Báo cáo khoa học: Cloning and expression of a tomato cDNA encoding a methyl jasmonate cleaving esterase pdf

Cloning and expression of a tomato cDNA encoding a methyl
jasmonate cleaving esterase
Christiane Stuhlfelder, Martin J. Mueller and Heribert Warzecha
Lehrstuhl fu
¨
r Pharmazeutische Biologie, Julius-von-Sachs-Institut fu
¨
r Biowissenschaften, Universita
¨
tWu
¨
rzburg, Germany
Jasmonic acid and its methyl ester are ubiquitous plant s ig-
nalling compounds nec essary f or the regulation of growth
and development, as well as for the response of plants to
environmental stress f actors. To date, it is not clear w hether
methyl jasmonate itself acts as a signal or if its conversion to
jasmonic acid i s mandatory pri or t o the induction of a def-
ense response. We have cloned a cDNA, e ncoding a m ethyl
jasmonate-cleaving enzyme, from tomato cell suspension
cultures. Sequence analysis revealed significant similarity
to plant esterases and to (S)-hydroxynitrile lyases with
an a/b-hydrolase fold structure. The coding sequence was
heterologously expressed in Escherichia coli and purified in
a c atalytically active form. Transcript levels, as well as en-
zymatic a ctivity, were determined in different tomato tissues.
High transcript levels and enzyme a ctivities w ere f ound in
roots and flowers, while the m RNA level and activity w ere
lowinstemsandleaves.Moreover,whentestedinmethyl
jasmonate- and elicitor-treated cell s uspension cultures,
transcript levels were found to decrease, i ndicating that this

particular enzyme might b e a regulator of jasmonate sig-
nalling.
Keywords: a/b-hydrolase; cell suspension culture; Lyco-
persicon e scule ntum; methyl jasmonate esterase; Solanaceae.
Jasmonic acid (JA) is a ubiquitous plant compound, which
plays a crucial role in t he response to w ounding or pathogen
attack, as w ell as i n developmental proce sses, such a s fruit
ripening, r oot growth, a nd fertility [ 1]. Most o f the en zymes
involved in JA biosynthesis have been characterized at a
biochemical and molecular level and the encoding genes
have been cloned. Biosynthesis takes place mainly in
chloroplasts and peroxisomes, initiating after the release
of the precursor linolen ic acid from membrane stores by
lipases. The enzymes lipoxygenase, allene oxide synthase,
and allene oxide cyclase form the b iosynthesic i ntermediate
12-oxo-phytodienoic a cid (OPDA). Subsequent action of
OPDA reductase and three cycles of b-ox idation lead to the
formation of JA [ 2,3]. Thereafter, JA may b e esterified to its
derivative methyl jasmonate (MeJA) [ 4], or c onjugated with
an amino acid or glucose [5].
For most of t he jasmonates it h as been shown that t hey
are capable of mediating a response by regulating gene
expression [6,7]. Analysis of Arabidopsis thaliana mutants
impaired in either JA biosynthe sis or signalling, gave a
deeper insight i nto the function of single oxylipin s [8]. T he
fad3–2fad7–2fad8 mutant, which forms almost no trienoic
fatty acids [9], is male sterile and fertility could only be
restored by application of linolenate or JA. Another mutant
– opr3 – a rrests jasmonate biosynthesis at the OPDA level
and is incapable of metabolizing exogenously applied

OPDA to JA [10]. opr3 mutants d isplayed a normal defense
response towards a variety of pathogens, indicating that
OPDA alone is sufficient to initiate an effective defense
response. However, mutant plants were male sterile and
fertility c ould be restored by th e exoge nous application of
JA. These experiments demonstrate that individual mem-
bers of the jasmonate family are involved – at least in
Arabidopsis – in different signalling pathways.
An Arabidopsis(jar1)mutantwithadefectinthe
jasmonate response has been described. The mutant is
insensitive to MeJA and does not show root growth
inhibition or vegetative storage protein (VSP) ind uction in
response to MeJA [11]. Recent analysis of the jar1 locus
revealed that its gene product modifies JA v ia adenylation,
which is a pparently a p rerequisite for downstream signaling.
The modification requires a free carboxyl group as the
enzyme does not accept MeJA as a substrate [12]. T herefore,
MeJA must be de methylated prior to becoming active.
Thus, root growth inhibition and VSP expression are
mediated by MeJA through JA, indicating that MeJA is not
amediatoronitsowninthisparticularsystem.
On the other hand, OPDA and JA can induce identical
genes a s w ell as d istinct s ets o f t arget genes, suggesting that
independent signalling pathways exist [13] and that the
combined action of different inducers might be necessary for
the full activation of responsive genes [14]. However, in
Correspondence to H. Warzecha , Lehrstuhl fu
¨
r Pharmazeutische
Biologie, Julius-von-Sachs-Institut, Julius-von-Sachs-Platz 2,

97082 Wu
¨
rzburg, Germany.
Fax: + 4 9 9318886182, Tel.: + 49 9318886162,
E-mail:
Abbreviations: HNL, ( S)-hydroxynitrile lyase; JA, j asmonic acid;
JMT, S-adenosyl-
L
-methionine jasmonic a cid carboxyl methyltrans-
ferase; MeJA, methyl jasmonate; MJE, methyl jasmonate esterase;
MeSA, methyl salicylate; OPD A , 12-oxo-phytodienoic acid; P I ,
proteinase inhibitors; PNAE, polyneuridine aldehyde esterase;
RACE, rapid amplification of c D NA ends; VSP, vege t ative storage
protein.
Note: The sequence reported h erein was deposited under GenBank
accession number AY455313.
Note: A website is available at http://132.187.108.6/
(Received 2 8 March 20 04, revised 1 9 May 2004,
accepted 25 May 2004)
Eur. J. Biochem. 271, 2976–2983 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04227.x
the case of MeJA and JA, the biological activities of the
exogenously administered compounds are apparently iden-
tical, possibly because rapid interconversion takes place
in vivo.
Another quality of JA-mediated plant defense is the
systemic spread of defense responses after local induction.
For instance, in tomato and potato plants the production of
proteinase inhibitors (PI) as an inducib le defense r esponse
against feeding C olorado potato beetle is not limited to the
site of their attack, but also appears in distant leaves o f the

plant [15]. The herbivore attack induces JA biosynthesis
locally via systemin, an 18 amino acid s ignal p eptide.
However, the induction of PI genes could be found also in
distant parts of the plant, which requires a long distance
signalling component. Grafting experiments with tomato
mutants deficient i n either J A biosynthesis or J A perception
proved that a jasmonate, rather than s ystemin, is the signal
which is translocated t hrough the plant [ 16]. Rootstocks of
the spr-2 mutant, which are impaired in JA biosynthesis,
were not capable of generating a transmissible signal that
could induce PI expression in wild-type scions, while
wounded wild-type root stocks did induce PI e xpression
in spr-2 scions.
A strong candidate for a transmissible jasmonate signal is
the JA-conjugate, MeJA. The volatile ester can diffuse
through membranes and c an be found in the headspace
above w ounded leaves [17], suggesting that M eJA m ight be
an interplant c ommunication signal [18]. M oreover, it has
been speculated that the physical role of MeJA is to
mobilize JA [ 19]. The proof of this hypothesis might come
from a more detailed understanding of how plants form
MeJA from JA, and vice versa. A recent study identified an
S-adenosyl-
L
-methionine:jasmonic acid carboxyl methyl-
transferase (JMT) from Arabidopsis, w hich conver ts JA to
MeJA [4]. Constitutive overexpression of JMT tripled the
MeJA content of transgenic plants and also induced JA-
responsive g enes. Therefore, t ransgenic plants with elevated
JMT levels s howed enhanced resistance against the necro-

trophic fungus Botrytis cinerea, suggesting a prominent role
for the enzyme in jasmonate-mediated defense.
It is not yet clear whether MeJA functio ns as a paracrine
signal that is re leased from sites of pathogen attack to
induce defense genes at distant sites. Moreover, i t r emains to
be clarified whether MeJA is a mediator on its own that
elicits JA responses without prior hydrolysis to JA. How-
ever, if M eJA is considered to be a s ignal, there must be a
way to regulate the signal by controlled formation and –
perhaps more importantly – by its controlled inactivation.
A candidate for performing the latter task is an est erase,
previously characterized in tomato [20]. E nzyme activity has
been found to be constitutively present in cell cultures of
many taxonomically distant plant species. In tomato cell
cultures, only one MeJA-hydro lysing enzyme could be
identified by activity-guided protein purification. The
tomato esterase has been purified and characterized from
tomato cell suspension cultures. Owing to its MeJA-cleaving
activity, we named the enzyme methyl jasmonate esterase
(MJE). Yet, it remains to be established w hether MJE has a
function in JA/MeJA signalling. As a first step to investigate
this, we cloned a cDNA from tomato encoding MJE.
Analysis of transcript incidence showed that MJE is
differently expressed in different organs as well as after
elicitation, indicating that this enzyme might be i nvolved i n
jasmonate signalling.
Experimental procedures
Plant material
Tomato plants (Lycopersicon e sculentum cv. M oneymaker)
were grown in the greenhouse under con ditions of 16 h light

and 8 h darkness. The growth temperature range was
16–22 °C with a relative humidity of 60–70%.
Cell suspension cultures (L. esculentum )weregrownin
1 L Erlenmeyer flasks in Linsmaier & Skoog media [21] for
7 d ays under continuous light (600 lux) on orbital s hakers
(100 r.p.m.) a t 24 ± 2 °C. Ce lls were harvested by suction
filtration,shockfrozeninliquidnitrogen,andstoredat
)80 °C until use.
Nucleic acid isolation and blot analyses
Plant material for the isolation of nucleic acid was either
4-day-old cell s uspension c ulture or 8-week-old plants. T otal
RNA from suspension cultures w as isolated either accord-
ing to the protocol described previously [22], or with the
TRIzol reagent ( Invitrogen), and used for RT-PCR or
Northern blots, respectively. Isolation of RNA from p lant
material was c arried out using the RNeasy Plant M ini Kit
(Qiagen). For DNA purification the protocol described
previously [23] was employed.
Standard protocols were used for the transfer of RNA
and DNA after electrophoretic sep aration [24]. Hybridiza-
tion of RNA or DNA transferred to n ylon membranes was
performed using a nonradioactive digoxigenin-probe label-
ling system (Roche).
RT-PCR and cloning of partial- and full-length cDNA
Two degenerated primers were designed according to
previously determined peptides. Sequences were YTTRTC
RCANACNACRTANACNCKRTGNACNSWNCCRTA
for MJErev4 and GAYATGGCNGCNWSNG GNATH
AAYCC for MJEfor3.1. With total RNA from cell suspen-
sion cultures and primer MJErev4 for first-strand synthesis,

cDNA was produced using the RT-PCR s ystem fr om Qiagen .
RT-PCR conditions were 30 min at 5 0 °C, 15 min at 9 5 °C;
five cycles of 30 s at 94 °C, 1 m in at 40 °Cand1minat
72 °C, followed by 30 cycles of 30 s a t 94 °C, 1 min at 45 °C
and 1 min at 72 °C. Gel electrophoresis, DNA elution a nd
modification was carried out according to standard protocols
[24]. A fter c loning o f th e P CR pr oduct into the v ector pG EM-
T ( Promega) and s equencing (automated s equencer LI-COR
4200), two homologous primers were designed for the rapid
amplification of cDNA ends (RACE) (RACEfor: GTGA
CAGCTTTCATGCCTGG; and RACErev: ATCCTGT
CCGTTGTTGTAAAC). 5¢-and3¢-RACE w as performed
using the SMART II system (BD Bioscience). Full-length
cDNA for expression was cloned by another RT-PCR with
primers f ullMJEforMQ (GCA TGCAGGGTGATAAAAA
TCACTTTGTA) and fullMJErev (AAGGATCCATAA
TATTTTTGCGAA ATC), adding rest rictio n sites for SphI
and BamHI, r espectively. Th e PCR pr oduct w as cloned i nto
vector pDRIVE (Qiagen), sequenced and subcloned into
Ó FEBS 2004 Methyl jasmonate esterase from tomato (Eur. J. Biochem. 271) 2977
expression vector pQE70 (Qiagen) via SphIandBglII
restriction s ites.
Overexpression and purification
Escherichia c oli M15 cells harbouring the MJE e xpression
plasmid w ere cultured a t 20 °C on a rotary shaker
(200 r.p.m.). Twenty-four hours after induction with
1m
M
isopropyl thio-b-
D

-galactoside, cells of a 5 L suspen-
sion were harvested and lysed by sonication. The crude
extract was cleared by centrifugation (10 000 g) and separ-
ated on Q-Sepharose fast-flow 26/20, gel-filtration on
Sephacryl S-100 HR 26/60, and MonoQ HR 5/5 (all
Amersham Biosciences), according t o a procedure described
previously [20]. For metal affinity chromatography, Talon
resin ( BD Bioscience) was utilized. A nalysis of proteins was
performed with SDS/PAGE (12.5%) under denaturating
conditions, a nd gels were silver stained as d escribed
previously [25]. For Western blot analysis, proteins were
blotted onto nitrocellulose filters and detected with
anti-6·His primary antibody, alkaline phosphatase-labeled
secondary antibody and chemoluminescent substrate
(CDP-Star; Roche) .
MJE activity was monitored according to a previously
published protocol [20].
Results
Isolation of MJE cDNA
We previously described an MJE, which is the only or at
least the predominant protein with MJE a ctivity i dentified in
tomato cell c ulture s. On the basis of the p artia l amino a cid
sequences obtained from the purified MJE [20], two
degenerate primers were developed. T his method has been
proven successful in several r eports [26,27] and s hould lead
to the identification of the encoding gene rather than
orthologous genes. Owing to the similarity of the peptide
fragments with sequences of known proteins, the sequence
PF18b (DMAASGINPK) was utilized to design a sense
primer, and sequence PF23 (RVYVVCDKD) w as used for

the generation o f an antisense prime r. Using tomato cDNA
as a template, a 498 bp fragment was amplified by PCR.
Sequencing revealed a DNA s tretch that e ncodes a peptide
with sequence similarity to a/b-hydrolase fold proteins (data
not shown), some of w hich have been p reviously aligned to
the internal fragments of purified MJE [20]. To obtain the
full-length cDNA by RACE, t wo seq u ence-specific prime rs
were synthesized, generating overlapping fragments after
5¢-and3¢-RACE, respectively. Sequencing and annealing of
the 5¢ and the 3¢ sequence revealed an ORF of 789 bp,
encoding a 262 amino acid protein (Fig. 1). All four p eptides
from the purified tomato protein could be identified in the
deduced amino acid sequence, which substantiates that the
cloned cDNA encodes the purified protein. The calculated
molecular m ass o f the encoded protein is 29 524.93 Da and
the pI ¼ 5.52. The calculated mass o f the encoded protein
corresponds well with the m olecular mass of  28 000
determined by SDS/PAGE for the purified plant protein.
Comparison of the N-termini showed that the protein
originally purified from tomato lac ked two amino acids:
Met and Glu. It could not be concluded if this was a result of
degradation of the protein during the purification process or
whether t he protein was modified in vivo .Nopeptidesignal
for subcellular targeting could b e i dentified.
Sequence alignment
Sequence analysis a nd alignment with known proteins f rom
GenBank showed a high similarity of MJE to ethylene-
induced esterase from Citrus sinensis (47% identity, 65%
positivity) [28], the tobacco salicylic ac id binding protein 2
(SABP2) (47% identity, 65% positivity) [29], the polyneu-

ridine aldehyde esterase (PNAE) from Rauvolfia serp entina
(44% identity, 65% positivity) [27], and s everal lyases
involved in the biosynthesis of cyanogenic compounds in
different plant species [36% identity to (S)-hydroxynitrile
lyase (HNL) from Hevea brasiliensis [30], 33% to (S)-
Fig. 1. Tomato methyl jasmonate esterase cDNA and deduced protein
sequence. Pe ptides determined by seq ue ncing of the p urified protein are
boxed and the names of the peptides are indicated in italic letters
above. Nucleic acids o f the ORF are shown in ca pital letters, while 5¢
and 3¢ untranslated regions are in lowercaseletters.Theputative
amino acid r esidues of t he catalytical t riad of a/b-hydrolase fold pro-
teins are marked with an asterisk.
2978 C. Stuhlfelder et al.(Eur. J. Biochem. 271) Ó FEBS 2004
acetone-cyanohydrin lyase from Manihot esculentum][31].
In addition, several putative proteins from the Arabidopsis
genome exhibited high sequence similarity to MJE. For
sequence alignment and analysis, only known or at least
partially characterized proteins were included (Fig. 2). As
all the aligned proteins belong to the extremely divergent
family of a/b-hydrolase fold proteins, i t c ould b e assumed
that MJE is a member of this protein family. As further
support of this assumption, MJE shows the highly con-
served amino acid residues forming the catalytic triad [32] –
nucleophile, acid, and a his tidine – represented b y s erine at
position 83, aspartic acid at position 2 11, and histidine at
position 240 (Fig. 2). Moreover, it has been shown that
MJE could be irreversibly in hibited by phenylmethanesulfo-
nyl fluoride [20], a specific inhibitor o f s erine hydrolases [33].
Bacterial expression and purification of tomato MJE
To obtain unequivocal evidence of the identity of the cloned

sequence, MJE cDNA was subcloned i nto a bacterial vector
for h eterologous expression. As amplification of the coding
sequence with a forward primer homologous to the 5¢-end
failed, the primer s equence had to be modified f or enhanced
binding and amplification. Primer design was carried out
using t he Vector NTI Software (Informax). Thereby, a
modified N-terminus of the encoded protein was created in
which Glu and Lys in positions 2 and 3, respectively, were
replaced with a single Gln.
At the C -terminus a 6 ·His extension was added to
simplify subsequent purification of the protein. Crude
extracts of E. coli M15 cells harbouring the pQE-MJE
plasmid showed MJE-esterase activity (1.64 pkatÆmg
)1
)
after isopropyl thio-b-
D
-galactoside induction, while wild-
type M15 cells did not show any MJE activity. This v alue
was comparable with the activity found in crude extracts
from tomato cell suspension culture in previous experiments
(1.77 pkatÆmg
)1
) [20], but was much less t han expected for
an enzyme from heterologous bacterial expression of the
cDNA. For visualization of proteins, bacterial extracts were
subjected to SDS/PAGE. In a comparis on of MJE-produ-
cing E. coli with wild-type cells, no p rominent protein w ith
the approximate size of MJE (29 kDa) could be detected
Fig. 2. Multiple sequence alignment. Alignment of methyl jasmonate esterase (MJE) with a/b-hydrolase fold proteins fr om different plant species.

EIE, ethylene-induced este rase from Citr us sinensis (GenBank accession number AAK58599); SABP2, salicylic acid-binding protein from Nic-
otiana tabac um (AY485932); PNAE, polyneuridine aldehyde esterase from Ra uv olfia serpentina (AAF22288); Pir7b, d efense-related ri ce ge ne from
Oryza sativa (CAA84024); H NL, (S)-hydroxynitrile lyase f rom Hevea brasiliensis (P52704).
Ó FEBS 2004 Methyl jasmonate esterase from tomato (Eur. J. Biochem. 271) 2979
after Coomassie Blue staining (data not shown). This
observation suggests that M JE is not abundantly produced
or that it is not stable in E. coli. Because of the low
abundance in E. coli, a four-step purification protocol was
employed to purify the enzyme (Table 1). Catalytically
active enzyme was obtained after anion exchange on
Q-Sepharose, gel filtration with Sephacryl S-100, further
anion-exchange chromatography on MonoQ, and finally
separation on immobilized metal affinity chromatography
(Talon resin). Starting from a 5 L bacterial s uspension
culture, MJE(His)
6
could be enriched 203-fold, resulting in
52 lg of protein. Figure 3 A s hows the silver-s tained poly-
acrylamide gel from the purified fraction. Although some
impurities are visible, we assume that solely the MJE is
responsible for MeJA-cleaving activity, as wild-type E. coli
is not capable of c leaving M eJA. A n a ntibody s pecific f or
hexa-histidine epitopes was used in an immunoblot experi-
ment to con firm the p resence and the size of the recombin-
ant protein. As shown in Fig. 3 B, extracts from E. coli
harbouring pQE-MJE showed a b and o f  29 kDa repre-
senting MJE(His)
6
, while wild-type bacteria did not.
Southern blot analysis

For Southern blot analysis, genomic DNA from cell
suspension cultures or greenhouse-grown tomato plants
(L. esculentum cv Moneymaker) was digested with BamHI ,
EcoRI, or HindIII restriction enzymes and probed with a
full-length cDN A of MJE at high stringency. It should be
noted that the MJE-coding sequence has a recognition site
for EcoRI at position 204 and therefore should show at least
two b ands in a Southern blot a nalysis. In addition to this ,
the probe hybridized with multiple bands (Fig. 4).
In the case of HNL from Cassava – which shows high
similarity to MJE – several gene copies were reported [ 34]. It
could not be concluded from our data whether tomato
contains several homologous genes, if some signals are a
result of probe hybridization with pseudogenes, or if
multiple bands occur o wing to the presence of recognition
sites for the utilized restriction enzymes within introns (as
assumed f or the HNL from Hevea) [30]. However, during
the purification of MJE there was n o evidence for the
expression of isoenzymes, although, if present, they might
have different catalytic properties.
Northern blot analysis and induction of MJE expression
in cell cultures
Northern blot analysis was u sed to determine MJE
transcript levels in different plant organs. Therefore, total
RNA from roots, leaves, stems, and flowers was probed
with full-length MJE cDNA. Transcripts of  1kbwere
present i n a ll plant tissues and significant v ariations in their
amounts could be d etected (Fig. 5A). High levels of MJE
mRNA could be found in roots a nd flowers, while low-to-
moderate amounts w ere present in the leaves and stems of

tomato plants. The RNA levels correspond well with the
MJE activity found in different plant organs, showing that
high enzymatic a ctivity c orrelates w ith a high transcript level
(Fig. 5 B).
Table 1. Purification of recombinant methyl jasmonate e sterase.
Purification step
Total protein
(mg)
Total activity
(pkat)
Specific activity
(pkat)
Purification
(fold)
Recovery
(%)
Crude extract 2044 3352 1.64 1 100
Q-Sepharose Fast Flow 712 1317 1.85 1.13 27.7
Sephacryl HR 26/60 67.5 652.7 9.67 5.89 19.4
Mono Q HR 5/5 1.9 100.6 54.39 33.16 3.0
Talon resin 0.052 24.1 464.3 283.11 0.8
Fig. 3. SDS/PAGE and Western blot a nalysis
of me thyl jasmonate esterase (MJE) purified
from rec ombinant bacteria. (A) Silver-stained
proteins after separation b y SDS/PAGE
(12.5% gel). M, marker proteins (sizes are
indicated on t he left); lane 1, purified f raction
after t he last pu rification step. MJE is indica-
tedbyanarrow.(B)Westernblotofcrude
bacterial extracts after transfer to n itrocellu-

lose. An antibody specific for 6·His modified
proteins was used. L ane M, pr otein standard;
lane 1, Escherichia coli harbouring expre ssion
plasmid p QE70-MJE; lane 2, E. coli without
the expression plasmid. MJE is indicated with
the arrow.
2980 C. Stuhlfelder et al.(Eur. J. Biochem. 271) Ó FEBS 2004
In order to evaluate whether transcript levels could be
regulated b y external stimuli, cell s uspension c ultures were
treated with MeJA, m ethyl salicylate (MeSA), or chitosan,
and mRNA levels were determined in a time-dependent
manner. While basal levels of m RNA w ere h igh in untreated
cultures, t he levels decreased 1 h after MeJA treatment and
returned to basal levels  8 h postinduction (Fig. 5C). A
similar time c ourse could b e observed a fter treatment w ith
the elicitor chitosan, although with a slightly delayed
response. No changes in mRNA levels were observed after
treatment with MeSA.
Discussion
Sequence alignment of the MJE revealed high similarity to
a/b-hydrolase fold proteins of different origin a nd with
diverse properties. Notably, all plant proteins of known
function with high sequence s imilarity to MJE appear to be
involved in the defense response and/or secondary metabo-
lism. Among the related proteins is the ethylene-induced
esterase from C. sinensis [28], a recently discovered SABP2
from tobacco [29], the Pir7b protein from Oryza sativa,
hydroxynitrile lyases of different origins and the PNAE
from the medicinal plant R. serpentina. Nevertheless, the
substrate acceptance and enzymatic activity of those

enzymes, if known, is highly diverse. With the increasing
number o f s pecified plant enzymes of this g roup of the a/b-
hydrolase f amily, i t might be assumed t hat they have arisen
from a common ancestral gene and the descendants
partially occupied species-specific niches in secondary
metabolism. Similar scenarios have been described for plant
O-methyl transferases [35,36] or dioxygenases [37]. It should
Fig. 4. Southern blot analysis of total genomic DNA from tomato
plants. Fifty micrograms of DNA was digested overnight with
restriction enzymes and separated on a 1% agarose gel. M, size
standard; lane 1, Bam HI-digested DNA; lane 2, EcoRI digest; l ane 3,
HindIII dig e st.
Fig. 5. Levels of methyl jasmonate esterase (MJE) t ranscripts in dif-
ferent tissues and after induction. (A)NorthernblotoftotalRNAfrom
different tissues hybridized with the full-length cDNA probe. RNA
was isolated from roots, leafs, stems and flowers. The lower panel
shows 17S rRNA as a loading control (after methylene-blue staining);
the results shown were consistent in three different experiments.
(B) Specific activity of MJE in different plant organs. (C) Time course
of MJE t ranscript a b undance after treatment with methyl jasmonate
(MeJA), methyl salicylate (MeSA), an d chitosan. Lo ading contr ols
show the ethidium b ro mide-stained gel p rior to transfer.
Ó FEBS 2004 Methyl jasmonate esterase from tomato (Eur. J. Biochem. 271) 2981
be noted that in the Arabidopsis genome at least 20 genes
with homologies to HNL and PNAE could b e found. It is
unlikely that they have similar properties to the a/b-
hydrolase fold proteins m entioned above, as they occur only
in distinct plan t families o r even s pecies and t heir substrates
are not present in Arabidopsis. O n the other hand, MJE
activity could be found in several plant systems. From the 1 8

plant cell suspension cultures of taxonomically distant
species tested to date, virtually all exhibited MJE activity
[20]. O ne might s peculate that the M JEs of different species
are encoded by orthologous genes.
Whenever analysed, plant cells and tissues contain JA
and its methyl ester, MeJA, side by side. In most reports,
authors have made little effort to distinguish between the
biological activity of the two compounds and usually only
JA content is measured. If values of bo th JA and MeJA are
published, the ratios depend on the plant species and the
tissue a nalysed a nd vary from 3 : 2 (JA/MeJA) i n Arabid-
opsis leaves [4] to a bout 10 : 1 in tomato flowers [38]. For
tomato cell suspension cultures, a JA/MeJA ratio of almost
1 : 1 was found [39]. To d ate there is no way of
distinguishing between the function of the two compound s
on a physiological basis as they can be rapidly converted
from one into the other. For the Arabidopsis jar1 mutant, i t
has been shown that the role of JAR1 is to modify JA via
adenylation of the carboxyl group [12]. As MeJA is not
accepted as a substrate in this putative essential step
involved in JA signalling, it is probable t hat MeJA has to
be hydrolyzed by MJE in order to become metabolically
activated. In this case, MeJA might represent a pool of inert
JA conjugates or plays a role as signalling molecule between
individual cells [19]. The inverse activity of JMT and MJE
suggests that both enzymes should be spatially separated.
MJE transcript levels were analyzed by Northern hybrid-
ization a nd revealed a high, constitutive expression in roots
and flowers and low RNA levels in leaves a nd stems,
suggesting that enzyme activity could b e found in all tissues.

These data were supported by activity assays of MJE in
different plant organs, which signify that high enzymatic
activity is contingent on high transcript levels.
Interestingly, undifferentiated tomato cell suspension
cultures accumulate both JA and MeJA at almost e qual
levels while displaying high MJE activity, suggesting that
individual cells may contain both JMT and MJE activity.
However, it is unlikely that a cell forms MeJA under the
expenditure of energy and degrades it immediately. Intra-
cellular s eparation of MeJA synthesis and hydrolysis would
be one way to avoid a treadmill situation, yet analysis of
tomato MJE and Arabidopsis JMT reveals no evidence for
subcellular targeting of the enzymes and, thus, both
enzymes should reside in the cytosol. Alternatively, sub-
strates may be presented for the enzymes in a highly
regulated manner.
To this end, it would be an a ppealing s cenario that a cell
could distinguish between endogenously formed and exo-
genous MeJA. Endogenously formed MeJA might be
exported and not hydrolysed, while MeJA coming from
outside the cell m ay be recognized as an alar m signal that –
after hydrolysis to JA – functions as intracellular defense
signal. In fact, a similar situation occurs in mammals.
Stimulated neutrophils may synthesize (within minutes)
leukotrienes, which are exported into the extracellular
environment where they act as autocrine and paracrine
signals. Ho wever, neu trophils are also capable of taking up
leukotrienes for intracellular catabolism, thereby locally
restricting and terminating the signal.
Transcript levels were also monitored after stimulation

of tomato cell cultures with exogenous MeJA or the
elicitor chitosan, which induces intracellular synthesis of
jasmonates in tomato [40]. Constitutively high MJE
accumulation transiently declined within 2 and 3 h after
the treatments, respectively. Decreasing transcript levels
may not immediately affect enzyme activity and thus
exogenous MeJA can still be hydrolysed to JA for some
time. H owever, downregulation of MJE by exogenou s
MeJA may limit MeJA hydrolysis and J A signalling when
cells are exposed to elicitors/jasmonates over longer time-
periods. Interestingly, basal transcript levels of JMT in
Arabidopsis leaves are low, and stimulation by exogenous
MeJA transiently increases JMT formation. Overexpres-
sion of JMT cDNA has been shown to increase the
synthesis o f MeJA [4], which, in turn, may leave producer
cells and function as an intercellular signal. As MJE
activity has been detected in all plant tissues examined so
far, MJE-harbouring cells may trap the volatile and highly
diffusible MeJA entering cells from the outside by
hydrolysis to JA anions inside the cells. Inc reasing JA
levels might t hen elicit specific responses. In the future,
generation and careful analysis of transgenic plants that
either constantly accumulate MJE or that are devoid of
MJE will help to solve the question of whether or not
MeJA is a paracrine or even a long-distance signal.
Acknowledgements
This work was supported by t he Sonderforschungsbereich (SFB) 567.
The a u thors thank Susanne Michel for performin g DNA sequen cing.
References
1. Creelman, R.A. & Mullet, J.E. (1997) Biosynthesis a nd action of

jasmonates in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48,
355–381.
2. Mueller, M. (1997) Enzymes i nvolved in j asmonic acid b iosyn-
thesis. Phys iol. Plant. 10 0, 653–663.
3. Schaller, F. (2001) Enzymes of the biosynthesis of octadecanoid-
derived s ignalling molecules. J. Exp. Bot. 52 , 11–23.
4. Seo, H .S., S o ng, J .T. , C heon g, J.J., L ee , Y .H., Lee, Y.W., H wang,
I., L ee, J.S. & Choi, Y .D. (2001) Jasmonic ac id carboxyl methyl-
transferase: a key enzyme for jasmonate-regulated plant r esponses.
Proc.NatlAcad.Sci.USA98, 4 788–4793.
5. Sembdner, G. & Parthier, B. (1993) The biochemistry and the
physiological and molecular actions of jasmonates. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 44, 569–568.
6. Wasternack, C . & Parthier , B. ( 1997) Jasmonate-signalled p lant
gene expression. Tre nds Pl ant Sci. 2, 302–307.
7. Kramell, R., Miersch, O., Atzorn, R., Parthier, B. & Wasternack,
C. (2000) Octadecanoid-derived alteration of gene expression and
the Ôoxylipin signatureÕ in stressed barley leaves. Implications for
different signali ng pathways. Plant Physiol. 123 , 177–188.
8. Berger, S. (2002) Jasmonate-related mutants of Arabidopsis as
tools f or studying s tress signaling. Planta 214, 497 –504.
9. McConn,M.&Browse,J.(1996)Thecriticalrequirementfor
linolenic acid is pollen development, not photosynthesis, in an
Arabidopsis mutant. Pl ant Cell 8, 403 –416.
2982 C. Stuhlfelder et al.(Eur. J. Biochem. 271) Ó FEBS 2004
10. Stintzi, A., Weber, H., Reymond, P., Browse, J. & Farmer,
E.E. (2001) Plant defense in the absence of jasmonic acid: the
role of cyclopentenones. Proc. Natl Acad. Sci. USA 98, 12837–
12842.
11. Staswick, P.E., Su, W. & Howell, S. H. (1992) Me thyl jas monate

inhibition of root growth and induction of a leaf protein are
decreased i n an Arabidopsis thaliana mutant. Proc. Natl A cad. Sc i.
USA 89, 6837–6840.
12. Staswick, P.E., Tiryaki, I. & Rowe, M.L. (2002) Jasmonate
response locus J AR1 a nd several related Arabidopsis genes enc ode
enzymes of the firefly luciferase superfamily that show activity on
jasmonic, salicylic, and indole-3-acetic acids in an a ssay for ade-
nylation. Plant Cell 14, 1405–1415.
13. Reymond,P.,Weber,H.,Damond,M.&Farmer,E.E.(2000)
Differential gene exp ression in respon se to mechanical wounding
and i nsect f eeding in Arabidopsis. Plant Cell 12, 707–720.
14. Howe, G.A. (2001) Cyclopentenone signals for plant defense:
remodeling the jasmonic acid response. Proc.NatlAcad.Sci.USA
98, 12317–12319.
15. Stratmann, J.W. (2003) Long distance run in the wound response
– j asmonic acid is p ulling ahead. Trends Plant Sc i. 8, 247 –250.
16. Li, L., Li, C., Lee, G.I. & Howe, G.A. (2002) Distinct roles for
jasmonate synthesis a nd ac tion in th e s ystemic w ound res ponse of
tomato. Proc. Natl Acad. Sci. USA 99, 6416–6421.
17. Meyer, R., Rautenbach, G.F. & D ubery, I.A. (2003) Identification
and q uantification of methyl jasm onate in leaf v olatiles o f Arabi-
dopsis thaliana usin g s olid-phase m i croextraction i n combination
with gas chromatography and mass spectrometry. Phytochem.
Anal. 14, 155–159.
18. Farmer, E.E. & Ryan, C.A. (1990) Interplant communication:
airborne methyl jasmonate induces synthesis of proteinase in-
hibitors in plant l eaves. Proc . Natl Acad. Sci. USA 87, 7713–7716.
19. Weber, H . (2002) Fatty acid-derived signals in plants. Trends Plant
Sci. 7, 217–224.
20. Stuhlfelder, C., Lottspeich, F. & Mueller, M .J. (2002) Purification

and partial amino acid sequences of an esterase from tomato.
Phytochemistry 60 , 233–240.
21. Linsmaier, E.M. & Skoog, F. (1965) Organic growth factor
requirements of tobacco tissue cultures. Physiol. Plant. 18, 100–
127.
22. Ausubel, F.M., Brent, R ., King ston, R.E., Moore, D.D., S mith,
J.A.,Seidman,J.G.&Struhl,K.E.(1994)Current Protocols in
Molecul ar Biology . Greene a nd. Wiley, New York.
23. Rogers, S.O. & B endich, A.J. ( 1985) Extraction o f D NA from
milligram amounts of fr esh, herbarium a nd mummified plant tis-
sues. Plant Mol. Biol. 5, 69–76.
24. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning – a Laboratory Manual, 2nd ed n. Co ld Spring H arbor
Laboratory Press, Cold Spring Harbor, New Y o rk.
25. Blum, H., Beier, H. & Gross, H.J. (1987) Improved silver staining
of prote ins, RNA, DNA in polyacrylamide gels. Electrophoresis 8,
93–99.
26. Warzecha, H., Gerasimenko, I., Kutchan, T.M. & Stockigt, J.
(2000) Molecular cloning and functional bacte rial expression of a
plant glucosidase specifically involved in alkaloid biosynthesis.
Phytochemistry 54 , 657–666.
27. Dogru,E.,Warzecha,H.,Seibel,F.,Haebel,S.,Lottspeich,F.&
Stockigt, J. (2000) The gene encoding polyneuridine aldehyde
esterase of monoterpenoid ind ole a lkaloid biosynthesis in p lan ts i s
an orth olog of the alpha/beta h ydrolase super family [In Process
Citation]. Eur. J. Biochem. 267, 1397–1406.
28. Zhong, G.Y., Goren, R., Riov, J., Sisler, E.C. & Holland, D.
(2001) Characterization of an ethylene-induced este rase gene iso-
lated from Citrus sinensis by competitive hybridization. Physiol.
Plant. 113, 2 67–274.

29. Kumar, D. & Klessig, D.F. (2003) High-affinity salicylic acid-
binding protein 2 i s req uired for p lan t innate imm unit y and has
salicylic acid-stimulated lipase activity. Proc. Natl Acad. Sci. USA
100, 1 6101–16106.
30. Hasslacher, M., Schall, M., Hayn, M., Griengl, H., Kohlwein,
S.D. & S chwab, H. (1996) Molecular cloning of the full-length
cDNA o f (S)-hydroxynitrile lyase f rom Hevea brasi lien sis . Func-
tional expre ssion in Escherichia c oli and Saccharomyces cerevisiae,
and identification of an active site residue. J. Biol. Chem. 271,
5884–5891.
31. Hughes, J., Carv alho, F.J. & Hughes, M .A. (1994) Purification,
characterization, and cloning of alpha-hydroxynitrile lyase f rom
cassava (Manihot esculenta Crantz). Arch. Biochem. Biophys. 311,
496–502.
32. Heikinheimo, P., Goldman, A., Jeffries, C. & Ollis, D.L. (1999) Of
barn owls and bankers: a lush variety of alpha/beta hydrolases.
Structure F old Des. 7, R141–R146.
33. Gold, A.M. (1965) Sulfonyl fluor ides as inhibitors of esterases.
Identification of serine as the site of sulfonation in phenyl-
methanesulfonyl alpha-chymotryp sin. Biochemistry 4, 897–901.
34. Hughes, J ., Keresztessy, Z ., Brown, K., Su handono , S . & Hughes,
M.A. (1998) Genomic organization and structure of alpha-
hydroxynitrile lyase in cassava (Manihot e sculenta Crantz). Arch.
Biochem. Biop hys. 356, 107–116.
35. Ibrahim, R.K., Bruneau, A. & Bantignies, B. (1998) Plant O-
methyltransferases: m olecular analysis, common signature and
classification. Plant Mol. Biol. 36, 1–10.
36. Pichersky, E. & Gang, D.R. (2000) Ge netics and b iochemistry o f
secondary metabolites in plants: an evolutionary perspective.
Trends Pla nt Sci. 5, 439–445.

37. Kliebenstein, D.J., Lambrix, V.M., Reichelt, M., Gershen zon, J. &
Mitchell-Olds, T. (2001) Gene dupl ication in the diversification of
secondary metabolism. Tandem 2-oxoglutarate-dependent dioxy-
genases control glucosinolate biosynthesis in arabidopsis. Plant
Cell 13 , 681–693.
38. Hause, B., Stenzel, I., Miersch, O., Maucher, H., Kramell, R.,
Ziegler, J. & Wasternack, C. (2000) Tissue-specific o xylipin sig-
nature of tomato flowers: allen e oxide cyclase is highly expressed
in dist inct flower o rgans a nd vascular b undles. Pla nt J . 24, 113–
126.
39. Gundlach, H., Mull er, M .J., Ku tcha n, T.M. & Z enk, M.H. (1992)
Jasmonic acid is a signal transducer i n elicitor-induced plant cell
cultures. Proc.NatlAcad.Sci.USA89, 2 389–2393.
40. Doares, S.H., Syrovets, T., Weiler, E.W. & Ryan, C.A. (19 95)
Oligogalacturonides a nd chitosan activate plant defensive genes
through the o ctadecanoid pathway. Proc. Natl Acad. Sc i. USA 92,
4095–4098.
Ó FEBS 2004 Methyl jasmonate esterase from tomato (Eur. J. Biochem. 271) 2983