Identification and Characterization of Substrates for the Arabidopsis thaliana RING-type E3 Ligases XBAT32 and KEG

Abstract

The ubiquitin proteasome system (UPS) challenges transcription as the primary regulatory mechanism in the plant kingdom, influencing many cellular processes by selectively degrading proteins. E3 ubiquitin ligases are the UPS elements responsible for endowing the UPS pathway with specificity, by selecting substrates to be targeted to the 26S proteasome for degradation. Many E3 ubiquitin ligases have critical functions related to plant hormone perception, signaling and response. Arabidopsis Keep on Going (KEG) and XB3 ortholog 2 in Arabidopsis thaliana (XBAT32) are E3 ubiquitin ligases intimately related to the hormones abscisic acid (ABA) and ethylene, respectively. I investigated the relationship between each of these E3 ligases and their substrates. Calcineurin B-like Interacting Protein Kinase 26 (CIPK26) is a substrate of KEG and the UPS, while N-MYC DOWNREGULATED-LIKE 1 (NDL1) is potentially a substrate of XBAT32. I further characterized the relationship between CIPK26, KEG and ABA using transgenic plant lines in a variety of assays, including cell free degradation assays and cycloheximide (CHX) chase assays. I demonstrated that activated CIPK26 promotes KEG degradation and showed that CIPK26 overexpression increases KEG turnover in the presence of ABA. This research supports a model where, in the presence of ABA, CIPK26 phosphorylates KEG in order to negatively regulate its’ activity and promote its degradation. Further, I provide evidence to support a role for XBAT32 and the UPS in regulating NDL1 stability, through the use of cell free degradation assays, a semi-in vitro ubiquitination assay, and a pull down assay. I offer support for an interaction between NDL1 and XBAT32, demonstrate that NDL1 is ubiquitinated and degraded by the 26S proteasome, and show that XBAT32 is likely to be required for this degradation. These findings broaden our understanding of the roles of XBAT32 related to lateral root development. Through this work we obtain a more complete understanding of the ways in which the UPS interacts with crucial plant hormones implicated in growth, development, and environmental stress response. This knowledge could contribute to the development of plants with a capacity to grow and thrive under adverse environmental conditions, of importance because climate change is having a major impact on crop viability and soil health.