Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling

Abstract

Jasmonates (JAs) are fatty acid-derived signaling compounds that control diverse aspects of plant growth, development and immunity. The F-box protein COI1 functions both as a receptor for jasmonoyl-l-isoleucine (JA-Ile) and as the component of an E3-ubiquitin ligase complex (SCF(COI1) ) that targets JAZ transcriptional regulators for degradation. A key feature of JAZ proteins is the C-terminal Jas motif that mediates the JA-Ile-dependent interaction with COI1. Here, we show that most JAZ genes from evolutionarily diverse plants contain a conserved intron that splits the Jas motif into 20 N-terminal and seven C-terminal (X(5) PY) amino acid submotifs. In most members of the Arabidopsis JAZ family, alternative splicing events involving retention of this intron generate proteins that are truncated before the X(5) PY sequence. In vitro pull-down and yeast two-hybrid assays indicate that these splice variants have reduced capacity to form stable complexes with COI1 in the presence of the bioactive stereoisomer of the hormone (3R,7S)-JA-Ile. cDNA overexpression studies showed that some, but not all, truncated splice variants are dominant repressors of JA signaling. We also show that strong constitutive expression of an intron-containing JAZ10 genomic clone is sufficient to repress JA responses. These findings provide evidence for functional differences between JAZ isoforms, and establish a direct link between the alternative splicing of JAZ pre-mRNA and the dominant repression of JA signal output. We propose that production of dominant JAZ repressors by alternative splicing reduces the negative consequences associated with inappropriate or hyperactivation of the JA response pathway.

Figure 1

Bipartite organization of the Jas domain-coding region in Arabidopsis JAZ genes. (a) Left, Phylogenetic tree constructed from the amino acid sequence of 12 full-length JAZs, showing four (A–D) subclades of proteins. Right, intron/exon organization of the corresponding genes. Thick green and blue bars indicate coding regions and non-coding untranslated regions in exons, respectively. The Jas motif coding-region is depicted in red. Introns are depicted by a thin black horizontal line or, in the case of the Jas intron, a red line. (b) Consensus sequence of the Jas motif in 12 Arabidopsis JAZ. The arrow indicates the location of homologous introns. The Jas intron is positioned invariably in phase 2 of the codon specifying Arg at position 20 of the motif. (c) Sequence of the Jas exon/intron junction in nine Arabidopsis JAZ genes that contain the intron. Exon and intron sequences are highlighted in black and gray, respectively, together with the predicted amino acid sequence. In the case of JAZ2 JAZ3 JAZ4 JAZ6 JAZ10, and JAZ11, an in-frame stop codon (red) is encountered close to the 5' end of the Jas intron, if the intron is retained.

Figure 2

RT-PCR-based detection of Jas intron retention in various JAZ transcripts. (a) Primer design strategy showing JAZ2 as an example. Structural elements of the gene include the Jas motif coding-region (black box) and Jas intron (black line). Light grey and dark grey bars indicate coding regions and non-coding untranslated regions in exons, respectively. Arrows indicate forward (FP) and reverse (RP) primers used for RT-PCR assays designed to amplify transcripts in which the Jas intron is spliced out or retained. The RP1 primer spans the exon/exon junction and thus hybridizes to transcripts in which the Jas intron is removed. The RP2 primer hybridizes to a gene-specific sequence near the 5’ end of the Jas intron, thereby amplifying transcripts in which the 5’ splice site of the intron is retained. Primer sequences are listed in Table S2. (b) RNA extracted from MeJA-treated Arabidopsis seedlings was used as a template for RT-PCR (see Experimental Procedures). Gene-specific FP-RP1 primer sets amplified fully-spliced transcripts (upper panel), whereas FP-RP2 primer sets amplified transcripts in which the 5’ end of the Jas intron was retained (lower panel). PCR-amplified products were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. For those RT-PCR assays yielding more than one main product, asterisks denote the transcript containing the Jas intron but not other introns, as determined by DNA sequencing of cloned products.

Figure 3

ΔPY JAZ isoforms differentially associate with COI1 in the presence of receptor ligands. (a) Hormone-dependent interaction of JAZ10 splice variants with COI1. Pull-down assays were performed using recombinant JAZ10 splice variants and crude leaf extracts from 35S-COI1-Myc leaves as a source of COI1. JAZ10.4 lacks the entire Jas motif. Reaction mixtures were supplemented with the indicated concentration of (3R,7S)-JA-Ile, or an equivalent volume of assay buffer (“0”). Protein bound to JAZ10-His was separated by SDS-PAGE and analyzed by immunoblotting with anti-Myc antibody for the presence of COI1-Myc. As a loading control, the immunoblotted membrane was stained with Coomassie blue to detect JAZ-His. (b) Effect of alternative splicing of the Jas intron on the sequence of the Jas motif in full-length (JAZ10.1, JAZ2.1, and JAZ3.1) and ΔPY (JAZ10.3, JAZ2.2, JAZ3.4) isoforms of JAZ10, JAZ2, and JAZ3. (c, d) Hormone-dependent interaction of full-length (JAZ2.1 and JAZ3.1) and ΔPY (JAZ2.2 and JAZ3.4) splice variants with COI1. Experiments were performed as described in panel a. (e) JA-Ile-dependent interaction of JAZ2 (upper) and JAZ3 (lower) splice variants with COI1 in the Y2H system. Yeast strains expressing both COI1 (as a DNA binding domain fusion) and the indicated JAZ splice variant (as an activation domain fusion) were plated on media containing X-gal and the indicated concentration (µM) of (3R,7S)-JA-Ile. LacZ-mediated blue-color formation is indicative of the strength of the COI1-JAZ interaction. (f) Coronatine-dependent interaction of JAZ2 (upper) and JAZ3 (lower) splice variants with COI1 in the Y2H system. Experiments were performed as described in panel e, except that coronatine (COR) was included at the indicated concentration (µM) in the yeast medium.

Figure 4

The PY submotif of JAZ2.1 and JAZ10.1 is not required for COI1 interaction. (a, b) Site-directed mutagenesis was used to change the PY diamino acid sequence in the Jas motif of JAZ2.1 (position 227–228) and JAZ10.1 (position 191–192) to AA. The resulting wild-type (WT) and mutant (PY→AA) proteins were expressed in E. coli as MBP-His fusions. Pull-down reactions containing purified JAZs and protein extract from leaves of the 35S-AtCOI1-Myc transgenic line were supplemented (lanes labeled “+”) with 2.5 µM (3R,7S)-JA-Ile (panel a) or 0.1 µM coronatine (panel b). Control reactions contained an equivalent amount of binding buffer (lanes labeled “−”). Reactions were incubated and processed as described in the legend for Figure 3a. The Coomassie blue-stained gel shows the input of MBP-JAZ-His fusion protein.

Figure 5

Ectopic expression of JAZ2.2 attenuates jasmonate responses. (a) Photograph of wild-type (WT), 35S-JAZ2.1, and 35S-JAZ2.2 seedlings grown for 9 d on MS medium containing 50 µM MeJA. Seedlings from two independent lines for each construct are shown. (b) Quantification of MeJA-induced root growth inhibition of seedlings shown in (a). Data show the mean ± SD (n = 15 seedlings per genotype). (c) JAZ2.1 and JAZ2.2 interact with MYC2 in yeast. Yeast strains co-transformed with plasmids encoding MYC2 (as an activation domain fusion; AD) and the indicated JAZ2 splice variant (as a DNA binding domain fusion; BD) were plated on media containing X-gal. LacZ-mediated blue-color formation is indicative of JAZ-MYC2 interaction. Yeast strains expressing BD-JAZ and an empty AD vector (AD-EV) control did not exhibit visible blue-color formation.

Figure 6

Overexpression of a JAZ10 genomic clone attenuates jasmonate signaling. (a) Photograph of wild-type (WT) and transgenic seedlings of the indicated genotype grown for 9 d on MS medium containing 50 µM MeJA. 10.1 10.3, and 10.4 refer to transgenic lines overexpressing cDNAs for JAZ10.1, JAZ10.3, and JAZ10.4, respectively (Chung and Howe, 2009), which were grown together with three independent 35S-JAZ10G lines (10G-2, 10G-4, and 10G-5). (b) Quantification of MeJA-induced root growth inhibition of seedlings shown in (a). Data show the mean ± SD of at least 11 seedlings per genotype. (c) Semi-quantitative RT-PCR analysis of JAZ10 transcripts in 2-week-old 35S-JAZ10G seedlings (line 2) grown on MS medium. Expression of transcripts derived from the 35S-JAZ10G transgene was assessed using primer sets (Table S2) that amplify all three JAZ10 transcripts (top panel), JAZ10.3 only (second panel), or JAZ10.4 only (third panel). PCR reactions contained a forward primer that hybridizes specifically to the 35S-JAZ10G transgene, and were limited to 22 cycles. The experiment was repeated with RNA isolated from two independent sets of 35S-JAZ10G-2 plants (P3 and P4). A primer set that amplifies an actin transcript (lower panel) was used as a control.