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Abstract:
The homeotic gene AGAMOUS (AG) plays a central role during Arabidopsis thaliana flower development, since it not only specifies the reproductive organs, but also terminates stem cell proliferation in the center of the flower. Multiple transcriptional inputs contribute to region specific AG activation via regulatory elements located in the second intron. We have shown previously that two transcription factors LEAFY (LFY) and WUSCHEL (WUS) are important direct activators, however their activity is not sufficient for correct AG expression. Detailed knowledge of the regulators involved in AG activation is a prerequisite for the understanding of patterning and stem cell control in emerging flowers. In an attempt to identify additional direct AG regulators, we performed yeast one-hybrid screens using cis-regulatory elements that had been shown to be evolutionary conserved within the family of Brasicacae. One of these highly conserved motifs was a 33 bp element termed AAGAAT-box that we could show to be important for activation of a AG::GUS reporter in Arabidopsis. In yeast we were able to identify nine putative transcription factors that interacted with this element. Five of them belong to the group of Myb-like transcription factors with a single MYB domain, the other four are closely related bZIP transcription factors. Close inspection of the AAGAAT-box sequence revealed consensus-binding sites both for Myb-like and bZIP transcription factors. Analysis of the nine transcription factors in a yeast transactivation assay revealed that all members of the MYB-like group were able to activate transcription from the AAGAAT-box trimer, whereas only one bZIP was a potent activator. Interestingly, we could observe cooperative effects between specific members of the two groups. Expression analysis by interrogating the AtGenExpress database indicated that all candidates were present in floral tissue, with most of them being transcribed also in other parts of the plant. In situ hybridization confirmed expression in floral tissue for five of the nine candidate genes. To asses the biological function of the newly identified candidate regulators, we currently examine the phenotype of knock out lines and plants carrying over- expression alleles. To get an insight into their role in AG regulation, we are analyzing in detail the response of various AG::GUS reporter lines, which will include binding site deletion lines, in loss and gain of function experiments.
