The Arabidopsis cell cycle F-box protein SKP2A binds to auxin

Abstract

Arabidopsis thaliana S-Phase Kinase-Associated Protein 2A (SKP2A) is an F-box protein that regulates the proteolysis of cell cycle transcription factors. The plant hormone auxin regulates multiple aspects of plant growth and development, including cell division. We found that auxin induces the ubiquitin-dependent degradation of SKP2A both in vivo and in vitro, suggesting that this hormone acts as a signal to trigger SKP2A proteolysis. In this article, we show that auxin binds directly and specifically to SKP2A. By TIR1-based superposition and docking analyzes, we identified an auxin binding site in SKP2A. Mutations in this binding site reduce the ability of SKP2A to bind to auxin and generate nondegradable SKP2A forms. In addition, these non-auxin binding proteins are unable to promote E2FC/DPB degradation in vivo or to induce cell division in the root meristem. Auxin binds to TIR1 to promote its interaction with the auxin/indole-3-acetic acid target proteins. Here, we show that auxin also enhanced the interaction between SKP2A and DPB. Finally, a mutation in SKP2A leads to auxin-resistant root growth, an effect that is additive with the tir1-1 phenotype. Thus, our data indicate that SKP2A is an auxin binding protein that connects auxin signaling with cell division.

Figure 1.

SKP2A Is Degraded in Response to Auxin in a TIR1-Independent Manner. (A) Immunoblotting analyses of the MYC-SKP2A levels in the tir1-1 mutant. Total protein from wild-type (wt), tir1-1, MYC-SKP2A–overexpressing plants (SKP2A^OE), and two different lines tir1-1/SKP2A^OE was extracted in the presence of MG132 and immunoblotted with anti-MYC. LC is the loading control, corresponding to the Ponceau-stained blot. The bottom panel shows the expression of the MYC-SKP2A transgene analyzed by RT-PCR. As a control, the expression of the ACTIN gene was analyzed. (B) Five-day-old PER8:SKP2A-GUS and tir1-1/PER8:SKP2A-GUS transgenic seedlings were incubated with 10 μM estradiol in a liquid medium for 16 h to induce the expression of the chimeric protein. After induction, these seedlings were incubated in MS medium for 2 h and then stained for GUS activity. Bar = 0.2 mm. (C) Expression of the SKP2A-GUS transgene analyzed by RT-PCR using primers from SKP2A and GUS coding regions in 5-d-old PER8:SKP2A-GUS and tir1-1/PER8:SKP2A-GUS transgenic seedlings that were incubated with 10 μM estradiol in a liquid medium for 16 h. As a control, the expression of the ACTIN gene was analyzed. [See online article for color version of this figure.]

Figure 2.

Auxin Directly Promotes SKP2A Degradation. (A) Degradation assay using crude plant protein extract from 5-d-old MYC-SKP2A^OE seedlings. The protein extracts were incubated in presence of the control ethanol solvent (EtOH) or 1 μM 2,4-D. These reactions were incubated at 30°C during the indicated time and then stopped by adding loading buffer and boiling the sample for 5 min. The levels of MYC-SKP2A were analyzed by immunoblotting with anti-MYC. (B) SKP2A is degraded in response to active auxins. Degradation assays using crude plant extract from 5-d-old MYC-SKP2A^OE seedlings. The protein extracts were incubated in presence of the control ethanol solvent (EtOH) or 1 μM 2,4-D, IAA, 1-NAA, and 2-NAA. These reactions were incubated at 30°C during the indicated time points and then they were stopped by adding loading buffer and boiling the sample for 5 min. The levels of MYC-SKP2A were analyzed by immunoblotting with anti-MYC. The bands were quantified by a Bio-Rad molecular imager, relative to a 100% value at time zero. The image presented is from one representative experiment of three, and the graph is a compilation of these experiments. Each value is the mean of the three degradation assays, and the error bars correspond to the sd.

Figure 3.

IAA Binds to SKP2A, but Not to SKP2B. (A) Pull-down reactions were performed with either MBP-SKP2A or MBP-SKP2B bound to amylase beads in the presence of 50 nM [³H]-IAA. The retained [³H]-IAA in the amylose beads after three washes was measured by scintillation counting. Each value is the mean of three independent measures, and the error bars correspond to the sd. d.p.m., disintegrations per minute. (B) Competitive binding of [³H]-IAA to MBP-SKPA and MBP-SKP2B in the presence of increasing concentrations of unlabeled IAA. Pull-down reactions were performed in the presence of 50 nM [³H]-IAA and the indicated amount of competitor. Each value corresponds to the mean of three independent experiments. Error bars represent the sd. (C) Competitive binding of [³H]-IAA to MBP-SKPA in the presence of increasing concentrations of unlabeled 1-NAA (black squares), 2-NAA (gray squares), or 2,4-D (black triangles). MBP alone was used as a control of nonbinding protein. Pull-down reactions were performed in the presence of 50 nM [³H]-IAA and the indicated amount of cold competitor. The inactive auxin 2-NAA does not compete with [³H]-IAA. Values are the mean of three independent experiments. Error bars represent the sd.

Figure 4.

Identification of the Auxin Binding Region in the SKP2A Structure. (A) An orthogonal view of a ribbon diagram of the modeled structure of SKP2A using the structure of human Skp2. Arrows indicate the different deletions generated for auxin binding analyses (T4, T3, T2, and T1), and the last amino acid of the truncated proteins is indicated by a number. (B) These truncated versions (MBP-SKP2A-T1, T2, T3, and T4), MBP, MBP-SKP2A, and MBP-SKP2B were expressed in bacteria and then incubated in the presence of 50 nM [³H]-IAA. The retained [³H]-IAA in the amylose beads after three washes was measured by scintillation counting. Each value is the mean of three independent measures, and the error bars correspond to the sd. d.p.m., disintegrations per minute.

Figure 5.

Model Structure of SKP2A and Identification of the Auxin Binding Site. (A) Superposition of the LRR domain of SKP2A (green) and the half side of TIR1 protein that binds auxin (gray) shown as ribbon diagrams. Residues of SKP2A in a neighborhood of 4.0 Å around auxin molecule (magenta) of the TIR1 structure are depicted as sticks. (B) Auxin molecule (green frame) docked to a binding site composed of the 11 residues (cyan frames) indicated. Numbers colored cyan refer to β-strands of the LRR domain, which is shown as a ribbon diagram in dark green. Lines represent bonds between backbone atoms, and sticks correspond to bonds between side chain atoms involved in the interaction. The bottom panel shows a close-up view of the nearest groups to auxin revealed by quantum calculations. Yellow dashed lines represent hydrogen bonds with numerical labels indicating H···X distances in Å. Cyan dashed lines represent hydrogen bonds that depend on internal rotation of S151 OH group and C177 SH group. Red line represents a putative interaction of the type N-H···π between a NH₂ side group of N202 and the π electron cloud of the aromatic ring of auxin. The red label gives the distance in Å between N atom and the center of this ring. Atoms are colored: C, light green and cyan; N, blue; O, red; S, yellow; H, white. (C) Recombinant MBP, MBP-SKP2A, and two SKP2A mutants, which have replaced the Ser-151 for Ala (MBP-SKP2A[S151A]), the Leu-128 for Ser (MBP-SKP2A[L128S]), or double mutant (MBP-SKP2A[L128S; S151A]), were incubated in the presence of 50 nM [³H]-IAA. The retained [³H]-IAA in the amylose beads after three washes was measured by scintillation counting. Each value is the mean of three independent measures, and the errors bars correspond to the sd. The results were normalized relative to the amount of [³H]-IAA retained in the MBP-SKP2A beads. (D) Recombinant MBP, MBP-SKP2A, MBP-SKP2B, and a SKP2B mutant that has replaced the Ser-128 for Leu (MBP-SKP2B[S128L]) were incubated in the presence of 50 nM [³H]-IAA. The retained [³H]-IAA in the amylose beads after three washes was measured by scintillation counting. Each value is the mean of three independent measures, and the errors bars correspond to the sd. The results were normalized relative to the amount of [³H]-IAA retained in the MBP-SKP2A beads and are presented as a percentage.

Figure 6.

Mutations in the Auxin Binding Site Stabilize SKP2A and DPB and Affect Cell Division. (A) Five-day-old PER8:SKP2A-GUS, PER8:SKP2Amut2-GUS, and the control PER8:GFP-GUS transgenic seedlings were transferred to MS plates containing 15 μM estradiol to induce the expression of the proteins for 16 h. After induction, these seedlings were stained for GUS activity (tp = 0) or incubated during 5 h in a MS medium (5 h MS) or MS containing 5 × 10⁻⁷ M 2,4-D (5 h+Aux) and then stained for GUS activity. Bar = 500 μm. (B) Higher magnification of PER8:SKP2A-GUS, PER8:SKP2Amut2-GUS, and the control PER8:GFP-GUS root tip shown in (A). Bar = 500 μm. (C) Expression of the SKP2A-GUS and SKP2Amut2-GUS transgenes analyzed by RT-PCR using primers from SKP2A and GUS coding regions. As a control, the expression of the ACTIN1 (ACT) gene was analyzed. (D) Immunoblotting analyses of the E2FC or MYC-DPB levels into the skp2a/MYC-DPB plants. Total protein was extracted from wild-type, MYC-DPB^OE, skp2a/MYC-DPB^OE, or skp2a/MYC-DPB^OE seedlings that overexpress HA-SKP2A, HA-SKP2Amut1 (two independent lines), or HA-SKP2mut2 (two independent lines) and immunoblotted (I-blot) with anti-E2FC or anti-MYC. LC is the loading control corresponding to the Ponceau-stained blot. Ø indicates skp2a/MYC-DPB^OE plants that overexpress the HA tag alone. The expression levels of both E2FC and MYC-DPB genes were analyzed by RT-PCR. As a control, the ACTIN gene levels were analyzed. (E) Root meristem cell number of control MYC-GFP, MYC-SKP2A^OE, or MYC-SKP2Amut2^OE plants grown in MS medium. For monitoring root meristem growth, cortex meristematic cells were counted at the indicated days. Error bars represent se (n = 15). Asterisks indicate statistically significant difference compared with wild-type meristems as determined by Student´s t test (*P < 0.02 and **P < 0.001, respectively). [See online article for color version of this figure.]

Figure 7.

Auxin Regulates SKP2A and DPB Interaction. (A) Recombinant MBP-SKP2A or MBP-SKP2A[L128S; S151A] proteins were incubated with GST-DPB bound to beads. When indicated, different concentrations of IAA were added during the incubation and the washing steps. The pulled-down proteins were analyzed by immunoblotting using purified IgGs against the SKP2A protein. The image presented is from one representative experiment of three. (B) Recombinant MBP-SKP2A or MBP-SKP2A[L128S; S151A] proteins were incubated with GST bound to beads. When indicated, 1 μM IAA was added during the incubation and the washing steps. The pulled-down proteins were analyzed by immunoblotting using purified IgGs against the SKP2A protein. (C) The bands detected in (A) and (B) were quantified using a Bio-Rad molecular imager in optical density units per square millimeter (OD/mm²). Each value is the mean of the three different experiments, and the error bars correspond to the sd. Numbers shown in the base correlate with the different bands labeled in (A) and (B).

Figure 8.

Mutation in SKP2A Enhances tir1 Auxin Resistance. (A) Wild-type (wt) and skp2a, tir1-1, and tir1-1 skp2a seedlings were grown in MS plates for 5 d. Afterwards, seedlings were transferred to a MS medium or MS medium containing different concentrations of 2,4-D. After 3 d, root elongation of at least 50 seedlings from two independent experiments was measured. The values are represented as the percentage of root growth inhibition. **P < 0.0001, statistically significant different for skp2a compared with the wild type as determined by Student’s t test. *P < 0.0005, statistically significant different for tir1-1 skp2a compared with tir1-1 as determined by Student’s t test. (B) Arabidopsis wild-type, MYC-SKP2A^OE, tir1-1, and tir1-1/MYC-SKP2A^OE seedlings were grown for 5 d in MS medium and then 1 extra day in MS. Afterwards, seedlings were collected and incubated for 24 h in a solution of chloral hydrate:glycerol:water (80:10:10) in ice. Root lengths were measured and the number of nonemerged LRP was counted using a Leica 2000 microscope. Error bars represent se (n ≥ 30). *P < 0.01, statistically significant difference for tir1-1 compared with wild-type values as determined by Student’s t test.**P < 0.0001, statistically significant difference for tir1-1/MYC-SKP2A^OE compared with tir1-1 values as determined by Student’s t test. (C) Arabidopsis wild-type, MYC-SKP2A^OE, tir1-1, and tir1-1/MYC-SKP2A^OE seedlings were grown for 4 d in MS medium and then 1 extra day in MS supplemented with 50 nM 2,4-D. Afterwards, seedlings were treated as in (A), and root lengths were measured and the number of nonemerged LRP was counted. Error bars represent se (n ≥ 30). In this case, statistically significant differences for tir1-1/MYC-SKP2A^OE compared with tir1-1 were not found. **P < 0.0001, statistically significant difference for MYC-SKP2A^OE compared with wild-type values as determined by Student’s t test. (D) Root length of 5-d-old seedlings grown in MS medium (gray bars) or 4 d in MS and 1 d in MS plus 50 nM 2,4-D (black bars). Error bars represent se (n ≥ 30).