In Arabidopsis thaliana, developmental arrest may be induced in young seedlings between 2 and 3 days after germination. During this time, seedlings that are desiccation stressed or exposed to abscisic acid (ABA) arrest development. ABI3, ABI5, and other ABA-insensitive (ABI) proteins are involved in ABA sensing and the onset of ABA-induced developmental arrest. However, the molecular mechanisms of how progression through this developmental arrest checkpoint is regulated have not yet been elucidated.

Our laboratory conducted a yeast two-hybrid screen of an Arabidopsis cDNA library that uncovered interactions between ABI3 and ABI3 Interactive Proteins (AIPs). The deduced amino acid sequence of one of these proteins, AIP6, predicts the presence of a RING-H2 domain, a motif common to eukaryotic E3 ubiquitin protein ligases. E3 enzymes catalyze the covalent attachment of ubiquitin tags to cellular proteins committing them for degradation by the 26S proteasome. Interestingly, the highest levels of AIP6 mRNA occur during the first 3 days of development after germination, at a time when ABI3 protein normally disappears from the seedling. These observations led us to propose that AIP6 protein is responsible for ABI3 degradation, and that it may be necessary to release young seedlings from developmental arrest. To test this hypothesis, transgenic plants expressing a tagged form of AIP6 will be used to identify AIP6-interaction partners in planta. The effect of AIP6 levels of ABI3 ubiquitylation and stability will also be tested in vivo. The ultimate test of whether AIP6 does function as an E3 ligase will be to show that the protein possesses E3 ligase activity in vitro, and that it is able to ubiquitylate ABI3.