Research Accomplishment Reports 2007

Mechanisms of Gene Regulation by the Embryo MADS-factor AGL15, and Roles in Glycine max Somatic Embryogenesis

S.E. Perry
Department of Plant and Soil Sciences

 

Project Description

One research focus in the lab has been to identify genes that are directly regulated by the MADS-domain factor AGL15 that accumulates to its highest amounts during embryogenesis and that promotes somatic embryogenesis in Arabidopsis when ectopically expressed. Some direct target genes are induced in response to AGL15, while others are repressed. Additionally, some direct targets do not have cis motifs that confer strong binding by AGL15 in vitro. These observations indicate that AGL15 may form complexes with other proteins that modulate AGL15s function at particular loci. We have utilized a yeast 2-hybrid approach to identify potential interacting proteins of AGL15 and confirmed that they are true interactors in yeast.

In the past year, most progress has been made on the AGL15-interacting protein SAP18 (for SIN3 Associated Polypeptide of 18 KDa). SAP18 is a member of the SWI-independent 3/Histone Deacetylase (SIN3/HDAC) complex and can interact with HDA6 and HDA19 in yeast. Futhermore, AGL15 can interact directly with HDA19. Histone deacetylation generally leads to repression of gene expression. Other experiments demonstrated that AGL15 can repress transcription in vivo, and a region essential to this repressive function contains a motif that is conserved among putative orthologs of AGL15 and present in other transcriptional regulators as a repression domain. This motif, referred to as an LxLxL or EAR motif in other transcriptional regulators, is involved in the association of AGL15 with SAP18 in yeast two-hybrid assays. When the leucines are mutated to alanines, AGL15 can no longer interact with the modified SAP18, yet other AGL15 interactions are not perturbed. Interaction of SAP18 via the LxLxL motif and recruitment of other HDAC components provides a possible mechanism for repression of gene expression by AGL15. These results are described in a manuscript that is currently submitted.

The other AGL15 interacting protein that has been under further investigation is GRP2, a protein that contains a cold shock domain and two CCHC-type zinc fingers. The K-domain of AGL15 and the cold shock domain of GRP2 are involved in the interaction in yeast. Unlike SAP18 that does not interact with other MADS-domain proteins, GRP2 can interact with a number of other MADS-factors that also contain K-domains. Preliminary co-precipitation studies support an interaction between GRP2 and AGL15 in planta. Furthermore, AGL15 may be involved in regulation of some of the CSD-containing proteins; chromatin immunoprecipitation studies have demonstrated binding of AGL15 to regulatory regions of GRP2, the close relative GRP2b, and possibly also CSD4. Further work is underway to understand the biological significance of this interaction.

We also proposed to test the effect of constitutive expression of AGL15 in Glycine max, specifically whether somatic embryogenesis and subsequent recovery of transgenic plants could be improved in this species. We have found an approximately three-fold increase in recovery of potential transformants when a 35S:GmAGL15 transgene was used. A manuscript is in preparation reporting on this work.

Impact

AGL15 is a DNA-binding transcriptional regulator that accumulates to highest amounts in embryo tissue and, when ectopically expressed, can promote development of tissue as embryo tissue. Identifying proteins with which AGL15 interacts to control gene expression, and how it interacts with these proteins will help us understand how AGL15 controls development as embryo tissue. This may aid in regeneration of recalcitrant species by somatic embryogenesis; an important tool for genetic engineering. Furthermore, a better understanding of molecular programs active during seed development may impact on many aspects of seed biology, including but not limited to, nutritional value, yield, viability and vigor, and engineering to allow new value-added products. Finally, demonstration that a motif within the C-terminal domain of AGL15 is involved in transcriptional repression contributes to our understanding of MADS-factors in general because little is known about this domain and several other MADS-factors contain a similar motif within the C-terminal domain.

Publications

Hill, K., H. Wang, and S.E. Perry. (2007) A transcriptional repression motif in the MADS-factor AGL15 is involved in recruitment of histone deacetylase complex components. submitted.

Thakare, D.R., W. Tang, K. Hill and S.E. Perry. (2007) Isolation and characterization of a full-length soybean (Glycine max) embryo MADS box gene. manuscript in preparation.