Rapid Degradation of Auxin/Indoleacetic Acid Proteins Requires Conserved Amino Acids of Domain II and Is Proteasome Dependent

Abstract

Auxin rapidly induces auxin/indoleacetic acid (Aux/IAA) transcription. The proteins encoded are short-lived nucleuslocalized transcriptional regulators that share four conserved domains. In a transient assay measuring protein accumulation, an Aux/IAA 13-amino acid domain II consensus sequence was sufficient to target firefly luciferase (LUC) for low protein accumulation equivalent to that observed previously for full-length PSIAA6. Single amino acid substitutions in these 13 amino acids, corresponding to known auxin response mutants, resulted in a sixfold to 20-fold increase in protein accumulation. Naturally occurring variant amino acids had no effect. Residues identified as essential by single alanine substitutions were not sufficient when all flanking amino acids were alanine, indicating the importance of flanking regions. Using direct protein degradation measurements in transgenic Arabidopsis seedlings, full-length IAA1, PSIAA6, and the N-terminal 73 PSIAA6 amino acids targeted LUC for rapid degradation with 8-min half-lives. The C-terminal 109 amino acids did not affect LUC half-life. Smaller regions containing domain II also targeted LUC for rapid degradation, but the rates were not equivalent to those of the full-length protein. A single domain II substitution in the context of full-length PSIAA6 increased half-life 30-fold. Proteasome inhibitors affected Aux/IAA::LUC fusion protein accumulation, demonstrating the involvement of the proteasome.

A role for phospholipase A in auxin‐regulated gene expression

“…Aux/IAA1:luciferase activity drops rapidly after auxin application, consistent with exogenous auxin increasing Aux/IAA1-luciferase degradation rate. PLA inhibitor had no effect on this rapid drop which is mediated by TIR1 [19][20][21][22][23]. However, examination of the steady-state levels of Aux/ IAA1:luciferase over a longer time course revealed an interesting aspect.…”

“…Transcription from this group of promoters is usually derepressed by proteolysis of Aux/IAA proteins [14,15]. Proteolysis is initiated by auxin-and SCF-dependent ubiquitination of IAA proteins, an E3 ubiquitin-ligase complex of Skp1-Cullin-F-box-Rbx proteins [16][17][18][19][20][21][22][23]. Central for auxin-dependent ubiquitination is a ternary complex of the F box protein, either TIR1 or a related AFB, Aux/IAA protein, and auxin in that these F box proteins are both an auxin receptor and SCF complex is the first hormone-regulated enzyme capable of modifying a transcription cofactor activity critical for gene activity.…”

Translating auxin responses into ovules, seeds and yield: Insight from Arabidopsis and the cereals

“…The Aux/IAA proteins can also bind TIR1/AFB proteins. When the TIR1/AFBs perceive and bind auxin, via their leucine rich repeat repeats, this strengthens their interaction with Aux/IAA proteins and leads to Aux/IAA polyubiquitination and degradation, via the 26S proteasome (Worley et al 2000;Ramos et al 2001;Mockaitis and Estelle 2008). Hence, when auxin is perceived, the repression of ARFs by Aux/IAA is lifted, and the ARF proteins are able to regulate transcription of auxin responsive target genes in a positive or negative manner depending on the ARF, the promoter sequence of the target gene and the interaction with additional co-activators or co-repressors ( Figure 2; Lee et al 2009;Farcot et al 2015).…”

“…The Aux/IAA proteins contain four key domains (Paul et al 2016;Wu et al 2017;Luo et al 2018); domain I recruits TPL/TPR co-repressors to enhance repression of ARF target genes, domain II facilitates interaction with the TIR1/AFB proteins (Worley et al 2000;Ramos et al 2001;Lee et al 2009), and domains III and IV facilitate interaction with ARF activator proteins and dimerization between Aux/IAA proteins (Ulmasov et al 1997;Guilfoyle et al 1998a). These domains are sometimes absent, depending on the gene and species ).…”

Developmental specificity of auxin response by pairs of ARF and Aux/IAA transcriptional regulators

“…Degradation of Aux/IAA proteins involves their conserved domain II, which mediates interaction with the SCF TIR1 ubiquitin-ligase complex for targeting of Aux/IAAs to the proteasome (Gray et al, 2001). Amino-acid exchanges in conserved residues of domain II affect the interaction with the SCF TIR1 ubiquitin-ligase complex, stabilizing mutant Aux/IAA proteins (Ramos et al, 2001). Such stabilizing mutations have been reported for 10 Aux/IAA genes (Reed, 2001;Hellmann and Estelle, 2002;Tatematsu et al, 2004;Yang et al, 2004).…”