THE ROLE OF ALTERNATIVE SPLICING IN THE REGULATION OF JASMONATE
SIGNALING
By
Lalita Chimanlal Patel

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Cell and Molecular Biology Program
2011

ABSTRACT
THE ROLE OF ALTERNATIVE SPLICING IN THE REGULATION OF JASMONATE
SIGNALING
By
Lalita Chimanlal Patel

The plant hormone jasmonate (JA) is studied primarily for its role in plant defense. In
response to attack by pathogens and herbivores, the JA-amino acid conjugate JA-isoleucine (JAIle) accumulates and promotes the association of the E3 ubiquitin ligase SCF

COI1

with its

substrates, the Jasmonate-ZIM domain (JAZ) repressor proteins. JAZ proteins bind the F-box
protein COI1 in a hormone-dependent manner and are targeted for ubiquitination and
degradation by the 26S proteasome pathway. JAZ degradation releases transcription factors from
repression, thereby promoting transcription of JA-responsive genes. Among the earliest response
genes to be transcribed are the JAZ genes themselves. Many JAZ genes are subject to alternative
splicing events that reduce the ability of JAZ proteins to interact with COI1 in a hormonedependent manner. These truncated JAZ proteins are hypothesized to attenuate JA responses. In
this study, JAZ transcripts were quantified in response to JA elicitation and in various tissues.
The results show that the relative abundance of JAZ splice variants is not significantly affected
by JA elicitation or tissue type, thus suggesting that alternative splicing of JAZ pre-mRNA is
controlled by the efficiency with which cis-acting splice sites are selected by the spliceosome.
Additionally, transgenic plants ectopically expressing cDNAs encoding truncated splice variants
of JAZ2 and JAZ4 have reduced sensitivity to JA, indicating that these proteins act as dominant
repressors of JA signaling. This research highlights the importance of alternative splicing as an
adaptive response of plants to environmental stress.

ACKNOWLEDGEMENTS

First and foremost, I would like to express my sincere gratitude to my advisor, Dr. Gregg
Howe. Throughout my years of working in his lab, I have come to truly admire and respect his
extraordinary abilities as a scientist, a professor, and a mentor. His continuous support and
encouragement have been critical to my successes and my development as a scientist.
I would also like to thank members of my thesis committee: Dr. David Arnosti, Dr. Eva
Farre, Dr. Beronda Montgomery-Kaguri, and Dr. Curtis Wilkerson, for their critical insight and
their understanding.
My unforgettable experience in the Howe lab was largely attributed to the interaction and
collaboration among members of the lab. First, I would like to thank a former graduate student,
Dr. Hoo Sun Chung, for providing much of the preliminary work on JAZ10. Second, I would like
to thank Dr. Marco Herde for his invaluable assistance in deciphering the results of our RNA-seq
data. I am indebted to Dr. Javier Moreno for his assistance in designing and troubleshooting
cloning projects. I would like to thank Dr. Yuki Yoshida for his preliminary analysis of the
jaz10-1 root length phenotype. I would like to thank Dr. Abe Koo, Dr. Jin Ho Kang, and
Christine Shyu for their advice, cooperation, and constructive criticism. Finally, I would like to
thank my beloved comrades, Dr. Ian Major and Marcelo Campos, for their unlimited support,
wholehearted sympathy, and friendship.
I am extremely grateful to my family and friends for their compassion and assistance
throughout my graduate career. Their constant care and appreciation is the basis for all of my
achievement.

iii

TABLE OF CONTENTS
LIST OF TABLES …………………………………………………………………………..……v
LIST OF FIGURES ……………………………………………………………………………...vi
LIST OF ABBREVIATIONS ………………………………………………………………......vii
INTRODUCTION ………………………………………………………………………………..1
JA Biosynthesis and Metabolism…………………………………………………………2
The Role of the Ubiquitin-Proteasome Pathway in JA Signaling……………...……....…8
Jasmonate-ZIM Domain (JAZ) Proteins………………………...………………….……10
JA Perception ……………………………………………………………………………11
Mechanism of Transcriptional Repression by JAZ Proteins …………………...……….12
JAZ-Interacting Transcription Factors …………………………………………………..13
Alternative Splicing in the Arabidopsis Genome ………………………………….....…15
Alternative Splicing of JAZ Genes ………………………………………………...……16
The Role of JA in Fertility ………………………………………………………………19
The Role of JAZ Proteins in the Integration of Other Hormone Signaling Pathways.…..20
JA-Auxin…………………………………………………………………………20
JA-GA…………………......……………………………………………….…….21
JA-Ethylene………………………………………………………………………21
JA-Phytochrome …………………...……………………………………………22
RESULTS ……………………………………………………………………………………….24
Regulation of JAZ Alternative Splicing …………………………………………………24
Alternative Splicing of JAZ Genes in Flowers and Roots ………………………………30
Detection of JAZ Alternative Splice Variants Using RNA Sequencing Data …………..36
Ectopic Expression of JAZ2.2 and JAZ4.2 Results in Decreased Sensitivity to JA.........40
DISCUSSION ………………………………………………………………………………...…45
MATERIAL AND METHODS …………………………………………………………………53
REFERENCES …………………………………………………………………………….……57

iv

LIST OF TABLES

Table 1. Evidence of JAZ Gene Induction and Jas Intron Retention in RNA-seq Data…………39
Table 2. qPCR Primers for the Quantification of JAZ Alternative Splice Variants………….….51

v

LIST OF FIGURES

Figure 1. The JA Signaling Pathway………………………………………………...……………3
Figure 2. The JA Biosynthetic Pathway…………………………………………………………..5
Figure 3. Gene Models of JAZ10 Alternative Splice Variants…………………….…………….17
Figure 4. Levels of JAZ10 Alternatively Spliced Transcripts in MeJA-Treated Seedlings……..25
Figure 5. Levels of JAZ Alternatively Spliced Transcripts in Wounded Leaves………...………28
Figure 6. Levels of JAZ10 Alternatively Spliced Transcripts in WT and 35S-JAZ10G Flowers..31
Figure 7. Levels of JAZ2 and JAZ6 Alternatively Spliced Transcripts in WT and 35S-JAZ10G
Flowers……………………………………………………………..…………………………....32
Figure 8. Quantification of JAZ10 Alternatively Spliced Transcripts in WT and 35S-JAZ10G
Roots…………..…………………………………………………………………………………34
Figure 9. Quantification of JAZ2 and JAZ6 Alternatively Spliced Transcripts in WT and 35SJAZ10G Roots…………………………………...……………………………………………….35
Figure 10. Evidence of JAZ10 Alternatively Spliced Transcripts in RNA-seq Data…………….37
Figure 11. Intron/Exon Structure for Select JAZ Genes Containing the Jas Intron…………….. 41
Figure 12. The MeJA-Induced Root Growth Phenotype of 35S-JAZ2.2 Seedlings is Similar to
That of 35S-JAZ10.3 Seedlings…………………...……………………………………………...43
Figure 13. MeJA-Induced Root Growth Phenotype of 35S-JAZ4.2…..…………………...…….44

vi

LIST OF ABBREVIATIONS

35S-JAZ10G
AFP
bHLH
BiFC
COI1
Col-0
COR
EAR
EMS
GA
GFP
GUS
JA
JAR1
JAZ
JMT
LRR
MeJA
NINJA
NPR1
OPCL1
OPDA
OPR3
PAMP
PTC
PTI
qPCR
RPKM
SA
SCF
TAP
TIR1
TPL
YFP

35S-JAZ10-genomic
ABI-FIVE BINDING PROTEIN
basic helix loop helix
Bimolecular fluorescent complementation assays
Coronatine Insensitive-1
Arabidopsis thaliana ecotype Columbia-0
Coronatine
ETHYLENE RESPONSIVE FACTOR-associated amphiphilic repression
ethyl methanesulfonate
Gibberellic acid
Green Fluorescent Protein
β-Glucuronidase
Jasmonic Acid
JASMONATE RESISTANT 1
Jasmonate-ZIM domain
JASMONIC ACID CARBOXYLMETHYLTRANSFERASE
Leucine-rich repeat
Methyl Jasmonate
Novel Interactor of JAZ
NONEXPRESSOR of PR GENES1
OPC-8:0 Co-A LIGASE 1
12-oxo-phytodienoic acid
12-OXOPHYTODIENOIC ACID REDUCTASE
Pathogen associated molecular patterns
Premature Termination Codon
Pathogen triggered immunity
Quantitative reverse-transcriptase polymerase chain reaction
Reads per kilobase pair per million mapped reads of model
Salicylic Acid
Skp/Cullin/F-box protein complex
Tandem Affinity Purification tag
Transport Inhibitor Response 1
TOPLESS
Yellow Fluorescent Protein

vii

INTRODUCTION

As sessile organisms, plants have evolved complex yet efficient means of responding to a
variety of abiotic and biotic stresses to optimize their fitness. Plants are routinely exposed to
stress, which often requires significant remodeling of transcriptional and metabolic networks.
Upon pathogen infection or herbivory, plants activate a diverse array of direct and indirect
defense responses, which are often adapted to specific pathogens or pests (Howe and Jander,
2008). Direct defense responses include production of anti-nutritive enzymes and toxic
secondary metabolites (Wittstock and Burow, 2010; Gonzales-Vigil et al., 2011). Some of the
best studied defensive enzymes, protease inhibitors, are effective in the insect midgut to directly
prevent nutrient accessibility and digestion (Ryan, 1990). Indirect defenses include the release of
volatile compounds that attract nearby predators of the attacking herbivore (Herde et al., 2008).
Although defensive mechanisms vary amongst plant species, a number of these defense
responses are inducible and regulated by the plant hormone jasmonate (JA).
In unperturbed tissue, JA regulates a variety of physiological processes such as cell division,
carbon partitioning, photomorphogenesis, as well as the development of reproductive tissues,
trichomes and vascular cambium (McConn and Browse, 1996; Creelman and Mullet, 1997; Li et
al., 2004; Feys et al., 1994; Yoshida et al., 2009; Sehr et al., 2010). Plants also utilize the JA
signaling pathway to respond to abiotic stresses such as ozone, ultraviolet irradiation, and high
salinity (Conconi et al., 1996; Rao et al., 2000; Glazebrook, 2005; Browse and Howe, 2008) JA
is primarily studied for its involvement in defense responses (Conconi et al., 1996; Rao et al.,
2000; Glazebrook, 2005; Browse and Howe, 2008; Howe and Jander, 2008; Acosta and Farmer,
2010).

1

A key aspect of the JA signaling pathway is the regulation of transcriptional repressors by
the Skp1-Cul1-Fbox (SCF)-ubiquitin-proteasome pathway in response to environmental stimuli
(Figure 1) (Howe, 2010). During optimal growth conditions, levels of bioactive JA-isoleucine
conjugate (JA-Ile) are low, and Jasmonate-ZIM domain (JAZ) proteins suppress defense
responses through repressive interaction with transcription factors such as MYC2, MYC3 and
MYC4 (Lorenzo et al., 2004; Chini et al., 2007; Fernandez-Calvo et al., 2011). Under these
conditions, JAZ proteins also interact with Novel Interactor of JAZ (NINJA), which recruits the
corepresseor TOPLESS, creating a multi-protein, repressor complex (Pauwels et al., 2010;
Pauwels et al., 2011). Upon pathogen or insect attack, JA-Ile is massively produced and enables
the interaction of SCF
COI1

SCF

COI1

and the JAZ proteins (Koo et al., 2009; Sheard et al., 2010). The

complex acts as an E3 ubiquitin ligase that targets JAZ transcriptional repressors for

degradation by the 26S proteasome (Chini et al., 2007; Thines et al., 2007; Katsir et al., 2008a;
Chung and Howe, 2009). The degradation of JAZ proteins allows the transcription factors to
promote expression of JA responsive genes. Further studies of JA signal transduction will reveal
its role in balancing growth and defense, and uncover basic principles of hormone action in
plants.

JA Biosynthesis and Metabolism

Mechanical wounding and herbivory stimulate the biosynthesis and accumulation of JA
(Reymond et al., 2000; Koo et al., 2009). JA biosynthesis begins with the release of α-linolenic
acid from the plastid membrane through the action of phospholipases and fatty acid desaturases

2

Figure 1. The JA Signaling Pathway. (A) During optimal growth conditions, levels of
bioactive JA-Ile are low and JAZ proteins (domain 1- D1, ZIM- Z, and Jas- J) suppress defense
responses. The Jas domain (J) of JAZ proteins interact with transcription factors such as MYC2
(activation domain-AD, binding domain- BD). JAZ proteins also interact with NINJA through
the ZIM domain (Z), which recruits the corepresseor TOPLESS, creating a multi-protein
repressor complex. (B) Upon wounding or pathogen attack, JA-Ile is massively produced and
enables the interaction of COI1 and the JAZ proteins. The SCF

COI1

complex acts as an E3

ubiquitin ligase that targets JAZ transcriptional repressors for degradation by the 26S
proteasome. The degradation of JAZ proteins allows MYC2 and RNA polymerase (RNAP) to
promote expression of JA responsive genes, including JAZ genes themselves. Many JAZ genes
produce alternative splice variants in which the Jas motif is removed or truncated (J with dotted
lines). (C) JAZ splice variants have reduced affinity for COI1 and thus are more stable in the
presence of high levels of JA-Ile. As a consequence, JAZ splice variants continue to repress
MYC2 in the presence of high JA-Ile levels as part of a negative feedback mechanism to
attenuate JA responses. For interpretation of the references to color in this and all other figures,
the reader is referred to the electronic version of this thesis.

3

FIGURE 1 (cont’d)

Figure 1. The JA Signaling Pathway. Modified from Howe (2010).

4

Figure 2. The JA Biosynthetic Pathway. Wounding and other stress-related conditions activate
the biosynthetic pathway for the production of JA-Ile. The process begins with the release of
linolenic acid from the chloroplast membrane, which is further metabolized by the chloroplastresiding enzymes LOX, AOS and AOC to form OPDA. OPDA is transported from the
chloroplast to the peroxisome through the ATP-binding cassette (ABC) COMATOSE
transporter, but small amounts may also be transported passively. Within the peroxisome, OPDA
is reduced by OPR3 and conjugated to CoA by OPCL1. Three cycles of β-oxidation yield
jasmonic acid (JA). Conjugation of JA to isoleucine by JAR1 yields the bioactive hormone, (3R,
7S)-JA-Ile.

5

FIGURE 2 (cont’d)

Figure 2. The JA Biosynthetic Pathway. Modified from Koo and Howe (2007).

6

(Figure 2) (McConn and Browse, 1996; Ishiguro et al., 2001). Linolenic acid is further
metabolized by the chloroplast-residing enzymes lipoxygenase (LOX), allene oxide synthase
(AOS) and allene oxide cyclase (AOC) to form oxo-phytodienoic acid (OPDA). OPDA is
transported from the chloroplast to the peroxisome through an ATP-binding cassette transporter
COMATOSE, but small amounts may also be transported passively (Theodoulou et al., 2005).
Once OPDA is in the peroxisome, it is reduced by 12-OXOPHYTODIENOIC ACID
REDUCTASE (OPR3), and conjugated to CoA by OPC-8:0 Co-A LIGASE 1 (OPCL1) for three
cycles of β-oxidation yielding JA (Stintzi and Browse, 2000; Koo et al., 2006; Schaller and
Stintzi, 2009). In plant tissues, JA occurs as the (3R,7S) stereoisomers. This isomer can be
converted to the more thermodynamically stable (3R,7R) isomer during extraction from plant
tissues (Creelman and Mullet 1997; Fonseca et al., 2009).
JA can be further metabolized and conjugated by various enzymes. Methylation of JA occurs
through the action of JASMONIC ACID CARBOXYLMETHYLTRANSFERASE (JMT).
Transgenic plants ectopically expressing JMT have increased expression of JA response genes
and enhanced resistance to B. cinerea infection (Seo et al., 2001). Ectopic expression of JMT
results in increased production of MeJA in Arabidopsis and tobacco (Seo et al., 2001; Stitz et al.,
2011). JMT is expressed in rosette leaves, cauline leaves, and flowers, and is induced in local
and systemic tissues upon wounding or exogenous MeJA treatment (Seo et al., 2001).
Additionally, ectopic expression of Arabidopsis JMT in Nicotiana attenuata suggested a
metabolic source-sink relationship in regulating the production and conjugation of JA (Stitz et
al., 2011). This study also provided evidence that JA metabolism is regulated in a tissue-specific
manner.

7

Another important enzyme in the JA biosynthesis pathway is JASMONATE RESISTANT 1
(JAR1), which conjugates (3R,7S)-JA to isoleucine (Staswick and Tiryaki, 2004) to yield (3R,
7S)-JA-Ile, which is the bioactive hormone (Fonseca et al., 2009). The jar1 mutant shows JA
insensitivity in roots and increased susceptibility to the soil fungus Pythium irregulare (Staswick
et al, 1998). The jar1 mutant also has reduced levels of JA-Ile (Staswick and Tiryaki, 2004).
Silencing of JAR4, the homolog of JAR1 in Nicotiana attenuata, generated plants with increased
susceptibility to herbivory by Manduca sexta larvae and this effect could be restored to wild-type
levels by supplementation of JA-Ile (Kang et al., 2006). Clearly, impaired production of JA-Ile
affects JA-mediated defense responses, supporting its role as the bioactive hormone.
Recent research has implicated the cytochrome P450 CYP94B3 and closely related members
of the family in the inactivation of JA-Ile by 12-hydroxylation. A T-DNA insertion mutant
(cyp94b3) hyper-accumulates JA-Ile, has reduced levels of 12-hydroxy-JA-Ile, and increased
expression of JA responsive genes. Ectopic expression of CYP94B3 causes male sterility, root
insensitivity to JA and increased susceptibility to insect feeding (Koo et al., 2011). Thus,
CYP94B3 likely plays a role in the attenuation of JA responses by reducing levels of available
bioactive JA-Ile.

The Role of the Ubiquitin-Proteasome Pathway in JA Signaling

Plant hormones utilize the ubiquitin-proteasome pathway as an essential part of hormone
perception and signaling. Until a receptor was identified, the mechanism by which JA affected
downstream changes in gene expression remained elusive. The bacterial phytotoxin coronatine
(COR), which is produced by the plant pathogen Pseudomonas syringae, is a structural mimic of

8

(3R,7S)-JA-Ile (Brooks et al., 2004; Katsir et al., 2008b; Fonseca et al., 2009). Ethyl
methanesulfonate (EMS), a chemical mutagen, was used to introduce mutations in Arabidopsis
seedlings, which were then screened for resistance to COR (Feys et al. 1994). Using this forward
genetics approach, mutants were identified that had a suite of JA-insensitive phenotypes,
including male sterility due to defects in anther dehiscence, root insensitivity to JA, and
decreased anthocyanin accumulation. Using a map-based cloning approach, the mutation was
found to be in a gene encoding the F-box protein CORONATINE INSENSITIVE1 (COI1) (Xie
et al., 1998). COI1 associates with the SKP1-CUL1 protein complex and functions as the
specificity determinant of the E3 ubiquitin ligase SCF

COI1

(Xie et al., 1998; Turner et al., 2002;

Xu et al., 2002).
The identification of COI1 as an F-box protein implied the existence of a negative regulator
that is ubiquitinated by SCF

COI1

in response to JA. For nearly a decade after the discovery of

COI1, much research was dedicated to finding the COI1-interacting substrates by various
experimental methods. The mechanism by which COI1 regulates JA responses remained elusive
until the discovery of Jasmonate-ZIM domain (JAZ) proteins (Chini et al., 2007; Thines et al.,
2007; Yan et al., 2007). Transcriptional profiling experiments revealed that expression of JAZ
genes is rapidly induced upon JA treatment, suggesting that these genes might be important for
the JA response pathway (Thines et al., 2007). In addition, a dominant negative mutation (jai13) that causes insensitivity to JA was shown to map to a JAZ gene (Chini et al., 2007).
Subsequent studies showed that JAZ proteins interact with COI1 in the presence of (3R,7S)-JAIle and COR, and that this interaction results in JAZ degradation via the 26S proteasome
pathway (Thines et al., 2007; Chini et al., 2007; Chung and Howe, 2009; Pauwels et al., 2011).

9

Jasmonate-ZIM domain (JAZ) Proteins

Arabidopsis has 12 JAZ proteins that belong to the larger family of TIFYXG-containing
proteins (Vanholme et al., 2007). JAZ proteins share three conserved regions: Domain 1, the
ZIM domain and the Jas domain (Shikata et al., 2004; Thines et al., 2007; Chung et al., , 2009).
The N-terminal Domain 1 is loosely conserved and contains an acidic region with unknown
functional significance (Thines et al., 2007). The ZIM domain is a 28-amino-acid sequence
containing the highly conserved TIFYXG motif (Vanholme et al., 2007). The ZIM domain is
necessary for protein-protein interactions, including the formation of homo- and heteromeric
JAZ-JAZ complexes (Chini et al., 2009; Chung and Howe, 2009). JAZ proteins have a Cterminal Jas motif with the highly conserved ELPIARRASLX2FX2KRX2RX5PY sequence
(Chini et al., 2007; Thines et al., 2007; Yan et al., 2007; Chung et al., 2010). In the case of nine
of the 12 JAZ proteins in Arabidopsis, the genomic DNA encoding the 27-amino-acid Jas
domain is encoded by two exons that are separated by an intron, referred to as the Jas intron
(Figure 3) (Chung et al., 2010). The N-terminal 20 amino acids of the Jas domain are necessary
and sufficient for hormone-dependent binding to COI1 and have been designated as the JAZ
degron (Sheard et al., 2010). Analyses of JAZ truncation mutants have also implicated the Jas
domain in binding of the transcription factor MYC2 (Chini et al., 2007; Thines et al., 2007;
Katsir et al., 2008b; Chini et al., 2009; Fonseca et al., 2009). Furthermore, analysis of JAZ1
truncation mutants fused to GFP suggests that the conserved PY sequence at the C-terminal end
of the Jas motif plays a role in nuclear localization (Grunewald et al., 2009).
Although there are 12 JAZ proteins in Arabidopsis, the role of each protein in the regulation
of JA responses is unclear. Biochemical approaches have revealed that differences in amino acid

10

sequence may alter the ability of JAZ proteins to interact with other proteins. For example, JAZ
proteins vary in their ability to homo- or heterodimerize, or participate in protein-protein
interactions with MYC2 or COI1 (Chini et al., 2009; Chung and Howe, 2009; Pauwels and
Goossens, 2011). With the exception of the jaz10-1 mutant, all available jaz T-DNA insertion
mutants have no phenotype (Thines et al., 2007). jaz10-1 shows a phenotype of increased cell
growth in the stem base and altered JA responses in interfascicular-derived cambium tissues
(Sehr et al., 2010). Recently, jaz10-1 was also shown to have a JA-hypersensitive phenotype in
roots, and increased susceptibility to Pseudomonas syringae infection, similar to a JAZ10 RNAi
line (Yan et al., 2007; Demianski et al., 2011). The general paucity of phenotypes in jaz single
mutants suggests there is functional redundancy amongst the gene family.

JA Perception

The recent elucidation of the x-ray crystal structure of the COI1-JAZ complex revealed the
molecular mechanism of JA-Ile and COR binding to the receptor. COI1 shares structural
similarity to the auxin receptor TIR1 (TRANSPORT INHIBITOR RESPONSE 1) (Katsir et al.,
2008a; Tan et al., 2007). Both COI1 and TIR1 have an N-terminal F-box motif and 18 leucinerich repeats (LRR) that are arranged in a solenoid structure and bind inositol polyphosphates
(Tan et al., 2007; Sheard et al., 2010). In vitro ligand binding assays performed with recombinant
ASK1, COI1, and JAZ proteins were used to define the biochemical features of the receptor
(Sheard et al., 2010). The highly conserved N-terminal region of the JAZ degron consists of
ELPIARR residues that are critical for high affinity binding of (3R,7S)-JA-Ile or COR (Sheard et
al., 2010). Using mass spectrometry and nuclear magnetic resonance data, inositol

11

pentakisphosphate was found to co-purify with the COI1-ASK1 complex (Sheard et al., 2010). It
was concluded that the high-affinity receptor for (3R,7S)-JA-Ile and COR is composed of a
complex of COI1-JAZ and inositol pentakisphosphate (Sheard et al., 2010). Evidence that COI1
is required for E3 ubiquitin ligase activity was shown by the detection of ubiquitinated JAZ6
(Saracco et al., 2009).

Mechanism of Transcriptional Repression by JAZ Proteins

In a screen for JAZ-interacting proteins, a tandem affinity purification (TAP) tagged
derivative of JAZ1 was found to interact with Novel Interactor of JAZ (NINJA) (Pauwels et al.,
2010). Analysis of the NINJA sequence revealed three conserved domains, named A, B, and C.
The C domain was previously described in members of the ABI-FIVE BINDING PROTEIN
(AFP) family as being necessary and sufficient for binding to the transcription factor ABI5
(Garcia et al., 2008). Yeast two-hybrid assays showed that domain C of NINJA interacts with the
TIFY motif in the ZIM domain in all JAZ proteins except JAZ7 and JAZ8 (Pauwels et al., 2010).
Because the TIFY motif is also involved in JAZ-JAZ dimerization and the C domain of NINJA
binds ABI5, it is unclear whether these domains compete for binding with other proteins or
create large, multi-protein repressor complexes (Geerinck et al., 2010).
The A domain in NINJA contains an ETHYLENE RESPONSIVE FACTOR-associated
amphiphilic repression (EAR) motif that has been previously shown to recruit the Groucho/Tup1
type co-repressor TOPLESS (TPL) (Geerinck et al., 2010; Kagale et al., 2010; Pauwels et al.,
2010). Previous research showed that EAR motifs are present in many Aux/IAA proteins, and
that the EAR motif recruits TPL to repress the expression of auxin-responsive genes (Szemenyei

12

et al., 2008). NINJA interacts with TPL in yeast and in plant nuclei, as determined by in vivo
bimolecular fluorescent complementation (BiFC) assays (Pauwels et al., 2010).
The TPL co-repressor is involved in multiple hormone pathways (Kagale et al., 2010;
Pauwels et al, 2010). TPL was first studied for its role in auxin signaling in the determination of
apical polarity (Long et al., 2006). A tpl1-1 mutant was identified in which the shoot pole is
transformed into a root pole during the transition stage of early embryogenesis, generating a
double-rooted plant that is “topless” (Long et al., 2006). TPL is a 1131-amino-acid protein
containing C-terminal WD40 repeats, a lissenchephaly type 1-like homolog domain (LiSH) and a
C-terminal to LiSH domain (CTLH), that latter of which interacts with EAR motifs (Long et al.,
2006, Pauwels et al., 2010). TPL also has a proline-rich region that is similar to the known
repressors in the Groucho/TUP1 family (Long et al., 2006). TPL may actively repress
transcription by recruiting the histone deacetylase HDA19 (Long et al., 2006).

JAZ-interacting Transcription Factors

Genetic screens for JA response mutants resulted in the identification of a JA-insensitive 1
(jin1) mutant (Lorenzo et al., 2004). Using a map-based cloning approach, the JIN1 locus was
found to encode the bHLH transcription factor MYC2, which positively regulates JA responses
in a COI1-dependent manner (Lorenzo et al., 2004; Chini et al., 2007). The JA-insensitivity of
jin1 mutants is relatively weak in comparison to the fully insensitive and male sterile coi1
mutant, suggesting that additional transcription factors are involved in promoting JA responses
(Lorenzo et al., 2004).

13

MYC2 belongs to a family of bHLH transcription factors that also includes MYC3 and
MYC4 (Boter et al., 2004; Fernandez-Calvo et al., 2011; Niu et al., 2011). The myc2myc3myc4
triple mutant is more insensitive to JA than the single myc2 (jin1) mutant, but still not as
insensitive as the COI1 null mutant coi1-1 (Fernandez-Calvo et al., 2011). The myc2myc3myc4
triple mutant is more susceptible to S. littoralis feeding than single or double myc mutants, and is
as susceptible as coi1-1 (Fernandez-Calvo et al., 2011). Additionally, ectopic expression of
MYC3 and MYC4 resulted in increased accumulation of anthocyanins, which are known to be
regulated by the JA pathway (Niu et al., 2011).
MYC2, MYC3 and MYC4 share approximately 56% sequence similarity, with the highest
degree of similarity in the N-terminus (Niu et al., 2011). JAZ proteins interact with both MYC3
and MYC4 in yeast two-hybrid and in vitro pull down assays (Niu et al., 2011; Fernandez-Calvo
et al., 2011). Structure-function studies further showed that a 67-amino-acid region in the ND93-160

terminus of MYC2 (MYC2

) is conserved in MYC3 and MYC4, and is sufficient to bind

JAZ proteins (Fernandez-Calvo et al., 2011). Genome-wide analysis of MYC2 binding sites
indicated preferential binding to the G-box motif CACGTG, which is overrepresented in
promoters of JA-responsive genes (Dombrecht et al., 2007; Chini et al., 2007). Interestingly,
MYC3 and MYC4 have identical preference for binding G-box motifs (Fernandez-Calvo et al.,
2011)
Analysis of promoter-GUS fusions of MYC3 showed that MYC3 is expressed in most
vegetative tissues, whereas MYC4 is expressed primarily in vasculature tissues (Fernandez-Calvo
et al., 2011). MYC2 promoter-GUS fusions show high basal expression in roots (Chen at al.,
2011). It was proposed that MYC3 and MYC4 work in concert with MYC2 to regulate JA
responses and are not fully functionally redundant. MYC2, MYC3 and MYC4 may also form

14

homo- and heterodimers in vivo (Fernandez-Calvo et al., 2011). Other transcription factors may
also play a synergistic role in regulating JA responses.

Alternative Splicing in the Arabidopsis Genome

Many intron-containing plant genes undergo alternative splicing (Filichkin et al., 2010;
Reddy, 2007). More than 80% of genes in the Arabidopsis genome harbor one or more introns
(Reddy, 2007). It is estimated that 42% of Arabidopsis intron-containing genes are alternatively
spliced, and further suggested that splicing might be regulated by abiotic stress (Filichkin et al.,
2010). Among 18 Arabidopsis accessions, the most differentially expressed genes are those
involved in defense responses and stress responses (Gan et al., 2011). To date, there are
relatively few examples of functionally relevant alternative splicing events in plants. But among
these few examples, many are involved in stress responses (Kazan et al., 2003). An example of
this is the Arabidopsis R gene, RPS4, which undergoes intron retention and splicing of a cryptic
intron within the 3

rd

exon (Zhang and Gassmann, 2003; Gassmann et al., 2008). Ectopic

expression of RPS4 cDNA confers susceptibility to Pseudomonas syringae pv tomato strain
DC3000, suggesting that intron retention and splicing of the cryptic intron is critical to
conferring defense responses (Zhang and Gassmann, 2003). Increasing evidence suggests that
alternative splicing may be an important mechanism for regulating the expression of stressresponsive genes (Ali and Reddy, 2008; Gassmann, 2008).

15

Alternative Splicing of JAZ Genes

A key feature of most JAZ genes is their strong expression in response to JA treatment or
stress-related cues that activate JA synthesis. JA-induced expression of JAZ genes may serve as a
mechanism of negative feedback regulation (Thines et al., 2007; Chung et al., 2009). Upon
methyl-jasmonate (MeJA) treatment or wounding, JAZ genes are highly induced and
alternatively spliced transcripts of several JAZ genes are produced (Yan et al., 2009; Chung and
Howe, 2009). Nine of the 12 Arabidopsis JAZ genes are predicted to produce alternative splice
variants in which the Jas domain is truncated due to Jas intron retention (Chung et al., 2010).
JAZ10 (At5g13220) naturally produces three alternative splice variants in which the Jas domain
is truncated or missing (Figure 3) (Chung and Howe, 2009). JAZ10.1 encodes the full-length
protein isoform. JAZ10.2 is produced by retention of the Jas intron. A premature termination
codon (PTC) within the Jas intron results in the loss of 12 amino acids from the C-terminus.
JAZ10.3 also retains the Jas intron. Unlike JAZ10.2, however, JAZ10.3 has another alternative
splicing event at the first GT within the retained Jas intron, resulting in a 3’ UTR that is shorter
than that of JAZ10.2. JAZ10.2 and JAZ10.3 encode identical proteins. A fourth JAZ10 splice
variant, JAZ10.4, is produced by use of an alternate splice donor site in the third exon. This
splicing event causes a frame-shift mutation that results in the deletion of the entire Jas domain
(Chung and Howe, 2009). In addition to JAZ10, several other JAZ genes undergo alternative
splicing events involving retention of the Jas intron. Similar to the case of JAZ10.2/JAZ10.3,
these transcripts are predicted to produce proteins that lack the C-terminal end (X5PY) of the Jas
motif (Chung et al. 2010).

16

Figure 3. Gene Models of JAZ10 Alternative Splice Variants. JAZ10.1 (A), consists of five
exons (black boxes) and four introns (lines) and encodes the full-length isoform containing a
complete Jas domain, which is encoded by the last two exons. JAZ10.2 retains the last intron,
referred to as the Jas intron. A premature termination codon located near the 5’ end of the Jas
intron results in production of a truncated protein lacking seven amino acids from the C-terminal
end of the Jas motif. JAZ10.3 retains a portion of the Jas intron but uses an alternative splice
acceptor site within the Jas intron to generate a transcript that encodes a protein identical to
JAZ10.2. JAZ10.4 is produced from the use of an alternative splice donor site located in the third
exon. This splicing event causes a frameshift and truncation of the entire Jas domain. Open
boxes represent 5’ and 3’ untranslated regions. Schematic representation of proteins produced for
each JAZ10 alternative splice variant (B). For interpretation of the references to color in this and
all other figures, the reader is referred to the electronic version of this thesis.
* indicate unique splicing events

17

FIGURE 3 (cont’d)

Figure 3. Gene Models of JAZ10 Alternative Splice Variants.

18

JAZ proteins in which the Jas domain is truncated or missing are more stable than their fulllength counterparts, have decreased affinity for COI1 and thus are stable in the presence of JA
(Chung and Howe, 2009; Chung et al., 2010; Sheard et al., 2011). Transgenic plants
overexpressing these truncated isoforms exhibit decreased sensitivity to JA (Chini et al., 2007;
Thines et al., 2007; Yan et al., 2007; Chung and Howe, 2009; Chung et al., 2010). Interestingly,
the Jas intron is also conserved in JAZ genes found in all land plant species, including the
evolutionarily distant Physcomitrella patens (Chung et al., 2010). Thus, alternative splicing
events affecting the Jas domain may be an important control point in regulating JA responses.

The Role of JA in Fertility

Previous research has revealed an essential role for JA in the development of male
reproductive tissues in Arabidopsis (Park et al., 2002; Stintzi and Browse, 2000). The JA
perception mutant coi1, as well as various JA biosynthetic mutants, are male-sterile due to
defects in anther dehiscence and anther filament elongation (Park et al., 2002; Stintzi and
Browse, 2000; McConn and Browse, 1996; Feys et al., 1994). Transcriptional profiling
experiments of JA treated Arabidopsis stamens revealed strong induction of flower-specific
transcription factors MYB21 and MYB24 in comparison to untreated samples (Mandaokar et al.,
2006). The myb21myb24 double knockout is male sterile in a similar manner to JA biosynthetic
and JA perception mutants, suggesting a regulatory role of these transcription factors in male
fertility (Mandaokar et al., 2006). The ability of MYB21 and MYB24 to interact with several
JAZ proteins suggests a role for JAZs in regulating male fertility (Song et al., 2011). In support
of this idea, ectopic expression of MYB21 in the coi1 mutant partially restored fertility (Song et

19

al., 2011). Overexpression of various JAZ derivatives that lack the Jas domain (e.g., JAZ10.4)
also results in male sterility (Thines et al., 2007; Chung et al., 2009; Song et al., 2011). The
mechanism by which MYB transcription factors and JAZ proteins regulate stamen and anther
development remains to be determined.

The Role of JAZ Proteins in the Integration of Other Hormone Signaling Pathways

JA-Auxin
There are many similarities between auxin and JA signaling that enable cross-regulation
between these two hormones. Among the ~700 F-box proteins encoded by the Arabidopsis
genome, the most closely related to COI1 is TIR1 and TIR1-like proteins (Katsir et al., 2008a;
Tan et al., 2007). At the protein level, COI1 and TIR1 are approximately 33% similar and share
overall structural features, including 18 tandem LRRs arranged in a solenoid array (Tan et al.,
2007; Sheard et al., 2010). Both COI1 and TIR1 bind inositol polyphosphates, which may be
involved in regulating receptor function (Tan et al., 2007; Sheard et al., 2010). Both TIR1 and
COI1 are components of SCF-type E3 ubiquitin ligases and share other subunits of the complex
(Hoffmann et al., 2011). Some mutants affected in the formation of SCF complexes, such as the
cul1 mutant, show both auxin- and JA-related phenotypes (Hoffmann et al., 2011). Upon
hormone perception, SCF

TIR1

COI1

and SCF

function as E3 ubiquitin ligases, targeting Aux/IAA

and JAZ repressor proteins, respectively, for degradation by the 26S proteasome (Pauwels et al.,
2010). Auxin and JA pathways utilize EAR motif-containing proteins to recruit TPL (Pauwels et
al., 2010).

20

JA-GA:
The gibberellic acid (GA) signaling pathway is involved in numerous aspects of plant
growth and development and has recently been integrated in the JA signaling network. GA
signaling is similar to that of auxin and JA. The hormone receptor SCF

GID1

binds DELLA

transcriptional repressors and targets them for degradation by the ubiquitin-26S proteasome
pathway (Ueguchi-Tanaka et al., 2007). Recently, it was shown that DELLA proteins compete
with MYC2 for interaction with JAZ proteins (Hou et al., 2010). The DELLA quintuple mutant
exhibits reduced expression of JA responsive genes, which can be even further reduced when
treated with a GA biosynthesis inhibitor (Hou et al., 2010). Chromatin immunoprecipitation and
in vitro transactivation assays provide evidence that MYC2 can bind DNA more efficiently when
levels of DELLA proteins are high and that this process is dependent on COI1 (Hou et al., 2010).
This suggests a model in which DELLAs bind JAZ proteins under low GA or high JA levels to
allow MYC2 to bind DNA and promote transcription (Hou et al., 2010).

JA-Ethylene:
Ethylene is a plant hormone involved in a number of plant developmental processes and is
also known to be important for defense against necrotrophic fungi (Dong, 1998; Browse, 2009).
Ethylene and JA induce defense genes more strongly in combination with each other, suggesting
a synergistic effect between these two pathways (Lorenzo et al., 2003). The transcription factors
EIN3 and EIL1 are necessary and sufficient for induction of ethylene responses (Alonso et al.,
1999). EIN3 and EIL1 interact directly with JAZ1, JAZ3 and JAZ9 in yeast two-hybrid and in
vivo BiFC assays (Zhu et al., 2011). The ein3 eil1 double mutant is partially insensitive to JA in
root length assays, suggesting that EIN3 and EIL1 are positive regulators of JA responses. This

21

study also showed that the ability of EIN3 to activate ethylene responsive genes depends in part
on JAZ1 (Zhu et al., 2011). These results provide a molecular mechanism for the role of JAZ
proteins in the integration of JA and ethylene hormone signaling pathways.

JA-Phytochome:
Arabidopsis possesses five types of phytochromes, phyA-phyE, that mediate different
aspects of light signaling (Sharrock and Quail, 1989). PhyA is responsible for the detection of
far-red light in the range of 600-750 nm and controls the expression of genes leading to
inhibition of hypocotyl elongation, opening of the apical hook, expansion of cotyledons,
accumulation of anthocyanin, and blockage of greening (Chory et al., 1996; Yang et al., 2005).
There is much experimental evidence supporting the crosstalk between the JA and light signaling
pathways. A mutant screen for suppressors of the constitutive photomorphogenesis 1 mutation
identified a mutant allele of JAR1 (Hsieh et al., 2000). The myc2 mutant was also discovered in a
screen for proteins binding specific elements of light responsive promoters (Yadav et al., 2005).
The ecological importance of this was demonstrated in an experiment in which plants grown in
high density or under far-red light had reduced JA responses and were more susceptible to
herbivores (Moreno et al., 2009). Interestingly, the phyA mutant is insensitive to JA in root
length assays, and JAZ1 transcript is more abundant in plants grown under far-red light (Robson
et al., 2010). Ectopic expression of JAZ1-GUS in the phyA mutant has revealed that PhyA is
required for the degradation of JAZ1 in response to wounding and JA treatment (Robson et al.,
2010).

22

In summary, the transcriptional regulation of alternative splicing of JAZ genes in response to
JA is not fully understood. It is necessary to determine whether splicing occurs constitutively or
whether splicing is regulated by stress-induced changes in the spliceosome machinery. Truncated
JAZ proteins that are stable in the presence of JA may serve to attenuate defense responses and
JA responsive gene expression. This research aims to clarify the regulation of alternative splicing
of JAZ genes, as well as characterize their functional significance. Information gained from these
studies will help to elucidate the role of alternative splicing in JA signaling and related responses
to pathogen attack and herbivory.

23

RESULTS

Regulation of JAZ Alternative Splicing

Upon MeJA treatment or wounding, JAZ gene expression is highly induced and may serve
as a mechanism of negative feedback regulation. In addition to high expression of JAZ genes,
alternatively spliced transcripts of several JAZ genes are detected (Thines et al., 2007; Chung and
Howe, 2009). Nine of the 12 Arabidopsis JAZ genes are predicted to produce alternative splice
variants in which the Jas domain is truncated due to Jas intron retention (Chung et al., 2010).
Several JAZ genes, including JAZ2, JAZ6 and JAZ10 are subject to Jas intron retention, and
produce similarly truncated proteins (Figure 11).
To determine whether the relative levels of each JAZ splice variant remains constant during
induction of JAZ expression or if there is a change in splice-site preference, the level of fulllength (JAZ10.1) and alternative splice variants (JAZ10.2, JAZ10.3, JAZ10.4) were quantified
using quantitative real time polymerase chain reaction (qPCR) and splice variant-specific primer
sets. In mock treated seedlings (Figure 4A, “0”), JAZ genes were expressed at a low basal level,
where the full-length transcript (JAZ10.1) predominates and the alternatively spliced transcripts
are present at very low to undetectable levels. The level of all four JAZ10 splice variant mRNAs
increased in response to MeJA treatment, with JAZ10.1 being the predominant form. The relative
abundance of each transcript, however, was generally maintained throughout the time course
(Figure 4A). For example, levels of JAZ10.3 and JAZ10.4 remain at approximately 3-10% of the

24

Figure 4. Levels of JAZ10 Alternatively Spliced Transcripts in MeJA-Treated Seedlings.
Col-0 seedlings were grown in liquid MS media for nine days and then treated with 100 μM
MeJA or a mock control (0.007% ethanol) for two hours. At the indicated time after treatment,
seedlings in each biological replicate (a pool of 15-20 seedlings per sample) were harvested for
isolation of RNA and qPCR analysis of JAZ10 (A), JAZ2 (B), and JAZ6 (C) transcript levels.
Relative expression refers to the level of a specific alternatively JAZ transcript after
normalization to the reference genes PP2A and YLS8. Data points show the mean and standard
error of three biological replicates. Due to the lack of a unique region in JAZ10.1, qPCR primers
for JAZ10.1 detect both JAZ10.1 and JAZ10.4 splice variants.

25

FIGURE 4 (cont’d)

Figure 4. Levels of JAZ10 Alternatively Spliced Transcripts in MeJA-Treated Seedlings.

26

levels of JAZ10.1. One notable exception to this was JAZ10.2, which accumulated to
approximately 35% of the level of JAZ10.1.
The same RNA samples were used to quantify alternatively spliced forms of JAZ2 and
JAZ6, which are subject to JAZ10.3-like splicing events involving retention of the Jas intron.
Similar to the results obtained with JAZ10, the full-length JAZ2.1 and JAZ6.1 transcripts
predominated over the alternatively spliced JAZ2.2 and JAZ6.2 transcripts in response to MeJA
treatment (Figure 4B and C). Relative levels of JAZ2.2 were approximately 2-5% that of JAZ2.1.
The basal expression of JAZ6.1 in unwounded leaves was relatively high in comparison to that
observed in the MeJA-treated samples (Figure 5C). Variation in the basal level expression
among the JAZ genes has been previously reported, including a high basal expression of JAZ6
(Chung et al., 2008). After MeJA treatment, relative levels of JAZ6.2 were approximately 5-9%
that of JAZ6.1.
To determine whether the results obtained for MeJA-treated seedlings extends to other
stages of plant development, the levels of alternatively spliced JAZ transcripts were also
measured in adult plants subject to mechanical wounding (Figure 5). In unwounded leaf tissue
(Figure 5A, “0”), JAZ genes are expressed at a low basal level, where the full-length transcript
(JAZ10.1) predominates and the alternatively spliced transcripts are present at very low to
undetectable levels. The level of all four JAZ10 mRNAs increased in response to wounding, with
JAZ10.1 being the predominant form. Similar to the results obtained with MeJA treatment
(Figure 4), the relative abundance of each transcript was maintained throughout the time course
(Figure 5A). Levels of JAZ10.3 and JAZ10.4 remained at approximately 3-11% of the levels of
JAZ10.1, similar to MeJA-treated seedlings. The peak level of JAZ10.2 was nearly 40% of
JAZ10.1.

27

Figure 5. Levels of JAZ Alternatively Spliced Transcripts in Wounded Leaves. Col-0 plants
were grown for 25 days on soil. Each biological replicate is a pool of three wounded leaves from
two plants. Wounded leaves were harvested for isolation of RNA and qPCR analysis of JAZ10
(A), JAZ2 (B), and JAZ6 (C) transcript levels. Data points show the mean and standard error of
four biological replicates. Due to the lack of a unique region in JAZ10.1, qPCR primers for
JAZ10.1 detect both JAZ10.1 and JAZ10.4 splice variants.

28

FIGURE 5 (cont’d)

Figure 5. Levels of JAZ Alternatively Spliced Transcripts in Wounded Leaves.

29

Alternative Splicing of JAZ Genes in Flowers and Roots

In animals, alternative splicing is often regulated by tissue-specific factors that alter the
spliceosome machinery (Grabowski and Black, 2001). To test the hypothesis that alternative
splicing of JAZ10 is regulated in a tissue-specific manner, qPCR was utilized to measure the
levels of JAZ10 alternative splice variants in floral tissue. These experiments were performed
with RNA isolated from Col-0 (wild-type; WT) flowers, as well as flowers from a transgenic line
(35S-JAZ10G) that overexpresses a JAZ10 genomic clone (Chung et al., 2010). As shown in
Figure 6, the level of all JAZ10 alternative splice variants in 35S-JAZ10G flowers was much
higher (approximately 30-fold) than that in WT flowers. The relative abundance of each splice
variant in WT and 35S-JAZ10G flowers, however, was similar. JAZ10.1 was the predominant
transcript in both genotypes. The level of JAZ10.2 was approximately 50% of the level of
JAZ10.1 Levels of JAZ10.3 and JAZ10.4 ranged from 5-15% of the level of JAZ10.1, similar to
the results obtained with RNA isolated from vegetative tissues (Figures 4 and 5).
Using the same RNA, levels of full-length and alternatively spliced JAZ2 and JAZ6 were
also measured in WT and 35S-JAZ10G flowers (Figure 7). Similar to the results obtained for
JAZ10, the full-length JAZ2.1 and JAZ6.1 transcripts were the predominately expressed forms,
with JAZ2.2 and JAZ6.2 accounting for less than 10% of the total transcript level. The level of
each JAZ2 and JAZ6 transcript in WT and 35S-JAZ10G flowers was similar, indicating that
ectopic expression of JAZ10 does not alter the expression of the endogenous JAZ2 and JAZ6
genes.

30

Figure 6. Levels of JAZ10 Alternatively Spliced Transcripts in WT and 35S-JAZ10G
Flowers. (A) Wild-type (WT) and 35S-JAZ10G plants were grown for seven weeks on soil. Data
show the mean and standard error of three biological replicates. Each replicate of RNA were
derived from flowers obtained from four plants. (B) Re-plotting of WT data shown in panel A,
using a different scale.

31

Figure 7. Levels of JAZ2 and JAZ6 Alternatively Spliced Transcripts in WT and 35SJAZ10G Flowers. The same RNA used for the experiment described in Figure 6 was analyzed
by qPCR for the expression of alternatively spliced forms of JAZ2 (A) and JAZ6 (B). Data points
show the mean and standard error of three biological replicates.

32

In addition to flowers, JAZ transcripts were also quantified in root tissues of WT and 35SJAZ10G seedlings. As seen in Figure 8, the 35S-JAZ10G transgene was highly expressed in
roots. Similar to results obtained with flowers, increased expression of the 35S-JAZ10G
transgene in roots did not significantly alter the ratio of JAZ10 splice variants. However, the
level of all JAZ10 splice variants was several-fold higher in roots compared to flowers. The
difference in 35S-JAZ10G expression may explain the previous observation that 35S-JAZ10G
roots are strongly insensitive to JA, whereas 35S-JAZ10G flowers do not exhibit JA-related
reproductive phenotypes typically observed in strong JA-insensitive mutants (Chung et al.,
2010).
JAZ2 and JAZ6 transcripts were also quantified in root tissues (Figure 9). Similar to the
results obtained for JAZ10, the full-length JAZ2.1 and JAZ6.1 transcripts were the predominantly
expressed forms, with JAZ2.2 and JAZ6.2 accounting for less than 5% of the total transcript
level. Interestingly, the levels of all JAZ2 and JAZ6 transcripts were greatly reduced in roots of
35S-JAZ10G seedlings compared to WT seedlings. Consistent with the proposed role of JAZ10
in negative regulation of JA responses (Yan et al., 2007; Chung and Howe, 2009; Sehr et al.,
2010; Demianski et al., 2011), these data suggest increased expression of one or more JAZ10
splice variants in roots represses the expression of JAZ2 and JAZ6.

33

Figure 8. Quantification of JAZ10 Alternatively Spliced Transcripts in WT and 35SJAZ10G Roots. (A) Wild-type (WT) and 35S-JAZ10G seedlings were grown for nine days on
MS plates (supplemented with 0.8% sucrose) in continuous light prior to RNA isolation from
root tissue. Data points show the mean and standard error of three biological replicates. (B) Replotting of WT data shown in panel A, using a different scale.

34

Figure 9. Quantification of JAZ2 and JAZ6 Alternatively Spliced Transcripts in WT and
35S-JAZ10G Roots. The same RNA used for the experiment described in Figure 8 was analyzed
by qPCR for the expression of alternatively spliced forms of JAZ2 (A) and JAZ6 (B). Data points
show the mean and standard error of three biological replicates.

35

Detection of JAZ Alternative Splice Variants Using RNA Sequencing Data

Illumina sequencing was performed on cDNA samples from MeJA- and coronatine-treated
seedlings, as well as from wounded leaves. Tophat was used to map reads to Arabidopsis TAIR 9
full-length cDNAs. Under all treatment conditions, JAZ genes were highly induced, as shown by
the increase in the total number of reads for each gene, compared to sequencing of samples from
control plants (Table 1A). In this experiment, coronatine treatment was generally the most
effective in inducing JAZ gene expression. Some JAZ genes, including JAZ6 and JAZ12, showed
a relatively high basal level of expression in mock-treated samples, which is consistent with
previous RNA blot analyses (Chung et al., 2008) (Table 1B).
To determine the relative expression levels of each JAZ splice variant, reads were aligned
and normalized to a unique region for each splice variant. Using Illumina data, the number of
reads per kilobase pair per million mapped reads of model (RPKM) was calculated. Only one
biological replicate was available for each treatment condition, thus excluding the ability to
perform statistical analysis for each treatment condition.
For JAZ genes in which Jas intron retention was predicted but were not annotated in TAIR 9
reference cDNAs, we created a new gene model in which the Jas intron was retained in an
otherwise fully spliced transcript. In some cases, the number of unique reads for the full-length
transcript could not be calculated because of the lack of a unique region distinct from alternative
splice variants. For example, no unique reads align to JAZ10.1 because it lacks a region that is
distinct from JAZ10.2, JAZ10.3 or JAZ10.4 (Figure 10). In order to obtain the number of reads
for JAZ10.1, the total reads were normalized to the entire cDNA, and unique reads for

36

Figure 10. Evidence of JAZ10 Alternatively Spliced Transcripts in RNA-seq Data. Gene
models for alternative splicing events are supported by reads that align to unique regions of each
JAZ10 splice variant. No reads are available for JAZ10.1 due to lack of sequence region that is
specific for JAZ10.1 Colored vertical bars indicate coverage over a unique region, whereas
brown horizontal bars are reads that align to the unique region. Data are from Dr. Marco Herde.
For interpretation of the references to color in this and all other figures, the reader is referred to
the electronic version of this thesis.
37

Table 1. Evidence of JAZ Gene Induction and Jas Intron Retention in RNA-seq data. (A)
JAZ genes are highly induced in response to JA elicitation compared to mock or unwounded
samples. (B) Reads per kilobase pair per million mapped reads of model were calculated for gene
models with alternative splicing events. Identification of alternative splicing events is supported
by reads that align to unique regions for each splice variant. JAZ9.1 and JAZ10.1 were calculated
from normalization of total reads to the size of the full-length cDNA and subtracting the unique
reads from unique regions of other splice variants, and is outlined in dots. * indicates strong
evidence for Jas intron retention.

38

Table 1. Evidence of JAZ Gene Induction and Jas Intron Retention in RNA-seq Data.

39

JAZ10.2, JAZ10.3 and JAZ10.4 were subtracted from this value. This approach was also used in
the case of JAZ9.1, where a unique region distinct from JAZ9.2 and JAZ9.3 was not found.
Based on the number of unique reads found in the data set, JAZ gene models in which there
was Jas intron retention were separated into those with “strong” or “weak” evidence (Table 1B).
JAZ genes with strong evidence of producing alternative splice variants by Jas intron retention
include: JAZ2, JAZ5, JAZ6, JAZ9, and JAZ10. In the case of JAZ10, JAZ2 and JAZ6, Jas intron
retention is also supported by qPCR results (Figures 4 and 5). Similar to qPCR results obtained
for JAZ10, JAZ2 and JAZ6, the full-length transcript is by far the most abundant form produced.
JAZ5.2 and JAZ9.3 do not have a PTC close to the intron retention site, but are much farther into
the Jas intron sequence than other JAZs (Chung et al., 2010).
Jas intron retention is weakly supported for JAZ3, JAZ4 and JAZ12. In these cases, it is
difficult to determine whether Jas intron retention is occurring or not due to low expression of
the gene and lack of replicates. For example, JAZ4.2 was not supported in the data set, likely due
to low expression levels.

Ectopic Expression of JAZ2.2 and JAZ4.2 Results in Decreased Sensitivity to JA

Seven of the 12 JAZ genes in Arabidopsis produce alternative splice variants in which the
Jas domain is truncated as a result of Jas intron retention (Chung et al., 2010). Because of the
presence of a PTC at the 5’-end of the Jas intron, these transcripts are predicted to encode
proteins that lack the conserved X5PY sequence that defines the C-terminal end of the Jas
domain (Figure 11).

40

Figure 11. Intron/Exon Structure for Select JAZ Genes Containing the Jas Intron. Exon
sequence is highlighted in black, Jas intron sequence is highlighted in grey, with predicted amino
acid sequence. In-frame premature stop codons are highlighted in red. Modified from Chung et
al., 2010. For interpretation of the references to color in this and all other figures, the reader is
referred to the electronic version of this thesis.

41

Previous studies showed that one such alternative splice variant, JAZ10.3, functions as a
dominant repressor of JA signaling when ectopically expressed in Arabidopsis (Yan et al., 2007;
Chung and Howe, 2009). To determine whether other truncated ΔPY splice isoforms exert
similar effects in vivo, transgenic plants expressing 35S-JAZ2.2 (Figure 12) were constructed and
tested for altered sensitivity to JA-induced root growth inhibition. The results showed that 35SJAZ2.2 plants have a JA-insensitive phenotype that is quantitatively similar to that of 35SJAZ10.3 plants (Figure 12).
Ectopic expression of JAZ alternative splice variants containing a PTC at similar positions
in the Jas intron as JAZ10.3 may aid in understanding the functional physiological role of
splicing in regulating JA responses. Besides JAZ10 and JAZ2, other JAZ genes may produce
splice variants with variable C-termini, depending on the location of the PTC in the retained Jas
intron. The splice variant JAZ4.2 retains a Jas intron that harbors a PTC one codon 3’ of where it
occurs for JAZ10.3 and JAZ2.2 (Figure 11) (Chung et al., 2010). Transgenic plants expressing
the full-length JAZ4.1 and splice variant JAZ4.2 from the 35S promoter were generated. 35SJAZ4.1 seedlings were as sensitive to JA as WT plants in the root length assay (data not shown).
Ten independent 35S-JAZ4.2 lines (T2 generation) that were segregating for the transgene were
screened for JA insensitivity in the root growth assay; all lines segregated for individuals whose
root length was significantly greater than that of JA-treated WT roots (Figure 13). These findings
provide genetic evidence that JAZ2.2, like JAZ10.3 and JAZ4.2, acts as a dominant repressor of
JA signaling.

42

Figure 12. The MeJA-induced Root Growth Phenotype of 35S-JAZ2.2 Seedlings is Similar
to that of 35S-JAZ10.3 Seedlings. Root length measurements were made with seedlings of the
indicated genotype. (A) All seedlings were grown for nine days on MS medium containing 0.8%
sucrose and 50 μm MeJA. (B) Data points represent the mean ± SD of the following number of
seedlings per genotype: wild type (WT), n = 17; 35S-JAZ10.3, n = 8; 35S-JAZ10.4, n = 7; 35SJAZ2.2, n = 28. The mean root length of 35S-JAZ2.2 seedlings was most similar to that of 35SJAZ10.3 seedlings, and was significantly different from WT (P < 0.0001), 35S-JAZ10.4
(P < 0.0001), and 35S-JAZ10.3 (P < 0.025) seedlings. Modified from Chung et al., 2010. For
interpretation of the references to color in this and all other figures, the reader is referred to the
electronic version of this thesis.

43

Root length (mm)

20
18
16
14
12
10
8
6
4
2
0

Figure 13. MeJA-Induced Root Growth Phenotype of 35S-JAZ4.2. All seedlings were grown
for nine days on MS media. Seedlings from independently transformed lines (T2 generation)
grown in the presence of 20 μm MeJA. Root length measurements were made for each indicated
genotype: wild type (WT), n = 34; (7-1), n=21; (7-2), n=11; (7-4), n=9; (7-6), n=16; (9-5), n=32;
(14-1), n=22; (14-2), n=12; (15-3), n=22; (15-4), n=17; (15-5), n=24. The mean root length of all
transgenic lines was statistically greater (p<0.0005) than WT.

44

DISCUSSION

The data presented here describe the analysis of alternative splicing of JAZ genes in specific
tissues and in response to stress-related cues (JA treatment and wounding). Prior to these studies,
there were two major hypotheses to explain how alternative splicing of JAZ genes is regulated.
First, alternative splicing may occur constitutively through splice site competition. A second
hypothesis is that alternative splicing is regulated by trans-acting factors, such as components
within the spliceosome that alter splice-site selection in response to developmental or
environmental cues. If the latter hypothesis is true and there is a change in splice site preference
in JA stimulated cells, then the ratio of various alternative splice variants should change in
response to JA treatment. The results of this research supports the first hypothesis, namely that
the absolute level of all JAZ alternative splice variants increase in response to JA treatment and
wounding. The abundance of all JAZ alternatively spliced transcripts increased upon MeJA
treatment and wounding. For all JAZ genes analyzed, the full-length transcript was the most
abundant form at all time points during the time course. On average, alternatively spliced JAZ
transcripts represent approximately 3-10% of the level of the full-length transcript. In some cases
(e.g., JAZ10.2), alternatively spliced transcripts accumulated to higher levels. Maintenance of
full-length and splice variant ratios in mock and treated samples suggest that spliceosome
machinery does not alter splice site preference upon JA treatment. Similar results were obtained
for plants subject to mechanical wounding.
Wound- and JA-induced transcription of JAZ genes results in increased levels of JAZ premRNA, which is alternatively spliced in a constitutive manner that does not cause major changes
in the relative abundance of each transcript. It is likely that differences in splice acceptor sites are

45

responsible for determining the relative amount of each splice variant for a particular gene.
Differences in the sequence of splice donor and acceptor sites may provide more or less
competition for the spliceosome machinery, leading to a constant ratio of each alternative splice
variant. The results do not exclude a role for altered mRNA stability in determining the
accumulated level of each transcript. In summary, the data suggest that the relative abundance of
splice variants for a particular JAZ gene is determined by the efficiency with which a particular
splice site is selected by the spliceosome apparatus, and does not result from changes in stressinduced splice-site preference. In general, full-length splice variants are the predominantly
expressed forms, which is consistent with previous studies on alternative splicing in plants (Ali
and Reddy, 2008).
The qPCR results showed that 35S-JAZ10G is expressed at much higher levels in roots than
flowers. This observation may explain why this transgenic line shows JA insensitivity in roots
but not in flowers, as determined by the absence of reproductive defects. Based on normalized
expression to references genes, JAZ10.1 and other JAZ10 splice variants accumulated to
much higher levels in roots as compared to flowers. Reduced expression of 35S-JAZ10G in
flowers compared to roots may be related to the developmental stage of the plants or the
efficiency of transgene expression in these tissues. Consequently, expression levels of 35SJAZ10G, principally the production of JAZ10.4, in flowers may not be sufficient to cause male
sterility. Expression of JAZ10.1 in WT roots and flowers are similar, suggesting endogenous
JAZ10.1 levels are not regulated in a tissue-specific manner.
Stress responsive genes may be expressed in a tissue specific manner (Zhang and
Gassmann, 2003; Ali and Reddy 2008). It was therefore of interest to compare the level of JAZ
alternative splice variants in different tissues, including seedlings, leaves, roots and flowers. In

46

general, there was much higher expression of JAZ10 and JAZ2 in roots than in flowers, whereas
JAZ6 was expressed at similar levels in these tissues. Similar to the results obtained for analysis
of basal JAZ expression in untreated seedlings and unwounded leaves, JAZ transcripts encoding
full-length proteins were the predominant form. Variation in JAZ expression patterns was
observed in different tissues. This could reflect variation in JA levels, the amount of stress
perceived by different tissues, or disparity in transcriptional activity in different tissue types. For
example, the expression of JAZ6.1 was similar in roots, flowers, and unwounded leaves, but was
much lower in liquid grown seedlings. As previously reported, the level of expression of each
JAZ gene varies (Chung et al., 2008). Nonetheless, this data indicate that JAZ genes are
alternatively spliced in the same manner that is independent of tissue type or level of
transcriptional induction within a given tissue.
Many alternatively spliced JAZ transcripts, including JAZ10.3, JAZ2.2, JAZ6.2 and JAZ4.2,
are produced through retention of the Jas intron. The JAZ transcripts encode proteins lacking the
C-terminal X5ΔPY amino acids. In all tissues examined, JAZ10, JAZ2 and JAZ6 full-length
transcripts are the predominant form and alternative splice variants are produced at low to
undetectable levels. It is interesting to note that in all treatment conditions and tissues examined,
JAZ10.2 was the most abundant of these transcripts. Since JAZ10.2 and JAZ10.3 essentially
produce the same protein, this distinction in transcript levels and abundance provides insight as
to which splice variant is involved in the attenuation of JA responses. The ΔPY form of JAZ10 is
also likely to be abundant because the protein products of JAZ10.2 and JAZ10.3 are identical.
Retention of the entire Jas intron in JAZ10.2 results in a long 3’ UTR. It is unknown what role, if
any, the 3’ UTR may have in JAZ mRNA stability and translation. Microarray studies suggest
that some JAZ transcripts are highly unstable, but it remains to be determined how transcript

47

instability impacts JAZ protein levels (Gutierrez et al., 2002). In summary, this data suggest ΔPY
JAZ variants are relatively abundant in JA stimulated tissues, have increased stability in response
to JA, and that these isoforms play an important role in attenuation of JA responses.
High throughput sequencing was used to determine whether JAZ genes produce alternative
splice variants in response to various stress-related treatments. JAZ gene expression increases in
response to MeJA treatment, COR treatment and wounding (Table 1A). Reads aligned to unique
splice junctions for each splice variant of JAZ10, but the full-length transcript (JAZ10.1) was
confirmed to be the predominant form in all conditions. Similarly, the RNA-seq data support the
existence of JAZ2 and JAZ6 transcripts in which the Jas intron is retained (Table 1B).
Interestingly, predictions of Jas intron retention in JAZ5 and JAZ9 splice variants are strongly
supported by the Illumina data, which has not been previously reported. Confirmation of JAZ5.2
and JAZ9.3 splice variants by qPCR, together with ectopic expression of these variants in
transgenic plants, may reveal their functional role in the regulation of JA signaling.
Some JAZ transcript models were weakly supported by the RNA-seq data, which may occur
for several reasons. Jas intron retention was identified by mapping reads to all JAZ isoforms in
TAIR9 and additional predicted retention events in Chung et. al., 2010. The inclusion of
additional predicted splice variants as mapping templates to identify unique reads often
prevented reads mapping to the full-length transcript due to sequence redundancy. Evidence of
Jas intron retention for JAZ3 and JAZ12 were supported weakly by the RNA-seq data. Some
TAIR9-supported gene models, such as JAZ4.2, also were not supported in this data set. In the
case of JAZ3, this is likely attributed to the low expression of the gene. It is also important to
note these data require replication. Comparison across treatments is difficult due to different
developmental stages, tissue types and treatment conditions. Weakly supported gene models

48

maybe be further confirmed by sequencing additional replicates and may require validation by
other methods such as qPCR.
The availability of a jaz10-1 null mutant provides a useful tool for further analysis of the
function of JAZ10 alternative splice variants (Sehr et al., 2010; Demianski et al., 2011). It is
currently unknown which JAZ10 splice variants are involved in attenuation of JA responses. To
address this question, transgenic plants expressing each JAZ10 splice variant from the native
JAZ10 promoter may be used to complement the JA hypersensitive phenotype of jaz10-1 plants.
It would also be interesting to measure the level of JAZ proteins and their splice variants, and to
determine the extent to which protein levels correlate with transcript levels.
Ectopic expression of alternative splice variants provides insight into the function of
alternatively spliced JAZ proteins. The transgenic lines 35S-JAZ10.3, 35S-JAZ10.4 and 35SJAZ10G were previously shown to exhibit JA insensitivity in roots and, in the case of 35SJAZ10.4, male sterility (Chung and Howe, 2009; Chung et al., 2010). In addition to JAZ10,
JAZ2.2 also showed JA insensitivity in root length assays (Chung et al., 2010). 35S-JAZ2.2 and
35S-JAZ10.3 confer similar levels of JA insensitivity in root length assays. Other overexpression
constructs, such as 35S-JAZ3.4, did not confer reduced sensitivity to JA in root length assays
(Chung et al., 2010). Consistent with this finding, JAZ3.4 interacts with COI1 in a similar
manner as full-length JAZ3.1 in yeast two hybrid assays and in vitro pull down assays (Chung et
al., 2010). The JA responsiveness of 35S-JAZ4.1 seedlings was identical to that of WT, whereas
35S-JAZ4.2 lines showed a significant reduction in JA sensitivity. Validation of this conclusion
will require further analysis of homozygous lines. Preliminary data suggest that 35S-JAZ4.2
seedlings are not as insensitive as seedlings that overexpress JAZ10.3, JAZ10.4 or JAZ2.2.

49

Variation in JA insensitivity among transgenic lines that ectopically express different JAZ
splice variants appears to correlate with differences in position of the PTC within the retained Jas
intron (Figure 11). It is possible that the position of the PTC in the Jas intron affects the stability
of the α-helix that is part of the JAZ degron, thereby altering interactions with COI1 (Sheard et
al., 2010). It is possible that removal of the X5PY motif through alternative splicing disrupts the
integrity of the degron, thereby impeding efficient hormone-dependent COI1-JAZ binding.
Analysis of other truncated JAZ proteins, such as JAZ6.2, may provide a means to test this
hypothesis. Additional experiments in which the length of the C-terminus is systematically
altered may also be used to test this hypothesis.
The phenomenon of alternative splicing as a mechanism to regulate stress responses may be
widespread in plants (Ali and Reddy, 2008; Gassmann, 2008; Filichkin et al., 2010). In addition
to JAZ genes, there may be numerous, functionally relevant alternative splicing events that are
involved in biotic stress response. Understanding the role of alternative splicing in the regulation
of JA signaling will provide valuable insight into how plants cope with biotic and abiotic stress.

50

AT LOCUS NAME
AT5G13220 JAZ10.1 FP
JAZ10.3 FP
JAZ10.1/10.3 RP
JAZ10.4 FP
JAZ10.4 RP
JAZ10.2 FP
JAZ10.2 RP

PRIMER SEQUENCE
5'-GAAGCGCAAGGAGAGATTAG-3'
5'-AAGGAGAGGTAATGATTCTTCAACAAT-3'
5'-AGCCAAATCCAAAAACGAACA-3'
5'-GCTAATGAAGCAGCATCTAAGAAAGA-3'
5'-GCGATGGGAAGATCGAAAGA-3'
5'-CCCCCAAATAATTAAAGAAAGGTTTTT-3'
5'-AAGCATGTGCGTTGTTGAACA-3'

AT1G74950 JAZ2.1/2.2 FP
JAZ2.1 RP
JAZ2.2 RP

5'-CAAAAACCGCAGCACAAGAG-3'
5'-CCTTTGATGTGATCCTATCCTTCCT-3'
5'-CAAGATATTATGTTTTCATTAAAATGCATTAC-3'

AT1G72450 JAZ6.1/6.2 FP
JAZ6.1 RP
JAZ6.2 RP

5'-CCGGGAACAATGAAGATCAAG-3'
5'-CCACAGCCCTGTCTTTTCGT-3'
5'-AGTTTCGGAGTTTAGTTTACCTGTCTTT-3'

AT1G13320 PP2A FP
PP2A RP

5'-AAGCAGCGTAATCGGTAGG-3'
5'-GCACAGCAATCGGGTATAAAG-3'

AT5G08290 YLS8 FP
YLS8 RP

5'-CTCTCAAGGACAAGCAGGAGTTCATT-3'
5'-CGGTATTTGGTGGAGTAATCTTTTGG-3'

Table 2. qPCR Primers for the Quantification of JAZ Alternative Splice Variants. Primer
pairs were designed across unique splice junctions for each JAZ alternative splice variant. In the
case of JAZ10.1 the primer pair detects two transcripts, JAZ10.1 and JAZ10.4. Primer pairs were
confirmed to have over 90% PCR efficiency.

51

MATERIALS AND METHODS

Plant material and growth conditions

Arabidopsis (ecotype Columbia-0; Col-0) was used as the wild type (WT) for all experiments.
Seeds were surface sterilized with 40% (v/v) bleach for ten minutes, washed with water ten
o

times, and stratified at 4 C for three days. Seedlings were inoculated in baffled, 125-ml flasks
containing 50 ml MS medium supplemented with 0.8% sucrose. Flasks were incubated with mild
o

-2 -1

shaking (200 rpm) under long-day conditions (16 h light/8 h dark) at 21 C and 100 μE m s of
light. For plants grown on soil, seeds were surface sterilized and stratified as described above
prior to sowing on autoclaved soil. 35S-JAZ10G, 35SJAZ2.2, 35S-JAZ4.2 transgenic lines were
previously described in Chung et al., 2010.

Plant treatments

Liquid-grown seedlings were treated with either 100 μm MeJA (Sigma-Aldrich) or a mock
control (0.007% ethanol) as described in the legend of Figure 4. For experiments involving
wounding, leaves of similar size on 25-day-old soil-grown plants were mechanically wounded
twice along the leaf midvein with a hemostat. Wounded and unwounded control leaves
harvested, and pooled from two plants per biological replicate. Primary and secondary
inflorescences were harvested from soil grown WT and 35S-JAZ10G plants. Flowers at various
developmental stages (stages 1-12 as defined by Smyth et al., 1990) prior to bud opening were
pooled from four, seven-week old plants. Root tissue was collected from vertically growing

52

seedlings on MS agar (% 0.8 agar) plates for nine days in continuous light. Autoclaved nylon
mesh (Sefar America Inc.) was placed on top of the agar prior to sowing seeds. Prior to
harvesting, a razor blade was used to excise the roots below the hypocotyl. Approximately 100
mg of tissue was collected and frozen in liquid nitrogen in fast-prep tubes (MP biomedical)
containing metal balls.

RNA extraction and cDNA synthesis

Prior to RNA extraction, tissue was homogenized in frozen blocks using the Qiagen
TissueLyser® II at a frequency of 25 s

-1

for 1.5 min. RNA was extracted with the RNeasy®

Plant Mini RNA kit (Qiagen) according to manufacturer’s protocol. To ensure removal of
genomic DNA, RNA was treated with RNase Free DNase (Qiagen) for 15 min prior to elution
with diethlypyrocarbonate treated, RNase-free water. RNA was quantified with a Nanodrop
spectrophotometer. Assessment of RNA quality was done by determining the A260/A280 ratio,
and only those samples with a ratio of 1.8 or higher were used. RNA samples for submission to
Illumina sequencing were also analyzed for integrity on a microfluidics chip (Agilent
Bioanalyzer). Only those RNA samples with and RNA integrity number higher than seven were
used for Illumina sequencing (Bustin et al., 2009).
Approximately 100 ng of RNA was used as a template for cDNA synthesis using the High
Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). cDNA was diluted 1:10 for
qPCR assays. In the case RNA isolated form roots, 500 ng of RNA was used at a 1:5 dilution.
Linearity of the reverse transcriptase (RT) reaction was tested using serial dilutions of RNA for
cDNA synthesis and showed no inhibition of the RT reaction with R2 values above 90%. Primer

53

efficiency was calculated by testing serial dilutions of cDNA concentrations and performing
qPCR and showed primers were efficient in amplifying transcripts with R2 values above 90%.

Quantitative real-time polymerase chain reaction (qPCR)

Primer pairs were optimized for Power SYBR Green (Applied Biosystems®) using the
PrimerExpress® software provided by Applied Biosystems®. All primer sets were checked for
specificity against the Arabidopsis genome using BLAST. For each primer set, dissociation
curves were generated to confirm the presence of a single product. Primers were designed to
unique splice-site junctions to ensure specificity. In the case of JAZ10.1, it was not possible to
identify a unique region that is not shared by the other splice variants. To resolve this problem, a
primer set was designed to detect both JAZ10.1 and JAZ10.4.
qPCR was performed using SYBR Green chemistry and the 7500 Fast Real-Time PCR
(Applied Biosystems) system with default settings. Ct values were determined using default auto
Ct settings. In the case of JAZ10.1, values shown are a sum of JAZ10.1 and JAZ10.4. JAZ
transcript levels were normalized to the reference genes PP2A (AT1G13320.1) and YLS8
(AT5G08290), using the geometric mean of the two reference genes and the ΔCt method
(Czechowski et al., 2005; Livak and Schmittgen, 2001). The expression level of these reference
genes did not change in response to JA treatment or in different tissues. Unless otherwise
indicated, error bars indicate standard error of at least three technical and biological replicates.

54

Illumina sequencing

Six RNA samples were analyzed by Illumina sequencing. One set of samples was obtained
from nine-day-old seedlings treated with 100 μm MeJA or a mock control (0.007% ethanol) for
two hours. The second set of RNA samples was obtained from -day-old seedlings treated with 5
μm coronatine (vendor) or a mock control (0.007% ethanol) for one hour. The third set of
samples was obtained from control (unwounded) or wounded leaves, which were harvested one
hour after mechanical wounding (see above). RNA was extracted as described above. All
samples were sequenced (55 nt paired-end reads) on the Illumina Genome Analyzer II. The
program Tophat was used to map unique, forward reads to the TAIR9 cDNAs of Arabidopsis.

Transgenic plants

Col-0 plants were grown on soil for 4 to 6 weeks until flowering. Plants were transformed
with Agrobacterium tumefaciens strain C58C1 as previously described (Clough and Bent, 1998).
Transformed T1 seeds were screened for resistance to kanamycin (50 μg/ml) and vancomycin
(100 μg/ml) on MS agar plates. T2 seedlings were screened for altered sensitivity to JA in the
root growth assays

55

Root length assays

Seeds of each genotype were surface sterilized with 40% (v/v) bleach for 10 min, washed
o

ten times with autoclaved water, and then stratified for three days at 4 C. Seeds were sown on
MS media supplemented with 0.8% sucrose with or without 50 μM MeJA. Seedlings were grown
vertically for nine days in continuous light. Photographs of roots were taken with a Nikon D80
DSLR camera, and root lengths were measured with ImageJ software.

56

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