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. 1999 May 11;96(10):5844-9.
doi: 10.1073/pnas.96.10.5844.

Activation and repression of transcription by auxin-response factors

T Ulmasov  1 , G Hagen, T J Guilfoyle
Affiliations

Activation and repression of transcription by auxin-response factors

T Ulmasov et al. Proc Natl Acad Sci U S A. .

Abstract

Auxin-response factors (ARFs) bind with specificity to TGTCTC auxin-response elements (AuxREs), which are found in promoters of primary/early auxin-response genes. Nine different ARFs have been analyzed for their capacity to activate or repress transcription in transient expression assays employing auxin-responsive GUS reporter genes. One ARF appears to act as a repressor. Four ARFs function as activators and contain glutamine-rich activation domains. To achieve transcriptional activation on TGTCTC AuxREs in transient expression assays, ARFs require a conserved dimerization domain found in both ARF and Aux/IAA proteins, but they do not absolutely require their DNA-binding domains. Our results suggest that ARFs can activate or repress transcription by binding to AuxREs directly and that selected ARFs, when overexpressed, may potentiate activation further by associating with an endogenous transcription factor(s) (e.g., an ARF) that is bound to AuxREs. Transfection experiments suggest that TGTCTC AuxREs are occupied regardless of the auxin status in cells and that these occupied AuxREs are activated when exogenous auxin is applied to cells or when ARF activators are overexpressed. The results provide new insight into mechanisms involved with auxin regulation of primary/early-response genes.

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Figures

Figure 1
Figure 1
Predicted sequences of Q-rich ARF MRs in Arabidopsis. Sequences are presented for MRs of ARF5, ARF6, ARF7, and ARF8. Glutamines are shaded with a black background and serines and leucines are shaded with a gray background. pI values for the middle regions are indicated for the sequences shown.
Figure 2
Figure 2
ARF1 functions as a repressor, and ARF6 functions as an activator. (A) Diagrams of the ARF1 and ARF6 effector genes are shown at the top. The CaMV 35S promoter and TMV 5′ leader used to drive expression of the effector genes have been described previously (5). The DBD, MRs (i.e., P-rich in ARF1 and Q-rich in ARF6), and conserved motifs III and IV in the CTD are indicated in the diagrams. Transient assays were carried out in carrot protoplasts with a P3(4X) promoter-GUS reporter gene or a GH3 promoter-GUS reporter gene and the effector plasmid indicated or no effector plasmid (“none”). (B) Titration of the ARF1 effector plasmid with the P3(4X) promoter-GUS reporter plasmid. A CaMV 35S promoter-LUC reporter gene (11, 12) was used as a control to show that the effector was specific for TGTCTC auxin-responsive reporter genes. min−35S is a −46 minimal CaMV promoter-GUS reporter gene. (Bars = SE.)
Figure 3
Figure 3
The MRs of ARF5, ARF6, ARF7, and ARF8 contain ADs. (A) Transfections with truncated ARFs containing MRs plus the CTD. Effector plasmids consisted of the GAL4 DBD fused to the VP16 acidic AD from herpes virus or to ARF proteins that had their DBDs truncated. (B) Transfections with truncated ARFs containing only CTDs. Effector plasmids consisted of the GAL4 DBD fused to ARF proteins that had their DBDs plus MRs truncated. The reporter gene used in both A and B was the GAL4(4X) promoter-GUS gene (12). Effector genes contained the same promoter as those effectors tested in Fig. 2. The amino acid position of the amino termini in the truncated ARFs is indicated by N.
Figure 4
Figure 4
ARF5, ARF6, ARF7, and ARF8 that lack DBDs activate transcription on TGTCTC AuxREs. (A) ARFs with glutamine-rich MRs do not require a DBD to activate transcription of a P3(4X) promoter-GUS reporter gene (2). Diagrams of the effector plasmids are shown at the top. ARF1, ARF2, ARF5, ARF6, ARF7, and ARF8 contained their MRs and CTDs (MR-CTD). The amino termini of the truncated ARF proteins are identical to those shown in Fig. 3. (B) In the absence of a DBD, the CTD of ARF7 fused to homologous or heterologous ADs or RDs can modulate TGTCTC AuxRE promoter-GUS reporter gene expression. Diagrams of the effector plasmids are shown at the top. ARF7MR-CTD is ARF7 with its DBD truncated. ARF7MR is ARF7 with both its DBD and CTD truncated. ARF7MR-CTDm is identical to ARF7MR-CTD with the exception that point mutations were introduced into motif IV of the CTD, resulting in two conserved amino acid substitutions (PW → RS). ARF1MR-ARF7-CTD is derived from ARF7MR-CTD, with the ARF1 MR swapped for the ARF7 MR. VP16-ARF7-CTD has the VP16 AD swapped for the ARF7 MR. VP16-ARF1-CTD has the VP16 AD swapped for the ARF1 MR. The reporter plasmid was the GH3 promoter-GUS reporter gene, m3-P3(4X)promoter-GUS reporter gene, and P3(4X) promoter-GUS reporter gene, as indicated. The amino termini of the truncated ARF1 and ARF7 constructs is the same as that in Fig. 3. Details on the constructs are presented in Materials and Methods. Effector genes contained the same promoter as those effectors tested in Fig. 2.
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