Abscisic acid increases Arabidopsis ABI5 transcription factor levels by promoting KEG E3 ligase self-ubiquitination and proteasomal degradation

Abstract

The Arabidopsis thaliana RING-type E3 ligase KEEP ON GOING (KEG) is a negative regulator of abscisic acid (ABA) signaling. Seedlings homozygous for T-DNA insertions in KEG accumulate high levels of the ABA-responsive transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5). Here, we demonstrate that KEG E3 ligase activity is required for the regulation of ABI5 abundance. KEG ubiquitinates ABI5 in vitro, and a functional KEG RING domain is required to restore the levels of ABI5 in keg-1 to that of the wild type. Overexpression of KEG leads to ABA insensitivity, which correlates with KEG protein levels. In the presence of ABA, ABI5 levels increase drastically via a decrease in ubiquitin-meditated proteasomal degradation. Our results indicate that ABA promotes ABI5 accumulation by inducing the ubiquitination and proteasomal degradation of KEG. A functional RING domain is required for the ABA-induced degradation of KEG, suggesting that the loss is due to self-ubiquitination. Mutations within KEG's kinase domain or treatments with kinase inhibitors prohibit the ABA-induced ubiquitination and degradation of KEG, indicating that phosphorylation, possibly self-phosphorylation, is involved in the ABA regulation of KEG protein levels. We discuss a model for how ABA may negatively regulate KEG protein abundance, leading to accumulation of ABI5 and ABA-dependent cellular responses.

Figure 1.

Rescue of Growth Arrest and ABI5 Protein Level in the keg-1 Mutants. (A) Growth of transgenic plants, control (empty transformation vector in Col-0 background), keg-1 (data not shown for the plants in soil), keg-1/35S:HA-KEG (35S:HA-KEG in keg-1 background), and keg-1/35S:HA-KEG^AA (RING mutant 35S:HA-KEG^AA in keg-1 background) plants on MS medium (7 d old, top panel) and in soil (4 weeks old, bottom panel). (B) Level of HA-KEG in transgenic plants as determined by immunoblot analysis using anti-HA antibody (top panel). Coomassie blue staining was used to confirm equal loading (middle panel). PCR was used to confirm presence of the T-DNA insertion and homozygosity of the keg-1 mutation (bottom panel). Gene-specific primers were used for the wild-type allele (lane 1) and in combination with T-DNA–specific primers for the mutant allele (lanes 2 to 4). (C) Levels of ABI5 protein in 7-d-old keg-1, control, keg-1/35S:HA-KEG, and keg-1/35S:HA-KEG^AA seedlings as detected by ABI5 antibodies (top panel). Arrowheads indicate the different forms of ABI5. Coomassie blue staining shows levels of loading in each lane (bottom panel). (D) GST-KEG (E3) is capable of ubiquitinating Flag-ABI5 in vitro in the presence of yeast E1 and Arabidopsis UBC8 (an E2). Omission of GST-KEG, UBC8, or ubiquitin (Ub) from the ubiquitination assay abolishes Flag-ABI5 ubiquitination. KEG with a mutant RING domain, GST-KEG^AA, does not ubiquitinate Flag-ABI5. GST protein was use as control. Asterisk indicates a nonspecific band. IB, immunoblot.

Figure 2.

Effects of KEG Overexpression on ABA and Salt Sensitivity. (A) Three independent lines of Col-0/35S:HA-KEG and control transgenic plants were grown for 7 d on MS medium with or without 0.5 μM ABA. (B) Levels of HA-KEG fusion protein in Col-0/35S:HA-KEG transgenic lines grown for 7 d on MS medium (top panel). Coomassie blue staining confirms equal loading (bottom panel). (C) Phenotype of Col-0/35S:HA-KEG transgenic and control plants germinated and grown for 5 d on MS growth medium with (bottom panel) or without (top panel) 100 mM NaCl. Line #3 shown in (A) and (B) were used.

Figure 3.

ABA Promotes KEG Degradation via the Ubiquitin-Dependent 26S Proteasome Pathway. (A) Eight-day-old keg-1/35:HA-KEG seedlings were incubated in liquid MS medium supplemented with 500 μM cycloheximide (CHX) followed by treatment with or without 50 μM ABA for the indicated amounts of time. The levels of HA-KEG at each time point were determined by immunoblot with HA antibody (top panel). Coomassie blue staining shows levels of loading in each lane (bottom panel). (B) Eight-day-old keg-1/35:HA-KEG seedlings were treated with 500 μM CHX and the proteasome inhibitor MG132 (30 μM) or DMSO (control) before treatment with 50 μM ABA for the indicated amounts of time. The levels of HA-KEG protein at indicated time points were determined in the total protein extracts by immunoblot with HA antibody (top panel). Coomassie blue staining shows levels of loading in each lane (bottom panel). (C) Eight-day-old control and keg-1/35S:HA-KEG seedlings were treated with MG132 followed treatment with (+) or without (−) 50 μM ABA for 9 h. HA-KEG was isolated using anti-HA affinity beads. HA and ubiquitin antibodies were used to detect HA-KEG (top panel) and ubiquitinated HA-KEG (bottom panel), respectively. IP, immunoprecipitation; IB, immunoblot.

Figure 4.

ABA-Induced KEG Degradation Requires a Functional KEG RING Domain. Eight-day-old keg-1/35S:HA-KEG and keg-1/35S:HA-KEG^AA (RING mutant) transgenic seedlings were incubated with 500 μM cycloheximide (CHX) followed by treatment with 50 μM ABA for the indicated amounts of time. For each time point, equal amounts of protein were analyzed by immunoblot using HA antibody to determine the levels of HA-KEG and HA-KEG^AA (top panel). Coomassie blue staining shows loading levels (bottom panel).

Figure 5.

Phosphorylation Is Required for ABA-Induced Degradation of KEG. (A) Eight-day-old keg-1/35:HA-KEG transgenic seedlings were treated with the kinase inhibitor STAU (1 μM) or DMSO (control) in the presence of cycloheximide (CHX) followed by 50 μM ABA for the indicated amounts of time. The levels of HA-KEG at each time point were determined by immunoblot analysis using HA antibody (top panel). Coomassie blue staining confirms equal loading (bottom panel). (B) Phenotype of 7-d-old keg-1/35S:HA-KEG and keg/35S:HA-KEG^K/R (kinase mutant) transgenic seedlings grown on MS growth medium. (C) Levels of ABI5 and HA-KEG as detected by anti-ABI5 and anti-HA for keg-1/35S:HA-KEG and keg/35S:HA-KEG^K/R transgenic plants grown for 7 d on MS growth medium without ABA. Coomassie blue staining confirms equal loading. (D) Eight-day-old keg-1/35S:HA-KEG and keg-1/35S:HA-KEG^K/R seedlings were treated with 500 μM CHX followed by 50 μM ABA for the indicated amounts of time. The levels of HA-KEG and HA-KEG^K/R at each time point were determined by immunoblot analysis using anti-HA antibody (top panel). Coomassie blue staining shows loading levels (bottom panel).

Figure 6.

Phosphorylation Promotes KEG Self-Ubiquitination. (A) Eight-day-old keg-1/35S:HA-KEG and keg-1/35S:HA-KEG^K/R transgenic seedlings were treated with MG132 for 16 h followed by treatments with (+) or without (−) 50 μM ABA for 9 h. HA-KEG and HA-KEG^K/R proteins were isolated from total protein extracts using anti-HA affinity beads and subjected to immunoblotting using HA and ubiquitin (Ub) antibodies to detect HA-KEG/KEG^K/R (top panel) and ubiquitinated HA-KEG/HA-KEG^K/R (bottom panel), respectively. IP, immunoprecipitation; IB, immunoblot. (B) HA-KEG and HA-KEG^K/R proteins were immunoprecipitated from 8-d-old keg-1/35S:HA-KEG and keg-1/35S:HA-KEG^K/R transgenic seedlings, respectively, using anti-HA affinity beads. Immunoprecipitated proteins were used in vitro kinase assays with (+) or without (−; control) ATP followed by ubiquitination assays. Ubiquitin (Ub) was omitted from assays as a control. Anti-HA and anti-Ub were used to detect HA-KEG/KEG^K/R (top panel) and ubiquitinated HA-KEG/KEG^K/R (bottom panel), respectively. IP, immunoprecipitation; IB, immunoblot.

Figure 7.

Model for ABA Regulation of KEG Activity to Determine ABI5 Abundance. In the absence of ABA, KEG is stable and continually ubiquitinates ABI5. Ubiquitinated ABI5 is degraded by the 26S proteasome. ABA promotes KEG autoubiquitination and subsequent degradation by the 26S proteasome. As KEG is phosphorylated in the presence and absence of ABA, ABA-induced KEG degradation may involve a rearrangement in phosphorylation status. This may be accomplished by KEG phosphorylating itself or another kinase. Reduction in KEG abundance leads to the accumulation of ABI5 and subsequent growth arrest.