AUX/IAA proteins are active repressors, and their stability and activity are modulated by auxin

Abstract

Aux/IAA genes are early auxin response genes that encode short-lived nuclear proteins with four conserved domains, referred to as I, II, III, and IV. Arabidopsis Aux/IAA proteins repressed transcription on auxin-responsive reporter genes in protoplast transfection assays. Mutations in domain II resulted in increased repression, whereas mutations in domains I and III partially relieved repression. Aux/IAA proteins fused to a heterologous DNA binding domain were targeted to promoters of constitutively expressed reporter genes and actively repressed transcription in an auxin-responsive and dose-dependent manner. In comparison with an unfused luciferase protein, luciferase fused to Aux/IAA proteins displayed less luciferase activity, which further decreased in the presence of auxin in transfected protoplasts. Domain II mutations increased and domain I mutations decreased luciferase activity with the fusion proteins. These results suggested that Aux/IAA proteins function as active repressors by dimerizing with auxin response factors bound to auxin response elements and that early auxin response genes are regulated by auxin-modulated stabilities of Aux/IAA proteins.

Figure 1.

Schematic Diagrams of Effector and Reporter Constructs. Expression of all effector constructs was driven by the CaMV 35S promoter, and each effector construct contained a nopaline synthase (nos) 3′ untranslated sequence (Ulmasov et al., 1997b). Effector plasmids encoding Aux/IAA proteins contained a HA epitope tag (see Methods) fused in-frame to their N termini and are diagrammed with conserved domains I, II, III, and IV. Effector plasmids encoding GAL4 DBD–Aux/IAA fusion proteins contained a yeast GAL4 DBD (amino acids 1 to 147) fused in-frame at the N termini of Aux/IAA proteins. An effector gene expressing only the GAL4 DBD is also shown. The P3(4X):GUS reporter gene consisted of the auxin responsive P3(4X) sequence (four tandem copies of the P3 AuxRE) fused to a CaMV minimal −46 promoter driving expression of the GUS reporter gene (Ulmasov et al., 1997a). The GH3:GUS reporter gene contained a 592-bp auxin-responsive soybean GH3 promoter-driving expression of the GUS reporter gene (Liu et al., 1994). The GAL4(4X)-D1-3(4X):GUS reporter gene consisted of four tandem copies of the GAL4 DNA binding site (see Methods) fused immediately upstream of four tandem copies of the constitutive D1-3 element, which in turn was fused to a CaMV minimal −46 promoter-driving expression of the GUS reporter gene. The GAL4(4X)-35S:GUS reporter gene consisted of four tandem copies of the GAL4 DNA binding site fused immediately upstream of the CaMV 35S promoter-driving expression of the GUS reporter gene. Reporter plasmids encoding Aux/IAA-LUC fusion proteins contained a LUC protein fused in-frame at the C termini of Aux/IAA proteins.

Figure 2.

Repression of Auxin-Responsive Reporter Genes by Arabidopsis Aux/IAA Effector Plasmids. (A) Repression with the P3(4X):GUS reporter gene. Effector plasmids encoding different Aux/IAA proteins were cotransfected with the auxin-responsive P3(4X):GUS reporter gene into carrot protoplasts, and protoplasts were incubated with or without 25 μM auxin (1-NAA). IAA effector plasmids are indicated using the nomenclature of Abel et al. (1995) and Kim et al. (1997). (B) Repression of the soybean GH3:GUS reporter gene with IAA7 and IAA17 effector plasmids. Effector plasmids encoding either IAA7 or IAA17 were cotransfected into carrot protoplasts and assayed as described in (A). GUS activities were measured 24 hr after transfections. Standard errors are indicated. None indicates GUS activities with the reporter gene in the absence of an effector gene.

Figure 3.

Repression of Auxin-Responsive Reporter Genes by Wild-Type and Domain II Mutant Versions of Arabidopsis Aux/IAA Proteins. (A) Repression by IAA effector plasmids in transfected carrot protoplasts with the P3(4X):GUS reporter gene. Effector genes encoding wild-type and domain II mutated versions (see Table 1) of IAA3/SHY2, IAA7/AXR2, IAA17/AXR3, and IAA19 were assayed for GUS activity with the reporter gene in the presence and absence of 25 μM auxin (1-NAA). (B) Repression by IAA7 effector plasmids in Arabidopsis protoplasts with the soybean GH3:GUS reporter gene. Effector genes encoding the wild type and a domain II mutated version of IAA7/AXR2 were assayed for GUS activity with the reporter gene in the presence and absence of 1 μM auxin (1-NAA). The ratios of reporter gene to effector gene in the assays are indicated below the effector lanes. Because of the relatively large standard errors for GUS assays with Arabidopsis protoplasts, t tests were used to confirm that reporter gene activity was significantly different for wild-type and domain II mutant effector genes at each reporter:effector ratio; 1:0.2 (P < 0.05), 1:0.4 (P < 0.05), 1:0.8 (P < 0.05), and 1:1 (P < 0.1). GUS activities were measured 24 hr after transfections. Standard errors are indicated. None indicates GUS activities with the reporter gene in the absence of an effector gene.

Figure 4.

Reversal of Repression by Mutations in Domains I and III of Aux/IAA Proteins. (A) Natural mutations in domains I and III partially reverse repression conferred by wild-type and domain II mutant IAA7/AXR2 proteins. The natural mutations correspond to those found in axr2-1, axr2-1-r3, and axr2-1-r4. Domain I, II, and III mutations in IAA7/AXR2 are described in Table 1. (B) Additional mutations in domains I and III, that have not been found in mutant screens, partially reverse repression by wild-type and domain II mutant Aux/IAA proteins. P3(4x):GUS was used as the reporter gene in carrot protoplasts. GUS activities were measured 24 hr after transfections. Standard errors are indicated. None indicates GUS activities with the reporter gene in the absence of an effector gene.

Figure 5.

Aux/IAA Proteins Repress Constitutive Reporter Genes in an Auxin-Responsive Manner When Targeted to DNA Binding Sites in Promoters. Effector genes encoding GAL4 DBD and GAL4 DBD–Aux/IAA fusion proteins were cotransfected into carrot protoplasts along with a constitutively expressed GUS reporter gene containing four tandem GAL4 DNA binding sites (see Methods for details on effector and reporter genes). GUS activities were measured 24 hr after transfections. Transfected protoplasts were incubated in the presence or absence of 25 μM auxin (1-NAA). Transfections were performed with effectors encoding GAL4 DBD fused to wild type (wt) and domain I, II, and III mutant versions (denoted with as mI, mII, and mIII; see Table 1) of IAA7 (indicated with a 7) and IAA17 proteins (indicated with a 17). G4 indicates GAL4 DBD; G4-DBD indicates an effector encoding the unfused GAL4 DBD. IAA7wt and IAA17wt refer to effector genes encoding IAA7 and IAA17 proteins that were not fused to a GAL4 DBD. None, no response to auxin. The effector:reporter ratio was 1:1. (A) Transfections with a GAL4D1-3:GUS reporter gene. (B) Transfections with a GAL4CaMV35S:GUS reporter gene. Standard errors are indicated.

Figure 6.

Auxin Decreases the Stability of Wild-Type and Mutant Aux/IAA-LUC Fusion Proteins in Transfected Carrot Protoplasts. (A) Relative luciferase activities with effector genes encoding luciferase alone (LUC) and luciferase fused to wild-type and domain I and II mutant versions of IAA17 and IAA19. Reporter genes encoding fusion proteins consisted of wild-type (wt) and mutant versions (mI and mII; see Table 1) of IAA17 and IAA19 fused to the N terminus of LUC (see Methods). Activities are relative to the reporter gene encoding an unfused LUC protein (100%). (B) An expanded scale of data from (A) showing results with reporter genes encoding IAA–LUC fusion proteins that displayed low luciferase activities. Standard errors are indicated.

Figure 7.

Auxin Dose Response for Reporter Genes Encoding the Wild Type and a Domain II Mutant Version of Arabidopsis IAA17. The auxin (1-NAA) was applied at the concentration indicated below each bar. Luciferase activity is relative to the reporter gene expressing the unfused LUC protein in transfected carrot protoplasts (100%). (A) Dose response for the 35S:IAA17mII-LUC reporter plasmid encoding IAA17 with a domain II mutation fused to LUC. (B) Dose response for the 35S:IAA17-LUC reporter plasmid encoding wild-type IAA17 fused to LUC. (C) Dose response for repression by effector genes encoding wild-type IAA17 (G4-IAA17wt) and domain II mutant IAA17 (G4-IAA17mII) with the GAL4D1-3:GUS reporter gene. Transfection assays were conducted as described in Figure 5 with a effector:reporter ratio of 1:1 at different NAA concentrations. GUS activity with the reporter gene in the absence of effector was considered 100% activity and 0% repression. Percentage of repression was calculated by subtracting the percentage of activity of the reporter gene observed with the effector gene from the percentage of activity of the reporter gene without the effector gene at each NAA concentration. There was no response of the reporter gene to auxin in the absence of effector. Standard errors are indicated.

Figure 8.

Model for the Auxin-Responsive Repression of Early Auxin Response Genes by Aux/IAA Proteins. The model predicts that ARF transcriptional activators (e.g., ARF5, -6, -7, -8) with glutamine-rich (Q) middle regions are bound via their DBDs (gray oval; Ulmasov et al., 1997a, 1999b) to AuxRE target sites in the promoters of early auxin response genes independent of auxin levels within cells (see Ulmasov et al., 1999a). When auxin levels are low (small IAA in brackets), primary/early auxin response genes are actively repressed by Aux/IAA proteins that dimerize with ARF transcriptional activators. Dimerization occurs via interactions between domains III and IV, which are found in both Aux/IAA and ARF proteins. When auxin levels increase (large IAA in brackets), Aux/IAA proteins dissociate from the DNA-bound ARF proteins and are rapidly degraded through the proteasome pathway (reviewed by Gray and Estelle, 2000). Dissociation of the Aux/IAA proteins from the ARF DNA complexes results in rapid transcriptional derepression/activation of early response genes, some of which encode Aux/IAA proteins. The transcription of these Aux/IAA and other early response genes continues at high levels for up to several hours as long as auxin concentrations remain high (Hagen et al., 1984; Theologis et al., 1985; Abel et al., 1995). The Aux/IAA proteins synthesized at high auxin concentrations are rapidly degraded and only accumulate to levels sufficient for repression when auxin levels decline, ultimately resulting in feedback inhibition of their own transcription.