Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana

Abstract

The plant hormone auxin regulates diverse aspects of plant growth and development. We report that in Arabidopsis, auxin response is dependent on a ubiquitin-ligase (E3) complex called SCFTIR1. The complex consists of proteins related to yeast Skp1p and Cdc53p called ASK and AtCUL1, respectively, as well as the F-box protein TIR1. Mutations in either ASK1 or TIR1 result in decreased auxin response. Further, overexpression of TIR1 promotes auxin response suggesting that SCFTIR1 is limiting for the response. These results provide new support for a model in which auxin action depends on the regulated proteolysis of repressor proteins.

Figure 1

Analysis of TIR1 expression. (A) Northern blot analysis of TIR1 with total RNA isolated from various tissues. TIR1–gus expression in 3-day-old seedling with staining apparent in the root tip, hypocotyl, and cotelydons (B); root tip of 10-day-old seedling (C); shoot of 10-day-old seedling with staining visible in stipules (arrowheads) and the apical meristem (D); an early lateral root primoridium (between arrowheads) (E). Vascular tissues are also stained; (F) an emerging lateral root; (G) emerged lateral root; (H) unfertilized flower. In situ RNA hybridization with TIR1 of 3-day-old seedling (I); 3-day-old seedling (J); root tip of 5-day-old seedling (K); shoot of 5-day-old seedling (L); longitudinal sections (10 μm) through shoot apical meristem (M,N); and heart-stage embryo (O,P). Sense strand control hybridizations are shown in J, N, and P. Size bars, 0.5 mm in B, D, H, I, and J, and 0.1 mm in all other panels.

Figure 2

TIR1 functions prior to the expression of cyc1At in lateral root development. (A) Number of emerged lateral roots (LR, solid bars) and the number of cyc1At–gus foci (CGF, open bars) in wild-type and tir1-1 seedlings in the presence and absence of exogenous auxin. For this study CGF are defined as the number of foci of cyc1At–gus staining observed along the root excluding the primary root meristem. This ranges from single cells expressing the cyc1At–gus reporter to mature lateral root meristems. Presumably these foci are all lateral root meristems at various stages of development. (B) cyc1At–gus expression in the roots of 10 day-old wild-type and tir1-1 seedlings. Nine-day-old seedlings were treated with 0.25 μm 2,4-D for various times and stained for gus activity. Root segments shown are located ∼5 mm from the root tip. At this location in wild-type roots, auxin treatment induces a high percentage of pericycle cells to differentiate into lateral root primordia. (Inset) High magnification of a single pericycle cell expressing the cyc1At–gus reporter prior to the first cell division of lateral root development.

Figure 3

TIR1 interacts with the Skp1-like proteins ASK1 and ASK2. (A) Sequence alignment of the A. thaliana ASK1 and ASK2 (GenBank accession no. AF059295) proteins as well as human and S. cerevisiae Skp1p with PileUp (Genetics Computer Group, Inc., Madison, WI). Identical amino acids are boxed in black. Similar amino acids are boxed in gray. (B) Serial dilutions of yeast strain YPB2 carrying the indicated two-hybrid plasmids. Cells were plated onto 3-AT selection medium (left) or control (right) medium and incubated at room temperature. The 3-AT plate was photographed after 10 days and the control plate after 3 days. (C) Western blot analysis of seedling extracts with polyclonal antisera raised against ASK1 (lanes 2–4) or ASK2 (lanes 6–8). (D) Western blot analysis of crude seedling extracts (lanes 1,2,5,6) or α-myc immunoprecipitates (lanes 3,4,7,8). (Bottom lanes 1–4) was probed with α-ASK1 antisera. (Bottom, lanes 5–8) Probed with α-ASK2 antisera.

Figure 4

The A. thaliana cullin, AtCUL1, interacts with TIR1 in planta. (A) Sequence alignment of AtCUL1 with the human and S. cerevisiae cullins, Cul4A and Cdc53p, with PileUp (Genetics Computer Group, Inc., Madison,WI). Identical amino acids are boxed in black. Similar amino acids are boxed in gray. (B) Western blot analysis of crude seedling extracts (lanes 1–4) or α-myc immunoprecipitates (lanes 6–7).

Figure 5

The ASK1 gene is required for normal auxin response. (A) ask1-1 and wild-type Ler seedling were grown on nutrient medium for 4 days and then transferred to medium supplemented with 0.085 μm 2,4-D and grown an additional 5 days. (Asterisk) Position of the root tips at the time of transfer. Size bars, 5 mm. (B) Mean number of lateral roots formed by wild-type and ask1-1 seedlings grown on unsupplemented medium for 11 days. (Open bar) ASKI; (hatched bar) ask1. Bars, standard error. (C) Dose response curve for wild-type, tir1-1, ask1-1, and tir1-1 ask1-1 mutants. Inhibition of root growth is expressed relative to growth on unsupplemented medium. Because of the incomplete penetrance of the ask1-1 mutation, only auxin resistant seedlings are included in this data set. Each value represents the mean of 10 seedlings. (□) Ler; (♦) tir1-1; (○) ask1-1; (▴) tir1-1, ask-1. For each data point, s.e≤10%.

Figure 5

The ASK1 gene is required for normal auxin response. (A) ask1-1 and wild-type Ler seedling were grown on nutrient medium for 4 days and then transferred to medium supplemented with 0.085 μm 2,4-D and grown an additional 5 days. (Asterisk) Position of the root tips at the time of transfer. Size bars, 5 mm. (B) Mean number of lateral roots formed by wild-type and ask1-1 seedlings grown on unsupplemented medium for 11 days. (Open bar) ASKI; (hatched bar) ask1. Bars, standard error. (C) Dose response curve for wild-type, tir1-1, ask1-1, and tir1-1 ask1-1 mutants. Inhibition of root growth is expressed relative to growth on unsupplemented medium. Because of the incomplete penetrance of the ask1-1 mutation, only auxin resistant seedlings are included in this data set. Each value represents the mean of 10 seedlings. (□) Ler; (♦) tir1-1; (○) ask1-1; (▴) tir1-1, ask-1. For each data point, s.e≤10%.

Figure 5

The ASK1 gene is required for normal auxin response. (A) ask1-1 and wild-type Ler seedling were grown on nutrient medium for 4 days and then transferred to medium supplemented with 0.085 μm 2,4-D and grown an additional 5 days. (Asterisk) Position of the root tips at the time of transfer. Size bars, 5 mm. (B) Mean number of lateral roots formed by wild-type and ask1-1 seedlings grown on unsupplemented medium for 11 days. (Open bar) ASKI; (hatched bar) ask1. Bars, standard error. (C) Dose response curve for wild-type, tir1-1, ask1-1, and tir1-1 ask1-1 mutants. Inhibition of root growth is expressed relative to growth on unsupplemented medium. Because of the incomplete penetrance of the ask1-1 mutation, only auxin resistant seedlings are included in this data set. Each value represents the mean of 10 seedlings. (□) Ler; (♦) tir1-1; (○) ask1-1; (▴) tir1-1, ask-1. For each data point, s.e≤10%.

Figure 6

TIR1 overexpression analysis. (A) Northern analysis of T2 seedlings transformed with the glucocorticoid inducible TIR1 or TIR1_P10A expression constructs. TIR1 expression was induced by treating with dexamethazone for 24 hr. (B) TIR1 expression was induced in 7-day-old transgenic P_GVG–TIR1 and P_GVG–TIR1_P10A seedlings by transferring seedlings to nutrient medium supplemented with dexamethazone and grown an additional 2 days. Untransformed Columbia seedlings are shown as a control. (Asterisk) Position of the root tips at the time of transfer. Root tips shown in insets are not from the same seedling pictured. Size bars, 10 mm. (C) Transgenic and control seedlings were germinated and grown for 4 days in the dark on nutrient medium containing dexamethazone. Size bars, 1 mm. (D) Wild-type seedlings grown as described in B and C with auxin in the growth medium instead of dexamethazone. The light-grown seedling (top) was transferred to medium containing 1 μm IAA. Size bar, 10 mm. The dark-grown seedling (bottom) was grown on medium supplemented with 1 μm 2,4-D. Size bar, 1 mm. (E) pIAA4–gus expression in P_GVG–TIR1 transgenic seedlings in the absence (top) and presence (center) of dexamethazone. Wild-type pIAA4–gus seedlings treated with 0.2 μm 2,4-D are shown at bottom. Seedlings were stained for β-glucuronidase activity 48 hr after induction with dexamethazone or 2,4-D.

Figure 7

Model for auxin response. In this model, TIR1 functions in an SCF ubiquitin ligase that targets one or more negative regulators of the auxin response pathway for ubiquitination and degradation. SCF^TIR1 activity requires the RUB modification of AtCUL1 via the AXR1–ECR1 pathway. The ubiquitin–mediated degradation of the repressor(s) derepresses the response pathway resulting in the activation of the early auxin response genes (AUX/IAA genes) that may control downstream events resulting in auxin-regulated growth and development. E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E2R, RUB-conjugating enzyme.