Identification of differentially expressed genes involved in amino acid and lipid accumulation of winter turnip rape (Brassica rapa L.) in response to cold stress

Yan Fang; Jeffrey A. Coulter; Junyan Wu; Lijun Liu; Xuecai Li; Yun Dong; Li Ma; Yuanyuan Pu; Bolin Sun; Zaoxia Niu; Jiaojiao Jin; Yuhong Zhao; Wenbo Mi; Yaozhao Xu; Wancang Sun

doi:10.1371/journal.pone.0245494

Peer Review History

Original SubmissionJune 4, 2020
23 Nov 2020 Decision Letter - Kun Lu, Editor PONE-D-20-16938 Identification of differentially expressed genes involved in amino acid and lipid accumulation of winter turnip rape (Brassica rapa L.) in response to cold stress PLOS ONE Dear Dr. Sun, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Dec 22, 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: 1. Why the author choose seedling stage sample to interpret the lipid accumulation. I think the cold tolerance have more relation with the sugar metabolism than the lipid metabolism. 2. line 217-218, rewrite it and the writing needs to be polished for the whole text. 3. In figure 1，the phenotype is not obvious under the cold stress, and the soil looks different in the pod, it would be better show readers the root phenotype since the whole research is on root.. 4. The figures quality are poor. 5. Since there are 9 amino acids were different between L7 and T2, which include Proline, and please use it as a example by using the transcriptome data to interpret this difference. 6. line 365, correct 4°; 7. How about the DEG involved in phytohormone synthesis or signaling, please add this analysis to the MS; 8. Table1 move to the supplimentary file; 9. Correct brassica rapa in italic; Reviewer #2: The manuscript deals with the analysis of genes related in response to cold stress in Brassica rapa L. The study is approached and contain intersting results, however, the manuscript needs revisions before accepting for publication. Following main points need due consideration. 1. P3-line 105-106, “until the plants grew to the six leaf stage, then the seedlings were treated with different conditions including control (CK) and cold stress”, various crop varieties have different growth periods, “grew to the six leaf stage”, the time statement can be more clearer, such as “three-week-old”, or “30 day old” and so on. 2. In materials and methods, the author should indicate photosynthetic photon flux density and relative humidity, it has been reported that light intensity affects the tolerance of plants to low temperature. 3. In Figure 1, the authors may consider the possibility of statistical analysis of phenotypic results, such as total leaf area, leaves fresh weight, root fresh weight, leaves dry weight, or root dry weight. 4. In Figure 3, transcript id should be added the annotation of gene name, such as “FAD3 (Bra××××××)”, and the author's analysis of Figure 3 is deficient, should make further analysis. 5. In transcriptome results, GO_enrichment and the KEGG map of amino acid metabolism and fatty acid synthesis should be added to the article or supporting information, it is helpful for readers to analyze the function of key genes. 6. Through out the manuscript lack of possible raising mechanisms, authors are encouraged to include some recent reports discussing the signalling regulation mechanisms. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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Revision 1
18 Dec 2020 Author Response Response to reviewers Reviewer #1: 1. Why the author choose seedling stage sample to interpret the lipid accumulation. I think the cold tolerance have more relation with the sugar metabolism than the lipid metabolism. Response: Thank you for your comments. There two reason why we choose seedling stage sample to interpret the lipid accumulation. At first, in cold and arid areas of northern China, the winter rapeseed was sown from later August to early September. Generally, seedlings grew to the fourth or fifth leaf stage, which experienced a significant cooling environment. When the seedings grew to the six-leaf stage, entered the overwintering stage. The cold resistance of the plants in the six-leaf stage determined whether the winter rapeseed could survive in the winter safely. Secondly, the plants show complex adaptations to freezing that prevent cell damage caused by cellular dehydration. Lipid remodeling of cell membranes during dehydration is one critical mechanism countering loss of membrane integrity and cell death. Therefore, the seedling samples were selected for sequencing. In addition, as you said, the cold resistance is strongly related to sugar metabolism in plant. However, the focus of our work is on amino acid and lipid accumulation, which is very different from other work, and this is also the major contribution of our work. 2. line 217-218, rewrite it and the writing needs to be polished for the whole text. Response: According to the comment of the reviewer, we have made correction in our revised paper (line 216-217). 3. In figure 1, the phenotype is not obvious under the cold stress, and the soil looks different in the pod, it would be better show readers the root phenotype since the whole research is on root. Response: We are sorry for our negligence. We checked it and changed the figure in our revised paper (Figure 1). Moreover, we added the root phenotype in revised paper. 4. The figures quality are poor. Response: We are sorry for our negligence. We did our best effort to modify the characters of figures in our revised paper. 5. Since there are 9 amino acids were different between L7 and T2, which include Proline, and please use it as a example by using the transcriptome data to interpret this difference. Response: Thank you for your comments. According to your suggestion, we conducted this analysis, and added this part in our revised paper (line 390-425). 6. line 365, correct 4°; Response: We are sorry for our negligence, we checked it and made correction (line 364 in revised paper). 7. How about the DEG involved in phytohormone synthesis or signaling, please add this analysis to the MS; Response: Thank you for pointing this out. In our previous study, we made analysis of DEG involved in phytohormone synthesis and signaling. In this work, we focus on the relation between amino acid, lipid accumulation and cold stress in winter rapeseed. We still appreciate your advice. 8. Table1 move to the supplimentary file; Response: Thank you for your comments. Table 1 reflected the whole RNA-seq characteristic in each sample. We think that it is important to further study in this area. So, we were insisted on putting table 1 in the body of text. 9. Correct brassica rapa in italic; Response: We are sorry for our negligence, we checked whole text and made correction. Reviewer #2: The manuscript deals with the analysis of genes related in response to cold stress in Brassica rapa L. The study is approached and contain intersting results, however, the manuscript needs revisions before accepting for publication. Following main points need due consideration. 1. P3-line 105-106, “until the plants grew to the six leaf stage, then the seedlings were treated with different conditions including control (CK) and cold stress”, various crop varieties have different growth periods, “grew to the six leaf stage”, the time statement can be more clearer, such as “three-week-old”, or “30 day old” and so on. Response: We are sorry for our negligence. In cold and arid areas of northern China, the winter rapeseed was sown from later August to early September. Generally, seedlings grew to the fourth or fifth leaf stage, which experienced a significant cooling environment. When the seedings grew to the six-leaf stage (60 days after sown), entered the overwintering stage (probably October to November). So, we're used to writing “six-leaf stage”. According to your advices, we were rewrite it within 60 days in revised paper (line 104). 2. In materials and methods, the author should indicate photosynthetic photon flux density and relative humidity, it has been reported that light intensity affects the tolerance of plants to low temperature. Response: We are sorry for our negligence. We added the photosynthetic photon flux density and relative humidity in our revised paper (line 108-109). 3. In Figure 1, the authors may consider the possibility of statistical analysis of phenotypic results, such as total leaf area, leaves fresh weight, root fresh weight, leaves dry weight, or root dry weight. Response: Thank you for your advices and we appreciated it. In our previous study, the fresh matter, dry matter, root/shoot ratio and main root diameter of L7 and T2 under cold treatment were described in detail [Morphology and physiological characteristics of cultivars with different levels of cold-resistance in winter rapeseed (Brassica campestris L.) during cold acclimation. Sci Agric Sin 2013, 22]. In this study, the cold treatment was decreased from 22℃ to -4℃ at a cooling rate of 2℃ per hour. After treated at low temperature, we selected the leaves firstly showed wilting symptoms as time point of sampling. So, in figure 1 (old), there are no visible significant different between L7 and T2. This caused a misunderstanding to the reader. In our revised paper, we replaced the figure 1 (old) with new picture and added the root morphology of two cultivars (revised paper Figure 1A and 1B). 4. In Figure 3, transcript id should be added the annotation of gene name, such as “FAD3 (Bra××××××)”, and the author's analysis of Figure 3 is deficient, should make further analysis. Response: We are very sorry for the misunderstanding caused by our incorrect expression. The 19 genes used for validated were randomly selected from RNA-seq data, were not selected from differentially expressed genes (DEGs). In our paper, we were mentioned “Nineteen genes were selected for qRT-PCR validation to verify the RNA-seq data, involving genes in the amino acid, sugar, lipid, and plant hormone signal metabolic pathways which have already been reported to be related with cold stress.”, it is because of the annotation information of these 19 genes suggested that these genes are annotated into these metabolic pathways. Moreover, we found that the genes of Bra011511, Bra004136, Bra008792, Bra007142 and Bra036828 respectively annotated in aldehyde dehydrogenase, glutamate decarboxylase 2, chalcone synthase, chalcone-flavanone isomerase 1 protein, flavanone 3-hydroxylase 1 protein were related cold stress by looking at the references. Since the purpose of the qRT-PCR experiment is verify the accuracy of sequencing data, and our results also show that our sequencing data are reliable. We think that this analysis is also feasible for this part. As for your suggestions, we also added the annotation information of the 19 transcripts in revised paper, which is shown in Table S5. 5. In transcriptome results, GO_enrichment and the KEGG map of amino acid metabolism and fatty acid synthesis should be added to the article or supporting information, it is helpful for readers to analyze the function of key genes. Response: Thank you for your comments. According to your suggestion, we conducted this analysis, and added this part (Figure 4 and 5) in our revised paper. 6. Through out the manuscript lack of possible raising mechanisms, authors are encouraged to include some recent reports discussing the signalling regulation mechanisms. Response: Thank you for pointing this out. In our previous study, we made analysis of DEG involved in signaling regulation [Transcriptome Analysis Reveals Key Cold-Stress-Responsive Genes in Winter Rapeseed (Brassica rapa L.). Int J Mol Sci 2019, 20, doi:10.3390/ijms20051071]. In this work, we focus on the relation between amino acid, lipid accumulation and cold stress in winter rapeseed. We still appreciate your advice. https://doi.org/10.1371/journal.pone.0245494.r002
4 Jan 2021 Decision Letter - Kun Lu, Editor Identification of differentially expressed genes involved in amino acid and lipid accumulation of winter turnip rape (Brassica rapa L.) in response to cold stress PONE-D-20-16938R1 Dear Dr. Sun, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Kun Lu, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No https://doi.org/10.1371/journal.pone.0245494.r003
Formally Accepted
21 Jan 2021 Acceptance Letter - Kun Lu, Editor PONE-D-20-16938R1 Identification of differentially expressed genes involved in amino acid and lipid accumulation of winter turnip rape (Brassica rapa L.) in response to cold stress Dear Dr. Sun: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Kun Lu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0245494.r004

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