The plant hormone auxin (indole-3-acetic acid or IAA) regulates plant 
development by inducing rapid cellular responses and changes in gene expression. 
Auxin promotes the degradation of Aux/IAA transcriptional repressors, thereby 
allowing auxin response factors (ARFs) to activate the transcription of 
auxin-responsive genes. Auxin enhances the binding of Aux/IAA proteins to the 
receptor TIR1, which is an F-box protein that is part of the E3 ubiquitin ligase 
complex SCF(TIR1). Binding of Aux/IAA proteins leads to degradation via the 26S 
proteasome, but evidence for SCF(TIR1)-mediated poly-ubiquitination of Aux/IAA 
proteins is lacking. Here we used an Arabidopsis cell suspension-based 
protoplast system to find evidence for SCF(TIR1)-mediated ubiquitination of the 
Aux/IAA proteins SHY2/IAA3 and BDL/IAA12. Each of these proteins showed a 
distinct abundance and repressor activity when expressed in this cell system. 
Moreover, the amount of endogenous TIR1 protein appeared to be rate-limiting for 
a proper auxin response measured by the co-transfected DR5::GUS reporter 
construct. Co-transfection with 35S::TIR1 led to auxin-dependent degradation, 
and excess of 35S::TIR1 even led to degradation of Aux/IAAs in the absence of 
auxin treatment. Expression of the mutant tir1-1 protein or the related F-box 
protein COI1, which is involved in jasmonate signaling, had no effect on Aux/IAA 
degradation. Our results show that SHY2/IAA3 and BDL/IAA12 are 
poly-ubiquitinated and degraded in response to increased auxin or TIR1 levels. 
In conclusion, our data provide experimental support for the model that 
SCF(TIR1)-dependent poly-ubiquitination of Aux/IAA proteins marks these proteins 
for degradation by the 26S proteasome, leading to activation of auxin-responsive 
gene expression.