Research Accomplishment Reports 2007

Regulation of Gene Expression during Plant Embryogenesis

S.E. Perry
Department of Plant and Soil Sciences

 

Project Description

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.

Impact

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.