The roles of auxin response factor domains in auxin-responsive transcription

Abstract

Auxin response factors (ARFs) are transcription factors that bind to TGTCTC auxin response elements in promoters of early auxin response genes. ARFs have a conserved N-terminal DNA binding domain (DBD) and in most cases a conserved C-terminal dimerization domain (CTD). The ARF CTD is related in amino acid sequence to motifs III and IV found in Aux/IAA proteins. Just C terminal to the DBD, ARFs contain a nonconserved region referred to as the middle region (MR), which has been proposed to function as a transcriptional repression or activation domain. Results with transfected protoplasts reported here show that ARFs with Q-rich MRs function as activators, whereas most, if not all other ARFs, function as repressors. ARF DBDs alone are sufficient to recruit ARFs to their DNA target sites, and auxin does not influence this recruitment. ARF MRs alone function as activation or repression domains when targeted to reporter genes via a yeast Gal4 DBD, and auxin does not influence the potency of activation or repression. ARF CTDs, along with a Q-rich MR, are required for an auxin response whether the MRs plus CTDs are recruited to a promoter by an ARF DBD or by a Gal4 DBD. The auxin response is mediated by the recruitment of Aux/IAA proteins to promoters that contain a DNA binding protein with a Q-rich MR and an attached CTD.

Figure 1.

Activation and Repression by ARFs on Auxin-Responsive Reporter Genes. Effector genes, which consisted of the CaMV 35S promoter encoding full-length ARF proteins, were cotransfected into protoplasts with a GUS reporter gene, which contained an auxin-responsive promoter. Reporter genes are diagrammed above the graphs. GUS activities were measured from protoplasts that were treated (+ auxin) or not treated (− auxin) with 10 μM 1-NAA. (A) The P3(4X)-GUS reporter gene was cotransfected into carrot suspension cell protoplasts with the ARF effector genes indicated. (B) Same as (A), but with a DR5(7X)-GUS reporter gene. (C) Same as (B), but with Arabidopsis suspension cell protoplasts.

Figure 2.

The DBD Is Sufficient for Targeting ARF1 and ARF5 to an Auxin-Responsive P3(4X)-GUS Reporter Gene in Protoplast Transfection Assays, and This Targeting Is Independent of Auxin. Effector and reporter genes (diagrammed at top) were cotransfected into carrot suspension cell protoplasts and assayed in the presence (+ auxin) or absence (− auxin) of 10 μM 1-NAA. ARF1 and ARF5 were tested as full-length constructs, as constructs that lacked a CTD dimerization domain, and as constructs that lacked both their normal MR and CTD but contained a heterologous MR or VP16 activation domain.

Figure 3.

ARF MRs Function as Repression or Activation Domains in an Auxin-Independent Manner. Effector genes containing a yeast Gal4 DBD (GD) fused in frame to ARF MRs are diagrammed at top. ARF1, -2, -3, -4, and -9 MRs are enriched for P and S, and ARF5, -6, -7, and -8 MRs are enriched for Q. The GD effector gene contained only the yeast Gal4 DBD. Carrot suspension cell protoplasts cotransfected with a reporter gene and an effector gene were assayed in the presence (+ auxin) or absence (− auxin) of 10 μM 1-NAA. (A) To measure repression by ARF effector genes, transfection assays were performed with a constitutive Gal4(4X)-D1-3(4X)-GUS reporter gene, which contains four Gal4 DNA binding sites upstream of four tandem copies of the constitutive D1-3(4X) element (Tiwari et al., 2001). (B) To measure activation by ARF effector genes, transfection assays were performed with the minimal promoter Gal4(4X)-GUS reporter gene, which contains four Gal4 DNA binding sites upstream of the CaMV −46 minimal promoter element (Tiwari et al., 2001).

Figure 4.

The ARF CTD Dimerization Domain Confers Auxin Responsiveness to an ARF5 MR. Effector and reporter genes (diagrammed at top) were cotransfected into carrot suspension cell protoplasts and assayed in the presence (+ auxin) or absence (− auxin) of 10 μM 1-NAA. The IAA17 and IAA17mII effector genes encode a full-length wild-type IAA17 and a full-length box II mutant IAA17mII protein, respectively. The IAA17mII mutant protein is more stable and is a stronger repressor than the wild-type IAA17 protein (Tiwari et al., 2001).

Figure 5.

Dimerization of ARF and Aux/IAA Proteins Occurs on the Promoter to Bring about Repression. Effector genes (diagrammed at top) were cotransfected along with a reporter gene into carrot suspension cell protoplasts and assayed in the presence (+ auxin) or absence (− auxin) of 10 μM 1-NAA. The GD-5MC and GD-1MC effector genes contain the yeast Gal4 DBD fused in frame to the ARF5 MR + CTD and the Gal4 DBD fused in frame to the ARF1 MR + CTD, respectively. The IAA17mI effector gene encodes an IAA protein with a mutation in conserved motif I, resulting in an unstable protein that is a weaker repressor than wild-type IAA17 (Tiwari et al., 2001). The VP16-IAA17mI effector gene contains a VP16 activation domain fused in frame to IAA17mI. The IAA17mI/mII effector gene encodes an IAA protein with a mutation in both conserved motifs I and II, resulting in a protein that is more stable than the wild-type IAA17 protein but a weaker repressor than the motif II mutant protein, IAA17mII (Tiwari et al., 2001). The GD-VP16-IAA17mI/mII effector gene encodes a protein containing a yeast Gal4 DBD fused in frame to the VP16 activation domain, which in turn is fused in frame to IAA17mI/mII. The VP16-IAA17mI/mII effector gene is identical to the GD-VP16-IAA17mI/mII effector gene but lacks the Gal4 DBD. (A) Transfection assays were performed with a minimal promoter Gal4(4X)-GUS reporter gene (diagrammed at top) (B) Transfection assays were performed with an auxin-responsive P3(4X)-GUS reporter gene (diagrammed at top). (C) Transfection assays were performed with a constitutive promoter Gal4(4X)-D1-3(4X)-GUS reporter gene (diagrammed at top).

Figure 6.

Model for the Repression and Activation of Auxin Response Genes. When auxin concentrations are low or below a threshold, early auxin response genes containing TGTCTC AuxREs are actively repressed, because Aux/IAA repressor proteins are dimerized to ARF transcriptional activators, preventing gene transcription. When auxin concentrations are increased, Aux/IAA proteins turn over more rapidly as a result of their being degraded more rapidly through the proteasome pathway (Rogg and Bartel, 2001; reviewed by Kepinski and Leyser, 2002). This more rapid degradation of Aux/IAA proteins effectively relieves the repression of early auxin response genes, resulting in gene activation. Gene activation might be enhanced further by the dimerization of ARF transcriptional activators to ARFs that are bound to AuxRE target sites. In this model, the auxin-sensitive target is the CTD dimerization domain, and the ARF DNA binding domain and activation/repression domain function independently of auxin.