Global analysis of differentially expressed genes between two Japonica rice varieties induced by low temperature during the booting stage by RNA-Seq

As one of the abiotic stresses, low temperature severely threatens rice production during its entire growth period, especially during the booting stage. In the present study, transcriptome analysis was performed comparing Longjing (LJ) 25 (chilling-tolerant) and LJ 11 (chilling-sensitive) rice varieties to identify genes associated with chilling tolerance in rice spikelets. A total of 23 845 expressed genes and 13 205 differentially expressed genes (DEGs) were identified, respectively. Gene ontology (GO) enrichment analyses revealed ‘response to cold’ (containing 180 DEGs) as the only category enriched in both varieties during the entire cold treatment period. Through MapMan analysis, we identified nine and six DEGs related to the Calvin cycle and antioxidant enzymes, respectively, including OsRBCS3, OsRBCS2, OsRBCS4, OsAPX2 and OsCATC, that under chilling stress were markedly downregulated in LJ11 compared with LJ25. Furthermore, we predicted their protein–protein interaction (PPI) network and identified nine hub genes (the threshold of co-expressed gene number ≥ 11) in Cytoscape, including three RuBisCO-related genes with 14 co-expressed genes. Under chilling stress, antioxidant enzyme activities (peroxidase (POD) and catalase (CAT)) were downregulated in LJ11 compared with LJ25. However, the content of malondialdehyde (MDA) was higher in LJ11 compared with LJ25. Collectively, our findings identify low temperature responsive genes that can be effectively used as candidate genes for molecular breeding programmes to increase the chilling tolerance of rice.


Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The aim of this work is to elucidate the molecular mechanisms underlying the response to chilling stress during the booting stage in Japonica rice cultivars. The authors' strategy in the present work technically sounds, RNA sequencing and bioinformatic analyses appear to be conducted properly, and the data obtained are presented nicely. I agree to the main conclusion that several chilling stress responsible genes, such as CATC, Trx23, POX1, and MAP1, can be useful for breeding of cold-tolerant rice cultivars. Also, I believe that the present work will be potentially of interest to many scientists in a range of research areas such as plant stress physiology, signaling, and molecular biology. I have only a few minor comments to improve the manuscript.
1. L139. It is better to explain more about genetic relationship between LJ25 and LJ11. How was LJ25 produced from LJ11? 2. L175-179. Experimental procedures for enzyme activity assay and MDA determination should be explained in detail. It is not fair only to cite a paper provided by another research group.
3. There are many grammatical/typographical errors. The authors should have checked more carefully before submission.

22-Apr-2020
Dear Dr ZHENHUA, The editors assigned to your paper ("Global analysis of differentially expressed genes between two Japonica rice varieties induced by low-temperature during the booting stage by RNA-Seq") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. The work presents transcriptomic analysis of two rice varieties (one chilling-tolerate and chillingsensitive) in order to identify global changes in gene expression, thus genetic response that leads to chilling-tolerance. It provides useful new information that could lead to improved rice varieties. However, the manuscript needs to be improved before acceptance. Specifically 1) more and proper references need to be provided 2) more detailed methods need to be provided 3) Spelling and grammatical errors and language/style overall needs to be improved 4) Figure legends need more detailed description 5) In addition to show transcriptome differences, the authors should analyse/discuss differences between genetic differences between the two strains and relate the differences to the differences in transcriptome profiling Comments to Author: Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The aim of this work is to elucidate the molecular mechanisms underlying the response to chilling stress during the booting stage in Japonica rice cultivars. The authors' strategy in the present work technically sounds, RNA sequencing and bioinformatic analyses appear to be conducted properly, and the data obtained are presented nicely. I agree to the main conclusion that several chilling stress responsible genes, such as CATC, Trx23, POX1, and MAP1, can be useful for breeding of cold-tolerant rice cultivars. Also, I believe that the present work will be potentially of interest to many scientists in a range of research areas such as plant stress physiology, signaling, and molecular biology. I have only a few minor comments to improve the manuscript.
1. L139. It is better to explain more about genetic relationship between LJ25 and LJ11. How was LJ25 produced from LJ11? 2. L175-179. Experimental procedures for enzyme activity assay and MDA determination should be explained in detail. It is not fair only to cite a paper provided by another research group.
3. There are many grammatical/typographical errors. The authors should have checked more carefully before submission.

01-Jun-2020
Dear Dr ZHENHUA, It is a pleasure to accept your manuscript entitled "Global analysis of differentially expressed genes between two Japonica rice varieties induced by low-temperature during the booting stage by RNA-Seq" in its current form for publication in Royal Society Open Science.
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Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/. Thank you for arranging a timely review of our manuscript (RSOS-192243.R1), entitled Global analysis of differentially expressed genes between two Japonica rice varieties induced by low-temperature during the booting stage by RNA-Seq. We are pleased to know that our study received highly favorable comments. We have carefully evaluated the reviewers' comments and thoughtful suggestions, responded to these suggestions point-by-point, and revised the manuscript accordingly. Here, we attached a revised manuscript in Word format with tracked changes for your approval.
We sincerely hope our revised manuscript meets the standard of Royal Society Open Science and will be accepted for publication.

Yours sincerely Zhenhua Guo
Associate Editor's comments (Professor Xiaodong Zhang): Associate Editor: 1 Comments to the Author: The work presents transcriptomic analysis of two rice varieties (one chilling-tolerate and chilling-sensitive) in order to identify global changes in gene expression, thus genetic response that leads to chilling-tolerance. It provides useful new information that could lead to improved rice varieties. However, the manuscript needs to be improved before acceptance. Specifically 1) more and proper references need to be provided Response: Thank you for your meticulous and considerate comment. We have added additional, appropriate references containing the methods to measure the activities of SOD, POD, CAT and the MDA content and deleted the less relevant references in our revised manuscript.
2) more detailed methods need to be provided Response: Thank you for this comment. We have described the methods used to measure the activities of SOD, POD, CAT and the MDA content in more detail in our revised manuscript.
3) Spelling and grammatical errors and language/style overall needs to be improved Response: Thank you for your comment. We have carefully examined the spelling and grammatical errors and the improper descriptions throughout the manuscript carefully and, with the help of the 'MJ Language Editing Services', we have corrected the spelling and grammatical errors in our revised manuscript, and the editorial certificate was attached below.

4) Figure legends need more detailed description
Response: Thanks very much for your comment. According to your advice, we have added more details in the figure legends of Fig.1, Fig.2, Fig.4, Fig.5, Fig.6, Fig.7, Fig.8, and Fig.9 in our revised manuscript.
5) In addition to show transcriptome differences, the authors should analyse/discuss differences between genetic differences between the two strains and relate the differences to the differences in transcriptome profiling Response: Thank you very much for this comment. According to your advice, we have discussed the genetic differences between LJ25 and LJ11 and analyzed the relationship of the genetic differences and the transcriptome differences between LJ25 and LJ11 in the discussion part of our revised manuscript.

Comments to Author:
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The aim of this work is to elucidate the molecular mechanisms underlying the response to chilling stress during the booting stage in Japonica rice cultivars. The authors' strategy in the present work technically sounds, RNA sequencing and bioinformatic analyses appear to be conducted properly, and the data obtained are presented nicely. I agree to the main conclusion that several chilling stress responsible genes, such as CATC, Trx23, POX1, and MAP1, can be useful for breeding of cold-tolerant rice cultivars. Also, I believe that the present work will be potentially of interest to many scientists in a range of research areas such as plant stress physiology, signaling, and molecular biology.
I have only a few minor comments to improve the manuscript. Heilongjiang Province. Therefore, the two cultivars have a similar genetic background and share a close genetic relationship. Moreover, they are very similar in growth and plant type in the field and in many other agronomic traits. In spite of the similarities in those agronomic traits, there is a significant difference in the chilling tolerance at the booting stage between these two cultivars, since LJ25 has acquired chilling tolerance while LJ11 has remained chilling sensitive. Based on this, LJ25 and LJ11 are widely used for chilling tolerance analysis, and treated as "mutant" and "wild type", respectively, though they are not technically in that relationship. Again, we apologize for this inaccurate statement and we have made appropriate changes in our revised manuscript.
2. L175-179. Experimental procedures for enzyme activity assay and MDA determination should be explained in detail. It is not fair only to cite a paper provided by another research group.
Response: Thanks for reviewer's reminding. I realize that it was really too rough to explain the experimental procedures for enzyme activity assay and MDA determination. Now I've rewritten this part and given more details of experimental procedures in the revised manuscript.
3. There are many grammatical/typographical errors. The authors should have checked more carefully before submission.
Response: Thank you very much for this comment. We have checked the grammatical/typographical errors carefully throughout the manuscript and corrected them in our revised manuscript.