CHARACTERIZATION AND EXPLORING THE ROLE OF BRASSICA AP2/ERF TRANSCRIPTION FACTORS AS CUL3-DEPENDENT E3 LIGASE SUBSTRATES

Raed Ahmad Moh'd Al saharin

Because increasing human population and global climate change are rapidly impacting agricultural productivity, it is imperative that new and adaptable solutions be created. The work in this thesis describes how a highly conserved regulatory system in plants could be utilized to target a broad range of responses to abiotic stresses that will address crop yield in the developing environmental shifts. Targeted protein degradation through an E3 ligase-26S proteasome is a primary and flexible way that plants regulate growth, develop, and response. One of these E3 ligase complexes, called Cullin-based E3 Ligases(CRL3BPM), contains BTB/POZ-MATH (BPM) substrate adapter proteins and has been described to regulate the degradation of a wide range of transcription factors (TFs) in the model plant Arabidopsis thaliana. Some of these TFs are critical for stress tolerance and metabolism, like the APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) family, which contains the TFs WRINKLED1 (WRI1), the master regulator of fatty acid biosynthesis in developing seeds, and RELATED TO APETALA2.4 (RAP2.4), which is important in abiotic stress response. Many, if not all, CRL3BPM substrates contain two protein-binding motifs, called SPOP BINDING CONSENSUS (SBC) and PEST. v These domains are critical for CRL3BPM-substrate interaction, which facilitates the subsequent degradation of the substrate. The Brassicaceae family contains several important vegetable crops such as B. rapa (Chinese cabbage), B. oleracea (broccoli, cabbage, and cauliflower), and oilseed crops (B. napus, B. rapa). Collectively, they provide 12% of the world’s vegetable oil production. In this work, we characterized three Brassica rapa RAP2.4 proteins that are closely related to Arabidopsis thaliana AtRAP2.4. The characterization showed tissue and stress-dependent expression patterns of these related proteins. Cloning and expression of the Brassica genes showed that the corresponding proteins can assemble with BPM and CULLIN3 proteins, and that they are instable in a 26S proteasome-dependent manner. Based on the two conserved substratebinding motifs, SBC and PEST, we have begun developing novel tools that are not only deepening our understanding of the regulatory roles of E3 ligase mediated protein degradation, but also providing novel insights into how this critical system might be employed to improve crop productivity.

CHARACTERIZATION AND EXPLORING THE ROLE OF BRASSICA AP2/ERF TRANSCRIPTION FACTORS AS CUL3-DEPENDENT E3 LIGASE SUBSTRATES

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