From the Center for Structural and Functional
Genomics, Biology Department, Concordia University, Montreal H3G 1M8,
Canada and the § Leibniz Institute of Plant Biochemistry,
Weinberg 3, D-06120 Halle/Saale, Germany
Received for publication, November 22, 2002, and in revised form, March 5, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
12-Hydroxyjasmonate, also known as tuberonic
acid, was first isolated from Solanum tuberosum and was
shown to have tuber-inducing properties. It is derived from the
ubiquitously occurring jasmonic acid, an important signaling molecule
mediating diverse developmental processes and plant defense responses.
We report here that the gene AtST2a from Arabidopsis
thaliana encodes a hydroxyjasmonate sulfotransferase. The
recombinant AtST2a protein was found to exhibit strict specificity for
11- and 12-hydroxyjasmonate with Km values of 50 and 10 µM, respectively. Furthermore, 12-hydroxyjasmonate
and its sulfonated derivative are shown to be naturally occurring in
A. thaliana. The exogenous application of methyljasmonate
to A. thaliana plants led to increased levels of both
metabolites, whereas treatment with 12-hydroxyjasmonate led to
increased level of 12-hydroxyjasmonate sulfate without affecting the
endogenous level of jasmonic acid. AtST2a expression was
found to be induced following treatment with methyljasmonate and
12-hydroxyjasmonate. In contrast, the expression of the
methyljasmonate-responsive gene Thi2.1, a marker gene in
plant defense responses, is not induced upon treatment with
12-hydroxyjasmonate indicating the existence of independent signaling
pathways responding to jasmonic acid and 12-hydroxyjasmonic acid. Taken
together, the results suggest that the hydroxylation and sulfonation
reactions might be components of a pathway that inactivates excess
jasmonic acid in plants. Alternatively, the function of AtST2a might be
to control the biological activity of 12-hydroxyjasmonic acid.
Jasmonic acid (JA)1 and
related cyclopentanones are important signaling molecules involved in
plant defense responses and in the control of key developmental
processes such as senescence, tuber formation, and anther development
(1). Plants respond to the exogenous application of JA or
methyljasmonate (MeJA) by the development of senescence-like symptoms
and by the up-regulation of a large number of genes including genes
coding for plant defense proteins such as thionins and proteinase
inhibitors or enzymes involved in phytoalexin synthesis (2).
The biosynthesis of JA occurs through the octadecanoid pathway and
starts with the oxidation of Some of these reactions might lead to the irreversible inactivation of
JA, whereas others might be reversible and used for transport and/or
storage. Alternatively, the metabolism of JA might generate molecules
with new properties. For instance, the 12-O- A sulfated conjugate of 12-OHJA was found to accumulate in the plant
species Tribulus cistoides, a member of the
Zygophylaceae family (14). This is the only report on the occurrence of
a conjugate of 12-OHJA in a plant species that does not produce tubers.
Furthermore, it is the only report of the occurrence of a jasmonate
conjugated with a sulfonate group. In mammals, it is well recognized
that the sulfonation reaction plays an important role in the modulation
of the biological activity of a number of compounds, such as steroids
and thyroid hormones, and catecholamine neurotransmitters (15-18).
Sulfonate conjugation not only facilitates transport and excretion of
hydrophobic molecules by increasing their water solubility, it
abolishes the biological activity of hormones such as estrogens.
Recently, the characterization of a brassinosteroid sulfotransferase
(ST) from Brassica napus demonstrated that plants like
animals use the sulfonation reaction to control the biological activity
of hormones (19). The sulfonation reaction can also lead to the
production of biologically active molecules. For example, the
sulfonation of gallic acid glucoside generates the periodic leaf
movement factor 1 involved in the seismonastic response in Mimosa
pudica (20).
Recently, we initiated a systematic investigation of the biochemical
functions of the 18 ST-coding genes from Arabidopsis thaliana. We report here the characterization of a ST (AtST2a) from this plant exhibiting strict specificity for hydroxyjasmonates. This is the first report of a ST involved in jasmonate metabolism. We
also demonstrate that 12-OHJA and its sulfate derivative are natural
constituents in A. thaliana. We propose a role for the hydroxyjasmonate ST in the regulation of the levels of JA and/or 12-OHJA in plants.
Materials
12-OHJA, JA isoleucine conjugate, CA, 7-iso-CA, 6-epi-CA,
6-epi-7-iso-CA, and their methyl esters, were from our laboratory collection. MeJA and JA were purchased from Bedoukian Research Inc. and
Sigma, respectively. Wild type A. thaliana seeds, ecotype Col-0, were obtained from Lehle Seeds. The enzymes used for cloning were from New England Biolabs and used under conditions recommended by
the manufacturer. All reagents were of analytical or molecular biology grade.
Cloning of AtST2a
cDNA clone of AtST2a (GenBankTM
accession number T43254) was obtained from the Arabidopsis
Biological Resource Center. Oligonucleotide 5'-CGGGATCCATGGCTACCTCAAGCATGAAG-3' was designed to introduce a
BamHI site at the 5'-end of the coding sequence.
AtST2a was amplified by polymerase chain reaction with Vent
DNA polymerase (New England Biolabs) using the above primer and
M13-20 primer. The amplified product was digested with BamHI
and ligated into the BamHI site of the bacterial expression
plasmid pQE30 (Qiagen). Clones containing AtST2a in the
proper orientation were determined by restriction enzyme analysis and
by sequencing the 5' junction.
DNA Sequence Analysis
DNA and protein sequence alignments were performed using the
ClustalW program2 and the
similarity/identity values determined from the pairwise comparisons of
all the ST genes. Boxshade was used to shade identical residues in the
alignments.3
Expression of Recombinant AtST2a in Escherichia coli
A culture of E. coli, strain XL1-blue harboring
AtST2a (A600 = 0.7) was induced with
1 mM isopropylthio- Partial Purification of Hydroxyjasmonate Sulfotransferase from A. thaliana
15-Day-old plants grown on MS medium in magenta boxes were
treated with 100 µM 12-OHJA or MeJA for a period of
8 h. At the end of the treatment, rosette leaves (about 2 g)
were harvested, ground in liquid nitrogen, and homogenized in 10 ml of
ice-cold 0.5 M bis-Tris (pH 6.5). The homogenate was
filtered through nylon mesh and the filtrate was centrifuged at
12,000 × g for 15 min. The supernatant was gently
stirred for 10 min with 0.5 ml of PAP-agarose pre-equilibrated in the
same buffer. The mixture was centrifuged for 5 min at 1000 × g on a benchtop microcentrifuge. The supernatant was
discarded and replaced with 1 ml of 0.5 M bis-Tris (pH 6.5) containing 1.0 M NaCl. The solution was gently stirred for
5 min followed by centrifugation at 1000 × g for 5 min. The supernatant was used for enzyme activity.
Sulfotransferase Assays
Analysis of substrate specificity was performed by testing
enzymatic activity with three different concentrations of acceptor substrates: 1, 10, and 100 µM. The reaction mixture (50 µl) contained 50 pmol of [35S]PAPS (PerkinElmer Life
Sciences) and ~0.25 µg of PAP-agarose-purified recombinant AtST2a
in 50 mM Tris (pH 7.5). For substrate interaction kinetic
experiments, concentrations of PAPS were 0.2, 0.6, 1.0, 2.5, and 5.0 µM. Concentrations of 11-OHJA were 10, 30, 50, 80, and
150 µM and 12-OHJA were 2, 6, 10, 15, and 50 µM. The reactions were allowed to proceed for 10 min at
25 °C. Under the assay conditions, the reaction was linear with time
from 0 to 40 min and with protein concentrations ranging from 0 to 2.0 µg/assay. The AtST2a-sulfated reaction product was extracted with
1-butanol saturated with water and an aliquot was counted for
radioactivity in scintillation fluid.
Detection and Quantification of JA and 12-OHJA from A. thaliana
Rosette leaves from fresh plant material (1 g) were homogenized
with 10 ml of methanol containing 100 ng of
[2H6]JA and
12-[2H3]OAc-JA as internal standards. The
filtrate was evaporated and acetylated with pyridine/acetic acid
anhydride (2:1) at 20 °C overnight. The reaction mixture was
evaporated, resuspended in ethyl acetate, and loaded on a silica (SiOH)
column (500 mg; Machery-Nagel). The flow-through containing JA and the
acetylated form of 12-OHJA was collected and evaporated. The evaporated
mixture was resuspended in 5 ml of methanol and loaded on a 3-ml
DEAE-Sephadex A25 column (acetylated-form in methanol). The column was
washed with 3 ml of methanol followed by 3 ml of 0.1 M
acetic acid in methanol. The jasmonates were eluted with 5 ml of 1 M acetic acid in methanol (Fraction A), evaporated, and
separated on preparative HPLC for GC-MS analysis.
The SiOH column was washed with methanol and the flow-through (Fraction
B) was collected for analysis of 12-OHJA sulfate. Fraction B was
evaporated, resuspended in 10% acetonitrile, and chromatographed by
reverse phase HPLC (method gradient, 10-90% acetonitrile in 15 min at
a flow rate of 1 ml/min). Fractions were collected from 4.5 to 7 min,
evaporated, and resuspended in 50 µl of methanol and analyzed by
LC-MS.
Preparative HPLC--
Fraction A eluted from the DEAE-Sephadex
A-25 column was subjected to preparative HPLC column, Eurospher 100-C18
(5 µm, 250 × 4 mm). Jasmonates were eluted with
methanol, 0.2% acetic acid in H2O (1:1) at a flow
rate of 1 ml/min and UV detection at 210 nm. Fractions between
Rt 9.15 and 11 min containing 12-OAc-JA and between
12 and 13.30 min containing JA were collected and evaporated. The
samples were dissolved in 200 µl of
chloroform/N,N-diisopropylethylamine (1:1) and
derivatized with 10 µl of pentafluorobenzylbromide at 20 °C
overnight. The evaporated derivatized samples were dissolved in 5 ml of
n-hexane and passed through a SiOH column (500 mg; Machery-Nagel). The pentafluorobenzyl esters were eluted with 7 ml of
n-hexane/diethyl ether (2:1), evaporated, dissolved in 100 µl of acetonitrile, and analyzed by GC-MS.
GC-MS--
The following parameters were used for GC-MS:
GCQ Finnigan, 70 eV, NCI, ionization gas NH3, source
temperature 140 °C, column Rtx-5 (30 m × 0.25 mm, 0.25 µm
film thickness), injection temperature was 250 °C, interface
temperature was 275 °C; helium 40 cm s
The retention time of
12-[2H3]OAc-JA-pentafluorobenzyl ester was
20.61 min and 12-OAc-JA-pentafluorobenzyl ester was 20.66 min, using
fragments m/z 270 (standard) and
m/z 267 for quantitation. The retention time of
[2H6]JA-pentafluorobenzyl ester was 14.66 min, and JA-pentafluorobenzyl ester was 14.72 min, using fragments
m/z 215 (standard) and
m/z 210 for quantitation.
Detection of 12-OHJA Sulfate by LC MS/MS--
The
negative ion electrospray (ES) mass spectra were obtained from a
Finnigan MAT TSQ 7000 instrument (electrospray voltage, 4 kV; heated
capillary temperature, 220 °C; sheath gas, nitrogen) coupled with a
Micro-Tech Ultra-Plus MicroLC system equipped with a RP18 column (4 µm, 1 × 100 mm, Ultrasep). For HPLC, a gradient was used
starting from H2O:acetonitrile (90:10; containing 0.2% acetic acid) to 10:90 in 15 min followed by a 10-min isocratic period
at a flow rate of 70 µl/min. The collision-induced dissociation (CID)
mass spectra during the HPLC run were performed with a collision energy
of 30 eV (collision gas, argon; collision pressure, 1.8 × 10 Northern Blot Analysis--
Rosette leaves of A. thaliana plants were pulverized in liquid nitrogen and total RNA
was extracted in buffer/phenol/chloroform as described (22). 10 µg of
total RNA was used for agarose gel electrophoresis after denaturing
with glyoxal and Me2SO (23). Northern blot analysis of
total RNA was achieved under high stringency conditions according to
standard procedures (24) using the 32P-labeled coding
sequence of AtST2a as a probe.
SDS-PAGE Electrophoresis--
To verify the solubility and
evaluate the level of purity of the recombinant protein after
chromatography on nickel-agarose and PAP-agarose, aliquots of the
recombinant enzyme were subjected to 12% polyacrylamide gel
electrophoresis according to the method of Laemmli (25). The proteins
were visualized by Coomassie Blue staining.
Reverse Transcriptase-Polymerase Chain Reaction--
2.5 µg of
total RNA was treated with 20 units of DNase I (Roche
Diagnostics) in 50 µl of 0.1 M sodium acetate, 5 mM MgSO4 (pH 5.0) for 10 min at 37 °C. DNase
I was heat inactivated at 95 °C for 5 min and RNA was ethanol
precipitated. cDNA was synthesized using Moloney murine leukemia
virus-reverse transcriptase (New England Biolabs) in a 25-µl reaction
volume as recommended by the manufacturer. 1 µl of room temperature
reaction product was used for PCR with Ex Taq DNA polymerase
(Takara Biochemicals).
Oligonucleotides (119G6) 5'-GGAGAGAGGATGGAGAAC-3' and (119-800)
5'-CGTCTTTGAGATCCTCGTACC-3' were used to amplify AtST2a in reverse transcriptase-PCR experiments. Oligonucleotides (Thi2.1 5')
5'-GGTGTATGCAAGTGAGTGGATG-3' and (Thi2.1 3')
5'-GACACATGCACACACACACAC-3' were used to amplify the Thi2.1
(GenBankTM accession number L41244) gene from A. thaliana.
Cloning of AtST2a--
The AtST2a cDNA
(GenBankTM accession number T43254) was obtained from the
Arabidopsis Biological Resource Center. AtST2a is
localized on chromosome V and can be retrieved from the
GenBankTM data base under accession number AB010697 from
nucleotides 53,936 to 55,015. It is flanked by an open reading frame
encoding another putative ST, AtST2b from nucleotides 50,627 to 51,670. AtST2a and AtST2b encode proteins of
359 and 347 amino acids corresponding to molecular masses of 41.3 and
39.6 kDa, respectively. The AtST2a and AtST2b
deduced protein sequences contain all the domains known to be involved
in the binding of the sulfonate donor PAPS and in catalysis (Fig.
1) (26). Amino acid sequence alignment
indicates that they share 85% amino acid sequence identity and 92%
similarity, suggesting that they might be isozymes with similar
substrate specificities. This hypothesis is further supported by the
fact that the amino acids known to interact with the sulfonate acceptor substrate are identical between the two sequences (26). The AtST2a and
-b proteins share 45% sequence identity with the A. thaliana and B. napus 24-epibrassinosteroid STs, 40%
sequence identity with the flavonol STs of Flaveria species,
and ~25% sequence identity with the mammalian STs.
Substrate Specificity--
To characterize the biochemical
function of AtST2a, we studied the activity of purified recombinant
AtST2a expressed in E. coli. The histidine-tagged AtST2a
enzyme could be purified to apparent homogeneity by a combination of
nickel- and PAP-agarose chromatography (Fig.
2) (27). The purified recombinant AtST2a enzyme was used to test a variety of acceptor molecules of plant and
mammalian origin. The substrate library comprises more than 100 compounds and includes phenolic acids, desulfoglucosinolates, flavonoids, steroids, gibberellic acids, phenylpropanoids,
hydroxyjasmonates, and coumarins, to mention a few. AtST2a was found to
exhibit strict specificity for 11- and 12-OHJA (Fig.
3). No activity could be detected with
the remaining compounds of the library. The enzyme did not accept
structurally related jasmonates such as 12-OHJA methyl ester, CA,
7-iso-CA, 6-epi-CA, 6-epi-7-iso-CA, and their methyl esters.
Furthermore, the AtST2a enzyme did not sulfonate the structurally
related mammalian prostaglandin E2, arachidonyl alcohol, and 11-eicosenol. AtST2b was also expressed in E. coli and could be recovered in a soluble form. No activity was
observed with all the substrates tested including 11- and 12-OHJA. This result is surprising when we consider the high level of sequence identity with AtST2a and the presence of all the amino acids known to
be required for cosubstrate binding and catalysis (28). Other substrates related in structure to 11- and 12-OHJA will have to be
tested to determine the biochemical function of this enzyme.
Enzyme Properties--
To determine the pH optimum of AtST2a
enzyme activity, the sulfonation of 12-OHJA was carried out at fixed
substrate (50 µM) and PAPS (5 µM)
concentrations in buffers ranging from pH 6 to 10. Highest enzyme
activity was observed at pH 7.5 in Tris buffer. However, the enzyme is
active in a broad pH range and retains ~80% activity at pH 9.5. The
kinetic parameters of AtST2a were determined from the results of
substrate interaction kinetic experiments. AtST2a exhibited preference
for 12-OHJA over 11-OHJA with Km values of 10 and 50 µM, respectively. Double reciprocal plots with 12-OHJA as
the variable substrate at several fixed concentrations of PAPS resulted
in an intersecting pattern (data not shown). A similar pattern was
obtained with PAPS as the variable substrate and the secondary slope,
and intercept replots were linear. As calculated from the replots, the
Vmax for the conversion of 12-OHJA to 12-OHJA
sulfate was 37.5 pkatal/mg of protein. The Km value
for PAPS was found to be 1 µM. Because JA is structurally similar to 12-OHJA, we tested if it could act as a competitive inhibitor in enzyme assays. The assays were performed with a fixed concentration of 12-OHJA (50 µM) and variable
concentrations of JA (0.1-100 µM). Addition of JA had no
effect on the amount of product formed (data not shown), indicating
that JA does not interfere with the binding of 12-OHJA at the enzyme
active site.
Identification and Quantification of Jasmonates in A. thaliana--
To date, the presence of 12-OHJA has been reported to
occur only in tuber-producing plant species (29) and in the fungus Botryodiplodia theobromae (30). To validate the results of
the in vitro studies, GC-MS was used to analyze A. thaliana extracts for the presence of 12-OHJA. To minimize the
effects of biological variability, pools consisting of 30-50 plants
were used for metabolite extraction. Internal standards were used to
compensate for differences in extraction efficiencies. The technical
variability of the GC-MS and LC-MS/MS upon repeated injections was
found to be smaller than 5%. Table I
shows that 12-OHJA is present at a level ~4-fold lower than JA.
Preliminary results indicate that 12-OHJA is also present in barley,
tomato, and tobacco leaves suggesting that it might have a wide
distribution in the plant kingdom (data not shown).
Identification of the Enzyme Reaction Product Formed in Vitro and
in Vivo--
To identify the reaction product formed by recombinant
AtST2a using LC-MS/MS, the enzyme reaction was carried out using
unlabeled PAPS. The product of the enzymatic reaction was found to
elute at the same retention time and gave the same fragmentation
pattern as authentic 12-OHJA sulfate (Fig. 3) (14).
12-OHJA sulfate has only been reported to occur in T. cistoides, a member of the Zygophylaceae family (14). To
determine whether it is present in A. thaliana,
methanolic extracts were subjected to LC-MS/MS. 12-OHJA sulfate was
identified as an endogenous compound in A. thaliana (Table
I). The lack of internal standard precluded the quantification of this
compound in the plant extracts.
Quantification of JA, 12-OHJA, and 12-OHJA Sulfate in MeJA- and
12-OHJA-treated Plants--
Jasmonates were quantified in plants
treated for 36 h with MeJA and 12-OHJA (Table I). Treatment with
MeJA led to 20- and 4-fold increases of the endogenous levels of
12-OHJA and 12-OHJA sulfate, respectively. Treatment with 12-OHJA has
no effect on the accumulation of JA. However, a 9-fold increase in
12-OHJA sulfate level is observed following this treatment.
Regulation Studies--
Rosette leaves from 15-day-old A. thaliana plants expressed very low levels of AtST2a
mRNAs when grown under normal conditions (Fig.
4, lane C). This result is
consistent with the absence of detectable hydroxyjasmonate
sulfotransferase activity in protein extracts from plants growing under
the same conditions. However, treatment with the jasmonates 12-OPDA,
JA, and JA-Ile resulted in strong induction of AtST2a
expression (Fig. 4). Interestingly, AtST2a expression is
also induced in a dose-dependent manner by 12-OHJA (Fig.
4). The steady state AtST2a mRNA levels increased within
30 min following 12-OHJA treatment (Fig.
5A). Similar kinetics of
induction were obtained when plants were treated with MeJA (Fig.
5B). The specific up-regulation of AtST2a by
12-OHJA and MeJA was confirmed by RT-PCR using
AtST2a-specific primers (Fig. 6). Furthermore, the induction of
AtST2a expression by 12-OHJA and MeJA was found to correlate
with increased hydroxyjasmonate ST activity in partially purified
protein preparations from plants treated for 8 h with 12-OHJA
(about 9 pkatal/mg of protein) or MeJA (about 14 pkatal/mg of protein).
To test if the accumulation of AtST2a transcript is specific
to jasmonates or a general stress response, Arabidopsis
plants were subjected to a number of physical and chemical treatments.
AtST2a transcript level is not altered in rosette leaves
from plants subjected to cold, heat, hypoxia, salt stress, or treatment
with salicylic acid. In addition, treatments with phytohormones like
gibberellins, abscisic acid, zeatin, and indole 3-acetic acid did not
affect the AtST2a transcript level (data not shown).
Thi2.1 is a marker gene for MeJA treatment or wounding (31,
32). As expected, Thi2.1 expression is induced when plants are treated with MeJA (Fig. 6). However, the expression of
Thi2.1 is not induced in rosette leaves from plants treated
with 12-OHJA. In contrast, AtST2a expression is induced by
both MeJA and 12-OHJA.
A cDNA clone encoding an enzyme catalyzing the sulfonation of
12-OHJA has been characterized in this study. The
Arabidopsis gene AtST2a is a member of a small
family comprising 18 members. It shares 85% amino acid identity with
AtST2b and between 40 and 45% identity with other members of the
family. To characterize the biochemical function of AtST2a, a large
number of substrates were tested. The substrate library comprises the
desulfo-derivatives of most of the known plant-sulfated metabolites as
well as a collection of metabolites for which no sulfated derivatives
have been reported in the literature. AtST2a was found to exhibit
strict specificity for 11- and 12-OHJA with Km
values of 50 and 10 µM, respectively. However, these
values are probably not reflecting the real affinity of the enzyme for
its substrates, because the assay mixture contained four steroisomers
as a result of the presence of two chiral carbons at C-3 and C-7 of the
cyclopentanone ring. Substrate-dependent stereoselectivity has
been reported for the rat hydroxysteroid and phenol STs and more
recently for the B. napus brassinosteroid ST (19, 33).
Although the stereochemistry of naturally occurring 12-OHJA has not
been established unambiguously, naturally occurring 12-OHJA seems to
have a cis conformation, and it was shown that cis-12-OHJA has a much stronger tuber-inducing activity than
the trans isomer (34). It will be interesting to see whether AtST2a functions in a stereoselective manner, because such a property has been
shown for other cyclopentenone-generating (allene oxide cyclase) and
converting (OPDA reductase 3) enzymes (35, 36).
The fact that the enzyme accepts 12-OHJA and not its methyl ester
derivative indicates a strict requirement for a carboxyl function at
carbon 1. Furthermore, the fact that JA is not a competitive inhibitor
of the reaction indicates that the presence of a hydroxyl group at
positions 11 or 12 on the side chain is required for binding at the
active site of the enzyme. The strict structural specificity exhibited
by AtST2a is a common feature of plant STs and has been observed with
the flavonoid, gallic acid glucoside, and brassinosteroid STs (19,
37).
Previous to this work, there were no reports on the occurrence of
12-OHJA or its sulfated derivative in A. thaliana or any other members of the Brassicaceae family. 12-OHJA was initially isolated as a tuber-inducing compound from Solanum
tuberosum (9). To date, its presence has also been reported
in Solanum demissum (38) and in the fungus B. theobromae (30). The glucoside of 12-OHJA has also been detected
in Helianthus tuberosus (39) and Astragalus
complanatus (40). The presence of 12-OHJA in A. thaliana indicates that its distribution can no longer be
considered to be restricted to tuber-producing plants and it raises
interesting questions about its function in plants that do not produce
tubers. This is the first report on the characterization of an enzyme that sulfonate hydroxylated jasmonates. Recently a mouse cDNA clone
encoding an ST that sulfonates compounds structurally similar to
jasmonates has been characterized (41). This enzyme, which is
predominantly expressed in the kidneys and the uterus, accepts eicosanoids (prostaglandins, thromboxanes, and leukotrienes) as substrates. However, the biological function and the regulation of this
enzyme have not been studied yet.
Treatments of Arabidopsis plants with JA, 12-OPDA, MeJA, or
JA-Ile lead to increased accumulation of the AtST2a
transcript (Fig. 4). It has been shown previously that 12-OPDA can
alter the expression of specific genes (42) and was found to exhibit a
strong activity in several JA bioassays (43, 44). However, we cannot
rule out the possibility that the effect of 12-OPDA on
AtST2a expression is caused by its metabolism to JA or
12-OHJA. JA-Ile was also shown to induce expression of specific genes
when applied exogenously to barley leaves (45). In this case, induction of gene expression could be attributed directly to the conjugate, because very little conversion to JA was observed. The significance of
AtST2a induction by the isoleucine conjugate of JA remains to be determined. Treatments with MeJA led to an increase in the amount
of AtST2a mRNA, 12-OHJA, and 12-OHJA sulfate. The metabolism of JA
to 12-OHJA sulfate might be a route leading to its inactivation. The
widespread occurrence of STs and sulfatases in mammals has led to the
hypothesis that the concerted action of both groups of enzymes might
regulate the levels of bioactive molecules (15). However, the
sulfonation of 12-OHJA might be irreversible considering the lack of
evidence for the presence of genes encoding sulfatases in the genome of
A. thaliana.
Alternatively, the function of AtST2a might be to control directly the
biological activity of 12-OHJA. Several lines of evidence suggest that
12-OHJA might have a biological activity different from JA or MeJA.
Unlike JA or MeJA, 12-OHJA does not exhibit inhibitory effects on plant
growth, such as promotion of senescence of oat leaves, inhibition of
soybean callus growth, and inhibition of lettuce seedling growth (34).
Furthermore, 12-OHJA was shown previously to have tuber-inducing
properties (9). It is possible that 12-OHJA controls developmental
processes other than tuberization in plants such as A. thaliana.
One of the most striking features of AtST2a regulation is
its induction following treatment with 12-OHJA (Fig. 5). Prior to this
work, the tomato gene encoding allene oxide cyclase was the only gene
known to be up-regulated by 12-OHJA (46). It is interesting to note
that in both Arabidopsis and tomato, treatments with 12-OHJA did not result in increased endogenous JA levels ruling out the possibility that the latter might be the mediator of the response (Table I) (46). The up-regulation of AtST2a expression in
response to 12-OHJA indicates the presence of a feed-forward mechanism that tightly controls the in vivo concentration of the free
acid by sulfonation. Similar feed-forward mechanisms have been shown to
regulate the level of the enzymes abscisic acid 8-hydroxylase and
gibberellin-2 oxidase involved in the catabolism of abscisic acid and
gibberellins, respectively (47, 48).
In contrast with AtST2a and allene oxide cyclase
(AOC), Thi2.1 expression is not induced by 12-OHJA (Fig. 6).
Thi2.1 encodes a thionin that is specifically induced by
wounding, pathogen infection, and following treatment with Me-JA (31).
Similarly, treatments of barley leaves with 12-OHJA did not induce the
expression of a multigene family of barley-encoding thionins (49). The
lack of induction of Thi2.1 following 12-OHJA treatment
suggests that Me-JA and 12-OHJA mediate their response via two
independent pathways. Taken together, the results suggest that 12-OHJA
might have a biological function different from JA and MeJA and that
the sulfonation reaction might be required to control its activity. In
the future, the construction of transgenic plants with altered levels
of AtST2a will allow to address the role of 12-OHJA in plant growth,
development, and defense responses.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-linolenic acid to yield 13-hydroperoxylinolenic acid (3). The hydroperoxide is then dehydrated
and cyclized to the cyclopentanone 12-oxophytodienoic acid (OPDA).
Reduction of OPDA by OPDA reductase 3 followed by three cycles
of
-oxidation yield JA. JA can undergo several modification reactions, the best studied being methylation at carbon 1 to yield MeJA
(4). In addition to the formation of the methyl ester, the
biotransformation of JA involves five major reactions (5): (i)
hydroxylation at carbon 11 or carbon 12 to form 11-hydroxyjasmonic acid
(11-OHJA) or 12-hydroxyjasmonic acid (12-OHJA); (ii) reduction of the
keto group at carbon 6 to form cucurbic acid (CA), which can also be
hydroxylated on the side chain; (iii) O-glycosylation of the
above hydroxylated compounds; (iv) conjugation of JA or its
hydroxylated derivatives with amino acids (5), tyramine (6), or
adenylate (7); and (v) degradation of the carboxylic acid side chain to
form cis-jasmone (8).
-D-glucopyranoside of 12-OHJA was shown
to induce tuber formation in vitro (9) and the degradation
of JA to cis-jasmone was found to affect the plant defense
response to herbivores by stimulating the activity of parasitic insects
(10). Furthermore, a distinct ratio of several octadecanoid-derived
compounds, the so-called "oxylipin signature" may have a biological
significance. For instance, such a signature was found to be different
among various plant species (11) and attributed to distinct defense status (12), and varied also in the same species during development (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for
10 h at 22 °C. Bacterial cells were collected by centrifugation and resuspended in 50 mM sodium phosphate buffer (pH 8.0)
containing 0.3 M NaCl and 14 mM
2-mercaptoethanol. The cells were lysed by sonication and the
recombinant proteins were recovered in the soluble fraction by
centrifugation at 12,000 × g for 15 min at 4 °C. The soluble recombinant AtST2a protein was purified by
affinity chromatography onto nickel-nitriloacetic acid-agarose matrix
as recommended (Qiagen). The Ni-agarose-purified protein was desalted on a PD-10 (Amersham Biosciences) column equilibrated in 25 mM bis-Tris (pH 6.5) (buffer A). The desalted protein was
chromatographed on a PAP-agarose column (0.5 × 10 cm) previously
equilibrated in buffer A. The bound protein was eluted with a linear
salt gradient of 0 to 1 M NaCl in the same buffer. Affinity
chromatography was performed on a Waters 625 LC HPLC system and protein
absorbance was monitored at 280 nm. AtST2a eluted at ~450
mM NaCl. Protein concentration was estimated using the
Bradford reagent (Bio-Rad) and bovine serum albumin as reference protein.
1; splitless
injection; column temperature program: 1 min 60 °C, 25°
min
1 to 180 °C, 5° min
1 to 270 °C,
1 min 270 °C, 10° min
1 to 300 °C, 25 min
300 °C.
3 torr). All mass spectra are averaged and background
subtracted. The following results were obtained: 12-OHJA sulfate,
Rt (LC-MS), 12.32 min, negative electrospray MS
m/z (relative intensity), 305 ([M
H]
, 100); CID spectrum, 225 (93), 147 (9), 97 (100), 59 (58). For the determination of 12-OHJA sulfate in plant material, the daughter ions at m/z 225, 97, and 59 were
measured in the selected ion monitoring mode. The CID spectrum of
12-OHJA sulfate displays significant ions at m/z
225, 97, and 59 reflecting the typical structural features of the
compound. The ion at m/z 97 represents a key ion
in the negative CID mass spectra of sulfated compounds (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (60K):
[in a new window]
Fig. 1.
Amino acid sequence alignment of AtST2a,
AtST2b, and the flavonol 3-ST from Flaveria chloraefolia
(accession number M84135). Deduced amino acid sequences were
aligned with CLUSTALW 1.8. Identical amino acids are
boxed in black and conservative changes in
gray. Hyphens indicate gaps introduced to
maximize alignment. Open arrows indicate amino acid residues
that are involved in catalysis in the flavonol 3-ST. Black
arrows identify amino acids involved in determining substrate
specificity in the flavonol 3-ST (26).
View larger version (82K):
[in a new window]
Fig. 2.
SDS-PAGE of fractions collected during
purification of recombinant AtST2a. Lane 1, molecular weight
markers in kDa; lane 2, E. coli crude extract;
lane 3, nickel-agarose-purified recombinant AtST2a;
lane 4, PAP-agarose purified AtST2a.
View larger version (13K):
[in a new window]
Fig. 3.
Mass spectrum of 12-OHJA sulfate obtained by
CID with 30 eV energy.
Quantification of JA, 12-OHJA, and 12-OHJA sulfate in A. thaliana
View larger version (60K):
[in a new window]
Fig. 4.
Northern blot analysis of AtST2a expression
in rosette leaves from 15-day-old A. thaliana plants
treated with OPDA, JA, 12-OHJA, and JA-Ile. Plants grown on MS
medium in magenta boxes were treated for 12 h with the indicated
concentrations of the different metabolites (C, control).
RNA gel blot analysis was performed using 10 µg of total RNA and
hybridized with 32P-labeled AtST2a cDNA as
probe. The same nylon membrane was probed with ACTIN
cDNA to evaluate RNA loading.
View larger version (87K):
[in a new window]
Fig. 5.
Kinetic of AtST2a mRNA
accumulation following treatment of A. thaliana with
12-OHJA and MeJA. 15-Day-old plants grown on MS medium in magenta
boxes were treated with 100 µM 12-OHJA (A) or
MeJA (B) and total RNA was extracted from rosette leaves at
the indicated time periods after treatment. RNA gel blot analysis was
performed using 10 µg of total RNA and hybridized with
32P-labeled AtST2a cDNA as probe. The same
nylon membranes were probed with ACTIN cDNA to evaluate
RNA loading.
View larger version (42K):
[in a new window]
Fig. 6.
RT-PCR of mRNA extracted from rosette
leaves of A. thaliana plants treated with 12-OHJA or
MeJA. 15-Day-old plants grown on MS medium in magenta boxes were
treated for 12 h with the indicated concentrations of 12-OHJA or
with 100 µM MeJA (C, control). PCR experiments
were performed with AtST2a- or Thi2.1-specific
primers. GDNA indicates amplification from genomic DNA. The
amplification products were loaded on ethidium bromide-stained agarose
gel. The size of DNA fragments in the 100-bp ladder is indicated
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
FOOTNOTES |
---|
* This work was supported by the Natural Sciences and Engineering Research Council of Canada and Forschungsgemeinschaft Grant SPP1067, WA875/3-2 of Germany.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom reprint requests should be addressed. Tel.: 514-848-3396; Fax: 514-848-2881; E-mail: varinl@clone.concordia.ca.
Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.M211943200
2 dot.imgen.bcm.tmc.edu:/multialign/multi-align.html.
3 www.ch.embnet.org/software/BOX_form.html.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: JA, jasmonic acid; ST, sulfotransferase; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PAP, 3'-phosphoadenosine 5'-phosphate; 12-OHJA, 12-hydroxyjasmonic acid; MeJA, methyljasmonate; OPDA, 12-oxo-phytodienoic acid; CA, cucurbic acid; CID, collision-induced dissociation; HPLC, high performance liquid chromatography; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; LC-MS, liquid chromatography-mass spectrometry.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Wasternack, C., and Hause, B. (2002) Prog. Nucleic Acid Res. Mol. Biol. 72, 165-221[Medline] [Order article via Infotrieve] |
2. | Wasternack, C., and Parthier, B. (1997) Trends Plant Sci. 2, 302-307[CrossRef] |
3. | Vick, B. A., and Zimmerman, D. C. (1984) Plant Physiol. 75, 458-461 |
4. |
Seo, H. S.,
Song, J. T.,
Cheong, J.-J.,
Lee, Y.-H.,
Lee, Y.-W.,
Hwang, I.,
Lee, J. S.,
and Choi, Y. D.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
4788-4793 |
5. | Sembdner, G., Atzorn, R., and Schneider, G. (1994) Plant Mol. Biol. 26, 1459-1481[Medline] [Order article via Infotrieve] |
6. | Miersch, O., Knofel, H. D., Schmidt, J., Kramell, R., and Parthier, B. (1998) Phytochemistry 47, 327-329[CrossRef] |
7. |
Staswick, P. E.,
Tiryaki, I.,
and Rowe, M. L.
(2002)
Plant Cell
14,
1405-1415 |
8. | Koch, T., Bandemer, K., and Boland, W. (1997) Helv. Chim. Acta 80, 838-850 |
9. | Yoshihara, T., Omer, E.-S. A., Koshino, H., Sakamura, S., Kikuta, Y., and Koda, Y. (1989) Agric. Biol. Chem. 53, 2835-2837 |
10. |
Birkett, M. A.,
Campbell, C. A.,
Chamberlain, K.,
Guerrieri, E.,
Hick, A. J.,
Martin, J. L.,
Matthes, M.,
Napier, J. A.,
Pettersson, J.,
Pickett, J. A.,
Poppy, G. M.,
Pow, E. M.,
Pye, B. J.,
Smart, L. E.,
Wadhams, G. H.,
Wadhams, L. J.,
and Woodcock, C. M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
9329-9334 |
11. |
Weber, H.,
Vick, B. A.,
and Farmer, E. E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10473-10478 |
12. |
Stintzi, A.,
Weber, H.,
Reymond, P.,
Browse, J.,
and Farmer, E. E.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
12837-12842 |
13. | Hause, B., Stenzel, I., Miersch, O., Maucher, H., Kramell, R., Ziegler, J., and Wasternack, C. (2000) Plant J. 24, 113-126[CrossRef][Medline] [Order article via Infotrieve] |
14. | Achenbach, H., Hübner, H., Brandt, W., and Reiter, M. (1994) Phytochemistry 35, 1527-1543[CrossRef][Medline] [Order article via Infotrieve] |
15. | Hobkirk, R. (1993) Trends Endocrinol. Metab. 4, 69-74 |
16. | Strott, C. A. (1997) Endocr. Rev. 17, 670-697 |
17. | Visser, T. J. (1994) Chem.-Biol. Interact. 92, 293-303[CrossRef][Medline] [Order article via Infotrieve] |
18. | Roth, J. A. (1986) Trends Pharmacol. Sci. 7, 404-407 |
19. |
Rouleau, M.,
Marsolais, F.,
Richard, M.,
Nicolle, L.,
Voigt, B.,
Adam, G.,
and Varin, L.
(1999)
J. Biol. Chem.
274,
20925-20930 |
20. | Varin, L., Chamberland, H., Lafontaine, J. G., and Richard, M. (1997) Plant J. 4, 831-837[CrossRef] |
21. | Boss, B., Richling, E., Herderich, M., and Schreier, P. (1999) Phytochemistry 50, 219-225[CrossRef] |
22. | Cashmore, A. R. (1982) in Methods in Chloroplast Molecular Biology (Edelman, M. , Hallick, R. B. , and Chua, N. H., eds) , pp. 387-392, Elsevier Biomedical Press, New York |
23. | McMaster, G. K., and Carmicheal, G. G. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 4835-4839[Abstract] |
24. | Sambrook, J. F., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
25. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
26. | Marsolais, F., Gidda, S. K., Boyd, J., and Varin, L. (2000) Rec. Adv. Phytochem. 34, 433-456 |
27. |
Marsolais, F.,
and Varin, L.
(1995)
J. Biol. Chem.
270,
30458-30463 |
28. | Marsolais, F., Laviolette, M., Kakuta, Y., Negishi, M., Pedersen, L. C., Auger, M., and Varin, L. (1999) Biochemistry 38, 4066-4071[CrossRef][Medline] [Order article via Infotrieve] |
29. | Yoshihara, T., Amanuma, M., Tsutsumi, T., Okumura, Y., Matsura, H., and Ichihara, A. (1996) Plant Cell Physiol. 37, 586-590 |
30. | Miersch, O., Schneider, G., and Sembdner, G. (1991) Phytochemistry 30, 4049-4051[CrossRef] |
31. |
Epple, P.,
Apel, K.,
and Bohlman, H.
(1995)
Plant Physiol.
109,
813-820 |
32. | Bohlmann, H., Vignutelli, A., Hilpert, B., Miersch, O., Wasternack, C., and Apel, K. (1998) FEBS Lett. 437, 281-286[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Banoglu, E.,
and Duffel, M. W.
(1997)
Drug Metab. Dispos.
25,
1304-1310 |
34. | Koda, Y. (1992) Int. Rev. Cytol. 135, 155-199[Medline] [Order article via Infotrieve] |
35. |
Ziegler, J.,
Stenzel, I.,
Hause, B.,
Maucher, H.,
Hamberg, M.,
Grimm, R.,
Ganal, M.,
and Wasternack, C.
(2000)
J. Biol. Chem.
275,
19132-19138 |
36. | Schaller, F., Biesgen, C., Mussig, C., Altmann, T., and Weiler, E. W. (2000) Planta 210, 979-984[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Varin, L.,
Marsolais, F.,
Richard, M.,
and Rouleau, M.
(1997)
FASEB J.
11,
517-525 |
38. | Helder, H., Miersch, O., Vreugdenhil, D., and Sambdner, G. (1993) Physiol. Plant. 88, 647-653[CrossRef] |
39. | Matsura, H., Yoshihara, T., Ichihara, A., Kikuta, Y., and Koda, Y. (1993) Biosci. Biotech. Biochem. 57, 1253-1256 |
40. | Cui, B., Nakamura, M., Kinjo, J., and Nohara, T. (1993) Chem. Pharm. Bull. 41, 178-182 |
41. | Liu, M. C., Sakakibara, Y., and Liu, C. C. (1999) Biochem. Biophys. Res. Commun. 254, 65-69[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Kramell, R.,
Miersch, O.,
Atzorn, R.,
Parthier, B.,
and Wasternack, C.
(2000)
Plant Physiol.
123,
177-188 |
43. |
Koch, T.,
Krumm, T.,
Jung, V.,
Engelberth, J.,
and Boland, W.
(1999)
Plant Physiol.
121,
153-162 |
44. | Blechert, S., Bockelmann, C., Füßlein, M. V., Schrader, T., Stelmach, B., Niesel, U., and Weiler, E. W. (1999) Planta 207, 470-479[CrossRef] |
45. | Kramell, R., Miersch, O., Hause, B., Ortel, B., Parthier, B., and Wasternack, C. (1997) FEBS Lett. 414, 197-202[CrossRef][Medline] [Order article via Infotrieve] |
46. | Stenzel, I., Hause, B., Maucher, H., Pitzschke, A., Miersch, O., Ziegler, J., Ryan, C. A., and Wasternack, C. (2002) Plant J. 33, 577-589 |
47. | Windsor, M. L., and Zeevaart, J. A. D. (1997) Photochemistry 45, 931-934[CrossRef] |
48. |
Thomas, S. G.,
Phillips, A. L.,
and Hedder, P.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4698-4703 |
49. | Miersch, O., Kramell, R., Parthier, B., and Wasternack, C. (1999) Phytochemistry 50, 353-361[CrossRef] |