Identifying cis-Regulatory Elements and trans-Acting Factors that Activate Transcription in the Suspensor of Plant Embryos
- Author(s): Henry, Kelli Frances
- Advisor(s): Goldberg, Robert B
- et al.
Seed crops, such as corn and soybean, are a major source of food for human and animal consumption. Understanding how genes are regulated in seeds is essential for the future development of genetically engineered seed crops that could significantly augment the food supply available for a rapidly growing human population. Given the importance of understanding the processes controlling seed development, it is surprising that the gene regulatory networks operating in seeds remain largely unknown. I have been using scarlet runner bean (SRB; Phaseolus coccineus), a close relative of soybean, to characterize a gene regulatory network active during early embryo development. Specifically, I have focused on gene activity in the suspensor, a specialized embryonic region involved in synthesizing and transporting nutrients to the growing embryo. To identify suspensor cis-regulatory sequences, I performed promoter dissection experiments on the SRB G564 gene, which is expressed in the suspensor early in embryo development. A 54-bp DNA fragment within the G564 upstream region is sufficient for suspensor-specific transcription in transgenic tobacco and Arabidopsis, indicating the suspensor transcriptional machinery is conserved in flowering plants. Mutagenesis of the 54- bp fragment identified five suspensor cis-regulatory elements: (i) three 10-bp motifs with the consensus 5'-GAAAAGCGAA-3', (ii) a Region 2 sequence 5'-TTG(A/G)(A/G/T)AAT-3' and (iii) a Fifth motif 5'-(A/G)AGTTA-3'. The Fifth motif sequence is a predicted MYB transcription factor binding site. A yeast one-hybrid screen identified three MYB transcription factors that bind to the 54-bp fragment and are expressed in the suspensor of Arabidopsis. Promoter deletion and mutagenesis experiments uncovered that sequences similar to these three types of suspensor motifs also activate suspensor transcription in the SRB GA 20-oxidase gene, which encodes an enzyme required for synthesis of the phytohormone gibberellic acid. The SRB G564 and GA 20-oxidase genes are activated by the same suspensor cis-regulatory elements and thus comprise a suspensor gene regulatory network, which is activated shortly after fertilization by transcriptional machinery that is conserved in the suspensors of flowering plants.