We have cloned a cDNA, encoding a methyl jasmonate-cleaving enzyme, from tomato cell suspension cultures.. Moreover, when tested in methyl jasmonate- and elicitor-treated cell suspension
Trang 1Cloning 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¨t Wu¨rzburg, Germany
Jasmonic acid and its methyl ester are ubiquitous plant
sig-nalling compounds necessary for the regulation of growth
and development, as well as for the response of plants to
environmental stress factors To date, it is not clear whether
methyl jasmonate itself acts as a signal or if its conversion to
jasmonic acid is mandatory prior to the induction of a
def-ense response We have cloned a cDNA, encoding a methyl
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 catalytically active form Transcript levels, as well as en-zymatic activity, were determined in different tomato tissues High transcript levels and enzyme activities were found in roots and flowers, while the mRNA level and activity were low in stems and leaves Moreover, when tested in methyl jasmonate- and elicitor-treated cell suspension cultures, transcript levels were found to decrease, indicating that this particular enzyme might be a regulator of jasmonate sig-nalling
Keywords: a/b-hydrolase; cell suspension culture; Lyco-persicon esculentum; methyl jasmonate esterase; Solanaceae
Jasmonic acid (JA) is a ubiquitous plant compound, which
plays a crucial role in the response to wounding or pathogen
attack, as well as in developmental processes, such as fruit
ripening, root growth, and fertility [1] Most of the enzymes
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 linolenic acid from membrane stores by
lipases The enzymes lipoxygenase, allene oxide synthase,
and allene oxide cyclase form the biosynthesic intermediate
12-oxo-phytodienoic acid (OPDA) Subsequent action of
OPDA reductase and three cycles of b-oxidation lead to the
formation of JA [2,3] Thereafter, JA may be esterified to its
derivative methyl jasmonate (MeJA) [4], or conjugated with
an amino acid or glucose [5]
For most of the jasmonates it has been shown that they are capable of mediating a response by regulating gene expression [6,7] Analysis of Arabidopsis thaliana mutants impaired in either JA biosynthesis or signalling, gave a deeper insight into the function of single oxylipins [8] The fad3–2fad7–2fad8mutant, 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 – arrests jasmonate biosynthesis at the OPDA level and is incapable of metabolizing exogenously applied OPDA to JA [10] opr3 mutants displayed 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 could be restored by the exogenous 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) mutant with a defect in the jasmonate response has been described The mutant is insensitive to MeJA and does not show root growth inhibition or vegetative storage protein (VSP) induction in response to MeJA [11] Recent analysis of the jar1 locus revealed that its gene product modifies JA via adenylation, which is apparently a prerequisite for downstream signaling The modification requires a free carboxyl group as the enzyme does not accept MeJA as a substrate [12] Therefore, MeJA must be demethylated prior to becoming active Thus, root growth inhibition and VSP expression are mediated by MeJA through JA, indicating that MeJA is not
a mediator on its own in this particular system
On the other hand, OPDA and JA can induce identical genes as well as distinct sets of target 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: + 49 9318886182, Tel.: + 49 9318886162,
E-mail: warzecha@biozentrum.uni-wuerzburg.de
Abbreviations: HNL, (S)-hydroxynitrile lyase; JA, jasmonic acid;
JMT, S-adenosyl- L -methionine jasmonic acid carboxyl
methyltrans-ferase; MeJA, methyl jasmonate; MJE, methyl jasmonate esterase;
MeSA, methyl salicylate; OPDA, 12-oxo-phytodienoic acid; PI,
proteinase inhibitors; PNAE, polyneuridine aldehyde esterase;
RACE, rapid amplification of cDNA ends; VSP, vegetative storage
protein.
Note: The sequence reported herein was deposited under GenBank
accession number AY455313.
Note: A website is available at http://132.187.108.6/
(Received 28 March 2004, revised 19 May 2004,
accepted 25 May 2004)
Trang 2the 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 inducible defense response
against feeding Colorado potato beetle is not limited to the
site of their attack, but also appears in distant leaves of the
plant [15] The herbivore attack induces JA biosynthesis
locally via systemin, an 18 amino acid signal peptide
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 in either JA biosynthesis or JA perception
proved that a jasmonate, rather than systemin, is the signal
which is translocated through 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 expression
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 can be found in the headspace
above wounded leaves [17], suggesting that MeJA might be
an interplant communication signal [18] Moreover, 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, which converts JA to
MeJA [4] Constitutive overexpression of JMT tripled the
MeJA content of transgenic plants and also induced
JA-responsive genes Therefore, transgenic plants with elevated
JMT levels showed 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 functions as a paracrine
signal that is released from sites of pathogen attack to
induce defense genes at distant sites Moreover, it remains to
be clarified whether MeJA is a mediator on its own that
elicits JA responses without prior hydrolysis to JA
How-ever, if MeJA is considered to be a signal, 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 esterase,
previously characterized in tomato [20] Enzyme activity has
been found to be constitutively present in cell cultures of
many taxonomically distant plant species In tomato cell
cultures, only one MeJA-hydrolysing 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 whether 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 involved in jasmonate signalling
Experimental procedures
Plant material Tomato plants (Lycopersicon esculentum cv Moneymaker) were grown in the greenhouse under conditions of 16 h light and 8 h darkness The growth temperature range was 16–22C with a relative humidity of 60–70%
Cell suspension cultures (L esculentum) were grown in
1 L Erlenmeyer flasks in Linsmaier & Skoog media [21] for
7 days under continuous light (600 lux) on orbital shakers (100 r.p.m.) at 24 ± 2C Cells were harvested by suction filtration, shock frozen in liquid nitrogen, and stored at )80 C until use
Nucleic acid isolation and blot analyses Plant material for the isolation of nucleic acid was either 4-day-old cell suspension culture or 8-week-old plants Total RNA from suspension cultures was 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 plant material was carried out using the RNeasy Plant Mini 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 separation [24] Hybridiza-tion of RNA or DNA transferred to nylon 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 GAYATGGCNGCNWSNGGNATH 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 system from Qiagen RT-PCR conditions were 30 min at 50C, 15 min at 95 C; five cycles of 30 s at 94C, 1 min at 40 C and 1 min at
72C, followed by 30 cycles of 30 s at 94 C, 1 min at 45 C and 1 min at 72C Gel electrophoresis, DNA elution and modification was carried out according to standard protocols [24] After cloning of the PCR product into the vector
pGEM-T (Promega) and sequencing (automated sequencer LI-COR 4200), two homologous primers were designed for the rapid amplification of cDNA ends (RACE) (RACEfor: GTGA CAGCTTTCATGCCTGG; and RACErev: ATCCTGT CCGTTGTTGTAAAC) 5¢- and 3¢-RACE was performed using the SMART II system (BD Bioscience) Full-length cDNA for expression was cloned by another RT-PCR with primers fullMJEforMQ (GCATGCAGGGTGATAAAAA TCACTTTGTA) and fullMJErev (AAGGATCCATAA TATTTTTGCGAAATC), adding restriction sites for SphI and BamHI, respectively The PCR product was cloned into vector pDRIVE (Qiagen), sequenced and subcloned into
Trang 3expression vector pQE70 (Qiagen) via SphI and BglII
restriction sites
Overexpression and purification
Escherichia coliM15 cells harbouring the MJE expression
plasmid were cultured at 20C on a rotary shaker
(200 r.p.m.) Twenty-four hours after induction with
1 mMisopropyl 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 to a procedure described
previously [20] For metal affinity chromatography, Talon
resin (BD Bioscience) was utilized Analysis of proteins was
performed with SDS/PAGE (12.5%) under denaturating
conditions, and gels were silver stained as described
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 activity identified in
tomato cell cultures On the basis of the partial amino acid
sequences obtained from the purified MJE [20], two
degenerate primers were developed This method has been
proven successful in several reports [26,27] and should 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) was used for
the generation of an antisense primer Using tomato cDNA
as a template, a 498 bp fragment was amplified by PCR
Sequencing revealed a DNA stretch that encodes a peptide
with sequence similarity to a/b-hydrolase fold proteins (data
not shown), some of which have been previously aligned to
the internal fragments of purified MJE [20] To obtain the
full-length cDNA by RACE, two sequence-specific primers
were synthesized, generating overlapping fragments after
5¢- and 3¢-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 peptides
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 mass of the encoded protein is 29 524.93 Da and
the pI¼ 5.52 The calculated mass of the encoded protein
corresponds well with the molecular 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 lacked 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 the protein was modified in vivo No peptide signal for subcellular targeting could be identified
Sequence alignment Sequence analysis and alignment with known proteins from GenBank showed a high similarity of MJE to ethylene-induced esterase from Citrus sinensis (47% identity, 65% positivity) [28], the tobacco salicylic acid binding protein 2 (SABP2) (47% identity, 65% positivity) [29], the polyneu-ridine aldehyde esterase (PNAE) from Rauvolfia serpentina (44% identity, 65% positivity) [27], and several 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 Peptides determined by sequencing of the purified protein are boxed and the names of the peptides are indicated in italic letters above Nucleic acids of the ORF are shown in capital letters, while 5¢ and 3¢ untranslated regions are in lower case letters The putative amino acid residues of the catalytical triad of a/b-hydrolase fold pro-teins are marked with an asterisk.
Trang 4acetone-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, it could be 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 histidine – represented by serine at
position 83, aspartic acid at position 211, and histidine at
position 240 (Fig 2) Moreover, it has been shown that
MJE could be irreversibly inhibited by
phenylmethanesulfo-nyl fluoride [20], a specific inhibitor of serine hydrolases [33]
Bacterial expression and purification of tomato MJE
To obtain unequivocal evidence of the identity of the cloned
sequence, MJE cDNA was subcloned into a bacterial vector
for heterologous expression As amplification of the coding
sequence with a forward primer homologous to the 5¢-end failed, the primer sequence had to be modified for enhanced binding and amplification Primer design was carried out using the 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 value 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 than expected for
an enzyme from heterologous bacterial expression of the cDNA For visualization of proteins, bacterial extracts were subjected to SDS/PAGE In a comparison of MJE-produ-cing E coli with wild-type cells, no prominent protein with 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 from different plant species EIE, ethylene-induced esterase from Citrus sinensis (GenBank accession number AAK58599); SABP2, salicylic acid-binding protein from Nic-otiana tabacum (AY485932); PNAE, polyneuridine aldehyde esterase from Rauvolfia serpentina (AAF22288); Pir7b, defense-related rice gene from Oryza sativa (CAA84024); HNL, (S)-hydroxynitrile lyase from Hevea brasiliensis (P52704).
Trang 5after Coomassie Blue staining (data not shown) This
observation suggests that MJE 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 suspension
culture, MJE(His)6could be enriched 203-fold, resulting in
52 lg of protein Figure 3A shows the silver-stained
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 cleaving MeJA An antibody specific for
hexa-histidine epitopes was used in an immunoblot
experi-ment to confirm the presence and the size of the
recombin-ant protein As shown in Fig 3B, extracts from E coli
harbouring pQE-MJE showed a band of 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 cDNA 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 bands in a Southern blot analysis 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 owing to the presence of recognition sites for the utilized restriction enzymes within introns (as assumed for the HNL from Hevea) [30] However, during the purification of MJE there was no 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 used 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 1 kb were present in all plant tissues and significant variations in their amounts could be detected (Fig 5A) High levels of MJE mRNA could be found in roots and flowers, while low-to-moderate amounts were 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 activity correlates with a high transcript level (Fig 5B)
Table 1 Purification of recombinant methyl jasmonate esterase.
Purification step
Total protein (mg)
Total activity (pkat)
Specific activity (pkat)
Purification (fold)
Recovery (%)
Fig 3 SDS/PAGE and Western blot analysis
of methyl jasmonate esterase (MJE) purified from recombinant bacteria (A) Silver-stained proteins after separation by SDS/PAGE (12.5% gel) M, marker proteins (sizes are indicated on the left); lane 1, purified fraction after the last purification step MJE is indica-ted by an arrow (B) Western blot of crude bacterial extracts after transfer to nitrocellu-lose An antibody specific for 6·His modified proteins was used Lane M, protein standard; lane 1, Escherichia coli harbouring expression plasmid pQE70-MJE; lane 2, E coli without the expression plasmid MJE is indicated with the arrow.
Trang 6In order to evaluate whether transcript levels could be
regulated by external stimuli, cell suspension cultures were
treated with MeJA, methyl salicylate (MeSA), or chitosan,
and mRNA levels were determined in a time-dependent
manner While basal levels of mRNA were high in untreated
cultures, the levels decreased 1 h after MeJA treatment and
returned to basal levels 8 h postinduction (Fig 5C) A
similar time course could be observed after treatment with
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 and with
diverse properties Notably, all plant proteins of known
function with high sequence similarity 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 of specified plant enzymes of this group of the
a/b-hydrolase family, it might be assumed that 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, BamHI-digested DNA; lane 2, EcoRI digest; lane 3,
HindIII digest.
Fig 5 Levels of methyl jasmonate esterase (MJE) transcripts in dif-ferent tissues and after induction (A) Northern blot of total RNA from 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 transcript abundance after treatment with methyl jasmonate (MeJA), methyl salicylate (MeSA), and chitosan Loading controls show the ethidium bromide-stained gel prior to transfer.
Trang 7be noted that in the Arabidopsis genome at least 20 genes
with homologies to HNL and PNAE could be found It is
unlikely that they have similar properties to the
a/b-hydrolase fold proteins mentioned above, as they occur only
in distinct plant families or even species and their substrates
are not present in Arabidopsis On the other hand, MJE
activity could be found in several plant systems From the 18
plant cell suspension cultures of taxonomically distant
species tested to date, virtually all exhibited MJE activity
[20] One might speculate that the MJEs 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 both JA and MeJA are
published, the ratios depend on the plant species and the
tissue analysed and vary from 3 : 2 (JA/MeJA) in
Arabid-opsisleaves [4] to about 10 : 1 in tomato flowers [38] For
tomato cell suspension cultures, a JA/MeJA ratio of almost
1 : 1 was found [39] To date there is no way of
distinguishing between the function of the two compounds
on a physiological basis as they can be rapidly converted
from one into the other For the Arabidopsis jar1 mutant, it
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 that 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 and revealed a high, constitutive expression in roots
and flowers and low RNA levels in leaves and stems,
suggesting that enzyme activity could be 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 equal
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 separation 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 appealing scenario that a cell
could distinguish between endogenously formed and
exo-genous MeJA Endoexo-genously formed MeJA might be
exported and not hydrolysed, while MeJA coming from
outside the cell may be recognized as an alarm 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 However, neutrophils 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 However, downregulation of MJE by exogenous MeJA may limit MeJA hydrolysis and JA signalling when cells are exposed to elicitors/jasmonates over longer time-periods Interestingly, basal transcript levels of JMT in Arabidopsisleaves are low, and stimulation by exogenous MeJA transiently increases JMT formation Overexpres-sion of JMT cDNA has been shown to increase the synthesis of 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 Increasing JA levels might then 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 the Sonderforschungsbereich (SFB) 567 The authors thank Susanne Michel for performing DNA sequencing.
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