PLANT U-BOX PROTEIN10 Regulates MYC2 Stability in Arabidopsis

Abstract

MYC2 is an important regulator for jasmonic acid (JA) signaling, but little is known about its posttranslational regulation. Here, we show that the MYC2 C-terminal region interacted with the PLANT U-BOX PROTEIN10 (PUB10) armadillo repeats in vitro. MYC2 was efficiently polyubiquitinated by PUB10 with UBC8 as an E2 enzyme and the conserved C249 in PUB10 was required for activity. The inactive PUB10(C249A) mutant protein retained its ability to heterodimerize with PUB10, thus blocking PUB10 E3 activity as a dominant-negative mutant. Both MYC2 and PUB10 were nucleus localized and coimmunoprecipitation experiments confirmed their interaction in vivo. Although unstable in the wild type, MYC2 stability was enhanced in pub10, suggesting destabilization by PUB10. Moreover, MYC2 half-life was shortened or prolonged by induced expression of PUB10 or the dominant-negative PUB10(C249A) mutant, respectively. Root growth of pub10 seedlings phenocopied 35S:MYC2 seedlings and was hypersensitive to methyl jasmonate, whereas 35S:PUB10 and jin1-9 (myc2) seedlings were hyposensitive. In addition, the root phenotype conferred by MYC2 overexpression in double transgenic plants was reversed or enhanced by induced expression of PUB10 or PUB10(C249A), respectively. Similar results were obtained with three other JA-regulated genes, TAT, JR2, and PDF1.2. Collectively, our results show that MYC2 is targeted by PUB10 for degradation during JA responses.

Figure 1.

PUB10 Self-Associates and Interacts with MYC2, MYC3, and MYC4 through Its ARM Repeats in Vitro. (A) Schematic diagrams of full-length PUB10, MYC2, and deletion derivatives used for in vitro pull-down assays. Amino acid alignment of a short sequence of the conserved U-box motif in Arabidopsis PUB10, PUB11, PUB12, and PUB13. Numbers refer to the positions of the first amino acid in the sequence. The asterisk indicates a conserved cysteine residue. In PUB10(C249A), Cys-249 of the PUB10 U-box was changed to Ala. (B) PUB10 interacts with MYC2, MYC3, and MYC4. GST, GST-PUB10, GST-PUB10(C249A), and deletion derivatives were used as baits. MBP, MBP-MYC2, MBP-MYC3, and MBP-MYC4 were used as target proteins. (C) PUB10 interacts with the C-terminal region of MYC2. GST, GST-PUB10, GST-PUB10(C249A), and GSTPUB10-ARM (G-ARM) were used as baits. MBP, MBP-MYC2-N, and MBP-MYC2-C were used as target proteins. (D) Self-association of PUB10. GST, PUB10, GST-PUB10(C249A), and GST-PUB10-ARM (G-ARM) were used as baits. MBP, MBP-PUB10, MBP-PUB10(C249A), and deletion derivatives were used as target proteins. (E) Input amounts of the bait and target proteins. SDS-PAGE gel was stained with Coomassie blue and used to monitor the load of bait and target proteins before pull-down assays. Asterisks indicate the bait and target proteins used in each experiment. For (B) to (D), target proteins (2 μg) were pulled down with the indicated bait proteins (2 μg each) and detected by anti-MBP antibody. aa, amino acids; UND, large U-box N-terminal domain; JID, JAZ-interacting domain; TAD, transactivation domain; G, GST; M, MBP.

Figure 2.

PUB10 Is an E3 Ubiquitin Ligase and Polyubiquitinates MYC2, MYC3, and MYC4 in Vitro. (A) PUB10 E3 activity depends on the integrity of its U-box motif. Epitope-tagged recombinant PUB10, PUB10(C249A), Arabidopsis UBA1, and Arabidopsis UBC8 proteins were purified from E. coli extracts. MBP-PUB10 and MBP-PUB10(C249A) were assayed for E3 activity in the presence or absence of Arabidopsis E1 (His-tagged UBA1) and Arabidopsis E2 (His-tagged UBC8). Polyubiquitinated PUB10 was detected by anti-MBP antibody. (B) Excess PUB10(C249A) protein functions as a dominant negative mutant by blocking PUB10 E3 activity. Numbers indicate the relative amounts of proteins in the reaction mixture, where 1 represents 100 ng of MBP, MBP-PUB10-myc, and MBP-PUB10(C249A)-HA. Polyubiquitinated PUB10 was detected by anti-myc antibody. (C) MYC2, MYC3, and MYC4 are substrates of PUB10 E3 ligase. Polyubiquitination of GST-MYC2, GST-MYC3, and GST-MYC4 was assayed in the presence of UBA1, UBC8, PUB10, and ubiquitin. MBP-PUB10(C249A) mutant protein and MBP-PUB23 were used as negative controls. Polyubiquitinated MYC2, MYC3, and MYC4 were detected by anti-GST antibody. Asterisks indicate the size of the unmodified substrates.

Figure 3.

PUB10 Colocalizes with MYC2, MYC3, and MYC4 and PUB10 Interacts with MYC2 in Vivo. (A) Colocalization of PUB10-YFP and MYC2-CFP in the nucleus. Fluorescence fusion genes, 35:PUB10-YFP and 35S:MYC2-CFP, were transiently expressed in N. benthamiana leaves in the presence of 50 µM MG132 (n = 6, biological replicates). (B) Colocalization of PUB10-CFP, MYC3-YFP, and MYC4-YFP in the nucleus. Fluorescence fusion genes, 35:PUB10-CFP, 35S:MYC3-YFP and 35S:MYC4-YFP, were transiently expressed in N. benthamiana leaves in the presence of 50 µM MG132. For (A) and (B), confocal images were taken 2 d after infiltration. Colocalization is shown by merging YFP, CFP, and differential interference contrast (DIC) images (Merge). Arrows indicate nuclei. Bars = 20 µm. (C) Coimmunoprecipitation of PUB10 with MYC2 in Arabidopsis. Two-week-old double transgenic Arabidopsis seedlings carrying XVE:HA-PUB10/35S:myc-MYC2 (line #8) were treated for 16 h with 50 μM MG132 in the absence or presence of 25 µM β-estradiol (β-est). (D) Coimmunoprecipitation of PUB10(C249A) with MYC2 in Arabidopsis. Two-week-old double transgenic Arabidopsis seedlings carrying XVE:HA-PUB10(C249A)/35S:myc-MYC2 (line #5) were treated for 16 h with 50 µM MG132 in the absence or presence of 25 µM β-estradiol. For (C) and (D), extracts were immunoprecipitated with anti-myc or anti-MBP antibodies. Input proteins and the immunoprecipitates were analyzed by immunoblots using anti-HA and anti-myc antibodies. Input refers to the starting protein amounts in extracts used for IP reactions.

Figure 4.

PUB10 Reduces Its Own Half-Life through Autoubiquitination. (A) Steady state level of overexpressed PUB10 and PUB10(C249A). Ten-day-old seedlings of 35S:PUB10-myc (line #1 and #4) and 35S:PUB10(C249A)-myc (line #3 and #6) were incubated in liquid MS medium with or without 50 μM MG132 for 16 h. Protein levels were detected by anti-myc antibody. (B) Degradation rate of overexpressed PUB10 and PUB10(C249A). Ten-day-old seedlings of 35S:PUB10-myc (line #4) and 35S:PUB10(C249A)-myc (line #6) were incubated in liquid MS medium with 50 μM MG132 for 16 h and washed five times before being transferred to liquid MS medium with 200 μM cycloheximide (CHX). Proteins were extracted at the indicated time points and detected by anti-myc antibody. (C) Degradation rate of induced PUB10 and PUB10(C249A). Ten-day-old seedlings of XVE:HA-PUB10 (line #13) and XVE:HA-PUB10(C249A) (line #3) were incubated in liquid MS medium with 50 μM MG132 and 25 μM β-estradiol for 16 h and washed five times before being transferred to liquid MS medium with 200 μM cycloheximide. Proteins were extracted at the indicated time points and detected by anti-HA antibody. For (A) to (C), α-tubulin levels as detected by anti-α-tubulin antibody were used as loading controls.

Figure 5.

MYC2 Is Stabilized in pub10 Mutant. (A) Real-time RT-PCR of MYC2-GFP and PUB10 transcript levels in 35S:MYC2-GFP/WT (line #2) and 35S:MYC2-GFP/pub10 (line #6) plants. Bars represent average values ± sd (n = 3 biological replicates). (B) Degradation rate of overexpressed MYC2-GFP in the wild type and pub10 mutant. Ten-day-old seedlings of 35S:MYC2-GFP/WT (line #2) and 35S:MYC2-GFP/pub10 (line #6) were incubated in liquid MS medium with 50 µM MG132 for 16 h and washed five times before being transferred to liquid MS medium with 200 µM cycloheximide. Proteins were extracted at the indicated time points and detected by anti-GFP antibody. β-Tubulin levels as detected by anti-β-tubulin antibody were used as loading controls.

Figure 6.

Wild-Type PUB10 Destabilizes MYC2, Whereas Dominant-Negative PUB10(C249A) Mutant Renders MYC2 More Stable in Vivo. Ten-day-old double transgenic Arabidopsis seedlings carrying XVE:HA-PUB10/35S:myc-MYC2 (line #8) (A) or XVE:HA-PUB10(C249A)/35S:myc-MYC2 (line #5) (B) were treated with 50 μM MG132 alone or 25 µM β-estradiol (β-est) plus 50 µM MG132 for 16 h, washed five times, and then transferred to MS medium with 200 µM cycloheximide (CHX). Proteins were extracted at the indicated time points and detected by anti-HA and anti-myc antibodies. For (A) and (B), α-tubulin levels as detected by anti-α-tubulin antibody were used as loading controls.

Figure 7.

The pub10 Mutant and PUB10 Overexpression Line Phenocopy MYC2 Overexpression Line and jin1 Mutant, Respectively, in MeJA Response. (A) Primary root growth of the wild type (Col-0), pub10, two independent lines of 35S:PUB10-myc (PUB10ox, line #1 and #4), jin1-9, and 35S:MYC2-GFP (MYC2ox, line #2) on MS medium with or without 10 μM MeJA for 10 d. Bars = 5 mm. (B) Quantitative effects of MeJA on root growth of various genotypes in (A). Average values of primary root length are given. The root length of each genotype in MS medium alone is designated as 100%. The relative root length with MeJA is shown as the percentage of that without MeJA. Bars represent average values ± sd of more than 50 seedlings of each genotype. Asterisks indicate significant change of root length in each genotype compared with that in Col-0. *P < 0.05; two-tailed t test. (C) Real-time PCR analysis of JA-responsive gene expression of TAT, JR2, and PDF1.2 in the absence or presence of 50 μM MeJA for 12 h. Transcript levels were normalized to ACT2 expression. Bars represent average ± sd (n = 3 biological replicates). Asterisks indicate statistically significant differences compared with the wild type (Col-0). *P < 0.05, **P < 0.01, and ***P < 0.001; two-tailed t test.

Figure 8.

Wild-Type PUB10 Induction Reverses the Root and Gene Expression Phenotype of MYC2 Overexpression Line in the Presence of MeJA. (A) Primary root growth of the wild type (Col-0), two independent lines of XVE:HA-PUB10/35S:myc-MYC2 (P10M2, line #8 and #10), and 35S:MYC2-GFP (MYC2ox line #2) on control medium (MS), 1 μM MeJA alone (MeJA), 10 μM β-estradiol alone (β-est), and 1 μM MeJA plus 10 μM β-estradiol (MeJA+β-est) for 15 d. Bars = 5 mm. (B) Quantitative effect of MeJA on root growth of various genotypes in (A). Bars represent average ± sd of more than 50 seedlings of each genotype. *P < 0.05 and **P < 0.01; two-tailed t test. (C) Real-time PCR analysis of JA-responsive gene expression of TAT and PDF1.2 in (A) and MYC2 expression in all genotypes grown on MS. Transcript levels were normalized to ACT2 expression. Bars represent average ± sd (n = 3 biological replicates). Asterisks indicate statistically significant differences compared with the wild type (Col-0). *P < 0.05 and **P < 0.01; two-tailed t test. For (B) and (C), P10M2 is XVE:HA-PUB10/35S:myc-MYC2 and MYC2ox is 35S:MYC2-GFP.

Figure 9.

PUB10(C249A) Induction Enhances the Root and Gene Expression Phenotype of MYC2 Overexpression Line in MeJA. (A) Primary root growth of the wild type (Col-0), two independent lines of XVE:HA-PUB10(C249A)/35S:myc-MYC2 (mP10M2, line #5 and #15), and 35S:MYC2-GFP (MYC2ox line #2) on control medium (MS), 1 μM MeJA alone (MeJA), 10 μM β-estradiol alone (β-est), and 1 μM MeJA plus 10 μM β-estradiol (MeJA+β-est) for 15 d. Bars = 5 mm. (B) Quantitative effects of MeJA on root length of various genotypes in (A). Bars represent average ± sd of more than 50 seedlings of each genotype. *P < 0.05 and **P < 0.01; two-tailed t test. (C) Real-time PCR analysis of JA-responsive gene expression of TAT and PDF1.2 in (A) and MYC2 expression in all genotypes grown on MS. Transcript levels were normalized to ACT2 expression. Bars represent average ± sd (n = 3 biological replicates). Asterisks indicate statistically significant differences compared with the wild type (Col-0). *P < 0.05 and **P < 0.01; two-tailed t test. For (B) and (C), mP10M2 is XVE:HA-PUB10(C249A)/35S:myc-MYC2 and MYC2ox is 35S:MYC2-GFP.