Identical Amino Acid Substitutions in the Repression Domain of Auxin/Indole-3-Acetic Acid Proteins Have Contrasting Effects on Auxin Signaling    

Abstract

Auxin/indole-3-acetic acid (Aux/IAA) proteins function as repressors of auxin response gene expression when auxin concentrations in a cell are low. At elevated auxin concentrations, these repressors are destroyed via the ubiquitin-proteasome pathway, resulting in derepression/activation of auxin response genes. Most Aux/IAA repressors contain four conserved domains, with one of these being an active, portable repression domain (domain I) and a second being an auxin-dependent instability domain (domain II). Here, we have analyzed the effects of amino acid substitutions in the repression domain of selected Aux/IAA proteins. We show that stabilized versions of Aux/IAA proteins with amino acid substitutions in domain I display contrasting phenotypes when expressed in transformed Arabidopsis (Arabidopsis thaliana) plants. An alanine-forleucine substitution in the LxLxL (where L is leucine and x is another amino acid) repression domain of IAA3, IAA6, or IAA19 confers enhanced auxin response gene expression and "high-auxin" phenotypes when expressed from the 35S or IAA19 promoter (as tested with IAA19) in transformed Arabidopsis plants. In marked contrast, a single alanine-for-leucine substitution in domain I of IAA12 or IAA17 confers repression of auxin response genes and "low-auxin" phenotypes. These results point to intrinsic differences in the repression domain(s) of IAA proteins and suggest that some IAA proteins have stronger or more complex repression domains than others.

The PB1 Domain in Auxin Response Factor and Aux/IAA Proteins: A Versatile Protein Interaction Module in the Auxin Response

“…Some Aux/IAA proteins lack an apparent domain II and have extended lifetimes compared with typical Aux/IAA proteins (Dreher et al, 2006), while others lack both domains I and II and might not function as repressors (Tiwari et al, 2004). Transgenic plants expressing stabilized Aux/IAA proteins with defective RDs display "high auxin" phenotypes, and it has been suggested that these phenotypes result from interactions of the stabilized defective repressors with activator ARFs, thus preventing association of wild-type Aux/IAA repressors and resulting in constitutive expression of auxin response genes (Li et al, 2011).…”

“…Aux/IAA proteins generally contain one (domain I) or two EARtype RDs in their N-terminal region, followed by a region containing a degron (domain II), and terminating in a PB1-type protein-protein interaction domain (domain III/IV) related to that in ARFs ( Figure 1A; Li et al, 2011;reviewed in Chapman and Estelle, 2009;Guilfoyle and Hagen, 2012;Salehin et al, 2015). Mutations in the conserved degron motif (GWPP) result in the stabilization of Aux/IAA proteins and generally result in plants having constitutive repression of auxin response genes and "low auxin" phenotypes.…”

“…ARFs are DNA binding proteins that are targeted to TGTCTC or related auxin response elements (AuxREs) in promoters of auxin response genes and function as transcriptional activators or repressors through an activation domain (AD) that is enriched in glutamine or a repression domain (RD) that lacks a glutamine-rich region (reviewed in Guilfoyle and Hagen, 2007). Aux/IAA proteins are shortlived nuclear-localized proteins without a DNA binding domain (DBD) and function as transcriptional repressors through a conserved D/E-L-X-L-X-L or related EAR-like (ethylene-responsive element binding factor-associated amphiphilic repression motif) RD (Tiwari et al, 2004;Li et al, 2011;reviewed in Chapman and Estelle, 2009). ARF and Aux/IAA proteins contain a similar PB1 (Phox and Bem1) protein-protein interaction domain (previously referred to as domain III/IV or domain III and domain IV) in their C termini that facilitates the formation of ARF-ARF, ARF-Aux/IAA, and Aux/IAA-Aux/IAA homo-and hetero-oligomers (Han et al, 2014;Korasick et al, 2014;Nanao et al, 2014; Phyre 2 fold library entry id c2m1mA [http://www.sbg.bio.ic.ac.uk/~phyre2/]; reviewed in Guilfoyle and Hagen, 2012).…”

Do some IAA proteins have two repression domains?

“…2). 4 The "low auxin" phenotypes include seedlings with short hypocotyls and petioles, agravitropic roots, reduced numbers of lateral roots artIcLe addendum 35S:IAA19mImII-1 transgene (Fig. 2A); however, the DR5:GUS staining in roots was much reduced in 35S:IAA19mIB/ IAA7mII, III, IV seedlings compared to 35S:IAA19mImII-1 seedlings (Fig.…”

“…When these stabilized proteins are constitutively expressed in transformed Arabidopsis plants, "high auxin" phenotypes are observed, and we have suggested a mechanism for how "high auxin" phenotypes arise in these plants. 4 In contrast, when an identical substitution is made in domain I of IAA17, plants expressing a stabilized version of this protein have "low auxin" phenotypes. We have suggested that these "low auxin" phenotypes are observed because IAA17 and proteins within the same clade have a second LxLxL repression domain, which continues to function when the LxLxL motif in domain I is rendered nonfunctional.…”