Degradation of the auxin response factor ARF1

Abstract

SummaryAuxin-mediated gene expression is largely controlled through a family of DNA-binding proteins known as auxin response factors (ARF). Previous studies on the role of proteolytic regulation in auxin signaling have focused on degradation of their interacting partner, the Aux/IAA proteins. Aux/IAA family members with domain II sequences are rapidly degraded, show auxin-enhanced degradation rates, and interact with the related F-box proteins TIR1 and AFB1-3, which indicates that they are ubiquitylated by a CUL1-dependent E3 ligase. To date, limited data have been generated regarding degradation of ARFs. Here, we focus on the degradation rate of one ARF family member, Arabidopsis thaliana ARF1, and find that the half-lives of N-terminally HA-tagged ARF1 and C-terminally luciferase-tagged ARF1 are both approximately 3-4 h. This halflife appears to be conferred by a component of the middle region (MR), and degradation of the luciferase fusion with the MR is more rapid when the fusion includes an additional nuclear localization signal. ARF1 degradation is proteasome-dependent and rates are not altered in a CUL1 mutant background, suggesting that this ARF is targeted for proteasomal degradation via an alternative set of machinery to that used for Aux/IAA degradation. Consistent with this, exogenous indole acetic acid does not affect the degradation of ARF1. Given increasing evidence that the relative ratio of Aux/IAAs to ARFs rather than the absolute quantity within the cell appears to be the mode through which auxin signaling is modulated, this half-life is likely to be biologically relevant.

Natural Variation of Transcriptional Auxin Response Networks inArabidopsis thaliana

“…For instance, both Aux/IAAs and ARFs are subjected to proteasomal degradation. In contrast to Aux/ IAAs, the degradation of ARFs seems to be auxin-and TIR1-independent, as shown in the case of ARF1 (Salmon et al, 2008). In this respect, auxin-independent factors could also contribute to the observed natural variation in expression profiles.…”

Salicylic Acid Suppresses Jasmonic Acid Signaling Downstream of SCFCOI1-JAZ by Targeting GCC Promoter Motifs via Transcription Factor ORA59

“…Samples were immediately frozen in liquid nitrogen and ground using a Retsch MM300 shaker. Subsequently, proteins were extracted using LUC extraction buffer (100 mM KPO 4 , pH 7.8, 1 mM EDTA, 7 mM b-mercaptoethanol, 1 mM PMSF, and 1 complete protease inhibitor tablet [Roche] per 10 mL) as described (Salmon et al, 2008). The supernatant was used for subsequent measurements of GFP fluorescence and LUC activity.…”

Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1

“…For expression of HIS 6x -HA 3x epitopetagged IAA1 KR mutants without additional Lys residues introduced by epitope tags or vector sequences, an HIS 6x -HA 3x epitope sequence was cloned into pLIT29 (New England Biolabs) to add the HIS 6x -HA 3x sequence 59 of IAA1 ORFs. An HA 3x ORF had been previously cloned NcoI/HindIII into pLIT29 by adding the sequence for the HA epitope into the forward primer with an NdeI site separating the HA 3x sequence from the ORF (Salmon et al, 2008). The HIS 6x sequence was added by annealing oligos 3-250 and 3-254 and ligating them into KpnI/NcoI restriction sites of HA 3x ORF pLIT29, making HIS 6x -HA 3x ORF pLIT29.…”