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. 2012 Feb;24(2):536-50.
doi: 10.1105/tpc.111.093005. Epub 2012 Feb 10.

JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis

Christine Shyu  1 , Pablo Figueroa, Cody L Depew, Thomas F Cooke, Laura B Sheard, Javier E Moreno, Leron Katsir, Ning Zheng, John Browse, Gregg A Howe
Affiliations

JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis

Christine Shyu et al. Plant Cell. 2012 Feb.

Abstract

The lipid-derived hormone jasmonoyl-L-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxL-type EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif-containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors.

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Figures

Figure 1.
Figure 1.
JAZ Proteins Differentially Interact with COI1 in the Presence of JA-Ile. (A) JA-Ile differentially promotes COI1 interaction with various JAZ proteins. Pull-down assays were performed using crude leaf extracts from 35S:COI1-Myc transgenic plants and purified recombinant JAZ-His proteins. Reactions were supplemented (+) with 1 μM (3R,7S)-JA-Ile or an equivalent volume of assay buffer (−). Protein bound to JAZ-His was separated by SDS-PAGE and analyzed by immunoblotting with anti-Myc antibody for the presence of COI1-Myc. The blotted membrane was stained with Coomassie blue to visualize the amount of JAZ-His loaded. (B) JAZ8 does not associate with COI1 in the presence of JA-Ile. Pull-down assays with purified JAZ8-His and JAZ10.1-His were performed as described in (A). Reaction mixtures were supplemented with the indicated concentration of (3R,7S)-JA-Ile. (C) JAZ8 weakly associates with COI1 in the presence of coronatine. Pull-down assays were performed as described in (A). Reactions were supplemented with the indicated concentration of coronatine (COR). (D) Saturation binding of 3H-labeled coronatine to COI1-JAZ8 (circle) and COI1-JAZ10.1 (triangle) complexes. Data show the mean ± se of two replicates performed in duplicate. CPM, counts per minute.
Figure 2.
Figure 2.
Overexpression of JAZ8 Results in Decreased Sensitivity to JA. (A) Root growth inhibition assay of wild-type (open bars) and 35S:JAZ8 (closed bars) seedlings grown on MS media containing the indicated concentration of MeJA. Root length measurements were made 6 d after seed germination. Data show the mean ± se (n > 20 seedlings per genotype for each concentration of MeJA except 100 μM, in which n = 6). Asterisks denote statistically significant differences (P < 0.05, Student’s t test) between the two plant genotypes at the indicated concentration of MeJA. (B) JA-responsive gene expression in wild-type (WT) and 35S:JAZ8 (JAZ8) seedlings. Seedlings were grown in liquid MS medium for 9 d and then treated for 2 h with the indicated concentration of MeJA or a mock control (0). Total RNA isolated from the treated seedlings was subjected to RNA gel blot analysis with the indicated probes. A JAZ8 probe was used to verify overexpression of JAZ8 in the transgenic line, and an ACTIN8 (ACT8) probe was used as a loading control. (C) Weight of S. exigua larvae reared on wild-type (open bars) or 35S:JAZ8 (closed bars) plants for 9 or 14 d. Data show the mean ± se (n = 56 to 65 larvae per plant genotype). The experiment was repeated four times with similar results. Data from a representative experiment are shown. (D) Photograph of representative S. exigua larvae recovered from wild-type and 35S:JAZ8 (JAZ8) plants after 14 d of feeding. [See online article for color version of this figure.]
Figure 3.
Figure 3.
JAZ8 Is Resistant to JA-Mediated Degradation in Vivo. (A) Differential stability of JAZ8 and JAZ10.1 in the presence of JA. Transgenic seedlings (7 d old) expressing JAZ8-YFP or JAZ10.1-YFP fusion proteins were treated either with water (mock) or with 50 μM MeJA for the indicated amount of time prior to imaging of roots by confocal fluorescence microscopy. Image settings used for seedlings of the same genotype at different time points were identical. (B) Coronatine stimulates JAZ8 turnover in a 26S proteasome-dependent manner. Transgenic seedlings (4 d old) expressing JAZ8-YFP were pretreated with water or the 26S proteasome inhibitor MG132 (100 μM) for 80 min, at which time seedlings were treated with coronatine (1 μM) for an additional 10 or 30 min. Seedlings treated with both coronatine and MG132 were imaged at the 30 min time point. YFP signal in root tissue was visualized by confocal microscopy. Microscope settings were identical for all images. [See online article for color version of this figure.]
Figure 4.
Figure 4.
A Jas Domain Swap Converts JAZ8 to a COI1-Interacting Protein That Does Not Exert Dominant Repression of JA Responses. (A) Amino acid alignment of the Jas motif in 12 Arabidopsis full-length JAZ proteins. Structural features within the Jas motif that physically associate with COI1 and JA-Ile (Sheard et al., 2010) are indicated. (B) Schematic diagram of chimeric proteins constructed by swapping of the Jas motif. Sequence regions derived from JAZ8 and JAZ10.1 are shown in gray and white, respectively. (C) Pull-down assays performed with parental (JAZ8 and JAZ10.1) and chimeric (JAZ10-Jas8, J10-J8; JAZ8-Jas10, J8-J10) proteins in the presence (+) or absence (−) of 2.5 μM JA-Ile. (D) JA-mediated root growth inhibition in wild-type (WT) and transgenic seedlings (35S:JAZ8, JAZ8; 35S:JAZ10.1, JAZ10.1; 35S:JAZ8-Jas10, J8-J10; and 35S:JAZ10-Jas8, J10-J8). Seedlings were grown for 6 d on MS medium supplemented or not supplemented with 20 μM MeJA. The root length ratio was calculated by dividing the average the root length of seedlings grown on MeJA-containing medium by the average root length of seedlings of the same genotype grown in the absence of MeJA. Data points show the mean ± se (n = 18 to 24 seedlings per data point). Asterisks denote significant differences (P < 0.05, Student’s t test) in comparisons between the indicated transgenic line and the wild type . [See online article for color version of this figure.]
Figure 5.
Figure 5.
The Canonical LPIAR Degron Promotes JAZ8 Binding to COI1, Destabilizes JAZ8, and Restores JA Responsiveness. (A) JAZ8LPIAR interacts with COI1 in a JA-Ile–dependent manner. Pull-down reactions were performed in the presence (+) or absence (−) of 2.5 μM JA-Ile as described in the legend for Figure 1A. Protein bound to the indicted JAZ-His fusion protein was separated by SDS-PAGE and analyzed by immunoblotting with anti-Myc antibody for the presence of COI1-Myc. (B) Transgenic lines overexpressing JAZ8LPIAR are sensitive to JA. Root growth assays were performed with wild-type (WT), 35S:JAZ10 (JAZ10.1), 35S:JAZ8 (JAZ8), and 35S:JAZ8LPIAR (JAZ8LPIAR) seedlings. Data show the mean ± se for each genotype (n = 20). Asterisk denotes significant differences (P < 0.05, Student’s t test) in comparisons between the indicated transgenic line and the wild type. (C) JAZ8LPIAR-YFP is degraded in vivo in response to JA treatment. Six-day-old transgenic seedlings expressing JAZ8-YFP (left) or JAZ8LPIAR-YFP (right) were pretreated with water or the 26S proteasome inhibitor MG132 (100 μM) for 75 min, at which time seedlings were treated with either MeJA (+MeJA, 50 μM) or water (mock control) for an additional 15 min. YFP signal in root tissue was visualized by fluorescence microscopy. Microscope settings were identical for all images. Bars = 50 μm. [See online article for color version of this figure.]
Figure 6.
Figure 6.
The EAR Motif of JAZ8 Is Required for Repression of JA Responses. (A) Consensus sequence of the Jas motif in JAZ8-like (top) and JAZ10-like (bottom) proteins from various plant species (see Supplemental Table 2 online). (B) Consensus sequence of the N-terminal 15 amino acids from JAZ8-like proteins from diverse plant species (see Supplemental Table 2 online). (C) Comparison of JA-mediated root growth inhibition in 35S:JAZ8 and 35S:JAZ8ΔEAR seedlings. Wild-type (WT), 35S:JAZ8 (line #24), and 35S:JAZ8ΔEAR (five independent homozygous lines) were grown for 8 d on medium supplemented or not supplemented with 20 μM MeJA. Root length ratios were calculated as described in the legend of Figure 4D. Data show the mean ± se for each genotype (n > 14 seedlings per genotype). Asterisks denote a significant difference (P < 0.05, Student’s t test) in comparisons between transgenic and wild-type seedlings. The mean root length ratio calculated for seedlings (n = 92) from all five independent 35S:JAZ8ΔEAR lines was not significantly different (P value = 0.068, Student’s t test) from that of the wild type. [See online article for color version of this figure.]
Figure 7.
Figure 7.
The EAR Motif Is Required for Transcriptional Repression by JAZ8. (A) Carrot protoplasts were transfected with the GUS reporter [35S(-46)LexA(2x)-Gal4(2x):GUS] alone or in combination with the indicated effector and LD-VP16 constructs as described in the text. GUS activities are expressed relative to the sample in which LD-VP16 was cotransfected with the GD effector (Gal4 DNA binding domain alone; set to 100%). GUS activities were normalized by cotransfection with a 35S:LUC construct. Asterisks denote statistically significant differences (P < 0.01, Student’s t test) in comparisons to the LD-VP16 + GD activation control. Data show the mean ± sd of three replicate assays. (B) Carrot protoplasts were cotransfected with the GUS reporter gene described in (A), LD-VP16, and the indicated effector construct. L9A, E10A, L11A, R12A, and L13A correspond to single Ala substitution mutations in the EAR motif of JAZ8-N (black boxes). The F14A mutation is located C-terminal to the EAR motif and serves as a control. GUS activities are expressed as described in (A). Asterisks denote statistically significant differences (P < 0.01, Student’s t test) in comparisons to the unmodified GD-JAZ8-N construct. Data points show the mean ± sd of three replicate assays. WT, wild type.
Figure 8.
Figure 8.
Protein–Protein Interaction Domains in JAZ8. (A) Y2H assay of JAZ8 and JAZ8 deletion derivatives with MYC2 and JAZ3. Yeast strains expressing both the bait (JAZ8) and prey (MYC2 or JAZ3) proteins were plated on media containing X-Gal. LacZ-mediated blue-color formation is indicative of protein–protein interaction. JAZ8 proteins were coexpressed with an empty prey vector (pB42AD) as a negative control. Photographic images of yeast cells were taken after 48 h of incubation at 30°C. (B) Y2H analysis of JAZ8 deletion proteins with MYC2, JAZ1, NINJA, and TPL. Yeast strains expressing both the bait (JAZ8 or JAZ8 deletion) and prey (MYC2, JAZ1, NINJA, or TPL) proteins were tested as described in (A). Empty bait (pGILDA) and prey (pB42AD) vectors were used as negative controls. As a positive control for NINJA interaction, yeast cells were cotransformed with pB42AD-NINJA and pGILDA-JAZ1 (bottom right). (C) JAZ8 interacts weakly with NINJA in vitro. Purified JAZ-His proteins (fused to MBP) were incubated with crude extract from yeast cells expressing HA-tagged derivatives of NINJA (N), JAZ1 (J), or an empty vector control (E). MBP-His was used as a control for specificity. Purified protein complexes were separated by SDS-PAGE and probed with an anti-HA antibody for the presence of HA-tagged NINJA (HA-NINJA) or JAZ1 (HA-JAZ1). The bottom panel shows a Coomassie blue (CB)–stained gel to visualize the amount of protein loaded. CE, crude yeast extracts that were used as an input control. Protein molecular weight markers were run in the fourth lane from the left.
Figure 9.
Figure 9.
The ZIM Domain Is Not Required for JAZ8-Mediated Repression of JA-Induced Root Growth Inhibition. (A) 35S:JAZ8ΔZIM plants are insensitive to JA-mediated root growth inhibition. Assays were performed with wild-type (WT), 35S:JAZ8 (JAZ8; line #24), and eight independent 35S:JAZ8ΔZIM (JAZ8ΔZIM; gray bars) lines that are homozygous for the transgene. Root length ratios were calculated as described in the legend of Figure 4D. Data show the mean ± se for each genotype (n = 12 to 24 seedlings per genotype). The mean root length ratio of seedlings (n = 127) from all eight 35S:JAZ8ΔZIM lines was not significantly different than that of 35S:JAZ8 seedlings (P value = 0.69, Student’s t test). Means with different italicized letters are significantly different at P < 0.001. (B) JA-mediated root growth phenotype of 35S:JAZ8ΔEZ lines expressing a JAZ8 derivative lacking both the EAR and ZIM motifs. Assays were performed as described in (A) and included 11 independent 35S:JAZ8ΔEZ T2 lines (JAZ8ΔEZ; gray bars). Data show the mean ± se for each genotype (n = 13 to 22 seedlings per genotype). The mean root length ratio of seedlings (n = 212) from all 11 35S:JAZ8ΔEZ lines was not significantly different than that of wild-type seedlings (P value = 0.62, Student’s t test). Means with different italicized letters are significantly different at P < 0.001.
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