Sumoylation of ABI5 by the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates abscisic acid signaling

SUMO (small ubiquitin-related modifier) conjugation (i.e., sumoylation) to protein substrates is a reversible posttranslational modification that regulates signaling by modulating transcription factor activity. This paper presents evidence that the SUMO E3 ligase SIZ1 negatively regulates abscisic acid (ABA) signaling, which is dependent on the bZIP transcription factor ABI5. Loss-of-function T-DNA insertion siz1-2 and siz1-3 mutations caused ABA hypersensitivity for seed germination arrest and seedling primary root growth inhibition. Furthermore, expression of genes that are ABA-responsive through ABI5-dependent signaling (e.g., RD29A, Rd29B, AtEm6, RAB18, ADH1) was hyperinduced by the hormone in siz1 seedlings. abi5-4 suppressed ABA hypersensitivity caused by siz1 (sizl-2 abi5-4), demonstrating an epistatic genetic interaction between SIZ1 and ABI5. A K391R substitution in ABI5 [ABI5(K391R)] blocked SIZ1-mediated sumoylation of the transcription factor in vitro and in Arabidopsis protoplasts, indicating that ABI5 is sumoylated through SIZ1 and that K391 is the principal site for SUMO conjugation. In abi5-4 plants, ABI5(K391R) expression caused greater ABA hypersensitivity (gene expression, seed germination arrest and primary root growth inhibition) compared with ABI5 expression. Together, these results establish that SIZ1-dependent sumoylation of ABI5 attenuates ABA signaling. The double mutant siz1-2 afp-1 exhibited even greater ABA sensitivity than the single mutant siz1, suggesting that SIZ1 represses ABI5 signaling function independent of AFP1.