain yield per plant, NGSR-5, NGSR-16, NGSR-21, and Kashimir Basmati were selected as tolerant materials. Based on harvest index, NGSR-2, NGSR-4, NGSR-19, NGSR-21, and Kashimir Basmati were selected. These data were not corresponded with these of heat susceptibility index. I guess that the traits selection used in this study might be not suitable for characterization of tolerant. Because the most important trait is the fertility spikelet No. (percentage) was not included in this study, and these growth stages among rice accessions might be not considered in this study.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

The global atmospheric temperature is increasing at a very fast pace every year, which cause the reduction of crop production. In this manuscript, 22 GSR lines and 4 local checks were evaluated under normal and heat-stress conditions for different Agro-Physiological parameters. As a result, two GSR lines were identified and could be utilized as parents for the development of heat-tolerant rice. The manuscript is well prepared. I have two minor questions. First, in this study, the results are simple and little molecular mechanism is introduced why some rice varieties are tolerance, some are not.  Second, I find two heat-tolerant rice NGSR-16 and NGSR-18 in the abstract, but GSR-13 and GSR-24 in the result part. They are not in consistent. 

Author Response

Reviewer 3 Comments Response

The global atmospheric temperature is increasing at a very fast pace every year, which cause the reduction of crop production. In this manuscript, 22 GSR lines and 4 local checks were evaluated under normal and heat-stress conditions for different Agro-Physiological parameters. As a result, two GSR lines were identified and could be utilized as parents for the development of heat-tolerant rice. The manuscript is well prepared. I have two minor questions.

Response: Dear Reviewer thank you for your appraisal comments on our article. No doubt, climate is changing at faster rate across the globe. Therefore, this study was designed accordingly. We have addressed your queries and provided a point-by-point response below:

First, in this study, the results are simple and little molecular mechanism is introduced why some rice varieties are tolerance, some are not.

Response: Thank you for raising this concern. Exactly, some rice genotypes were tolerant to heat stress and some were susceptible. In this scenario, this study evaluated the adaptability mechanisms of different GSR lines under heat stress. This study provided the factual investigations of responses for future adaptation of GSR under future projected heat stress in South Asia especially Pakistan.  

Second, I find two heat-tolerant rice NGSR-16 and NGSR-18 in the abstract, but GSR-13 and GSR-24 in the result part. They are not in consistent. 

Response: Dear reviewer thank you for nice suggestion. Most of the responses among all lines/genotypes for yield and yield components, and quality parameters depicted that the NGSR-13 performed poorly related to other GSR lines under consideration. Therefore, it is concluded that NGSR-13 was observed with least tolerance against heat stress. Please see the line 212, Figure 1B (PCA), Figure 3D (HSI), Figure 4 (PFP), and Figure 5B (NDVI).

Round 2

Reviewer 2 Report

Dear Authors

The manuscript was included several missing points which were lacks data of spikelet fertility, the relationship between pollen fertility and spikelet fertility, heading data, and relationship between heading data and spikelet fertility.

These data will need for the characterization of introgression lines for tolerant to heat stress.

These data in the manuscript will be useful for the primitive screening and characterizations of introgression lines as GSR. However, the data set will not suitable and not enough for scientific report of international journal.

I would like to request to collect the additional data which I indicated as lacked points.

Best regards 

 

Author Response

Dear Authors

The manuscript was included several missing points which were lacks data of spikelet fertility, the relationship between pollen fertility and spikelet fertility, heading data, and relationship between heading data and spikelet fertility. These data will need for the characterization of introgression lines for tolerant to heat stress. These data in the manuscript will be useful for the primitive screening and characterizations of introgression lines as GSR. However, the data set will not suitable and not enough for scientific report of international journal. I would like to request to collect the additional data which I indicated as lacked points.

Response: Dear Reviewer, Thank you for your careful reading of our manuscript and your helpful comments and questions. We agreed with the reviewer that the above-mentioned traits are very important for screening of heat tolerance but we don’t have the said data. We have presented all the data in the manuscript and hope that the presented data will be acceptable. Many previous studies such as Zafar et al., 2019 (http://dx.doi.org/10.1101/739433) and Hazra et al. (2016) / J. Biosci. Agric. Res. 07(01): 600-607  also used these traits for the screening of germplasm against high temperature. So, we believe that both the traits harvest index and heat susceptibility index are good indicators of heat tolerance.

Reviewer 3 Report

What is the relationship between GSR and NGSR? Also,  I can not find GSR-24 in the whole manuscript, Please explain it. Moreover, I have listed the recommendations for authors, please revise the manuscript throroughly. 

Author Response

What is the relationship between GSR and NGSR?  

Response: Thank you for your query. GSR and NGSR actually are the same. 552 GSR lines were obtained from CAAS, China, and their adaptability mechanisms were investigated by NIGAB, Pakistan through multi-locational trials in Pakistan for two years. On the basis of these investigations, 22 GSR lines were evaluated as more self-adaptable to varying climatic stresses such as drought and salinity. These 22 GSR lines were also used in the current study to evaluate the heat tolerance mechanisms. We used the word NGSR as a reference to these 22 GSR lines because they have been evaluated at NIGAB, Pakistan (DOIs:10.3390/plants11111461; 10.3389/fgene. 2022.832542).

 

Also,  I can not find GSR-24 in the whole manuscript, Please explain it.

Response: Thank you for pointing this out. There was a typo mistake in figure 4 legend. Now it is corrected, please see the figures (1-4) legends.

Moreover, I have listed the recommendations for authors, please revise the manuscript throroughly. 

Response: The manuscript has been thoroughly read by all authors and necessary changes have been made regarding traditional English editing, grammatical mistakes, and other scientific mistakes. Once again, all authors are thankful for your time to comprehensively review our manuscript which helped us a lot to make it worthy for the scientific community.

Round 3

Reviewer 2 Report

Dear authors

The dat ofr seed fertility for heat stress is the most important data, and the lack of them was a fatal mistake.

HI and pollen fertility were the secondally traits and direct data for torelant to heat stress.

And HSI was not explained in the Metereila and Methods. I could not understand the meaning.

And the Kashimir Basmati is the best material and not GSR line. It means that GSR lines ahowed the varietion for heat stress, but these were not so high.

I have to evaluated that the research paper was not included so important data for heat stress tolerance, and not suitable for international journal.

Best regards 

Author Response

Dear authors

The dat ofr seed fertility for heat stress is the most important data, and the lack of them was a fatal mistake. HI and pollen fertility were the secondally traits and direct data for torelant to heat stress.

Response: Dear Reviewer, thank you for your careful reading of our manuscript and your helpful comments and questions. We agreed with the reviewer that the traits mentioned above are essential for screening heat tolerance. Many previous studies such as Zafar et al., 2019 (http://dx.doi.org/10.1101/739433) and Hazra et al. (2016) / J. Biosci. Agric. Res. 07(01): 600-607  also used these traits for the screening of germplasm against high temperature. So, we believe that both the traits harvest index and heat susceptibility index are good indicators of heat tolerance.

And HSI was not explained in the Metereila and Methods. I could not understand the meaning.

Response: Thank you for your query. The heat susceptible index (HSI) is already explained in the text. Please see lines 185-208.

And the Kashimir Basmati is the best material and not GSR line. It means that GSR lines ahowed the varietion for heat stress, but these were not so high.

Response: Thank you for pointing out your concern. For some of the traits, Kashmir Basmati performed well, however overall GSR lines showed better performance for most of the traits. On the basis of this study, Kashmir Basmati was out performer among the checks, whereas among the GSR lines, NGSR-16 was the best.

I have to evaluated that the research paper was not included so important data for heat stress tolerance, and not suitable for international journal.

Response: Thank you for your query. We have revised the manuscript carefully and believed that th revised version will be acceptable.