KEEP ON GOING, a RING E3 Ligase Essential for Arabidopsis Growth and Development, Is Involved in Abscisic Acid Signaling

Abstract

Analysis of the Arabidopsis thaliana RING-ANK (for Really Interesting New Gene-Ankyrin) family, a subgroup of RING-type E3 ligases, identified KEEP ON GOING (KEG) as essential for growth and development. In addition to the RING-HCa and ankyrin repeats, KEG contains a kinase domain and 12 HERC2-like repeats. The RING-HCa and kinase domains were functional in in vitro ubiquitylation and phosphorylation assays, respectively. Seedlings homozygous for T-DNA insertions in KEG undergo growth arrest immediately after germination, suggestive of increased abscisic acid (ABA) signaling, a major phytohormone that plays a key role in plant development and survival under unfavorable conditions. Here, we show that KEG is a negative regulator of ABA signaling. keg roots are extremely sensitive to the inhibitory effects of ABA and exhibit hypersensitivity to exogenous glucose, consistent with the known interaction between glucose and ABA signaling. The observations that KEG accumulates high levels of ABSCISIC ACID-INSENSITIVE5 (ABI5) without exogenous ABA, interacts with ABI5 in vitro, and that loss of ABI5 rescues the growth-arrest phenotype of keg mutant seedlings indicate that KEG is required for ABI5 degradation. In this capacity, KEG is central to ABA signaling by maintaining low levels of ABI5 in the absence of stress.

The multifaceted role of ABA in disease resistance

“…Downstream of ABI1 and ABI2, the ABA response is channeled through a variety of transcription factors and other signaling components. Levels of the ABI5 transcription factor are controlled by the RING-type E3 ligase KEG (KEEP ON GOING) [36], which represses abiotic stress responses [36] and early post-invasive defense in Arabidopsis [35]. Furthermore, the early-acting transcription factor AIM1 (ABSCISIC ACID-INDUCED MYB1) has recently been shown to control positively abiotic stress tolerance and early post-invasive defense in tomato [22].…”

“…Furthermore, a genetic suppressor screen for mutations that block edr1-mediated disease resistance lead to the identification of the keg-4 (keep on going-4) mutant, which concomitantly blocked ABA hyper-responsiveness in edr1-1 [35]. KEG encodes an ubiquitin ligase that functions as a negative regulator in ABA signaling by targeting the ABI5 transcription factor for degradation [36]. Unlike other mutations in KEG, the keg-4 mutation is thought to enhance ubiquitin ligase activity, causing lower ABI5 levels and decreased ABA sensitivity [35].…”

E3 ubiquitin ligases and abscisic acid signaling

“…24 Loss of ABI5 in keg-1 background only partially rescues the keg mutant phenotype suggesting that KEG may also regulate other ABAresponsive transcription factors, such as the ABI5-related bZIP proteins. 33 We have begun to unravel the mechanism by which ABA protects ABI5 from degradation by KEG. 24 In the presence of ABA, KEG protein levels are reduced via ABA-induced selfubiquitination and subsequent degradation by the 26S proteasome, thus allowing ABI5 levels to rise.…”

“…30,32 KEG, a multi-domain RING-type E3 ligase, has been shown to be required for maintaining low levels of ABI5 in the absence of ABA. 24,33 Disruption of KEG gene expression results in growth arrest immediately after germination, accumulation of extremely high levels of ABI5 protein and hypersensitivity to ABA. 33 Whereas overexpression of KEG leads to ABA insensitivity.…”

“…24,33 Disruption of KEG gene expression results in growth arrest immediately after germination, accumulation of extremely high levels of ABI5 protein and hypersensitivity to ABA. 33 Whereas overexpression of KEG leads to ABA insensitivity. 24 Loss of ABI5 in keg-1 background only partially rescues the keg mutant phenotype suggesting that KEG may also regulate other ABAresponsive transcription factors, such as the ABI5-related bZIP proteins.…”