Progress 02/01/11 to 01/31/15
Outputs
Target Audience: Our main target audience currently includes other scientists and students (graduate and advanced undergraduates) working/studying in the public and private sector. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Both Associate Scientist Dong-Hoon Jeong (now Assistant Professor at Hallym University in S. Korea) and postdoc Pingchuan Li (now a postdoc in Canada) received valuable training working on this project. Related topics are part of lectures given by the PDs when they teach classes including to students from other disciplines and during a short course for high school teachers. How have the results been disseminated to communities of interest? The results have been disseminated in four publications so far and presented at meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Drought-responsive small RNAs (obj. 1 and obj. 3) Small RNAs can regulate genes at several levels including through epigenetic regulation. Our first objective involved the examination of small RNA populations at different time points with and without drought stress, after recovery, and in the next generation in rice. A total of 39 small RNA libraries have been constructed and sequenced exceeding the number proposed. An average of about 21M total and 3M distinct genome-matched small RNA sequences per library were obtained from a total of 821M sequence reads. These data provide a source for small RNA clustering and regulated miRNAs. The previously reported (Jeong et al., 2011) expression patterns of drought-responsive miR820 and its target, OsDRM2 (which encodes a DNA methytransferase), were validated by confirming that both miR820 and OsDRM2 were down-regulated by drought treatment using samples made for this project. These experiments included samples harvested during the re-hydration after drought stress, which indicated that miR820 and OsDRM2 expression levels recovered. The results imply that DNA methylation by OsDRM2 under drought stress and during the recovery may be dynamically regulated and fine-tuned by miR820. The development of transgenic plants that alter normal regulation exerted by miR820 or DRM2 is in progress to evaluate the effects on drought responses. To examine the RNA degradome, which is the population of RNA decay intermediates including those from small RNA guided guided cleavage, PARE libraries were made from samples selected from those used for small RNA libraries. RNA-Seq libraries have been made to evaluate mRNA levels in these samples. The sequencing data from these exceeded our objectives by more than 15 PARE libraries and 15 RNA-Seq libraries. In addition to drought-responsive miRNAs, examples of miRNA cleavage from the PARE data and siRNA clusters that are drought responsive were identified. These include both up-regulated and down-regulated small RNA clusters that may be associated with epigenetic control. Analysis of BS-Seq data (obj. 2) To detect methylation, ten whole-genome bisulfite sequencing (BS-seq) libraries were constructed and found to be of high quality based on the number of cytosines analyzed. The libraries included samples from the drought time course described above as well as a recovery sample and a panicle drought sample. These have been analyzed for the identification of differentially methylated regions (DMRs). Numerous DMRs were identified from analyses of the five BS-Seq comparisons between drought treatment or recovery and the corresponding controls in different cytosine contexts. Most of these were annotated as transposable elements in the CHH context and but many DMRs were also evident in protein-coding genes in this and other contexts. Future work should lead to a more detailed analysis and enable further comparisons with small RNA clusters, and PARE and RNA-Seq data to examine the potential impact of hypo- and hyper-methylated loci on gene expression patterns. Data release (obj. 4) Web viewers have been built to examine the different types of RNA data: Small RNA, PARE, RNA-Seq and BS-Seq. The databases and web interfaces developed for this project are the precursors for data release. Data will also be deposited into GEO (Gene Expression Omnibus) and release is expected in the coming year. Overall, the data and findings from this work should be of considerable value for elucidating the impact of epigenetic changes associated with drought stress and recovery in rice. Not only will itfacilitate global and single gene-level study of such changes but it will enable associations to be made with levels of small RNA, mRNA, and partially degraded mRNA to help elucidate complex mechanisms of gene regulation. Although studies in seedlings are easier and more common, this study provides an important added dimension relevant to yield by including panicle samples. Similarly, by going to the next generation, heritability of the regulation can be examined as well. The insights gained could suggest new strategies for breeding or engineering drought tolerance. Also, the results may be relevant to other plants, particularly the grasses. Finally, one of the lead researchers who received training under this project now leads his own research program devoted to improvement of rice.
Publications
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Progress 02/01/13 to 01/31/14
Outputs
Target Audience: Our main target audience currently includes other scientists and students (graduate and advanced undergraduates) working/studying in the public and private sector. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Both Associate ScientistDong-Hoon Jeongand postdoc Pingchuan Li received valuable training working on this project. How have the results been disseminated to communities of interest? The results have been disseminated in publications and presented at the PI meeting. What do you plan to do during the next reporting period to accomplish the goals? Transgenic plants to overexpress miR820 and miR820-resistant OsDRM2 will be generated. Further analysis of the data from drought-treated panicles will follow. In addition, the association of these regulated small RNA loci on DNA methylation and gene expression will be further examined. The association of regulated small RNA loci on DNA methylation and gene expression will be further examined. Future work will compareDMR data with small RNA clusters and RNA-Seq data to examine the potential impact of hypo- and hyper-methylated loci on gene expression patterns. Future efforts will seek to link RNA-seq data to the drought-regulated small RNA loci and DMRs.
Impacts
What was accomplished under these goals?
Drought-responsive miRNAs (obj. 1 and obj. 3) The previously reported (Jeong et al., 2011) expression patterns of drought-responsive miR820 and its target, OsDRM2, were validated by confirming that both miR820 and OsDRM2 were down-regulated by drought treatment using samples made for this project. These experiments included samples harvested during the re-hydration after drought stress, which indicated that miR820 and OsDRM2 expression levels recovered. These results imply that DNA methylation by OsDRM2 under drought stress and during the recovery may be dynamically regulated and fine-tuned by miR820. To evaluate the effects of miR820 and OsDRM2 on drought responses in transgenic plants, binary vectors to overexpress miR820 and miR820-resistant OsDRM2 are under construction. In the coming year, transgenic plants will be generated. During the reporting year, two more miRNAs were identified as candidates for drought responsive miRNAs. Both were up-regulated by drought stress treatment in panicle tissues. Their expression patterns were validated with one biological replicate and will be further confirmed with another biological replicate and the impact of these miRNAs on their targets will be examined. Analysis of small RNA clusters (obj. 1) During a previous reporting period, we identified several candidates for drought stress–regulated small RNA clusters. Over the past year, MySQL tables were established for genome-wide examination of drought-stress regulated small RNA clusters. From the initial global analysis of regulated small RNA clusters, we identified both up-regulated and down-regulated small RNA clusters in seedlings. Further analysis of the data from drought-treated panicles will follow. In addition, the association of these regulated small RNA loci on DNA methylation and gene expression will be further examined. Analysis of BS-Seq data (obj. 2) This past year, we further developed a pipeline for the identification of differentially methylated regions (DMRs). Specifically, the DMRs were defined by tiling the 12 rice chromosomes into hundreds of thousands of bins in the size of 100 base pairs. All the bins, with absolute methylation differences over 0.3, 0.2 and 0.1 for CG, CHG and CHH respectively, were compared by performing the Fisher’s Exact test within samples. The significant DMR pairs between control and drought treatment samples were further assessed by an FDR correction based on the Benjamini–Hochberg procedure at a p=0.01 cutoff. We identified 312,502 DMRs among the ten samples used for the five BS-Seq comparisons between drought treatment or recovery and corresponding controls, in different cytosine contexts. Among the comparisons, the one from panicles showed more sensitivity than those from seedlings to drought. In each of the comparisons, the majority of the DMRs were annotated as transposable elements (>60%) and genic DMRs averaged almost 10% among the comparisons Future work will compare these data with small RNA clusters and RNA-Seq data to examine the potential impact of hypo- and hyper-methylated loci on gene expression patterns. Analysis of RNA-Seq data (obj. 1 and obj. 4) This year, we built a web-viewer of RNA-Seq data for this project to examine well-known drought-responsive genes. The initial analysis supported the regulation of key genes by drought treatment. Future efforts will seek to link RNA-Seq data to the drought-regulated small RNA loci and DMRs. The databases and web interfaces developed for this project are the precursors for data release.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Jeong, D.-H. and Green, P.J. (2013) The Role of Rice microRNAs in Abiotic Stress Responses. J. Plant Biol. 56:187-197.
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Progress 02/01/12 to 01/31/13
Outputs
OUTPUTS: Our first objective is to examine small RNA populations at different time points with and without drought stress, after recovery, and in the next generation in rice. During the second year of our project, we made 12 more small RNA libraries from the next generation of drought treated seedlings. In total, 39 small RNA libraries have been constructed and sequenced. An average of about 21M total and 3M distinct genome-matched small RNA sequences per library were obtained from a total of 821M sequence reads. Using this set of small RNA data, we are currently investigating the drought stress-regulated small RNA clusters. The analysis is based on static clustering with 500-bp window. Our second objective is to examine the impact of drought on chromatin modifications and gene expression. Efforts on whole-genome bisulfite sequencing (BS-seq) have focused on improving the bisulfite conversion of our BS-seq preparation pipeline for sequencing via multiplexed Tru-Seq. Sequencing of the remaining two rice libraries was completed, resulting in a total of ten BS-seq libraries which were made from drought time courses of seedling and inflorescence tissues. We also worked on the development of an interactive MySQL based WebViewer for the BS-seq data. Several protocols for the ChIP-seq were tested. Other activities concentrated on the development of multiple tools for examining chromatin modification data. This year, we made eight PARE libraries and eight RNA-seq libraries from the next generation of drought-treated seedlings. In total, we have made 22 PARE libraries from samples selected from those used for small RNA libraries. Twenty eight RNA-Seq libraries have also been made to evaluate mRNA levels in these samples. The work was disseminated in a poster presented at the Project Director's meeting in May 2012 and an associated accomplishments report. PARTICIPANTS: Participants include Pam Green, PD; Blake Meyers, Co-PD; Dong-Hoon Jeong, Postdoc/Associate Scientist; Pingchuan Li, Postdoc, all from the University of Delaware. Both the postdoc and postdoc/Associate Scientist (UD equivalent of Research Assistant Professor) receive valuable training working on this project. TARGET AUDIENCES: Our main target audience currently includes other scientists and students (graduate and advanced undergraduates) working/studying in the public and private sector. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Initial analysis of our rice PARE libraries for a miRNA that has been reported to be up-regulated by drought stress showed that the precise target cleavage guided by this miRNA was increased during drought. This result indicates that future efforts to identify additional drought-regulated RNA decay events will be fruitful. The impact of the regulated small RNA clusters mentioned in outputs are being studied by comparing the data from PARE, RNA-seq, and BS-seq. For example, we have identified a gene which gives rise to drought-inducible secondary siRNAs from an exon. This gene also exhibited a drought-inducible expression pattern based on RNA-Seq data and a drought-inducible PARE accumulation pattern evident from our PARE data. Several tools were developed to advance our bioinformatics analysis of our BS-seq data. These include tools for metaviewer analysis and for whole genome comparison based on the Circos tool. In addition, we have advanced a tool for analysis of differentially methylated regions, aspects of which are still under development. The work to date is being developed for presentation in May of 2013 at the Project Director's meeting. The webviewer mentioned above is being developed to facilitate future data release. Two publications are also products of this research.
Publications
- Jeong, D.H., and Green, P.J. Methods for validation of miRNA sequence variants and the cleavage of their targets. Methods. 58: 135-43 (2012)
- Li, P., Demirci, F., Mahalingam, G., Demirci, C., Nakano, M., and B.C. Meyers (2013). An integrated workflow for DNA methylation analysis. J. Genetics & Genomics. In press.
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Progress 02/01/11 to 01/31/12
Outputs
OUTPUTS: Our first objective is to examine small RNA populations at different time points with and without drought stress, after recovery, and in the next generation in rice. In the first year of our project, we have made 27 of the proposed small RNA libraries, including two biological replicates of control and drought time course samples for seedling and inflorescence libraries, plus recovery samples for seedlings. We have also harvested samples for making libraries from the next generation. Our second objective is to examine the impact of drought on chromatin modifications and gene expression. We have made ten whole-genome bisulfite sequencing (BS-seq) libraries, of which eight have been sequenced. The libraries include the time course described above, from 8 hours to 7 days. The best libraries had 271 million reads, with 233 million distinct sequences, providing 8X genome coverage. The lowest level of genome coverage we obtained was 2X, but this will be remedied with additional sequencing. To examine the RNA degradome, we have made 14 PARE libraries from samples selected from those used for small RNA libraries. Twelve RNA-Seq libraries have been made to evaluate mRNA levels in these samples. PARTICIPANTS: Participants include Pam Green, PD; Blake Meyers, Co-PD; Dong-Hoon Jeong, Postdoc/Associate Scientist; Pingchuan Li, Postdoc, all from the University of Delaware. Both the postdoc and postdoc/Associate Scientist (UD equivalent of Research Assistant Professor) receive valuable training working on this project. TARGET AUDIENCES: Our main target audience now includes other scientists and students (graduate and advanced undergraduates) working/studying in the public and private sector. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Initial analysis has identified a drought-regulated siRNA cluster in an inverted repeat region. Our progress thus far exceeded our objectives by ten PARE libraries and eight RNA-Seq libraries. We have also made significant technical advances, including the development of a TruSeq (multiplexed) library construction protocol for BS-seq libraries, and advances in the informatics of BS-seq read mapping. The work from our first year is being prepared for a poster presentation at the Project Directors meeting taking place in May of 2012. This work contributed to one publication during the reporting year.
Publications
- Jeong, D.H., Park, S., Zhai, J., Gurazada, S.G., De Paoli, E., Meyers, B.C., and Green, P.J. Massive Analysis of Rice Small RNAs: Mechanistic Implications of Regulated MicroRNAs and Variants for Differential Target RNA Cleavage. Plant Cell 23: 4185-4207 (2011)
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