The Plant Cell, Vol. 21: 131–145, January 2009, www.plantcell.

org ã 2009 American Society of Plant Biologists

A Critical Role for the TIFY Motif in Repression of Jasmonate

Signaling by a Stabilized Splice Variant of the JASMONATE
ZIM-Domain Protein JAZ10 in Arabidopsis C W

Hoo Sun Chunga,b and Gregg A. Howea,b,1

a Department of Energy–Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824
b Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824

JASMONATE ZIM-domain (JAZ) proteins act as repressors of jasmonate (JA) signaling. Perception of bioactive JAs by the
F-box protein CORONATINE INSENSITIVE1 (COI1) causes degradation of JAZs via the ubiquitin-proteasome pathway, which in
turn activates the expression of genes involved in plant growth, development, and defense. JAZ proteins contain two highly
conserved sequence regions: the Jas domain that interacts with COI1 to destabilize the repressor and the ZIM domain of
unknown function. Here, we show that the conserved TIFY motif (TIFF/YXG) within the ZIM domain mediates homo- and
heteromeric interactions between most Arabidopsis thaliana JAZs. We have also identified an alternatively spliced form
(JAZ10.4) of JAZ10 that lacks the Jas domain and, as a consequence, is highly resistant to JA-induced degradation. Strong
JA-insensitive phenotypes conferred by overexpression of JAZ10.4 were suppressed by mutations in the TIFY motif that block
JAZ10.4–JAZ interactions. We conclude that JAZ10.4 functions to attenuate signal output in the presence of JA and further
suggest that the dominant-negative action of this splice variant involves protein–protein interaction through the ZIM/TIFY
domain. The ability of JAZ10.4 to interact with MYC2 is consistent with a model in which a JAZ10.4-containing protein complex
directly represses the activity of transcription factors that promote expression of JA response genes.

INTRODUCTION JA exerts its many effects through large-scale changes in gene

expression (Schenk et al., 2000; Sasaki et al., 2001; Goossens
The lipid-derived hormone jasmonate (JA) regulates many phys- et al., 2003; Reymond et al., 2004; Devoto et al., 2005; Suzuki
iological activities during the plant life cycle. JA is well known for et al., 2005; Sasaki-Sekimoto et al., 2005; Uppalapati et al., 2005;
its ability to promote plant defense responses against insect Mandaokar et al., 2006; Schmidt and Baldwin, 2006). Activation
herbivores, necrotrophic pathogens, and various abiotic of JA response genes is mediated in part by the basic helix-loop-
stresses as well (Pozo et al., 2004; Glazebrook, 2005; Browse helix transcription factor MYC2 (also known as JIN1) (Abe et al.,
and Howe, 2008; Howe and Jander, 2008). JA also plays an 2003; Lorenzo et al., 2004; Laurie-Berry et al., 2006; Chini et al.,
important role in normal plant growth and development, includ- 2007; Dombrecht et al., 2007; Takahashi et al., 2007). Current
ing cell division, cell fate determination, photomorphogenesis, models of JA signaling indicate that, in the absence of JA,
and sexual reproduction (McConn and Browse, 1996; Li et al., MYC2’s function as a transcriptional activator is repressed by
2004; Sineshchekov et al., 2004; Browse, 2005; Chen et al., members of the JASMONATE ZIM-domain (JAZ) family of pro-
2007; Balbi and Devoto, 2008). An important attribute of JA is its teins that physically interact with MYC2 (Chini et al., 2007;
ability to act both as a potent inhibitor of vegetative growth and Melotto et al., 2008). Elevated levels of JA trigger the release of
as a positive regulator of reproductive and defensive processes MYC2 from this repression by increasing the rate of JAZ turn-
(Yan et al., 2007; Howe and Jander, 2008; Mitra and Baldwin, over. Bioactive JAs promote interaction of JAZ proteins with the
2008; Pauwels et al., 2008). These antagonistic activities of JA CORONATINE INSENSITIVE1 (COI1) component of the ubiquitin
suggest a broader role for the hormone in dealing with the E3 ligase SCFCOI1 (Xie et al., 1998; Thines et al., 2007; Katsir
dilemma of plants to grow or defend (Herms and Mattson, 1992) et al., 2008b; Melotto et al., 2008). This interaction leads to
in rapidly changing environments. ubiquitination and degradation of JAZs by the 26S proteasome.
Ligand binding studies have shown that jasmonoyl-isoleucine
(JA-Ile) and structurally related JA–amino acid conjugates stim-
1 Address
ulate COI1–JAZ interactions through the formation of a stable
correspondence to howeg@msu.edu.
The author responsible for distribution of materials integral to the COI1/JA-Ile/JAZ complex (Katsir et al., 2008b). The bacterial
findings presented in this article in accordance with the policy described toxin coronatine is a structural mimic of JA-Ile and a potent
in the Instructions for Authors (www.plantcell.org) is: Gregg A. Howe agonist of this intracellular receptor system (Katsir et al., 2008b;
(howeg@msu.edu). Melotto et al., 2008).
C
Some figures in this article are displayed in color online but in black
and white in the print edition.
Several lines of evidence indicate that the C-terminal Jas
W
Online version contains Web-only data. domain plays a key role in destabilizing JAZ repressors in
www.plantcell.org/cgi/doi/10.1105/tpc.108.064097 response to increased JA levels. JAZ–b-glucuronidase (GUS)
132 The Plant Cell

(Thines et al., 2007) and JAZ–green fluorescent protein (Chini fail to interact with COI1 but retain the ability to bind MYC2
et al., 2007) fusion proteins are degraded in JA-treated cells, (Melotto et al., 2008). However, this model does not explain the
whereas truncated JAZ proteins (referred to here as JAZDJas) action of JAZDJas proteins that do not interact with MYC2. Chini
that lack the Jas domain are stable in the presence of the et al. (2007) reported that a truncated form of JAZ3 (also known
hormone. Ectopic expression of JAZDJas proteins results in as JAI3) that lacks the Jas domain interacts with COI1 in a JA-
various JA-insensitive phenotypes, including male sterility, re- independent manner but fails to interact with MYC2. These
sistance to JA-mediated inhibition of root growth, reduced workers proposed a poison complex model in which hormone-
expression of JA response genes and secondary metabolites, independent binding of JAZ3DJas (JAI3DC) to COI1 obstructs
susceptibility to insect feeding, and enhanced resistance to access of SCFCOI1 to endogenous JAZs, thereby allowing en-
coronatine-producing strains of Pseudomonas syringae (Chini dogenous JAZs to repress MYC2. Elucidation of how JAZDJas
et al., 2007; Thines et al., 2007; Chung et al., 2008; Melotto et al., proteins repress JA responses is critical for understanding the
2008; Shoji et al., 2008). Biochemical studies have shown that general mechanism of JA signaling.
the Jas domain is sufficient for formation of COI1/JAZ complexes JAZ proteins in Arabidopsis thaliana are encoded by 12 genes,
and associated ligand binding (Katsir et al., 2008b; Melotto et al., designated JAZ1 to JAZ12. The JAZ repressor model predicts
2008). In addition, point mutations in the Jas domain that block that loss-of-function jaz mutations should result in constitutive or
hormone-induced COI1/JAZ interaction confer dominant JA hypersensitive JA responses. Analysis of various jaz null mutants
insensitivity (Melotto et al., 2008). suggests functional redundancy between family members (Chini
A simple hypothesis to explain the dominant action of JAZD et al., 2007; Thines et al., 2007). Interestingly, however, trans-
Jas proteins is that they interact with and repress the activity of genic plants silenced for the expression of JAZ10 (also called
MYC2. This idea can account for the JA-insensitive phenotype of JAS1) were more sensitive than the wild type to JA (Yan et al.,
plants that overexpress JAZ variants (e.g., JAZ1-A205A206) that 2007). These workers identified an alternatively spliced form of

Figure 1. Homo- and Heteromeric Interaction of Arabidopsis JAZ Proteins.

(A) Y2H assay for homomeric JAZ interactions. Yeast strains coexpressing AD and BD fusions to each of the 12 full-length JAZ proteins were plated on
media containing X-gal. Based on the intensity of LacZ-mediated blue-color formation, the strength of each interaction was rated as strong (e.g., JAZ1),
medium (e.g., JAZ5), weak (e.g., JAZ6), or undetectable (e.g., JAZ8). See Table 1 for ratings on all interactions.
(B) Immunoblot analysis of JAZ proteins in yeast strains used for Y2H assays shown in (A). Each lane contains total protein extracted from cells
expressing AD and BD fusions of the indicated JAZ protein (e.g., “1” corresponds to JAZ1). AD-JAZ and BD-JAZ fusion proteins were detected with
anti-HA (left panel) and anti-LexA (right panel) antibodies, respectively.
(C) BiFC assay of JAZ3-JAZ3 homomeric interaction in planta. YFP fluorescence was detected in N. tabacum leaves coinfiltrated with Agrobacterium
strains expressing JAZ3-nYFP and cYFP-JAZ3. 4’,6-Diamidino-2-phenylindole (DAPI) staining shows the location of nuclei. The merged image shows
colocalization of DAPI and YFP fluorescence.
 Role of the ZIM/TIFY Domain in JA Signaling 133

JAZ10 (referred to here as JAZ10.3) that lacks seven amino acids JAZ10.4 overexpression are suppressed by mutations in the
from the C-terminal end of the Jas domain. Overexpression of TIFY motif that block JAZ10.4–JAZ interactions. These results
this truncated protein, but not the Jas domain–containing full- indicate that JAZ10.4 functions to dampen signal output in the
length protein (JAZ10.1), conferred partial insensitivity to JA. presence of JA and that the repressive activity of this splice
These important findings strengthen the rationale for studies variant depends on the ZIM/TIFY domain. The data are consis-
aimed at determining how JAZDJas proteins regulate JA signal tent with a model in which a JAZ10.4-containing protein complex
output. directly impairs the activity of transcription factors (e.g., MYC2)
JAZ proteins are classified as members of the tify family, also that control expression of JA response genes.
known as ZIM (Vanholme et al., 2007). The defining feature of all
family members is the highly conserved TIFY motif (TIFF/YXG)
located within a larger conserved region referred to as the ZIM (or RESULTS
TIFY) domain (Chini et al., 2007; Thines et al., 2007; Vanholme
et al., 2007; Yan et al., 2007). The tify family in Arabidopsis Homo- and Heteromeric Interaction of JAZ Proteins
includes the so-called ZIM and ZIM-like (ZML) transcription
factors that are implicated in cell expansion (Shikata et al., 2004) We used the yeast two-hybrid (Y2H) system to determine the
as well as PEAPOD (PPD) proteins that regulate leaf size and homomeric interaction potential of the 12 members of the
shape (White, 2006). Unlike the ZIM and ZML proteins that Arabidopsis JAZ family. Each full-length JAZ was fused sepa-
possess a zinc-finger DNA binding domain, JAZs do not contain rately to the LexA DNA binding domain (BD) and the B42
a known DNA binding domain. Rather, JAZs are proposed to activation domain (AD). Homodimeric interactions were as-
exert their effects on gene expression through interaction with sessed by coexpression of the BD and AD fusion proteins in
MYC2 (Chini et al., 2007) and perhaps other transcription factors. yeast cells containing a lacZ reporter gene under the control of a
This idea is supported by the nuclear location of several JAZ LexA-dependent operator. As shown in Figure 1A, JAZ1, JAZ2,
family members (Chini et al., 2007; Thines et al., 2007; Yan et al., JAZ3, and JAZ10.1 strongly interacted as homodimers, as
2007). The occurrence of the ZIM domain in proteins from higher determined by strong LacZ reporter activity. Weaker homomeric
and lower (i.e., moss) plants but not in green algae suggests an interactions were observed for JAZ4, JAZ5, and JAZ6, whereas
important role for JAZs in the evolution of land plants (Vanholme the remaining five family members (JAZ7, JAZ8, JAZ9, JAZ11,
et al., 2007; Chico et al., 2008; Katsir et al., 2008a). and JAZ12) failed to interact. Protein gel blot analysis showed
Here, we provide evidence that the ZIM domain mediates that all JAZ fusion proteins were expressed in yeast cells (Figure
formation of homo- and heteromeric complexes involving most 1B), indicating that the absence of interaction cannot be attri-
members of the Arabidopsis JAZ family. We identify a novel buted to a lack of protein expression.
alternatively spliced form of JAZ10 (JAZ10.4) that, unlike the We next used a bimolecular fluorescence complementation
previously characterized JAZ10.3, lacks the entire Jas domain (BiFC) assay to determine whether JAZ-JAZ homomeric com-
and confers severe JA insensitivity when overexpressed in plexes form in planta. JAZ3 was chosen for this analysis because
Arabidopsis. We demonstrate that JAZ10.4 is stabilized against it self-associates strongly in yeast (Figure 1A) and, when ex-
JA-induced degradation and, significantly, has the ability to pressed as a fusion with green fluorescent protein, is localized to
interact with MYC2 and with other JAZ proteins. We also show the nucleus (Chini et al., 2007). N- and C-terminal fragments of
that severe dominant JA-insensitive phenotypes resulting from yellow fluorescent protein (YFP) (nYFP and cYFP, respectively)

Table 1. Summary of JAZ–JAZ Interactions in Yeast

JAZ1 JAZ2 JAZ3 JAZ4 JAZ5 JAZ6 JAZ7 JAZ8 JAZ9 JAZ10.1 JAZ11 JAZ12

JAZ1 +++ +++ +++ +++ + ++ + + +++

JAZ2 +++ +++ +++ + + +
JAZ3 +++ +
JAZ4 +++ +
JAZ5 ++ ++ + ++
JAZ6 +++ + + ++ ++
JAZ7
JAZ8 +++ +++ +++ +++ +++ +++ + +++ +++
JAZ9 + +++ +
JAZ10.1 +++ +++ +++ +++ ++ + +++ +
JAZ11 + ++ +++ + + ++
JAZ12 +++
Each of the 12 full-length Arabidopsis JAZs was tested in both AD (top row) and BD (left column) orientations against the remaining 11 family members
(132 heteromeric combinations). A summary of results for JAZ homomeric interactions is also included. Based on the intensity of LacZ-mediated blue-
color formation, the strength of each interaction was rated as strong (+++), medium (++), weak (+), or undetectable ( ), as shown in Figure 1A.
Heteromeric interactions observed in both AD and BD orientations are presented in bold.
134 The Plant Cell

were fused to either the N or C termini of JAZ3 to produce four eral JAZ proteins that interacted strongly as homodimers tended
constructs (JAZ3-nYFP, JAZ3-cYFP, nYFP-JAZ3, and cYFP- to interact with a larger number of JAZs. For example, JAZ1 and
JAZ3). Agrobacterium tumefaciens–mediated transformation JAZ3 interacted with nine and seven other family members,
was used to coexpress each of the four possible combinations respectively, in one or both BD/AD orientations. We also found
of nYFP and cYFP JAZ fusion constructs in epidermal cells of examples of proteins (e.g., JAZ8) that did not form homomeric
Nicotiana tabacum. Coexpression of JAZ3-nYFP and cYFP- complexes but did interact with several other family members.
JAZ3, but not any other combination tested, resulted in a YFP We conclude that most JAZ proteins in Arabidopsis form
signal within the nucleus (Figure 1C). This finding confirms the homodimers and heterodimers in yeast.
Y2H results and further demonstrates that JAZ3 can self-
associate in the plant nucleus. Involvement of the TIFY Motif in JAZ–JAZ Interactions
We also employed Y2H assays to systematically test all
possible JAZ-JAZ heteromeric interactions in both BD/AD ori- As an initial approach to identify sequence determinants that
entations. Out of a total of 132 possible heteromeric combina- mediate JAZ-JAZ binding, we examined the ability of various
tions, we observed 47 interactions of varying strengths involving deletion mutants of JAZ3 (Figure 2A) to interact with full-length
all family members except JAZ7 (Table 1). Nine of these interac- JAZ3 and JAZ10.1. Deletion of 140 amino acids from the N ter-
tions (JAZ1-JAZ2, JAZ1-JAZ10.1, JAZ2-JAZ5, JAZ2-JAZ6, minus of JAZ3 (DN), including the weakly conserved N-terminal
JAZ3-JAZ4, JAZ5-JAZ6, JAZ6-JAZ10.1, JAZ6-JAZ12, and region observed by Thines et al. (2007), resulted in a slight
JAZ9-JAZ10.1) were detected in both BD/AD orientations. Sev- reduction in the strength of homomeric and heteromeric

Figure 2. Requirement for the TIFY Motif in Homo- and Heteromeric Interaction of JAZ Proteins.
(A) Schematic diagram of JAZ3 deletion constructs analyzed in (B) and (C). The diagram shows the highly conserved Jas (blue) and ZIM (pink, with TIFY
motif) domains as well as the weakly conserved sequence (orange, labeled “N”) at the N terminus (Thines et al., 2007).
(B) Y2H assay to assess the interaction of each JAZ3 deletion construct (AD fusion) shown in (A) with full-length JAZ3 and JAZ10.1 (BD fusions).
(C) Immunoblot analysis of JAZ3 deletion proteins in yeast strains tested in (B). AD-JAZ3 fusion proteins were detected with an anti-HA antibody.
(D) Amino acid sequence alignment of the ZIM domain in 12 Arabidopsis JAZs. The highly conserved TIFY motif (TIFF/YXG) is boxed. The in-color
alignment depicts amino acids with similar hydrophobicity and was generated with the software BioEdit v 7.0.9.
(E) Ala-scanning mutagenesis of the JAZ3 TIFY motif. Wild-type and mutant forms (e.g., T180A) of JAZ3 (AD fusions) were tested in the Y2H system for
interaction with wild-type JAZ3 and JAZ10.1 (BD fusions).
(F) Ala-scanning mutagenesis of the JAZ10.1 TIFY motif. Wild-type and mutant forms (e.g., T106A) of JAZ10.1 (AD fusions) were tested in the Y2H
system for interaction with wild-type JAZ10.1 and JAZ6 (BD fusions).
 Role of the ZIM/TIFY Domain in JA Signaling 135

interactions with JAZ3 and JAZ10.1, respectively (Figure 2B). A gel blot analysis showed that all Ala-substituted mutants of JAZ3
JAZ3 variant (Jas) that lacks both the N-terminal and ZIM do- and JAZ10.1 were expressed in yeast (see Supplemental Figure
mains was previously shown to interact with COI1 in a hormone- 1 online). These findings establish an essential role for the TIFY
dependent manner (Melotto et al., 2008). This C-terminal motif in JAZ–JAZ interactions.
fragment of JAZ3 failed to interact with either full-length JAZ3 The ZIM domain was originally identified in ZIM, ZML1, and
or JAZ10.1 (Figure 2B). A third deletion construct (DJas) that ZML2 members of the tify family (Shikata et al., 2004; Vanholme
lacks the Jas domain interacted strongly with full-length JAZ3 et al., 2007). All three of these proteins possess a GATA-type Zn-
and JAZ10.1. Protein gel blot analysis confirmed that the JAZ3 finger DNA binding motif and are thought to act as transcription
deletion variants used in these experiments were expressed in factors. If the ZIM domain functions as a protein–protein inter-
yeast cells (Figure 2C). Taken together, these results indicate that action determinant, we reasoned that ZIM, ZML1, and ZML2 may
the N-terminal region and the Jas domain are not required for interact with one another. Indeed, we found that each protein
JAZ3-JAZ interactions, thus implicating the ZIM domain as an homo- and heterodimerized in yeast (see Supplemental Figure 2
important determinant for JAZ-JAZ binding. online).
To further investigate the role of the ZIM domain in JAZ–JAZ
interaction, we performed Ala-scanning mutagenesis of the Characterization of a Novel JAZ10 Splice Variant That Lacks
highly conserved TIFY motif found in the ZIM domain of JAZ3 the Jas Domain
and all other family members (Figure 2D). We assessed the
interaction of the resulting mutant JAZ3 variants with both wild- During efforts to isolate a full-length cDNA for JAZ10, we iden-
type JAZ3 and JAZ10.1 in the Y2H system. Ala substitution of tified three distinct transcripts from this gene. These included the
Gly-185 abolished the interaction with wild-type JAZ3, whereas At5g13220.1 and At5g13220.3 transcripts that are predicted by
replacement of Thr-180, Ile-181, Phe-182, and Tyr-183 did not The Arabidopsis Information Resource (TAIR) to encode the
affect this interaction (Figure 2E). Heteromeric interaction of longest isoform (i.e., JAZ10.1) and the JAZ10.3/JAS1.3 isoform
JAZ3 with JAZ10.1 was also disrupted by the G185A mutation as containing a partially truncated Jas domain (Yan et al., 2007). The
well as by an I181A mutation in JAZ3 (Figure 2E). Site-directed third transcript we identified (designated At5g13220.4), which
mutagenesis of the TIFY motif in JAZ10.1 yielded similar results. was not annotated in the TAIR database, is predicted to encode a
In this case, Ala substitution of either Ile-107 or Gly-111 abro- 167–amino acid protein, designated JAZ10.4. The sequence of
gated JAZ10.1 binding to itself and to JAZ6 (Figure 2F). Protein At5g13220.4 indicates that it is produced by the use of an

Figure 3. Identification of Three JAZ10 Variants Produced by Alternative Splicing.

(A) Schematic diagram of alternatively spliced transcripts At5g13220.1, At5g13220.3, and At5g13220.4 encoding JAZ10.1, JAZ10.3, and JAZ10.4,
respectively. Thick lines (black, translated; gray, untranslated) represent exons (labeled A to E). Modified exons in At5g13220.3 and At5g13220.4 result
from use of alternative splice sites marked D9 and C9, respectively.
(B) Alternative splicing differentially affects the sequence of the C-terminal Jas domain in the three JAZ10 splice variants. Amino acid sequences
N-terminal to Ser-147 are identical in the three isoforms and are not shown. Amino acids comprising the Jas domain are underlined, whereas amino
acids encoded by exon D are in bold. A frame shift in exon D of At5g13220.4 (resulting from alternative splicing of exon C) removes the Jas domain and
adds 20 unrelated amino acid residues C-terminal to Ser-147. Asterisks indicate the stop codon.
(C) RT-PCR analysis of JAZ10 transcripts in RNA isolated from wounded leaves. The location of primers used for RT-PCR is indicated by arrows in (A).
PCR products were separated by gel electrophoresis, and the resulting gel was stained with ethidium bromide. Arrows denote transcripts encoding the
three JAZ10 splice variants.
136 The Plant Cell

alternative splice donor site in the third exon of the JAZ10 gene JAZ10.4 did not interact with COI1 at any concentration of
(Figure 3A). This splicing event causes a frame-shift mutation coronatine tested. Quantification of b-galactosidase activity
that deletes the entire Jas domain and adds 20 amino acids confirmed that JAZ10.3 weakly interacts with COI1 in compar-
C-terminal to Ser-147 (Figure 3B). RT-PCR was used to verify the ison to JAZ10.1 and that JAZ10.4 fails to bind COI1 in the
occurrence of At5g13220.4 in wounded Arabidopsis leaves. As presence of 200 mM coronatine (Figure 4B). Protein gel blot
shown in Figure 3C, three RT-PCR products ranging in size analysis showed that all three JAZ10 proteins were produced in
between ;500 and 650 bp were distinguishable by agarose gel yeast cells (Figure 4C). We also performed Y2H assays to
electrophoresis. Cloning and sequencing of these PCR products determine whether the JAZ10 splice variants interact with the
confirmed their identity as At5g13220.1, At5g13220.3, and basic helix-loop-helix transcription factor, MYC2. As shown in
At5g13220.4. RT-PCR experiments performed with transcript- Figure 4D, all three JAZ10 isoforms interacted with MYC2 in the
specific primers also detected the At5g13220.3 and absence of coronatine. These findings indicate that the Jas
At5g13220.4 splice variants in wounded leaves (see Supple- domain is critical for interaction of JAZ10 splice variants (e.g.,
mental Figure 3 online). JAZ10.1) with COI1 but is not required for binding of JAZ10
The role of the Jas domain as a JAZ destabilization element isoforms to MYC2.
(Katsir et al., 2008b; Melotto et al., 2008) led us to test the To further investigate potential functional differences between
hypothesis that JAZ10.1, JAZ10.3, and JAZ10.4 differentially the JAZ10 isoforms, we tested whether JAZ10.3 and JAZ10.4,
interact with COI1. We assessed the three isoforms for their like JAZ10.1 (Figure 1, Table 1), form homo- and heterodimers in
ability to interact with COI1 in a ligand-dependent manner in the yeast. As shown in Figure 4E, the JAZ interaction pattern of
Y2H system. Coronatine, a potent signal for COI1–JAZ interac- JAZ10.3 was identical to that of JAZ10.1, with both variants
tions (Katsir et al., 2008b; Melotto et al., 2008), stimulated partnering with eight of the 14 JAZ proteins tested. In this BD/AD
JAZ10.1 binding to COI1 in a dose-dependent manner (Figure orientation, JAZ10.4 interacted with the same set of eight JAZs
4A). JAZ10.3 interacted with COI1 in the presence of relatively and also formed heterodimers with JAZ2 and JAZ3 (Figure 4E).
high concentrations of coronatine (e.g., 200 mM), whereas Protein gel blot analysis of protein extracts from yeast strains

Figure 4. Protein–Protein Interaction Characteristics of JAZ10 Isoforms.

(A) Coronatine-dependent interaction of three JAZ10 splice variants with COI1 in the Y2H system. Yeast strains cotransformed with AD-JAZ10 isoforms
(JAZ10.1, JAZ10.3, or JAZ10.4) and BD-COI1 were plated on media containing the indicated concentration of coronatine (COR).
(B) Quantification of b-galactosidase activity in yeast strains shown in (A). Each strain was grown in the absence of coronatine (“0”) or in the presence of
30 or 200 mM coronatine (COR). Data show the mean 6 SD of triplicate technical replicates.
(C) Immunoblot analysis of JAZ10 and COI1 proteins in yeast cells tested in (A). JAZ10 splice variants and COI1 were detected with anti-HA and anti-
LexA antibodies, respectively.
(D) Interaction of three JAZ10 splice variants with MYC2 in the Y2H system. Yeast strains cotransformed with AD-MYC2 and BD-JAZ10 variants
(JAZ10.1, JAZ10.3, and JAZ10.4) were plated on media containing X-gal. A yeast strain cotransformed with AD-MYC2 and an empty BD vector
(pGILDA) is shown as a control (empty + MYC2).
(E) Homo- and heteromeric interaction of JAZ10 splice variants. Yeast strains cotransformed with one of the three JAZ10 isoforms (as an AD fusion) and
other members (as a BD fusion) of the JAZ family were plated on media containing X-gal.
(F) Protein gel blot analysis of JAZ10 isoforms and JAZ3 in yeast strains used for the Y2H experiment shown in (E). JAZ10 splice variants and JAZ3 were
detected with anti-HA and anti-LexA antibodies, respectively.
 Role of the ZIM/TIFY Domain in JA Signaling 137

cotransformed with JAZ3 and JAZ10 isoforms showed that each genic plants overexpressing JAZ10.1, JAZ10.3, or JAZ10.4. As
JAZ protein was expressed (Figure 4F). We conclude that the previously reported by Yan et al. (2007), overexpression of
three JAZ10 splice variants interact with a similar set of JAZs in JAZ10.1 and JAZ10.3 did not have overt effects on plant fertility.
the Y2H system. By contrast, we observed that ;30% (49 of 150 plants exam-
Previous studies have shown that several JAZ family mem- ined) of T1 plants expressing a 35S-JAZ10.4 transgene were
bers, including JAZ10.1 and JAZ10.3, are located in the nucleus male sterile as a result of defects in filament elongation and
(Chini et al., 2007; Thines et al., 2007; Yan et al., 2007). To anther dehiscence (Figures 6A to 6D). These flower phenotypes,
determine whether the modified C terminus of JAZ10.4 affects which are hallmarks of strong JA synthesis and perception
the targeting of the protein to the nucleus, we used confocal laser mutants (Feys et al., 1994; McConn and Browse, 1996; Sanders
microscopy to examine the subcellular location of a JAZ10.4- et al., 2000; Stintzi and Browse, 2000; Ishiguro et al., 2001; Park
YFP fusion protein transiently expressed in tobacco epidermal et al., 2002), were not rescued by exogenous methyl-JA (MeJA)
cells. Control experiments showed that whereas YFP alone (data not shown). Fertilization of sterile 35S-JAZ10.4 flowers with
partitions to both the cytosol and the nucleus, JAZ10.1-YFP wild-type pollen yielded F1 progeny in which the sterile pheno-
and JAZ10.3-YFP are located predominantly in the nucleus, as type strictly cosegregated with the 35S-JAZ10.4 transgene. To
previously reported (Yan et al., 2007) (Figure 5). JAZ10.4-YFP determine whether overexpression of JAZ10.4 affects other JA
exhibited a nuclear localization pattern that was similar to that of responses, we compared JA-mediated root growth inhibition in
the other two isoforms. We conclude that the COI1-noninteract- 35S-JAZ10.4 seedlings to that of wild-type, coi1-1, 35S-
ing splice variant JAZ10.4 localizes to the nucleus. JAZ10.1, and 35S-JAZ10.3 seedlings (Figures 6E and 6F). As
reported by Yan et al. (2007), root growth of 35S-JAZ10.3
seedlings was moderately resistant to JA, whereas 35S-
Overexpression of JAZ10 Splice Variants Differentially JAZ10.1 roots were as sensitive as wild-type seedlings to the
Affects JA Signal Output hormone. 35S-JAZ10.4 seedlings exhibited a strong JA-
insensitive root growth phenotype, which was comparable to
The absence of a Jas domain in JAZ10.4 indicated to us that this that of coi1-1 seedlings.
splice variant might exert dominant-negative effects on JA
signaling, as previously observed for JAZ10.3 (Yan et al., 2007)
and other JAZs in which the Jas domain was removed by Differential Stability of JAZ10 Splice Variants in Vivo
targeted deletion or mutagenesis (Chini et al., 2007; Thines
et al., 2007; Melotto et al., 2008; Shoji et al., 2008). To investigate The strong JA-insensitive phenotypes conferred by JAZ10.4
this possibility, we assessed JA-related phenotypes in trans- overexpression, together with the inability of this isoform to

Figure 5. Subcellular Localization of JAZ10 Splice Variants.

JAZ10-YFP fusion proteins were transiently expressed by Agrobacterium-mediated transformation of tobacco epidermal cells. YFP fluorescence was
detected with a confocal laser scanning microscope. DAPI fluorescence was used as a marker for the nucleus. YFP alone (not fused to JAZ10) is located
in both the nucleus and the cytosol.
138 The Plant Cell

Figure 6. Overexpression of JAZ10 Splice Variants Differentially Affects JA Responses.

(A) to (D) JAZ10.4-overexpressing plants are male sterile.

(A) Wild-type (left) and 35S-JAZ10.4 (right) inflorescence.
(B) to (D) Close-up view of a wild-type flower (B) and JAZ10.4 flowers ([C] and [D]) that have short filaments and nondehisced anthers.
(E) Differential effect of JAZ10 isoforms on JA-mediated inhibition of root growth. Germinated seedlings of the indicated genotype were grown for 7 d on
MS medium containing 50 mM MeJA. Bar = 5 mm.
(F) Quantification of JA-induced root growth inhibition of seedlings shown in (E). Data show the mean 6 SD (n = 15 seedlings per genotype).
[See online article for color version of this figure.]

interact with COI1, suggested that JAZ10.4 is resistant to JA- relative stability of the three JAZ10 splice variants in hormone-
induced protein degradation. To test this idea, we generated stimulated plants is JAZ10.4 > JAZ10.3 > JAZ10.1 and that
stable transgenic lines expressing JAZ10.1-YFP, JAZ10.3-YFP, JAZ10.4 is highly resistant to JA-induced degradation. Pretreat-
and JAZ10.4-YFP fusion proteins under control of the 35S ment of seedlings with MG132, a specific inhibitor of the 26S
promoter. Root growth inhibition assays showed that the JA proteasome, impaired JA-induced turnover of JAZ10.1-YFP
responsiveness of each line was essentially identical to that of (Figure 7B). This finding indicates that JA-induced degradation
JAZ10.1-, JAZ10.3-, and JAZ10.4-overexpresing plants; the root of this Jas domain–containing isoform is mediated by the 26S-
length of wild-type, 35S-JAZ10.1-YFP, 35S-JAZ10.3-YFP, and proteasome pathway.
35S-JAZ10.4-YFP seedlings grown on Murashige and Skoog
(MS) plates containing 50 mM MeJA was 5.6 6 1.0 mm, 5.4 6 0.8 The Dominant-Negative Effect of JAZ10.4 Requires a
mm, 11.5 6 0.5 mm, and 16.9 6 1.3 mm, respectively. To Functional TIFY Motif
investigate the hormone-dependent stability of each JAZ10
isoform in vivo, we monitored YFP fluorescence in roots of 7-d- We next investigated whether the strong repression of JA re-
old transgenic seedlings that were either treated (for 2 h) or not sponses by JAZ10.4 is dependent on a functional TIFY motif. To
treated with MeJA. In the absence of MeJA, a nuclear-localized address this question, we generated transgenic lines that over-
YFP signal was observed in all three genotypes (Figure 7A). produce JAZ10.4I->A and JAZ10.4G->A proteins harboring I107A
Analysis of several independent lines (at least five lines per or G111A point mutations, respectively, in the TIFY motif of
construct) showed that the strength of the YFP signal in 35S- JAZ10.4. Although these mutations abrogate JAZ10.1 interac-
JAZ10.1-YFP plants was consistently less than that in 35S- tion with other JAZs in the Y2H system (Figure 2F), it was
JAZ10.3-YFP and 35S-JAZ10.4-YFP seedlings (Figure 7A, necessary to determine the effect of these mutations on
Mock). Treatment with 1 mM MeJA eliminated nuclear YFP JAZ10.4–JAZ interactions. As shown in Figure 8A, JAZ10.4I->A
fluorescence in 35S-JAZ10.1-YFP roots but did not obviously lost the ability to interact with JAZ6, JAZ10.1, JAZ10.3, and
affect the signal in 35S-JAZ10.3-YFP and 35S-JAZ10.4-YFP JAZ10.4. JAZ10.4G->A also failed to bind JAZ10.4 but did retain
seedlings. Treatment of seedlings with 100 mM MeJA resulted in ability to interact with JAZ6, JAZ10.1, and JAZ10.3. Protein gel
loss of the JAZ10.3-YFP nuclear signal, although a diffuse blot analysis of protein extracts from yeast strains cotransformed
(presumably cytosolic) fluorescence pattern in roots was still with JAZ10.1 and mutant forms of JAZ10.4 showed that the JAZ
detected. The fluorescence pattern in 35S-JAZ10.4-YFP seed- fusion proteins were expressed (Figure 8B). These findings,
lings was unaffected by 100 mM MeJA. We conclude that the together with results of experiments to assess JAZ10.4G->A and
 Role of the ZIM/TIFY Domain in JA Signaling 139

JAZ10.4I->A roots were as sensitive to JA-induced growth arrest

as wild-type roots (Figures 8G and 8H). 35S-JAZ10.4G->A roots,
by contrast, were less sensitive to JA than wild-type roots but
significantly more sensitive (P < 0.0001; Student’s t test) than
35S-JAZ10.4 roots. These results show that the ability of
JAZ10.4 to repress JA signaling in various tissues is strongly
suppressed by I107A and moderately suppressed by G111A.
To test the possibility that normal JA responsiveness of 35S-
JAZ10.4I->A plants results from low expression of the JAZ10.4I->A
mutant protein in planta, we analyzed the level of GUS activity in
stable transgenic lines that constitutively express either
JAZ10.4-GUS or JAZ10.4I->A-GUS fusion proteins. In several
independent transgenic lines tested (at least five lines per con-
struct), seedlings expressing the 35S-JAZ10.4I->A-GUS trans-
gene exhibited a pattern of strong blue staining (in roots,
cotyledons, and leaves) that was similar to that of 35S-
JAZ10.4-GUS seedlings (Figure 9A), indicating that the
JAZ10.4I->A-GUS fusion protein is expressed. 35S-JAZ10.4-
GUS plants were male sterile and highly insensitive to JA-
induced root growth arrest (Figures 9B and 9C). This finding
Figure 7. Stability of JAZ10 Splice Variants in Vivo.
shows that JAZ10.4-GUS, like JAZ10.4, strongly represses JA
(A) Differential stability of three JAZ10 splice variants in response to JA. signal output. Plants overexpressing JAZ10.4I->A-GUS, how-
Transgenic seedlings expressing JAZ10.1-YFP, JAZ10.3-YFP, or ever, were fully fertile and exhibited normal sensitivity to exog-
JAZ10.4-YFP fusion proteins were treated either with water (Mock) or enous JA (Figures 9B and 9C). We also examined the subcellular
with the indicated concentration of MeJA (JA). Two hours after treatment,
localization of JAZ10.4-YFP and JAZ10.4I->A-YFP fusion pro-
YFP signal in root tissue was visualized by fluorescence microscopy. The
teins to determine whether I107A affects subcellular protein
exposure times for each image in (A) and (B) were identical.
(B) Proteasome-dependent degradation of JAZ10.1-YFP. Seedlings
distribution. Following transient Agrobacterium-mediated ex-
expressing the 35S-JAZ10.1-YFP transgene were pretreated with water pression of these proteins in tobacco leaf epidermal cells, laser
or the 26S proteasome–specific inhibitor MG132 (50 mM) for 2 h, at which scanning confocal microscopy revealed that both JAZ10.4-YFP
time seedlings were treated with either MeJA (JA; 10 mM) or water (Mock) and JAZ10.4I->A-YFP are targeted to the nucleus (Figure 9D).
for 2 h. YFP signal in root tissue was visualized by fluorescence These results indicate that the I107A mutant form of JAZ10.4 is
microscopy. expressed and targeted to the nucleus.

JAZ10.4I->A interaction with other members of the JAZ family DISCUSSION

(see Supplemental Figure 4 online), indicate that I107A is more
effective than G111A at disrupting JAZ10.4 heterodimerization Functional Diversification of JAZ Proteins by
with other JAZs. Alternative Splicing
Having established the effect of I107A and G111A on JAZ10.4–
JAZ interactions in yeast, we assessed JA-mediated fertility Alternative splicing plays an important role in increasing protein
phenotypes in transgenic plants that overexpress JAZ10.4I->A diversity and, ultimately, biological complexity. Our characteri-
and JAZ10.4G->A. Of 150 independent 35S-JAZ10.4I->A plants zation of functionally distinct JAZ10 splice variants adds to a
(T1 generation) examined, none showed defects in filament growing body of literature indicating that alternative splicing of
elongation, anther dehiscence, silique development, or viable pre-mRNAs provides a general mechanism to alter the proteome
seed production (Figures 8C and 8D), indicating that I107A in ways that optimize plant adaptation to stress (Reddy, 2007;
completely suppresses JAZ10.4-mediated male sterility. In a Barbazuk et al., 2008). In addition to the JAZ10.1 and JAZ10.3
population of 40 independent T1 35S-JAZ10.4G->A plants, 12 isoforms described by Yan et al. (2007), we identified a third
individuals exhibited reduced fertility; these plants produced JAZ10 splice variant (JAZ10.4) that lacks the entire Jas domain.
only a few mature siliques during the latter stage of reproductive Several observations indicate that differential association of
development (Figure 8C). Some flowers produced by this group JAZ10 variants with COI1 affects the stability and action of
of semifertile 35S-JAZ10.4G->A plants were indistinguishable each isoform in different ways. We showed that JAZ10.1 inter-
from wild-type flowers, whereas other 35S-JAZ10.4G->A flowers acts with COI1 in a ligand-dependent manner, whereas JAZ10.4
had anther filaments that failed to fully elongate (Figures 8E fails to bind COI1. JAZ10.3, which contains a partially truncated
and 8F). Jas domain, interacted weakly with COI1. Analysis of JAZ10-YFP
We also studied the effect of TIFY point mutations (I107A and reporter lines showed that JAZ10.1 is readily degraded via the
G111A) on the JA-resistant root growth phenotype that results 26S proteasome pathway in response to JA treatment, whereas
from JAZ10.4 overexpression. In agreement with the ability of JAZ10.4 is highly resistant to hormone-induced destruction
I107A to strongly suppress JAZ10.4-mediated sterility, 35S- (Figure 7). Consistent with COI1 interaction studies performed
140 The Plant Cell

in yeast, high concentrations of JA were required to induce

turnover of JAZ10.3-YFP in planta. The differential stability of
JAZ10 splice variants can explain the JA-related phenotypes
resulting from ectopic expression of each isoform. Overexpres-
sion of JAZ10.3 and JAZ10.4 conferred weak and strong JA-
insensitive phenotypes, respectively, whereas 35S-JAZ10.1
plants that produce the full-length protein did not exhibit altered
sensitivity to JA. We conclude that functional differences be-
tween JAZ10 splice variants can be attributed to sequence
alterations in the Jas domain that affect COI1 binding and, as a
consequence, the stability of each isoform. Gene prediction
programs (www.softberry.com) suggest that JAZ10 orthologs
in rice (Oryza sativa) (gi:115458122) and poplar (Populus tricho-
carpa) (Joint Genome Institute gene model 548076) are subject
to alternative splicing events that alter the Jas domain. Thus,
diversification of JAZ function through alternative splicing may
be widespread in the plant kingdom.
Several Jas domain–containing JAZ proteins have been
shown to be degraded in a JA-dependent manner (Chini et al.,
2007; Thines et al., 2007; this study). Presumably, these proteins
act to repress JA responses in unstimulated cells that contain
low levels of the hormone and are degraded in the presence of
high JA levels. By contrast, our results indicate that JAZ10.4 is an
endogenous repressor of JA signal output in JA-stimulated cells.
This hypothesis is consistent with the observation that JAZ10
RNA interference–silenced plants are hypersensitive to JA (Yan
et al., 2007), whereas null mutations in several other JAZ genes
apparently do not have this effect (Chini et al., 2007; Thines et al.,
2007). It seems likely that the increased sensitivity of JAZ10-
silenced lines to JA results from a deficiency in stabilized JAZs
(e.g., JAZ10.4) rather than from reduced expression of the more
labile JAZ10.1. Sequence variation in the Jas domain could
potentially give rise to JAZ family members having a wide range
of stabilities, which could provide a mechanism to modulate the
amplitude and duration of JA responses. The ability of JAZ10.4 to

(A) Y2H analysis of the effect of I107A and G111A TIFY mutations on the
interaction of JAZ10.4 with JAZ6, JAZ10.1, JAZ10.3, and JAZ10.4. Yeast
strains cotransformed with AD fusions of JAZ10.4 (or JAZ10.4I->A and
JAZ10.4G->A mutants) and BD-JAZ fusions (JAZ6, JAZ10.1, JAZ10.3,
and JAZ10.4) were plated on X-gal–containing medium.
(B) Immunoblot analysis of JAZ proteins in yeast cells cotransformed
with AD fusions of JAZ10.4 (or JAZ10.4I->A and JAZ10.4G->A mutants)
and BD-JAZ10.1, which were tested in (A). AD- and BD-JAZ fusions were
detected with anti-HA and anti-LexA antibodies, respectively.
(C) to (F) Mutations (I107A and G111A) in the TIFY motif differentially lack
the male-sterile phenotype conferred by overexpression of wild-type
JAZ10.4. Complete lack of sterility is shown by the inflorescence ([C],
left) and floral (D) phenotype of 35S-JAZ10.4I->A plants. Partial lack of
JAZ10.4-mediated sterility is indicated by development of a few mature
siliques on the inflorescence of 35S-JAZ10.4G->A plants ([C] and [D],
right). 35S-JAZ10.4G->A plants produced a mixture of wild-type-like
flowers (E) and flowers with shortened anther filaments (F).
(G) Differential effect of I107A and G111A mutations on JAZ10.4-
mediated changes in root growth in the presence of JA. Seedlings of
the indicated genotype were grown for 6 d on MS medium containing 50
mM MeJA. Bar = 5 mm.
Figure 8. The TIFY Motif Is Required for Repression of JA Responses by (H) Measurement of primary root length of seedlings shown in (G). Data
JAZ10.4. are the mean 6 SD (n = 19 seedlings per genotype).
 Role of the ZIM/TIFY Domain in JA Signaling 141

Figure 9. The I107A Mutant Form of JAZ10.4 Is Expressed and Targeted to the Nucleus.

(A) Histochemical GUS staining of 35S-JAZ10.4-GUS and 35S-JAZ10.4I->A-GUS transgenic seedlings. Seedlings were grown on MS medium
containing kanamycin (50 mg/mL) for 14 d prior to transferring to a 48-well microtiter plate for GUS staining.
(B) The male-sterile phenotype conferred by overexpression of JAZ10.4-GUS (left) is not shown by plants overexpressing the I107A mutant form of
JAZ10.4-GUS (right).
(C) The JA-insensitive root growth phenotype conferred by overexpression of JAZ10.4-GUS is not shown by plants overexpressing the I107A mutant
form of JAZ10.4-GUS.
(D) Subcellular localization of JAZ10.4-YFP and JAZ10.4I->A-YFP fusion proteins that were transiently expressed by Agrobacterium-mediated
transformation of tobacco epidermal cells. YFP and DAPI fluorescence was detected by confocal laser scanning microscopy.

strongly attenuate signal output, for example, may be important 2007). Here, we present several lines of evidence to indicate that
for reigning in JA responses that are energetically demanding the ZIM domain mediates homo- and heteromeric protein–
and potentially damaging to the cell. It is also possible that protein interactions among JAZ proteins. We have identified
JAZ10.4 or other stabilized JAZs attenuate JA-mediated growth nine Arabidopsis JAZs (including the three JAZ10 splice variants)
inhibition under environmental conditions where rapid growth is that self-associate in yeast. BiFC experiments showed that JAZ3
required to compete for limited resources (Izaguirre et al., 2006; homomeric complexes accumulate in the plant nucleus. Our Y2H
Yan et al., 2007, Zhang and Turner, 2008). results also revealed an extensive network of JAZ heterodimers;
An important question that remains to be addressed is how the in an experiment to test all 66 possible heterodimeric combina-
production of JAZ10.4 is regulated in wild-type plants. Our tions between 12 full-length Arabidopsis JAZs (Table 1), we
results support a scenario in which negative feedback control identified 38 heterodimeric interactions involving most but not all
of signal output by JAZ10.4 (and perhaps JAZ10.3) is initiated JAZs. Given that protein dimerization is a common regulatory
upon transcriptional activation of the JAZ10 gene in response to mechanism in signal transduction (Klemm et al., 1998), it is
wounding or other inductive cues that trigger JA synthesis (Yan conceivable that combinatorial interactions among various JAZs
et al., 2007; Chung et al., 2008). It is possible that wounding play a role in generating diverse JA signal outputs. Additional
regulates alternative splicing of JAZ10 pre-mRNA in a manner work is needed to determine the physiological relevance of these
that favors the accumulation of a specific transcript (Bove et al., interactions and to assess whether differences in the interaction
2008). However, our data and those of Yan et al. (2007) show that potential of various JAZs have functional significance.
all three transcripts accumulate in wounded leaves. Assuming Site-directed mutagenesis studies showed that the TIFY motif
that all three JAZ10 isoforms are synthesized at the same rate in is a key determinant of JAZ homo- and heteromerization. Be-
the same JA-stimulated cell, the relative abundance of each cause the ZIM domain contains several highly conserved resi-
isoform would be determined largely by the rate of JA-induced dues in addition to the TIFY motif (Figure 2D), it is likely that other
turnover. Based on the results of JAZ10-YFP degradation assays regions of the domain are also important for protein–protein
(Figure 7), we predict that JAZ10.4 (and perhaps JAZ10.3) should interaction. Y2H assays with JAZ3 deletion constructs and
accumulate to higher levels than JAZ10.1 and other labile JAZs in JAZ10.4 showed that JAZ-JAZ binding does not require the
JA-stimulated cells. N-terminal region or the C-terminal Jas domain. Although this
finding supports the notion that the ZIM domain is the primary
The ZIM Domain Mediates Protein–Protein Interaction determinant for intermolecular contact between JAZ proteins, it
remains to be determined whether the ZIM domain is a self-
The ZIM domain is the defining feature of the tify family that stabilizing structure that is sufficient for protein–protein inter-
contains JAZ, PPD, and ZIM/ZML proteins (Vanholme et al., action. The ability of ZIM and ZML proteins to homo- and
142 The Plant Cell

heterodimize in yeast (see Supplemental Figure 2 online) shows stiochiometry of JAZ-JAZ dimers (or higher order oligomers) in
that protein–protein interaction extends to other members of the any given cell type are (1) the rate of JAZ synthesis, (2) the rate of
tify family. This finding raises the possibility that JAZs functionally JAZ degradation, and (3) the interaction capacity of each JAZ
interact with PPD and ZIM/ZML proteins. expressed in that cell type. Inductive cues that increase the
Our results extend the functional analogy between JAZ proteins synthesis of bioactive JAs are predicted to remodel JAZ-JAZ
and auxin/indole-3-acetic acid (Aux/IAA) proteins that are sub- partnering by increasing the rate of de novo JAZ synthesis and
strates for the SCFTIR1 E3 ligase component of the auxin response the selective degradation of unstable JAZs. We suggest that
pathway. JAZ proteins appear to share several key features with complexes containing stabilized JAZs (e.g., JAZ10.4) will accu-
Aux/IAA repressor proteins, including hormone-induced rapid mulate in JA-stimulated cells and, eventually, attenuate expres-
degradation by the SCF/ubiquitin/26S proteasome pathway, in- sion of JA response genes.
teraction with transcription factors in the nucleus, and rapid Ectopic expression of JAZ10.4 likely perturbs the normal
induction of their corresponding genes in response to increased balance of JAZ proteins by creating a situation in which
hormone levels (Chini et al., 2007; Thines et al., 2007; Chico et al., JAZ10.4 predominates over more labile JAZ isoforms. A simple
2008; Chung et al., 2008; Katsir et al., 2008b). A role for the ZIM model to explain the requirement for the TIFY motif in JAZ10.4-
domain in JAZ–JAZ interactions is reminiscent of the ability of Aux/ mediated repression of signal output is that JAZ10.4 homo- or
IAA proteins to homo- and heterodimerize (Kim et al., 1997). heteromeric complexes are the functional unit for direct repres-
Sequence determinants (domains III and IV) that mediate these sion of MYC2. The ability of JAZ10.4 to interact with MYC2
interactions are conserved in AUXIN RESPONSE FACTOR tran- (Figure 4D) in the Y2H system is consistent with this model, as is a
scription factors that control expression of auxin response genes, site-directed mutagenesis study indicating that the COI1 and
which enables Aux/IAAs to bind to and repress AUXIN RE- MYC2 interaction surfaces of JAZ9 are likely not identical
SPONSE FACTOR activity (Kim et al., 1997; Ulmasov et al., (Melotto et al., 2008). This model may also explain the dominant-
1999). Available evidence indicates that the ZIM/TIFY domain is negative action of JAZ1DJas (Thines et al., 2007) and other
not conserved in MYC2 and is not required for MYC2 binding to JAZDJas proteins that have the capacity to form homo- and
JAZ proteins (Chini et al., 2007). Nevertheless, the possibility that heterodimers (Table 1). This scenario, however, would not appear
the ZIM domain mediates JAZ interaction with proteins outside the to explain the repressive action of JAZ3DJas, which was shown
tify family cannot be excluded. to interact with COI1 but not with MYC2 (Chini et al., 2007). These
workers proposed a model in which binding of JAZ3DJas to
A Role for the ZIM Domain in Regulating JAZ Function SCFCOI1 impairs the turnover of endogenous JAZs that would
accumulate and repress MYC2 activity. The necessary biochem-
We took advantage of the strong JA-insensitive phenotype of ical and genetic tools are now available to rigorously test these
35S-JAZ10.4 plants to study the functional significance of the models of JAZDJas action. Further study of naturally occurring
ZIM domain in JA signaling. The main conclusion of these JAZ isoforms that evade degradation by the JA/COI1 pathway
experiments is that mutations (e.g., I107A) in the TIFY motif promises to provide new insight into the molecular mechanisms
that disrupt JAZ10.4 interaction with other JAZs also abrogate underlying the control of JA responses.
the dominant-negative effects of JAZ10.4 on JA signal output.
Importantly, functional analysis of JAZ10.4 TIFY mutants METHODS
(JAZ10.4I->A and JAZ10.4G->A) showed that the capacity of
JAZ10.4 to interact with other JAZs in yeast is tightly correlated Plant Material and Growth Conditions
with the strength of the JAZ10.4-mediated dominant-negative
effect. Because mutation of the TIFY motif did not appear to Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild type
affect the accumulation or nuclear localization of JAZ10.4, the for all experiments. Soil-grown plants were maintained in a growth
chamber at 218C under 16 h light (100 mE m22 s21) and 8 h dark.
most straightforward interpretation of these results is that the
Arabidopsis was transformed with Agrobacterium tumefaciens (strain
repressive activity of JAZ10.4 depends on the ability of its ZIM
C58C1) using the floral dip method (Clough and Bent, 1998). A list of
domain to interact with another protein. Our Y2H analyses transgenic plants used in this study is provided in Supplemental Table
support the hypothesis that JAZ10.4 interacts in planta either 1 online. Transformed lines (T1 generation) were selected on MS plates
with itself or with another tify protein to repress JA signaling. containing kanamycin (50 mg/mL) and vancomycin (100 mg/mL). At least
Interestingly, Ouellet and colleagues (2001) showed that a mu- 40 independent T1 plants per genotype were transferred to soil for
tation (in domain III) that impairs the ability of an Aux/IAA protein subsequent phenotypic analysis. We identified homozygous lines by
to homo- and heterodimerize suppresses auxin-related pheno- testing T3 progeny for resistance to kanamycin. We propagated male-
types conferred by an intragenic mutation that stabilizes the Aux/ sterile 35S-JAZ10.4 lines by outcrossing to wild-type pollen. F1 progeny
IAA against auxin-induced degradation. Thus, the repressive containing the transgene were selected on MS medium plates containing
kanamycin (50 mg/mL). Nicotiana tabacum (cv Petit Havana) was grown in
activity of stabilized JAZ and Aux/IAA variants depends on their
a growth chamber maintained under 17 h of light (200 mE/m22/s) at 288C
ability to interact with other family members or with downstream
and 7 h of dark at 188C.
transcription factors.
Our results raise the possibility that a network of JAZ-JAZ
Y2H Assays
complexes differentially interact with transcription factors to
control the specificity and strength of JA signal output. Among Y2H assays were performed with the Matchmaker LexA system (Clon-
the important factors that would determine the composition and tech). Full-length JAZ cDNAs were isolated as previously described
 Role of the ZIM/TIFY Domain in JA Signaling 143

(Chung et al., 2008). cDNAs encoding JAZ10 splice variants were gen- BiFC Assays
erated from RNA prepared from rosette leaves collected 1.5 h after
The full-length JAZ3 coding sequence was cloned into the pSY728,
mechanical wounding. RT-PCR reactions were performed with Pfu Turbo
pSY738, pSY735, and pSY736 vectors (Bracha-Drori et al., 2004) to
DNA polymerase (Stratagene) and the primer sets listed in Supplemental
generate JAZ3-nYFP, JAZ3-cYFP, nYFP-JAZ3, and cYFP-JAZ3, respec-
Table 1 online. JAZ cDNAs were subcloned into the Matchmaker pGILDA
tively. See Supplemental Table 1 online for primer information. These
vector to generate fusions of the bait protein with the LexA DNA BD.
constructs were subcloned into the XhoI and SalI restriction sites of pBI-
These cDNAs were also subcloned into the pB42AD vector to generate
TS and transformed into Agrobacterium strain C58C1. Mixtures of two
fusions of the prey protein with the B42 AD. Yeast transformation and
Agrobacterium strains were coinfiltrated into tobacco leaves, such that
selection of transformants were conducted as described by Melotto et al.
each of the four possible combinations of nYFP and cYFP JAZ3-fusion
(2008). JAZ–JAZ interaction assays were performed as follows. A 1-mL
constructs was coexpressed. Imaging of protein–protein interaction was
culture of yeast transformants in SD-glucose medium (BD Biosciences)
performed by confocal laser microscopy as described above.
supplemented with -Ura/-His/-Trp dropout solution was grown to an
OD600 of 1.0 to 1.2. Cells were recovered by centrifugation and resus-
pended in 0.4 mL of distilled water. Four microliters of the resulting cell In Vivo Protein Degradation Assay
suspension was placed on SD-galactose/raffinose (-Ura/-His/-Trp)-
Seedlings of transgenic plants expressing JAZ10.1-YFP, JAZ10.3-YFP,
inducing media (in 96-well plates) containing 80 mg/mL of X-gal. To test
and JAZ10.4-YFP fusion proteins grown for 7 d on MS plates were
the interaction of JAZ10 variants with COI1, coronatine (Sigma-Aldrich) was
transferred to a 48-well microtiter plate for JA treatment. Seedlings were
added to the medium before the yeast cells were plated. All photographic
incubated with JA (0, 1, and 100 mM MeJA) for 2 h at room temperature on
images of Y2H plates were taken after 48 h of incubation of the plate at
an orbital shaker with low speed (70 rpm). To test the effect of proteasome
308C. Expression of the AD and BD fusion proteins was detected by
inhibition on protein stability, seedlings expressing JAZ10.1-YFP were
protein gel blot analysis using anti-hemagglutinin (HA; Covance) and anti-
pretreated with water or 50 mM MG132 (Sigma-Aldrich) for 2 h prior to 10
LexA (Upstate) antibodies, respectively. b-Galactosidase activity in yeast
mM MeJA treatment. Fluorescence of JAZ10-YFP fusion proteins was
cells was measured with liquid cultures as described by the manufacturer
analyzed with a Zeiss Axio Scope fluorescence microscope (Carl Zeiss).
(Clontech) using ortho-nitrophenyl-b-D-galactopyranoside as a sub-
Images were taken using the software AxioVision 4.7 provided by the
strate.
manufacturer. All images shown in a single panel in Figure 7 were taken
with the same exposure time and software settings.
Site-Directed Mutagenesis

Ala-scanning mutagenesis of the TIFY motif was performed with Pfu Root Growth Inhibition Assay
Turbo DNA Polymerase according to the instructions provided with the
QuikChange XL site-directed mutagenesis kit (Stratagene). PCR reac- Seeds were surface-sterilized with 30% (v/v) commercial bleach for 15
tions were performed with JAZ3, JAZ10.1, and JAZ10.4 cDNAs in min and washed 10 times with sterile distilled water. Seeds of each
pB42AD or pGEM-T Easy vectors, and the mutagenic primers are listed genotype were placed on square Petri plates (Fisher) containing MS
in Supplemental Table 1 online. The presence of the desired mutation was medium that was supplemented with 50 mM MeJA. Plates were incu-
confirmed by DNA sequencing. bated at 48C for 4 d in darkness and then incubated under normal growth
conditions for the remainder of the experiment. Plates were oriented in a
vertical position 6 to 7 d prior to measurement of primary root length.
Subcellular Protein Localization
Wild-type (Col-0) and coi1-1 seeds (or other appropriate lines) were
The pBI-eYFP binary vector was prepared by subcloning the eYFP coding included as controls for JA-sensitive and JA-resistant phenotypes, re-
sequence into the KpnI and SacI restriction sites of the pBI-TS binary spectively.
vector (Koo et al., 2006; Schilmiller et al., 2007), which is a modified
version of pBI121. The PCR-amplified open reading frames (without a
Histochemical Staining
stop codon) of JAZ10.1, JAZ10.3, JAZ10.4, and JAZ10.4I->A were
subcloned into pGEM-T Easy (Promega), followed by recloning into the Seedlings grown on MS medium as described in the legend to Figure 9
BamHI site of pBI-eYFP. The resulting constructs encode fusion proteins were transferred to a 48-well microtiter plate for GUS staining. Seedlings
in which the JAZ C terminus is fused in frame to the N terminus of eYFP. were vacuum-infiltrated with GUS staining buffer (50 mM KPO4, pH 7.2, 2
Oligonucleotide primers used to generate these constructs are listed in mM potassium ferricyanide, 2 mM potassium ferrocyanide, 0.1% [v/v]
Supplemental Table 1 online. Agrobacterium (strain C58C1) harboring Triton X-100, and 2 mM X-Gluc) for 15 min and then incubated for 12 h at
binary vector constructs were grown overnight at 288C in YEP medium 378C. A graded series of ethanol washes (up to 70% ethanol) was used to
containing 50 mg/mL kanamycin and 50 mg/mL rifampicin. Bacterial cells remove chlorophyll, as described previously (Schilmiller et al., 2007).
were resuspended in induction medium (10 mM MES, pH 5.6, 10 mM Photographic images were taken with a Leica MZ16 dissecting micro-
MgCl2, and 150 mM acetosyringone) and incubated for 1.5 h at room scope. Strong GUS staining was also observed in several (at least five per
temperature prior to infiltration. Leaves of 5-week-old Nicotiana tabacum construct) independent 35S-JAZ10.4-GUS and 35S-JAZ10.4I->A-GUS
plants were syringe-infiltrated with the bacterial suspension (OD600 = 0.2), lines within 2 to 3 h of incubation with the X-Gluc substrate.
and plants were maintained in a growth chamber for 2 d. Confocal
microscopy was performed with a Zeiss LSM 510 META microscope (Carl
Accession Numbers
Zeiss) and imaging software provided by the manufacturer. To visualize
nuclei, tobacco leaves that were previously infected with Agrobacterium The nucleotide sequence of At5g13220.4 encoding JAZ10.4 has been
were syringe-infiltrated 2 h prior to imaging with a solution containing 10 deposited in GenBank under accession number FJ417331. Arabidopsis
mg/mL DAPI (Sigma-Aldrich). YFP and DAPI fluorescence were moni- Genome Initiative numbers for genes described in this article are as
tored simultaneously by excitation at 514 nm (argon laser) and 405 nm follows: COI1 (At2g39940), JAZ1 (At1g19180), JAZ2 (At1g74950), JAZ3
(diode laser), respectively. YFP and DAPI fluorescence was detected after (At3g17860), JAZ4 (At1g48500), JAZ5 (At1g17380), JAZ6 (At1g72450),
passage through band-pass 530- to 600-nm and 474- to 525-nm emis- JAZ7 (At2g34600), JAZ8 (At1g30135), JAZ9 (At1g70700), JAZ10
sion filters, respectively. (At5g13220), JAZ11 (At3g43440), JAZ12 (At5g20900), MYC2
144 The Plant Cell

(At1g32640), ZIM (At4g24470), ZML1 (At3g21175), and ZML2 insensitive219 is involved in phytochrome A-mediated signaling in
(At1g51600). Arabidopsis. Plant Physiol. 143: 1189–1202.
Chico, J.M., Chini, A., Fonseca, S., and Solano, R. (2008). JAZ
Supplemental Data repressors set the rhythm in jasmonate signaling. Curr. Opin. Plant
Biol. 11: 486–494.
The following materials are available in the online version of this article. Chini, A., Fonseca, S., Fernandez, G., Adie, B., Chico, J.M., Lorenzo,
Supplemental Figure 1. Protein Gel Blot Analysis of JAZ3 and O., Garcia-Casado, G., Lopez-Vidriero, I., Lozano, F.M., Ponce, M.
JAZ10.1 Proteins with Point Mutations in the TIFY Motif. R., Micol, J.L., and Solano, R. (2007). The JAZ family of repressors is
the missing link in jasmonate signalling. Nature 448: 666–671.
Supplemental Figure 2. Y2H Analysis of Homo- and Heteromeric
Chung, H.S., Koo, A.J.K., Gao, X., Jayany, S., Thines, B., Jones, A.D.,
Interactions between Arabidopsis ZIM, ZIM-Like1 (ZML1), and ZML2
and Howe, G.A. (2008). Regulation and function of Arabidopsis
Proteins.
JASMONATE ZIM-domain genes in response to wounding and her-
Supplemental Figure 3. Detection of Transcripts Encoding JAZ10.3 bivory. Plant Physiol. 146: 952–964.
and JAZ10.4 Using Transcript-Specific Primers. Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for
Supplemental Figure 4. Yeast Two-Hybrid Analysis of the Effect of Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
I107A and G111A TIFY Mutations on the Interaction of JAZ10.4 with J. 16: 735–743.
Other Members of the JAZ Family. Devoto, A., Ellis, C., Magusin, A., Chang, H.S., Chilcott, C., Zhu, T.,
and Turner, J.G. (2005). Expression profiling reveals COI1 to be a key
Supplemental Table 1. List of Plasmid Constructs and Primers Used regulator of genes involved in wound- and methyl jasmonate-induced
in This Study. secondary metabolism, defence, and hormone interactions. Plant
Mol. Biol. 58: 497–513.
Dombrecht, B., Xue, G.P., Sprague, S.J., Kirkegaard, J.A., Ross,
ACKNOWLEDGMENTS
J.J., Reid, J.B., Fitt, G.P., Sewelam, N., Schenk, P.M., Manners,
We thank Maeli Melotto and Sheng Yang He (Michigan State University) J.M., and Kazan, K. (2007). MYC2 differentially modulates diverse
for assistance with Y2H experiments and for providing JAZ1, JAZ9, and jasmonate-dependent functions in Arabidopsis. Plant Cell 19: 2225–2245.
COI1 clones in yeast vectors. We also thank Chris Bergum (Michigan Feys, B., Benedetti, C.E., Penfold, C.N., and Turner, J.G. (1994).
State University) for technical assistance and John Browse (Washington Arabidopsis mutants selected for resistance to the phytotoxin coro-
State University) for providing ZIM and ZML cDNA clones. Plasmids natine are male sterile, insensitive to methyl jasmonate, and resistant
used for BiFC assays were obtained from the ABRC. This research was to a bacterial pathogen. Plant Cell 6: 751–759.
supported by the National Institutes of Health Grant R01GM57795 and Glazebrook, J. (2005). Contrasting mechanisms of defense against
by the U.S. Department of Energy Grant DE-FG02-91ER20021. biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43:
205–227.
Goossens, A., Hakkinen, S.T., Laakso, I., Seppanen-Laakso, T.,
Received October 29, 2008; revised December 22, 2008; accepted Biondi, S., De Sutter, V., Lammertyn, F., Nuutila, A.M., Soderlund,
January 2, 2009; published January 16, 2009. H., Zabeau, M., Inze, D., and Oksman-Caldentey, K.M. (2003). A
functional genomics approach toward the understanding of second-
ary metabolism in plant cells. Proc. Natl. Acad. Sci. USA 100: 8595–
8600.
REFERENCES
Herms, D.A., and Mattson, W.J. (1992). The dilemma of plants - To
Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K., and Yamaguchi- grow or defend. Q. Rev. Biol. 67: 283–335.
Shinozaki, K. (2003). Arabidopsis AtMYC2 (bHLH) and AtMYB2 Howe, G., and Jander, G. (2008). Plant immunity to insect herbivores.
(MYB) function as transcriptional activators in abscisic acid signaling. Annu. Rev. Plant Biol. 59: 41–66.
Plant Cell 15: 63–78. Ishiguro, S., Kawai-Oda, A., Ueda, J., Nishida, I., and Okada, K.
Balbi, V., and Devoto, A. (2008). Jasmonate signalling network in (2001). The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a
Arabidopsis thaliana: Crucial regulatory nodes and new physiological novel phospholipase A1 catalyzing the initial step of jasmonic acid
scenarios. New Phytol. 177: 301–318. biosynthesis, which synchronizes pollen maturation, anther dehis-
Barbazuk, W.B., Fu, Y., and McGinnis, K.M. (2008). Genome-wide cence, and flower opening in Arabidopsis. Plant Cell 13: 2191–2209.
analyses of alternative splicing in plants: Opportunities and chal- Izaguirre, M.M., Mazza, C.A., Biondini, M., Baldwin, I.T., and Ballaré,
lenges. Genome Res. 18: 1381–1392. C.L. (2006). Remote sensing of future competitors: Impacts on plant
Bove, J., Kim, C.Y., Gibson, C.A., and Assmann, S.M. (2008). Char- defenses. Proc. Natl. Acad. Sci. USA 103: 7170–7174.
acterization of wound-responsive RNA-binding proteins and their Katsir, L., Chung, H.S., Koo, A.J.K., and Howe, G.A. (2008a).
splice variants in Arabidopsis. Plant Mol. Biol. 67: 71–88. Jasmonate signaling: a conserved mechanism of hormone sensing.
Bracha-Drori, K., Shichrur, K., Katz, A., Oliva, M., Angelovici, R., Curr. Opin. Plant Biol. 11: 428–435.
Yalovsky, S., and Ohad, N. (2004). Detection of protein-protein Katsir, L., Schilmiller, A.L., Staswick, P.E., He, S.Y., and Howe, G.A.
interactions in plants using bimolecular fluorescence complementa- (2008b). COI1 is a critical component of a receptor for jasmonate and
tion. Plant J. 40: 419–427. the bacterial virulence factor coronatine. Proc. Natl. Acad. Sci. USA
Browse, J. (2005). Jasmonate: An oxylipin signal with many roles in 105: 7100–7105.
plants. Vitam. Horm. 72: 431–456. Kim, J., Harter, K., and Theologis, A. (1997). Protein-protein interac-
Browse, J., and Howe, G.A. (2008). Update on jasmonate signaling: tion among the Aux/IAA proteins. Proc. Natl. Acad. Sci. USA 94:
New weapons and a rapid response against insect attack. Plant 11786–11791.
Physiol. 146: 832–838. Klemm, J.D., Schreiber, S.L., and Crabtree, G.R. (1998). Dimerization
Chen, I.C., Huang, I.C., Liu, M.J., Wang, Z.G., Chung, S.S., and as a regulatory mechanism in signal transduction. Annu. Rev. Immu-
Hsieh, H.L. (2007). Glutathione S-transferase interacting with far-red nol. 16: 569–592.
 Role of the ZIM/TIFY Domain in JA Signaling 145

Koo, A.J.K., Chung, H.S., Kobayashi, Y., and Howe, G.A. (2006). Sasaki-Sekimoto, Y., et al. (2005). Coordinated activation of meta-
Identification of a peroxisomal acyl-activating enzyme involved in the bolic pathways for antioxidants and defence compounds by jas-
biosynthesis of jasmonic acid in arabidopsis. J. Biol. Chem. 281: monates and their roles in stress tolerance in Arabidopsis. Plant J. 44:
33511–33520. 653–668.
Laurie-Berry, N., Joardar, V., Street, I.H., and Kunkel, B.N. (2006). Schenk, P.M., Kazan, K., Wilson, I., Anderson, J.P., Richmond, T.,
The Arabidopsis thaliana JASMONATE INSENSITIVE1 gene is re- Somerville, S.C., and Manners, J.M. (2000). Coordinated plant
quired for suppression of salicylic acid-dependent defenses during defense responses in Arabidopsis revealed by microarray analysis.
infection by Pseudomonas syringae. Mol. Plant Microbe Interact. 19: Proc. Natl. Acad. Sci. USA 97: 11655–11660.
789–800. Schilmiller, A.L., Koo, A.J., and Howe, G.A. (2007). Functional diver-
Li, L., Zhao, Y., McCaig, B.C., Wingerd, B.A., Wang, J., Whalon, M.E., sification of acyl-CoA oxidases in jasmonic acid biosynthesis and
Pichersky, E., and Howe, G.A. (2004). The tomato homolog of action. Plant Physiol. 143: 812–824.
CORONATINE-INSENSITIVE1 is required for the maternal control of Schmidt, D.D., and Baldwin, I.T. (2006). Transcriptional responses of
seed maturation, jasmonate-signaled defense responses, and glan- Solanum nigrum to methyl jasmonate and competition: A glasshouse
dular trichome development. Plant Cell 16: 126–143. and field study. Funct. Ecol. 20: 500–508.
Lorenzo, O., Chico, J.M., Sanchez-Serrano, J.J., and Solano, R. Shikata, M., Matsuda, Y., Ando, K., Nishii, A., Takemura, M., Yokota,
(2004). JASMONATE-INSENSITIVE1 encodes a MYC transcription A., and Kohchi, T. (2004). Characterization of Arabidopsis ZIM, a
factor essential to discriminate between different jasmonate-regulated member of a novel plant-specific GATA factor gene family. J. Exp.
defense responses in Arabidopsis. Plant Cell 16: 1938–1950. Bot. 55: 631–639.
Mandaokar, A., Thines, B., Shin, B., Lange, B.M., Choi, G., Koo, Y.J., Shoji, T., Ogawa, T., and Hashimoto, T. (2008). Jasmonate-induced
Yoo, Y.J., Choi, Y.D., Choi, G., and Browse, J. (2006). Transcrip- nicotine formation in tobacco is mediated by tobacco COI1 and JAZ
tional regulators of stamen development in Arabidopsis identified by genes. Plant Cell Physiol. 49: 1003–1012.
transcriptional profiling. Plant J. 46: 984–1008. Sineshchekov, V.A., Loskovich, A.V., Riemann, M., and Nick, P.
McConn, M., and Browse, J. (1996). The critical requirement for (2004). The jasmonate-free rice mutant hebiba is affected in the
linolenic acid is pollen development, not photosynthesis, in an response of phyA’/phyA’’ pools and protochlorophyllide biosynthesis
Arabidopsis mutant. Plant Cell 8: 403–416. to far-red light. Photochem. Photobiol. Sci. 3: 1058–1062.
Melotto, M., Mecey, C., Niu, Y., Chung, H.S., Katsir, L., Yao, J., Zeng, Stintzi, A., and Browse, J. (2000). The Arabidopsis male-sterile mutant,
W., Thines, B., Staswick, P.E., Browse, J., Howe, G.A., and He, S. opr3, lacks the 12-oxophytodienoic acid reductase required for
Y. (2008). A critical role of two positively charged amino acids in the jasmonate synthesis. Proc. Natl. Acad. Sci. USA 97: 10625–10630.
Jas motif of Arabidopsis JAZ proteins in mediating coronatine- and Suzuki, H., Reddy, M.S., Naoumkina, M., Aziz, N., May, G.D.,
jasmonoyl isoleucine-dependent interactions with the COI1 F-box Huhman, D.V., Sumner, L.W., Blount, J.W., Mendes, P., and Dixon,
protein. Plant J. 55: 979–988. R.A. (2005). Methyl jasmonate and yeast elicitor induce differential
Mitra, S., and Baldwin, I.T. (2008). Independently silencing two pho- transcriptional and metabolic re-programming in cell suspension
tosynthetic proteins in Nicotiana attenuata has different effects on cultures of the model legume Medicago truncatula. Planta 220:
herbivore resistance. Plant Physiol. 148: 1128–1138. 696–707.
Ouellet, F., Overvoorde, P.J., and Theologis, A. (2001). IAA17/AXR3: Takahashi, F., Yoshida, R., Ichimura, K., Mizoguchi, T., Seo, S.,
Biochemical insight into an auxin mutant phenotype. Plant Cell 13: Yonezawa, M., Maruyama, K., Yamaguchi-Shinozaki, K., and
829–841. Shinozaki, K. (2007). The mitogen-activated protein kinase cascade
Park, J.H., Halitschke, R., Kim, H.B., Baldwin, I.T., Feldmann, K.A., MKK3-MPK6 is an important part of the jasmonate signal transduction
and Feyereisen, R. (2002). A knock-out mutation in allene oxide pathway in Arabidopsis. Plant Cell 19: 805–818.
synthase results in male sterility and defective wound signal trans- Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G.,
duction in Arabidopsis due to a block in jasmonic acid biosynthesis. Nomura, K., He, S.Y., Howe, G.A., and Browse, J. (2007). JAZ
Plant J. 31: 1–12. repressor proteins are targets of the SCFCOI1 complex during jas-
Pauwels, L., Morreel, K., De Witte, E., Lammertyn, F., Van Montagu, monate signalling. Nature 448: 661–665.
M., Boerjan, W., Inze, D., and Goossens, A. (2008). Mapping methyl Ulmasov, T., Hagen, G., and Guilfoyle, T.J. (1999). Dimerization and
jasmonate-mediated transcriptional reprogramming of metabolism DNA binding of auxin response factors. Plant J. 19: 309–319.
and cell cycle progression in cultured Arabidopsis cells. Proc. Natl. Uppalapati, S.R., Ayoubi, P., Weng, H., Palmer, D.A., Mitchell, R.E.,
Acad. Sci. USA 105: 1380–1385. Jones, W., and Bender, C.L. (2005). The phytotoxin coronatine and
Pozo, M.J., Van Loon, L.C., and Pieterse, C.M.J. (2004). Jasmonates - methyl jasmonate impact multiple phytohormone pathways in tomato.
Signals in plant-microbe interactions. J. Plant Growth Regul. 23: 211–222. Plant J. 42: 201–217.
Reddy, A.S.N. (2007). Alternative splicing of pre-messenger RNAs in Vanholme, B., Grunewald, W., Bateman, A., Kohchi, T., and
plants in the genomic era. Annu. Rev. Plant Biol. 58: 267–294. Gheysen, G. (2007). The tify family previously known as ZIM. Trends
Reymond, P., Bodenhausen, N., Van Poecke, R.M., Krishnamurthy, Plant Sci. 12: 239–244.
V., Dicke, M., and Farmer, E.E. (2004). A conserved transcript White, D.W.R. (2006). PEAPOD regulates lamina size and curvature in
pattern in response to a specialist and a generalist herbivore. Plant Arabidopsis. Proc. Natl. Acad. Sci. USA 103: 13238–13243.
Cell 16: 3132–3147. Xie, D.X., Feys, B.F., James, S., Nieto-Rostro, M., and Turner, J.G.
Sanders, P.M., Lee, P.Y., Biesgen, C., Boone, J.D., Beals, T.P., (1998). COI1: An Arabidopsis gene required for jasmonate-regulated
Weiler, E.W., and Goldberg, R.B. (2000). The Arabidopsis DELAYED defense and fertility. Science 280: 1091–1094.
DEHISCENCE1 gene encodes an enzyme in the jasmonic acid syn- Yan, Y., Stolz, S., Chetelat, A., Reymond, P., Pagni, M., Dubugnon,
thesis pathway. Plant Cell 12: 1041–1061. L., and Farmer, E.E. (2007). A downstream mediator in the growth
Sasaki, Y., et al. (2001). Monitoring of methyl jasmonate-responsive repression limb of the jasmonate pathway. Plant Cell 19: 2470–2483.
genes in Arabidopsis by cDNA macroarray: Self-activation of jasmonic Zhang, Y.I., and Turner, J.G. (2008). Wound-induced endogenous
acid biosynthesis and crosstalk with other phytohormone signaling jasmonates stunt plant growth by inhibiting mitosis. PLoS ONE:
pathways. DNA Res. 8: 153–161. e3699.