Components of the Ubiquitin/26S proteasome system regulate abscisic acid signaling and development in Arabidopsis thaliana

Protein turnover by the Ubiquitin (Ub)/26S proteasome System (UPS) is an important proteolytic pathway that selectively removes misfolded/abnormal and short-lived regulatory proteins in eukaryotes. Commitment to breakdown begins with the attachment of a Ub polymer onto a target protein in an ATP-dependent enzyme cascade. The poly-Ub chain marks the protein for degradation by the 26S proteasome, which cleaves the target into protein fragments, with the concomitant release of reusable Ub moieties by de-ubiquitinating enzymes (DUBs). The 26S proteasome is a multi-subunit, self-compartmentalized protease in the nucleus and cytoplasm that controls the turnover of numerous cellular constituents. The subunit RPN10 is a Ub receptor in the complex that identifies a subset of targets, including the abscisic acid (ABA) signaling transcription factor ABA-INSENSITIVE-5 (ABI5). ABI5 transcriptionally activates downstream genes that induce post-germinative growth arrest in response to adverse environmental conditions. The selective turnover of ABI5 is essential for seedlings to reestablish growth when suboptimal conditions are alleviated. To further characterize ABI5 breakdown by the UPS, I identified the RING Ub ligase KEEP ON GOING (KEG) as an effector of ABI5 degradation in Arabidopsis thaliana. When KEG expression was eliminated, ABI5 protein hyperaccumulated without sensing ABA, causing a severe growth arrest of seedlings. I found that phosphorylated forms of ABI5 were present in tissues of arrested seedlings, and I demonstrated that the known ABA-responsive SNF1-RELATED PROTEIN KINASE-2.2 (SnRK2.2) and SnRK2.3 coordinately promoted ABI5 phosphorylation. SnRK2.2/2.3 elimination partially rescued keg growth arrest, altered ABI5 phosphorylation, and inhibited ABI5 accumulation.;In yeast, targets are shuttled to the 26S proteasome by soluble Ub receptors such as the Ub-like (UBL)/Ub-associated (UBA) protein RADIATION SENSITIVE-23 (RAD23), which interacts with RPN10. I identified four conserved RAD23(a-d) orthologs in Arabidopsis that bind Ub-conjugates and associate with 26S proteasomes in vivo. Reverse genetic analyses revealed that RAD23b mutations generate growth defects throughout development, while a loss of all RAD23 expression results in embryonic lethality. Additionally, rad23 mutants are sensitive to mitomycin C, which cross-links DNA and inhibits mitosis, indicating a potential role for plant RAD23 proteins in DNA repair. This work provides the first detailed description of RAD23 function in plants.