Progress 07/01/07 to 06/30/12
Outputs
OUTPUTS: A graduate student working on this project completed her Ph.D. in late 2010 and she is currently a postdoctoral scholar at the University of Kentucky, Dept. of Horticulture. Another student working on the project graduated in 2007 and is now a researcher at the University of Nottingham. Both are using skills obtained from participation in this completed project in their new positions. A third graduate student working on an unrelated project has also gained experience in techniques to identify targets of transcription factors and he completed his Ph.D. in early 2010 and is currently a postdoctoral scholar at The Ohio State University. Two undergraduates in the Agricultural Biotechnology program performed their independent research projects in the lab. One graduated in Dec. 2010 and is currently in the Nursing program at Vanderbilt University. The other graduated in Dec. 2012 and hopes to attend a physician's assistant program. Two other undergraduates worked as student workers in the lab and gained hands on experience with a variety of techniques. Three postdoctoral scholars have received training supported by funds from the USDA (started 2005), National Science Foundation (started 2009) and the United Soybean Board (started 2010). This funding was made possible by results generated by the project (and prior project). One postdoc is now at the University of Arizona as a postdoctoral fellow. Two other postdocs are currently in the lab and presented their findings at the 22nd International Conference on Arabidopsis Research held in Madison, Wisconsin in 2011 and both have submitted manuscripts on their work in 2012. One manuscript is under review, a second was declined but resubmission encouraged (both Plant Physiology; experiments for the resubmission are in progress) and a third was deemed "well done" but not high impact enough for The Plant Cell (the top journal in the field) so we are splitting to two manuscripts and will submit one in the upcoming month. The project has also led to collaborations with manuscripts published or in preparation. The chromatin immunoprecipitation approach developed in the lab during this and the prior Hatch project has lead to more than 80 labs in more than 15 countries contacting us for a detailed protocol or technical advice. In 2011 the book Molecular Biology volume 754: Plant Transcription Factors: Methods and Protocols, Humana Press (J.M. Walker, Series Editor), co-edited by Ling Yuan and myself was published. Also in 2011 and 2012, workshops for 5th grade students were run as a collaboration with Raven Run Nature Sanctuary to teach students about using the scientific method to investigate environmental control of flowering time. In summary, the work has resulted in five peer reviewed publications, two book chapters, one book coedited by S.E. Perry and Dr. L. Yuan, and five published abstracts. Two theses based on the work were completed. Three graduate students, three postdocs, and four undergraduates received training. In addition five other publications are submitted, in revision or in preparation. PARTICIPANTS: Individuals: Perry, Sharyn E., principal investigator Zheng, Yumei, graduate student Hill, Kristine, graduate student Hartman, Jeanne, senior lab technician Wang, Fangfang, postdoctoral scholar Zheng, Qiaolin, postdoctoral scholar Thakare, Dhiraj, postdoctoral scholar Mueller, Rachel, undergraduate student worker Jones, Olivia, undergraduate student worker Burnie, Whitney, undergraduate researcher Brinker, Kory, undergraduate researcher Collaborators: Dr. A. Bruce Downie, University of Kentucky Dr. Donna E. Fernandez, University of Wisconsin-Madison Dr. Dale Karlson, Monsanto Opportunities for Professional Development: Yumei Zheng assisted in development and presentation of a workshop for 5th grade students on control of flowering time. Qiaolin Zheng and Jeanne Hartman are responsible for supervising the undergraduate student workers, Rachel Mueller and Olivia Jones. Qiaolin Zheng and Fangfang Wang assisted mentoring Kory Brinker and Whitney Burnie during their independent research projects. TARGET AUDIENCES: The research community is the target audience of information that we acquire in our research. This is disseminated to the community by publications and by deposition of large datasets into repositories. We also make regular reports to the agencies supporting the research (NSF and USB). PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The chromatin immunoprecipitation approach developed in the lab during this and the prior Hatch project has lead to more than 80 labs in more than 15 countries contacting us for a detailed protocol or technical advice. We have determined the regulatory network for the MADS-domain protein AGL15 during embryogenesis by identifying direct up- and down-regulated targets using chromatin immunoprecipitation (ChIP)-chip and expression microarrays. We have found AGL15 regulates genes encoding transcription factors important for embryo development and we have identified new genes with products playing roles in embryogenesis, in particular somatic embryogenesis. We have extended the regulatory network by looking at genes controlled by the protein encoded by one AGL15-target called FUSCA3. Unlike AGL15 that can directly repress and directly express direct target genes, FUSCA3 appears to act primarily or exclusively as a transcriptional activator including direct regulation of genes encoding miRNAs (Wang and Perry, submitted). This raises a question of how AGL15 has diverse roles at different loci Most likely AGL15 represses/expresses genes by interaction with other proteins and interaction with histone deacetylases complexes may explain repression. The AGL15-histone deacetylases complex interaction was published in Hill et al., (2008) Plant J. 53: 172. We have been continuing work to understand activation as well as repression. We have extended our work to a crop plant, Glycine max (soybean) and identified orthologs of AGL15 (GmAGL15) and the closest relative in Arabidopsis AGL18 (GmAGL18) from soybean. GmAGL15 enhances recovery of transgenics when overexpressed (reported in Thakare et al., (2008) Plant Physiol. 146:1663). We now have recovered transgenic lines overexpressing GmAGL15 and GmAGL18 and found that explants from these transgenics show increased somatic embryo development compared to controls. We have performed a pilot microarray study to determine changes between 35SGm:AGL15 and control tissue and found that a large number of stress related genes are upregulated in response to increased GmAGL15 in the 0 days after culture explants. These genes become up-regulated in the nontransgenic controls after 3 days on medium to initiate somatic embryo development. We have confirmed direct regulation of select genes by GmAGL15 by performing chromatin immunoprecipitation (ChIP) in soybean as well as expression analysis. We have found interactions between genes and hormones and will be shortly resubmitting work on ethylene and somatic embryogenesis (Zheng, Q., Y. Zheng and Perry, revision in preparation). In summary, we have contributed to an understanding of gene regulatory networks unpinning embryogenesis, and to the mechanisms of regulation by AGL15. We will continue work to understand molecular mechanisms controlling embryogenesis, knowledge of which can help develop strategies to optimize seed development and regeneration by somatic embryogenesis.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Two postdoctoral scholars have received training in the past year supported by funds from the National Science Foundation (started 2009) and the United Soybean Board (started 2010). Both postdocs presented their findings at the 22nd International Conference on Arabidopsis Research held in Madison, Wisconsin in 2011 and both will be preparing manuscripts on their work in the upcoming year. The project has also led to ongoing collaborations. In 2011 the book Molecular Biology volume 754: Plant Transcription Factors: Methods and Protocols, Humana Press (J.M. Walker, Series Editor), co-edited by Ling Yuan and myself was published. Also in 2011, a workshop for 5th grade students was run as a collaboration with Raven Run Nature Sanctuary to teach students about using the scientific method to investigate environmental control of flowering time. PARTICIPANTS: Individuals: Perry, Sharyn E., principal investigator; Zheng, Yumei, graduate student; Hartman, Jeanne, senior lab technician; Wang, Fangfang, postdoctoral scholar; Zheng, Qiaolin, postdoctoral scholar; Mueller, Rachel, undergraduate student worker; Collaborators: Dr. A. Bruce Downie, University of Kentucky; Dr. Donna E. Fernandez, University of Wisconsin-Madison; Opportunities for Professional Development: Yumei Zheng assisted in development and presentation of a workshop for 5th grade students on control of flowering time. Qiaolin Zheng and Jeanne Hartman are responsible for supervising the undergraduate student worker, Rachel Mueller. TARGET AUDIENCES: The research community is the target audience of information that we acquire in our research. This is disseminated to the community by publications and by deposition of large datasets into repositories. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We are continuing to use combinations of mutants, hormones and inhibitors of hormones to tease apart cross-talk between gene products and hormones in somatic embryogenesis. In the past year, we have made some interesting observations and will be preparing a manuscript in the coming year. The hope is that work in the model plant Arabidopsis will contribute to an understanding of embryogenesis that can be applied to crop plants to facilitate recovery of transgenic plants by somatic embryo development. In fact we have been performing work to test whether stable introduction of a soybean ortholog of a gene involved in embryogenesis in Arabidopsis will also promote somatic embryo formation in Glycine max and results look encouraging. This should allow extension of competency for regeneration by somatic embryogenesis to genotypes recalcitrant for the process. As part of this, in the past year we have used microarrays to measure transcript abundance in response to AGL15 (a transcriptional regulator expressed during embryogenesis) accumulation in soybean. We are able to compare these results to results already obtained in Arabidopsis. Furthermore, we have optimized a modification of the chromatin immunoprecipitation (ChIP) procedure to isolate DNA fragments directly bound by AGL15 in soybean and will be able to distinguish direct from indirect targets to understand the regulatory networks in which AGL15 is involved and that may promote embryo development. We have previously performed ChIP analysis in Arabidopsis and so can compare results between these two species. We continue to analyze downstream targets of AGL15 and are extending what we've learned about the regulatory network to other embryo-expressed factors. In the past year, we have optimized ChIP for one such regulator and will proceed with whole genome analysis early this year and we have developed materials and tools for another regulator. Our hope is to contribute to a basic understanding of gene regulation controlling embryogenesis in order to develop strategies to optimize 1.) seed development and 2.) regeneration by somatic embryogenesis.
Publications
- Yumei Zheng and Sharyn E. Perry. (2011) Chapter 16: Chromatin immunoprecipitation to verify or to identify in vivo protein-DNA interactions. In: Methods in Molecular Biology: Plant Transcription Factors: Methods and Protocols, volume 754. Edited by Ling Yuan and Sharyn E. Perry. Springer Science and Business Media, LCC; Humana Press. Pages 277-291.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: A graduate student who has been working on this project has completed and defended her thesis in late 2010, and has earned her Ph.D. A second graduate student working on an unrelated project has also gained experience in techniques to identify targets of transcription factors and he completed his Ph.D. in early 2010. He is currently a postdoctoral researcher at The Ohio State University. An undergraduate in the Agricultural Biotechnology program performed her independent research project in the lab and graduated in December 2010. The project has produced data that has resulted in funding from the National Science Foundation (started 2009 and continuing 2010) and the United Soybean Board (started 2010) and these funds are supporting two new postdoctoral scholars who joined the lab in March 2010. The project has also led to ongoing collaborations and one publication from one of these collaborations in the past year. PARTICIPANTS: PARTICIPANTS: Individuals: Perry, Sharyn E., principal investigator Zheng, Yumei, graduate student Hartman, Jeanne, senior lab technician Wang, Fangfang, postdoctoral scholar Zheng, Qiaolin, postdoctoral scholar Collaborators: Dr. A. Bruce Downie, University of Kentucky, Dr. Donna E. Fernandez, University of Wisconsin-Madison: TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We are continuing to use combinations of mutants, hormones and inhibitors of hormones to tease apart cross-talk between gene products and hormones in somatic embryogenesis. The hope is that work in the model plant Arabidopsis will contribute to an understanding of embryogenesis that can be applied to crop plants to facilitate recovery of transgenic plants by somatic embryo development. In fact we have been performing work to test whether stable introduction of a soybean ortholog of a gene involved in embryogenesis in Arabidopsis will also promote somatic embryo formation in Glycine max and preliminary results look encouraging. This will allow extension of competency for regeneration by somatic embryogenesis to genotypes recalcitrant for the process. We continue to analyze downstream targets of AGL15, a transcriptional regulator expressed during embryogenesis, and are extending what we've learned about the regulatory network to other embryo-expressed factors.
Publications
- Tingsu Chen, Nihar Nayak, Susmita Maitra Majee, Jonathan Lowenson, Kim R. Schafermeyer, Alyssa C. Eliopoulos, Taylor D. Lloyd, Randy Dinkins, Sharyn E.Perry, Nancy R. Forsthoefel, Steven G. Clarke, Daniel M. Vernon, Zhaohui Sunny Zhou,Tomas Rejtar, and A. Bruce Downie. (2010) Substrates of the Arabidopsis thaliana PROTEIN ISOASPARTYL METHYLTRANSFERASE1 Identified Using Phage Display and Biopanning. Journal of Biological Chemistry, 285: 37281-37292
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We have continued to probe mechanisms underpinning plant embryogenesis by identifying genes directly and indirectly regulated by the DNA-binding MADS-factor AGL15. AGL15 accumulation correlates with competence for somatic embryogenesis. Understanding somatic embryo development is fascinating from the basic viewpoint of determining how a cell can dedifferentiate and redifferentiate in embryo mode, as well as having practical aspects to promote regeneration of value-added transgenic plants by somatic embryogenesis. One route to contribute to determination of mechanisms of somatic embryo development is to determine genes controlled by transcriptional regulators that promote this process, such as AGL15. We have globally mapped where AGL15 associates with DNA in vivo, resulting in ~2000 binding sites. We have combined this information with expression microarray data to determine perturbations in the transcriptome in response to increased or decreased AGL15. This has allowed us to identify genes that are likely to be directly expressed in response to AGL15 as well as genes directly repressed by AGL15, resulting in ~200 genes with a direct, consistent and significant response to AGL15. Interestingly, other transcriptional regulators are overrepresented in the directly expressed list, but not the directly repressed list. DNA motifs that AGL15 recognizes are overrepresented in the fragments identified as bound by AGL15. We published this work in The Plant Cell in 2009. Currently we are focusing on objectives 3 and 4 of the project to confirm direct targets as regulated by AGL15 (and the redundant AGL18) and to determine the function of regulated genes. Finally we are developing tools to allow us to extend the gene regulation network underlying embryogenesis by looking at genes regulated by some AGL15 targets that encode transcriptional regulators. The data obtained has also led to collaborations resulting in a publication in the Journal of Experimental Botany in 2009 and an ongoing collaboration with a researcher at the University of Wisconsin-Madison. PARTICIPANTS: Individuals: Perry, Sharyn E., principal investigator Zheng, Yumei, graduate student Mitchell, Katherine, research analyst Collaborators: Dr. Donna E. Fernandez, University of Wisconsin-Madison Dr. Darl Karlson, West Virginia University Dr. Claudia Cosio, Geneva University, Switzerland Training: A graduate student has been trained and she will complete her thesis work in late 2010. A second graduate student working on an unrelated project has also gained experience in techniques to identify targets of transcription factors. He will complete his Ph.D. in early 2010. The project has produced data that has resulted in funding from the National Science Foundation (started 2009) and the United Soybean Board (started 2010) and these funds will support two new postdoctoral scholars who will join the lab in early 2010. TARGET AUDIENCES: The research community PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Limited number of studies have been performed to globally identify direct targets of plant transcription factors. Consequently not much is known about the number or types of targets that a specific factor controls. We have now published our results adding to an emerging picture of transcriptional control. We have found, as have others, that while many sites are occupied by a given transcription factor in vivo (generally 1000s of DNA binding sites), relatively few interactions between a transcriptional regulator and DNA result in significant changes in gene expression of the nearby gene. There are numerous possible reasons, but it seems important to assess the impact of DNA-protein interaction. Analysis of genes directly and indirectly controlled by AGL15 has led us to investigate hormone interactions involved in control of somatic embryo development. We are currently using combinations of mutants, hormones and inhibitor treatments to tease apart how different hormones cross-talk in control of somatic embryo development. We have extended our work in Arabidopsis and demonstrated that ectopic expression of AGL15 in soybean can promote somatic embryogenesis in this important crop.
Publications
- Zheng, Y., Ren, N., Wang, H., Stromberg, A.J. and Perry, S.E. 2009. Global Identification of Targets of the Arabidopsis MADS Domain Protein AGAMOUS-Like15. The Plant Cell 21, 2563-2577.
- Nakaminami, K., Hill, K., Perry, S.E., Sentoku, N., Long, J.A. and Karlson, D.T. 2009. Arabidopsis Cold Shock Domain Proteins: Relationships to Floral and Silique Development. Journal of Experimental Botany 60, 1047-1062.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: A means of regeneration, either by organogenesis or by somatic embryogenesis, is required for genetic engineering of most plants. However, somatic embryogenesis is poorly understood making it difficult to optimize the process in plants recalcitrant to this route of regeneration. A number of genes when ectopically expressed in Arabidopsis promote somatic embryo development and most of these encode transcriptional regulators. Among this group of genes is AGL15, a member of the MADS-domain transcription factor family. To understand how AGL15 functions in plant development, we proposed to identify genes directly as well as indirectly controlled by AGL15. In the past year we have used the Affymetrix ATH1 array to measure gene expression during somatic embryo development, comparing tissue with normal amounts of AGL15 to tissue with increased and decreased amounts of this protein. We have combined these results with whole-genome mapping of binding sites for AGL15 that we obtained by chromatin immunoprecipitation (ChIP)-on-chip using the Affymetrix whole genome tiling array. This has allowed us to identify genes directly regulated by AGL15 from those further downstream in the network. Two different approaches were used to analyze the ChIP-on-chip data. We have verified both binding by AGL15 and changes in expression for several targets by independent approaches. We have selected several targets to test function of the encoded proteins in embryogenesis. We are currently preparing a manuscript on this work. We have shared results pre-publication with other scientists who suspected AGL15 may control their genes of interest. This has led to productive collaborations with researchers at West Virginia University, the University of Wisconsin-Madison, and the University of Geneva resulting in one accepted manuscript, one in preparation, and one anticipated in the future. The ChIP protocol developed in my lab has received broad interest and we have sent a detailed protocol and provided assistance to over 70 labs in more than 15 countries. Last year, we hosted a visitor from Wisconsin to learn how to perform ChIP. PARTICIPANTS: Perry, Sharyn E., principal investigator Zheng, Yumei, graduate student Mitchell, Katherine, research analyst Collaborators: Dr. Donna E. Fernandez, University of Wisconsin-Madison Dr. Darl Karlson, West Virginia University Dr. Claudia Cosio, Geneva University, Switzerland TARGET AUDIENCES: The research community PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
There have been a limited number of studies to globally identify direct targets of plant transcription factors. Consequently not much is known about the number or types of targets that a specific factor controls. We have found ~2000 sites are bound by AGL15 in vivo and nearby a gene. The minority of these genes show significant changes in transcript accumulation in response to AGL15 amounts during somatic embryo development. This agrees with results from other labs studying other transcriptional regulators. We have examined expression of select genes in developing seeds that contain zygotic embryos and found good agreement with the somatic embryo system. Categorization of directly controlled genes revealed that other transcription factors may be overrepresented in the gene set directly expressed by AGL15, but underrepresented in the gene set directly repressed by AGL15. Genes encoding other transcription factors with key roles in embryo development were identified and confirmed as directly up-regulated targets of AGL15. Other genes were selected for further analysis, and found to impact upon somatic embryo development. The global mapping indicated hormone involvement and possible interaction between hormones in somatic embryo development and we are currently performing experiments to follow-up on these observations.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: A means of regeneration, either by organogenesis or by somatic embryogenesis, is required for genetic engineering of most plants. However, somatic embryogenesis is poorly understood making it difficult to optimize the process in plants recalcitrant to this route of regeneration. A number of genes when ectopically expressed in Arabidopsis promote somatic embryo development and most of these encode transcriptional regulators. Among this group of genes is AGL15, a member of the MADS-domain transcription factor family. To understand how AGL15 functions in plant development, we proposed to identify genes directly as well as indirectly controlled by AGL15. Expression microarrays are a common means to nearly globally assess gene expression changes. We used the Affymetrix ATH1 array and measured gene expression during somatic embryo development in tissue ectopically expressing AGL15, in tissue lacking AGL15 and the redundant MADS-factor AGL18 and in wild type tissue. Genes
differentially expressed may be direct or indirect targets. Chromatin immunoprecipitation (ChIP) is an increasingly used approach to identify DNA fragments with which a protein associates in vivo, indicating direct regulation. To determine, in a high-throughput manner, which genes may be directly controlled, we proposed a custom chromatin immunoprecipitation-chip approach. However, with the availability of the Affymetrix whole genome tiling array that would allow mapping over the genome rather than just on DNA fragments represented on the proposed custom chip, we opted to use the Affymetrix array. We isolated in vivo formed AGL15-DNA complexes, recovered the DNA and generated probe to hybridize to the tiling array. We have now completed three biological replicates of the ChIP-chip experiment and are currently integrating the expression and tiling array data to identify direct targets responsive to AGL15/18. We will select targets based on our data as well as information available in
databases to further test the role the products play in plant development and promotion of somatic embryogenesis.
PARTICIPANTS: Individuals: Perry, Sharyn E., principal investigator Thakare, Dhiraj R., postdoctoral scholar Hill, Kristine, graduate student Zheng, Yumei, graduate student Mitchell, Katherine, research analyst Collaborators: Dr. Donna E. Fernandez, University of Wisconsin-Madison Dr. Darl Karlson, West Virginia University Dr. Claudia Cosio, Geneva University, Switzerland Dr. John Harada, University of California-Davis Training: Two graduate students trained one of whom received her Ph.D. in 2007 (K. Hill, Ph.D., 2007. Identification and Characterization of Proteins that Interact with AGAMOUS-Like 15, a MADS domain Protein that Preferentially Accumulates in the Plant Embryo). One postdoctoral scholar trained.
TARGET AUDIENCES: The research community
Impacts
ChIP is increasingly used to identify direct targets of transcription factors, but it is still a relatively new approach. We optimized a protocol for plant tissue that has been requested by over 70 labs in more than 15 countries. With the advent of tiling arrays, it has become possible to map in vivo binding sites in a high-throughput manner. There have been a limited number of studies to globally identify direct targets of plant transcription factors. Consequently not much is known about the number or types of targets that a specific factor controls. We are very early on in the analysis of our data, but it appears that other transcription factors may be overrepresented in the gene set directly expressed by AGL15, but underrepresented in the gene set directly repressed by AGL15. Further analysis will contribute to an understanding of networks of gene regulation during plant development, and in particular during somatic embryogenesis. The information we obtained from the
array analysis has already led to a number of collaborations including with researchers in California, Wisconsin, West Virginia and Switzerland.
Publications
- Hill, K., Wang, H., and Perry, S.E. 2008. A transcriptional repression motif in the MADS-factor AGL15 is involved in recruitment of histone deacetylase complex components. Plant Journal, 53, 172-185.
- Thakare, D.R., Tang, W., Hill, K., and Perry, S.E. 2008. The MADS-domain transcriptional regulator AGAMOUS-Like 15 promotes somatic embryo development in Arabidopsis thaliana and Glycine max. Plant Physiology (accepted pending minor revision).
- Zheng, Y., Thakare, D., Tang, W., Hill, K., Ren, N., Stromberg A.J., and Perry S.E. 2007. Regulatory networks controlled by the MADS-factor AGL15 during embryo development. Plant Sciences Symposium: Translational Seed Biology: From Model Systems to Crop Improvement. UC Davis, California, September 17-20, 2007.
- Perry, S.E. 2007. Mechanisms of gene regulation by the embyro MADS-factor AGL15, and roles in Glycine max somatic embryogenesis. USDA PD meeting, Genes to Products: Agricultural Plant, Microbe, and Biobased Product Research, March 12-14, 2007.
- Hill, K., Wang, H., and Perry S.E. 2007. A transcriptional repression motif in the MADS-factor AGL15 is involved in recruitment of histone deacetylase complex components. ASPB Annual Meeting, Chicago, Illinois.
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