Progress 08/01/03 to 07/31/06
Outputs
EMBRYONIC FLOWER (EMF) genes are required to maintain vegetative growth in Arabidopsis. Global gene expression studies suggest that EMF1 and EMF2 genes down regulate the flower program directly independent of the floral inducers. In addition, EMF genes appear to regulate other major gene programs involved in growth and development processes. Interestingly, EMF1 seems to regulate the seed maturation program independent of EMF2. EMF2 is homologous to the Polycomb Group (PcG) proteins involved in epigenetic silencing in animals. Through protein interaction and genetic studies, we showed that EMF2 interacts with another PcG protein, CURLY LEAF (CLF), a homolog of histone methyltransferase, and proposed a multiprotein complex consisted of EMF2/CLF/FERTILIZATION INDEPENDENT ENDOSPERM DEVELOPMENT (FIE)/MULTICOPY SUPPRESSOR of IRA (MSI)1, which is equivalent to the Drosophila PcG Repressive Complex (PRC)2. Recently, we showed that a member of EMF2 complex interacts with EMF1,
suggesting that both EMF genes participate in the multi-step PcG silencing processes. Our results indicate that EMF2 is required for labeling the flower organ genes with histone methyl marks, allowing subsequent EMF1-mediated transcriptional inhibition. Phylogenetic investigation of the EMF2 homologs shows that in addition to the three homologous sequences that share a common VEF domain with EMF2 in Arabidopsis, i.e., VERNALIZATION (VRN)2, FERTILIZATION INDEPENDENT SEED DEVELOPMENT (FIS)2, VEF-L36, EMF2 homologs exist widely in land plants, from angiosperms to moss. Our studies indicate that EMF2 is likely the ancestral sequence that duplicated and diverged to generate VRN2, which may have duplicated and given rise to VEF-L36 and FIS2. In the course of this study, we identified 8 full length cDNA of EMF2 in rice, barley, wheat and maize. We generated a full length cDNA clone of OsEMF1 and knock down OsEMF1 in transgenic rice, which indeed flowered early. To study the biological and
biochemical functions of the EMF2 homologs, we introduced antisense OsEMF2 into rice, and OsEMF2 and tomato EMF2 into Arabidopsis emf2 mutants to investigate if the homologs can rescue emf2.
Impacts
Understanding the molecular mechanism of EMF2 mediated flowering and shoot development would enable the design of crops that flower at specific time, whereby increasing the geographical range of their cultivation. In this study we identified EMF2 homologs in major crops, e.g., tomato, soybean, rice, maize, wheat, barley, etc.. Understanding the biological and biochemical functions of these homologous genes will have significant impact in agriculture.
Publications
- Lertpiriyapong K. and Z. R. Sung. 2003. The elongation defective1 mutant of Arabidopsis is impaired in the gene encoding a serine-rich secreted protein. Plant Molecular Biology. 53: 581-595.
- Henderson J., H.-C. Li, S. Rider, A. P. Mordhorst, J. Romero-Severson, J.-C. Cheng, J. Robey, Z. Sung, Sacco C. de Vries, Joe Ogas. 2004. PICKLE acts throughout the plant to repress expression of embryonic traits and may play a role in GA-dependent responses. Plant Physiol. 134:995-1005.
- Chanvivattana, Y. A. Bishop, D. Schubert, C. Stock, Y.-H. Moon, Z. R. Sung and J. Goodrich. 2004. Interaction of Polycomb-group proteins controlling flowering in Arabidopsis. Development 131: 5263-5276.
- Sung Z. R. , L. Chen, and Y. Moon. 2003. Molecular mechanism of shoot determinacy and flowering in Arabidopsis. HortScience 38(7): 1325-1327.
- Calonje M. and Z. R. Sung. 2006. Complexity beneath the silence. Current Opinion in Plant Biology. 9: 530-537.
- Chen L. J. and Z. R. Sung. 2007. Evolution of VEF domain-containing PcG genes (submitted).
- Calonje M., R. Sanchez, L. Chen, and Z. R. Sung. 2007. EMF1 function like the PRC1 in Arabidopsis. (submitted).
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Progress 01/01/05 to 12/31/05
Outputs
EMF2 is a member of the PcG protein complex involved in floral repression during vegetative development. To study temporal and spatial pattern of EMF2 protein distribution, we employed protein tagging and immunological approaches. To this date, these techniques have been proven ineffective. For example, EMF2 cDNA tagged with 3X FLAG or HA expressed under the control of the EMF2 or the 35S promoter can rescue emf2 mutant phenotype, indicating their functionality. However, anti-FLAG antibody could not detect EMF2 protein by western blot or immunocytochemical analysis. Three different anti-EMF2 antibodies have been raised in animals, but they could not detect Arabidopsis EMF2 proteins either. While we continue to tag EMF2 with different tags, we are now studying the requirement of EMF2 gene activity during development by using tissue-specific and inducible promoter directed expression of EMF2 in transgenic plants. In addition, we conducted an evolutionary study to
investigate the origin and the ancestral function of EMF2 in plants. Identifying the conserved sequence among the EMF2 homologs would elucidate the molecular function of EMF2 in the multi-protein PcG complex.
Impacts
Understanding the molecular mechanism of EMF2 mediated flowering and shoot development would enable the design of crops that flower at specific time, whereby increasing the geographical range of their cultivation.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The objectives of the proposed research are to 1) Develop tools to detect EMF2 proteins and study their expression pattern, 2) Isolate and characterize EMF-protein complex, 3) Identify genes repressed by EMF proteins and study the mechanism of their repression by the EMF-protein complex. To detect EMF2 proteins, we tagged the EMF2 cDNA and the genomic sequences with different tags, e.g., HA, FLAG, GFP, and generated anti-EMF2 antibody against EMF2 peptides. The antibodies were able to detect EMF2 fusion proteins on western blots and we are in the process of using the antibody to detect endogenous EMF2 protein in Arabidopsis protein extracts. To identify EMF2IP, we studied EMF2 interaction with the likely candidates, and screened for unknown EMF2IPs as well. We found that EMF2 interact with two PcG proteins, CLF and CLK, but not with the like-heterochromatin protein1. Based on mutant phenotype and biochemical properties, CLF is the PcG protein most likely to interact
with EMF2. Using the yeast two hybrid system (Y2H), we constructed full length EMF2 and a series of EMF2 truncations, as prey fusions with the GAL4 transcriptional activation domain (AD), and tested for interaction with baits comprising a fusion of full length CLF or CLF truncations with a GAL4-DNA-binding domain (BD). Only truncations containing the VEF domain of EMF2 and the C5 domain of CLF, not full length or other truncations, interacted with each other (Chanvivattana et al, 2004). The same results were obtained using tagged fusion constructs and in vitro pull down assays. Genetic interaction between EMF2 and CLF was also demonstrated. EMF2 also interacted with CLK, a protein partially redundant to CLF that can partially substitute CLF function in clf mutants. Since CLF interacts directly with FIE and FIE directly with MSI1, it is proposed that the EMF2 repressive complex consists of EMF2/CLF/FIE/MSI1. To investigate the EMF2 target sequence, we are using studying the
transcriptional activity of AG gene in emf2 mutants. Using AG promoter deletion::GUS constructs, we found both AG enhancer and promoter sequences are necessary for EMF2-mediated repression. emf2 harboring pMD200 that includes both AG promoter and intron 2 showed reporter gene GUS activity strongly in seedlings, whereas harboring KB9R (AG intron 2 alone) and pMD222 (AG promoter, intron 1, exon 1 and 2, and partial intron 2 gave no ectopic GUS expression.
Impacts
Understanding the molecular mechanism of EMF2 mediated flowering and shoot development would enable the design of crops that flower at specific time, whereby increasing the geographical range of their cultivation.
Publications
- Chanvivattana Y., A. Bishopp, D. Schubert, C. Stock, Y.-W. Moon, Sung, Z.R. and Goodrich, J. 2004. Interaction of Polycomb-group proteins controlling flowering in Arabidopsis. Development 131:5263-5276.
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Progress 01/01/03 to 12/31/03
Outputs
EMF2 encodes a polycomb group (PcG) protein required for floral repression. In Drosophila PcG proteins form chromatin complexes to maintain the repression of homeotic gene expression. To investigate whether EMF2 is involved in a protein complex, we are studying its interaction with other PcG and non PcG proteins involved in flowering and found that EMF2 interacts with another PcG protein, CURLY LEAF (CLF). Mutants impaired in CLF flower early and display curly leaf. Like EMF2, CLF also functions to repress flowering; mutants impaired in CLF flower early and produce curly leaves. CLF is a SET domain protein. Some SET domain proteins have been shown to confer histone methyl transferase activity, which is instrumental in gene inactivation. CLF interacts with another PcG protein, and the protein complex could involve more than these three PcG proteins. Therefore, we carried out yeast-two-hybrid screen and identified candidates of EMF2 interacting proteins (EMF2IP). The
function of these proteins is being investigated by identifying knockout mutants that are impaired in their genes and by studying the phenotype of the mutants. The interaction between these EMF2IP and EMF2 are being confirmed by in vitro and in planta interaction experiments. In addition, we are using immunological analysis or protein-tagging approach and chromatin immunoprecipitation experiments to identify the EMF2 chromatin complex. Fusion protein between EMF2-GST was produced and used to generate an anti-EMF2 antibody. It detects a 71.7 kd protein from Arabidopsis nuclei and not emf2 mutant nuclei. The anti-EMF2 antibody will be used to immunoprecipitate proteins in an attempt to identify EMF2IPs. Characterization of the EMF2 chromatin complex that inactivates the flower homeotic genes will elucidate the mechanism of epigenetic change in plants.
Impacts
Understanding the molecular mechanism of EMF2 mediated flowering and shoot development would enable the design of crops that flower at specific time, whereby increasing the geographical range of their cultivation.
Publications
- No publications reported this period
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