The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis

Abstract

Xie and colleagues previously isolated the Arabidopsis COI1 gene that is required for response to jasmonates (JAs), which regulate root growth, pollen fertility, wound healing, and defense against insects and pathogens. In this study, we demonstrate that COI1 associates physically with AtCUL1, AtRbx1, and either of the Arabidopsis Skp1-like proteins ASK1 or ASK2 to assemble ubiquitin-ligase complexes, which we have designated SCF(COI1). COI1(E22A), a single amino acid substitution in the F-box motif of COI1, abolishes the formation of the SCF(COI1) complexes and results in loss of the JA response. AtRbx1 double-stranded RNA-mediated genetic interference reduces AtRbx1 expression and affects JA-inducible gene expression. Furthermore, we show that the AtCUL1 component of SCF(COI1) complexes is modified in planta, where mutations in AXR1 decrease the abundance of the modified AtCUL1 of SCF(COI1) and lead to a reduction in JA response. Finally, we demonstrate that the axr1 and coi1 mutations display a synergistic genetic interaction in the double mutant. These results suggest that the COI1-mediated JA response is dependent on the SCF(COI1) complexes in Arabidopsis and that the AXR1-dependent modification of the AtCUL1 subunit of SCF(COI1) complexes is important for JA signaling.

Figure 1.

COI1 Interacts with ASK1 and ASK2 in the Yeast Two-Hybrid System. COI1 was fused to the LexA DNA binding domain in pLexA and examined for interaction with ASK1 and ASK2 fused in frame to the activating domain in pB42AD. The two-hybrid reporter strain EGY48 coexpressing the indicated fusion proteins was grown on 2% Gal/1% raffinose/SD/-Ura/-His/-Trp/-Leu/X-β-Gal (top). Interactions of the indicated constructs were quantified by routine β-galactosidase assay and are expressed in Miller units (bottom).

Figure 2.

Alignment of Rbx1 Amino Acid Sequences of Arabidopsis, C. elegans, Drosophila, Mouse, and Human. Identical amino acid residues are shaded in black. Dashes denote gaps introduced by the alignment program. Protein names are indicated at left, and amino acid residue positions are indicated at right.

Figure 3.

Protein Gel Blot Analysis of Extracts Prepared from the Indicated Arabidopsis Plants. Blots were probed with polyclonal antisera against ASK1 (A), ASK2 (B), Rbx1 (C), or COI1 (D). The bands corresponding to each protein are indicated. The asterisk indicates the position of a nonspecific immunoreacting band. Molecular mass standards are given in kD (left). WT, wild type.

Figure 4.

Coimmunoprecipitation Assays. (A) The Flag-tagged COI1 forms an SCF complex in Arabidopsis. Protein extracts from coi1-1 (lane 1) and transgenic coi1-1 expressing Flag-tagged COI1 (coi1-1::Flag-COI1; lane 2) were immunoprecipitated with α-Flag antibody (lanes 3 and 4). (B) ASK2 associates physically with COI1 but not with ASK1 in planta. Protein extracts from a control wild-type plant (lane 5) and a transgenic plant expressing the Myc-tagged ASK2 (WT::Myc-ASK2; lane 6) were immunoprecipitated with α-Myc antibody (lanes 7 and 8). (C) COI1_E22A abrogates SCF^COI1. Protein extracts from coi1-1 plants transgenic for vector (coi1-1::Myc; lane 9), Myc-tagged COI1_E22A (coi1-1:: Myc-COI1_E22A; lane 10), or Myc-tagged COI1 (coi1-1::Myc-COI1; lane 11) were immunoprecipitated with α-Myc antibody (lanes 12 to 14). The resulting immunoprecipitates were resolved by SDS-PAGE and detected with the indicated antibodies. Molecular mass standards are given in kD. Arrows in (A) and (C) indicate the modified version of AtCUL1. The asterisk in (B) indicates the position of a nonspecific immunoreacting band. Hc, immunoglobulin heavy chain; IP, immunoprecipitate; Lc, immunoglobulin light chain; WT, wild type.

Figure 5.

COI1_E22A Abolishes JA Response. coi1-1 plants transgenic for Myc-tagged COI1_E22A, Myc-tagged COI1, or vector (see Figure 4C) were examined for JA-inhibitory root growth (top), JA-inducible expression of the defense gene PDF1.2 (middle), and JA-regulated pollen development and plant fertility (bottom). Seedlings were grown on Murashige and Skoog (1962) (MS) medium with (+) or without (−) 10 μM methyl jasmonate (MeJA; top). Total RNA was stained with ethidium bromide as an equal loading control (middle). The white arrow indicates the viable pollen grains in anther, which were stained with 1% fluorescein diacetate (catalog No. F-7378; Sigma) and visualized by fluorescence microscopy. The inflorescence was photographed in 6-week-old soil-grown plants (bottom).

Figure 6.

Analysis of Glucocorticoid-Inducible AtRbx1(RNAi) Mutant Line A2. (A) DEX treatment resulted in a decrease of both AtRbx1 mRNA and AtRbx1 protein in the glucocorticoid-inducible AtRbx1(RNAi) mutant line A2. Arabidopsis plants transgenic for the glucocorticoid-inducible AtRbx1(RNAi) construct (line A2) or vector were grown on MS medium for 3 weeks and treated with 0.5 μM DEX (+) (catalog No. D-1756; Sigma) or with water as a control (−) for 24 h. The plants were harvested for RNA gel blot analysis (top) and protein gel blot analysis (bottom). Total RNAs were stained with ethidium bromide as an equal loading control. The immunoblot was detected with α-actin antibody as a protein loading control. (B) DEX treatment led to a reduction of JA-inducible accumulation of AtVSP and AOS transcripts. Three-week-old Arabidopsis plants were mock treated (−) or treated with 1 μM DEX for 24 h and then followed by mock treatment (−) or treatment with MeJA (+) for 9 h. The RNA gel blot was hybridized with digoxigenin-labeled AtVSP and AOS probes. Total RNAs were stained with ethidium bromide as an equal loading control. Consistent with the results shown in (A), the blot was probed with digoxigenin-labeled AtRbx1 to demonstrate that the DEX treatment led to a decrease of AtRbx1 expression in these glucocorticoid-inducible AtRbx1(RNAi) mutant plants.

Figure 7.

The Abundance of the Modified AtCUL1 of SCF^COI1 Was Reduced in the axr1-3 Mutant. Protein extracts from axr1-3 (lane 1), axr1-3 expressing Flag-tagged COI1 (axr1-3::Flag-COI1) (lane 2), and wild-type AXR1 plants expressing Flag-tagged COI1 (AXR1::Flag-COI1) (lane 3) were immunoprecipitated with α-Flag antibody (lanes 4 to 6). The α-Flag immune complexes were probed with α-COI1, α-AtCUL1, or α-ASK1 antisera. The arrow indicates the modified version of AtCUL1.

Figure 8.

axr1-3 and axr1-12 Exhibited Reduced JA Response. (A) MeJA dose–response curve of root growth. axr1-3, axr1-12, tir1-1, coi1-1, and wild-type (WT) plants were grown for 1 week on MS medium containing various concentrations of MeJA. Root length of the seedlings grown on MS containing MeJA (0.1, 0.5, 1, 5, 10, 25, 50, or 80 μM) was expressed as a percentage of root length on MS medium. Each data point is the mean of >15 samples. The experiment was repeated four times, and the results were consistent. Error bars represent sd (n > 15). (B) Phenotype of 10-day-old seedlings grown on MS medium or MS with 25 μM MeJA. (C) RNA gel blot analysis of the JA-inducible expression of PDF1.2, Thi2.1, and AtVSP. Ethidium bromide staining of rRNAs shown at bottom indicates the equal loading amount of total RNA on the gel.

Figure 9.

Identification of Leaky Mutant Alleles coi1-2 and coi1-8 of COI1. (A) coi1-2 and coi1-8 exhibit insensitivity to JA-inhibitory root elongation compared with the wild type (WT), but it is less severe than that in coi1-1. (B) coi1-2 and coi1-8 display a partial fertility phenotype, whereas the wild type is fertile and coi1-1 is male sterile. Shown are inflorescences of 8-week-old plants grown in soil under 16-h-light (21–23°C)/8-h-dark (16–19°C) growth conditions. The white arrows indicate fertile siliques, and the red arrows indicate sterile flowers.

Figure 10.

The coi1-2 axr1-3 Double Mutant Exhibited a Severe Defect in JA Response. (A) MeJA dose–response curve of root growth. Root length of 1-week-old seedlings grown on MS medium containing 1, 5, or 25 μM MeJA is expressed as a percentage of root length on MS. Each data point represents the mean of >15 samples. The experiment was repeated three times. Error bars represent sd (n > 15). (B) Phenotype of 8-day-old seedlings grown on MS or MS with 25 μM MeJA. (C) The coi1-2 axr1-3 double mutation resulted in male sterility. Shown are inflorescences of 8-week-old plants. The white arrows indicate fertile siliques, and the red arrows indicate sterile flowers. (D) The coi1-2 axr1-3 double mutation led to a significant reduction of JA-inducible accumulation of AtVSP, PDF1.2, and Thi2.1 transcripts. Ethidium bromide staining of rRNAs shown at bottom indicates the equal loading amount of total RNA on the gel. WT, wild type.