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INTRODUCTION |
One of the most destructive diseases of potato (Solanum
tuberosum L.) is late blight caused by infection with the oomycete Phytophthora infestans. After the first appearance in the
United States and Europe 150 years ago, late blight of potato, along with that of tomato, re-emerged during the late 1980s and early 1990s
as an important disease in the United States and Canada (1). Thus,
research on the molecular mechanisms of the interaction between potato
and P. infestans is of current interest.
Plants have evolved various defense mechanisms to cope with pathogen
attack. One of these is the pathogen-induced accumulation of compounds
with antimicrobial activity (phytoalexins; for review see Ref. 2).
Accordingly, P. infestans infection of potato causes the
accumulation of rishitin and several structurally related sesquiterpenoid phytoalexins in tubers (3, 4). In contrast, P. infestans-infected leaves do not respond with elevated levels of
sesquiterpenoid phytoalexins (5) but show a strong stimulation of
enzyme activities of the phenylpropanoid metabolism, such as phenylalanine ammonia-lyase (EC 4.3.1.5) and 4-coumarate:CoA ligase (EC
6.2.1.12) (6-8). Among the accumulating phenylpropanoids, N-(hydroxycinnamoyl)-tyramines have been identified both in
P. infestans-infected leaves and suspension-cultured potato
cells (9). N-(hydroxycinnamoyl)-amines, such as
N-feruloyltyramine and 4-N-coumaroyltyramine, are
incorporated into cell walls and substantial amounts are also secreted
into the cell culture medium (9, 10). These compounds appear to be
involved in cell wall fortification in response to P. infestans attack. Cell wall incorporation of phenylpropanoids has
repeatedly been shown in other systems (9, 11-15) and is believed to
enhance the efficiency of the cell wall to act as a barrier against
pathogens by increasing rigidity and decreasing digestibility of the
cell wall (16-18). On the other hand, the fact that large amounts of
tyramine amides are secreted into the culture medium in potato cell
cultures may indicate an induced apoplastic accumulation in intact
leaves. The P. infestans-induced pathway in potato has also
been shown to occur in response to elicitor treatment in cultured cells
of Nicotiana glutinosa and Eschscholtzia
californica (19), as well as Nicotiana tabacum
(20).
The synthesis of N-(hydroxycinnamoyl)-tyramines is catalyzed
by hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (THT)1 (EC 2.3.1.110). THT
was first discovered in tobacco mosaic virus-inoculated tobacco leaves
(21) and has since been studied with regard to biotic and abiotic
elicitor- and stress-stimulated activity increases in tobacco and other
plants (Ref. 22 and literature cited therein). The enzyme catalyzes the
transfer of hydroxycinnamic acids from the respective CoA esters to
tyramine and other amines leading to the formation of hydroxycinnamic
acid amides: S-(hydroxycinnamoyl)-CoA + amine
N-(hydroxycinnamoyl)-amine + HS-CoA.
THT activity increases in suspension-cultured potato cells after
elicitor treatment (23), preceded by an increase in the activities of
phenylalanine ammonia-lyase and tyrosine decarboxylase (EC 4.1.1.25)
(10). Purification and characterization of THT was achieved with potato
(23, 24) and tobacco (22) suspension-cultured cells. In continuation of
our studies on the induction of the phenylpropanoid metabolism in
potato by P. infestans, we report here partial amino acid
sequencing of THT as well as molecular cloning and prokaryotic
expression of a potato cDNA clone encoding this enzyme. We show
that the recombinant enzyme possesses similar biochemical properties as
the native protein. Expression analyses revealed elicitor-induced
accumulation of THT mRNA in cultured potato cells.
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EXPERIMENTAL PROCEDURES |
Chemicals--
All chemicals and solvents were of analytical
grade and were obtained from Merck (Darmstadt), Serva (Heidelberg), or
Sigma (München).
Partial Amino Acid Sequencing of THT--
In gel digestion of
THT, peptide mapping, and peptide sequencing were essentially carried
out as described by Eckerskorn and Grimm (25).
Determination of THT Activity--
THT was assayed as described
(23). Determination of its activity was done by high-performance liquid
chromatography (HPLC) coupled with a photodiode array detection as
described previously (10). The hydroxycinnamoyl-CoAs were chemically
synthesized by the ester exchange reaction via the acyl
N-hydroxysuccinimide esters (26) and purified on polyamide
columns (27) followed by preparative HPLC (system Gold; Beckman
Instruments, München, Germany) equipped with a Nucleosil 100-10
C18 column (VarioPrep, 10 µm, 250 × 40 mm inner
diameter; Macherey-Nagel, Düren, Germany). The CoA esters were
eluted with a flow rate of 10 ml/min with a linear gradient within 50 min from solvent A (1% aqueous formic acid) to solvent B (methanol).
The eluates were dried, redissolved into water, and their molarities
adjusted by applying known absorbance coefficients (26, 28, 29).
Amplification and Cloning of THT-encoding Sequences--
Based
on the partial amino acid sequence, degenerate primers were designed
for peptides 7 (5'-CA(C/T)ATITA(C/T)CA(A/G)(C/T)TITT(C/T)TA(C/T)CA(A/G)ATIC-3') as
well as 3 and 4 (5'-C(C/T)TCIAC(C/T)T(G/C)ICC(C/T)TCIACIACIGG-3'). Reverse transcription with total RNA from elicitor-induced potato suspension-cultured cells was carried out using SuperscriptTM (Life Technologies, Inc.) according to the manufacturer's instructions. Subsequent PCR was performed with 35 cycles of 94 °C for 1 min, annealing temperature for 1 min, 72 °C for 1 min in a thermocycler (Stratagene, Heidelberg). A temperature gradient with 2°-intervals ranging from 42 to 64 °C was chosen to identify the optimal
annealing temperature. The 250-bp PCR fragment generated at
temperatures of 42 to 48 °C was subcloned into pPCRII (Invitrogen,
Leek, The Netherlands) according to the manufacturer's instructions.
Isolation of THT cDNA Clones--
RNA was isolated from
potato (S. tuberosum cv. Desirée) suspension cells
that had been treated with P. infestans elicitor for 5 h according to the method used by Dunsmuir et al. (30). From
total RNA, poly(A)+ RNA was isolated using Dynabeads
(Dynal, Hamburg). cDNA was produced using the Time Saver Kit
(Pharmacia, Freiburg) and cloned into the EcoRI site of
gt11 (Stratagene). In vitro packaging with Gigapack® III Gold (Stratagene) yielded a library of
7 × 106 plaque-forming units, which was subsequently
screened for THT sequences using the radioactively labeled 250-bp PCR
fragment as a probe. Thirty positive signals were chosen for the
isolation of single plaques.
DNA was digested with
EcoRI, and the resulting inserts were cloned into
EcoRI cut pUC18. Sequence analysis was carried out using the
T7-Sequencing Kit (Pharmacia) or with a LICOR automatic sequencer (MWG
Biotech, Ebersberg).
Expression of THT Sequences in Escherichia coli--
The coding
sequences of the cDNA clone pTHT3 were cloned into the expression
vector pQE30 (Qiagen, Hilden). After digestion of plasmid DNA of clone
pTHT3 with NcoI and treatment with Klenow enzyme, the DNA
was recut with HindIII and cloned into pQE30 that had been
cut with BamHI, filled-in, and re-cut with
HindIII. Positive clones were transferred into the E. coli strain M15(pREP4). Growth and induction of transformants were
performed as described by the manufacturer. Bacterial pellets were
resuspended in 1 M imidazole (pH 6.5) and used for
determination of enzyme activity as described by Schmidt et
al. (10). His-tagged protein was purified as described by the manufacturer.
Southern and Northern Analyses--
DNA was isolated from young
leaves of 8-week-old greenhouse-grown potato plants (S. tuberosum L. cv. Desirée) using a plant DNA isolation kit
(Boehringer Mannheim) and digested with restriction enzymes. After gel
electrophoresis, the DNA was transferred to nylon Hybond N membranes
(Amersham Buchler, Braunschweig) and these were hybridized to the
radioactively labeled 0.95-kb EcoRI fragment of the cDNA
clone pTHT3. Hybridization was performed in 5× saline/sodium
phosphate/EDTA, 5× Denhardt's, 0.1% SDS, 50% formamide, 100 µg/ml
denatured salmon sperm DNA. Filters were washed three times at 65 °C
with 3× SSC, 0.1% SDS. RNA isolation from different tissues of potato
plants and Northern analyses were performed according to Geerts
et al. (31).
Characterization of Recombinant THT--
Determination of
substrate specificity, molecular mass, pH optimum, and dependence on
cofactors were essentially performed as described (23); however, 0.5 M potassium-phosphate buffer (pH 6.8) was used instead of 1 M imidazole.
Product Identification and Accumulation in Potato Cell
Cultures--
Hydroxycinnamic acid amides accumulating in potato cell
cultures were identified by electrospray (ES) mass spectrometry and were further characterized by HPLC retention times and on-line UV
spectroscopy. ES mass spectra were obtained from a Finnigan MAT TSQ
7000 instrument (ES voltage 4.5 kV (positive ions), 3.5 kV (negative
ions); heated capillary temperature 220 °C; sheath and auxillary
gas: nitrogen) coupled with a Micro-Tech Ultra-Plus MicroLC system
equipped with a RP18-column (4 µm, 1 × 100 mm; SEPSERV). HPLC
runs were performed using a gradient system from 20 to 90%
CH3CN in H2O (containing 0.2% HOAc) within 15 min and held on 90% for 10 min; flow rate 70 µl min
1.
Positive ion ES mass spectra were run with a skimmer CID of 20 eV. All
spectra were averaged and background subtracted. Analysis of product
accumulation in potato cell walls and in the medium was carried out as
described previously (10).
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RESULTS |
Partial Amino Acid Sequence of THT--
THT was purified (see Ref.
24) from suspension-cultured cells of potato cv. Desirée (10).
For sequence analysis, the purified protein was subjected to
LysC-endopeptidase in gel digestion. THT peptides were separated by
reversed-phase HPLC and subjected to peptide sequence analysis (Fig.
1). The similarity of the sequences of
fragments 3 and 4 as well as that of 6 and 7 suggests the presence of
isoenzymes or different subunits of THT, as proposed earlier (24).

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Fig. 1.
Reversed-phase HPLC separation of the in
gel LysC-endopeptidase digest of THT. The identified
sequences of some peptides are assigned.
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Isolation and Characterization of PCR and cDNA Clones Encoding
THT--
A THT-specific cDNA fragment was obtained by reverse
transcription-PCR with RNA isolated from elicitor-treated potato
cells (see Experimental Procedures). Sequence analysis of the subcloned PCR fragment revealed that apart from the sequences coding for peptides
3 and 7, which were used for primer design, those encoding peptides 2, 5, 8, and 9 were present (Fig. 2).

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Fig. 2.
Nucleotide and deduced amino acid sequence of
potato THT cDNA. The nucleotide sequence of the
EcoRI insert of pTHT3 is shown as well as the deduced amino
acid sequence. Differences to the sequence of the PCR fragment are
shown above the cDNA sequence. The asterisk denotes the
stop codon. Amino acids representing the sequenced peptides are in
bold type and underlined. Numbers below these
sequences correspond to the peptides shown in Fig. 1.
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A cDNA library was constructed in
gt11 with RNA from potato
cells induced with P. infestans elicitor for 5 h. One
of the positive clones was shown to contain an insert of about 1 kb, which was subsequently subcloned as an EcoRI fragment into
pUC18 yielding the THT cDNA clone pTHT3. Fig. 2 shows the
nucleotide sequence of the THT cDNA as well as the deduced amino
acid sequence. The cDNA has a length of 938 bp with an open reading
frame ranging from position 53 to 796. There are 52 bp of 5'- and 142 bp of 3'-untranslated sequences; however, the cDNA clone pTHT3 does not contain a poly(A) tail. The encoded protein comprises 248 amino
acids and has a calculated molecular mass of 28.4 kDa. Peptide 6 could
not be found as a contiguous sequence but is contained in peptides 9 and 7.
Expression of THT in E. coli--
Expression of THT in E. coli was achieved using the vector pQE30, which adds an affinity
tag of six histidine residues to the N terminus. The insert of pTHT3
was cloned into pQE30, and the resulting plasmid pQE-THT3 was
transferred to M15(pREP4) cells. Bacterial extracts were prepared and
analyzed by SDS-polyacrylamide gel electrophoresis before and after
induction with IPTG (Fig. 3A).
A protein band with an apparent molecular mass of about 30 kDa was
detectable specifically after induction with IPTG (Fig. 3A,
lane 3). This protein was purified with Ni-NTA-agarose (Fig. 3A, lane 4). Both crude bacterial extracts and
the purified protein exhibited THT enzyme activity when measured by
incubation with 4-coumaroyl-CoA and tyramine as substrates and analysis
of the products by HPLC (Fig. 3B). Some THT activity was
found in extracts containing pQE-THT3 before the addition of IPTG (Fig.
3A, lane 2) indicating incomplete repression by the lac
repressor. Bacterial extracts without pQE-THT3 (Fig. 3A,
lane 1) or heat-treated extracts (data not shown) did not
show THT enzyme activity.

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Fig. 3.
Expression of THT in E. coli. Panel A, Coomassie stain of an
SDS-polyacrylamide gel electrophoresis with extracts from bacteria
expressing THT. Lane M, molecular mass marker; lane
1, extracts from bacteria containing pQE30; lane 2,
extracts from bacteria containing pQE-THT3 before addition of IPTG;
lane 3, extracts from bacteria containing pQE-THT3 5 h
after addition of IPTG; lane 4, purified recombinant
protein. Panel B, THT enzyme activity measured in extracts
of bacteria. THT enzyme activity was measured in extracts of bacteria
described in panel A. Activity measured for the purified
protein was set to 100% corresponding to 73 millikatal/kg. Background
activity in lane 2 was 8 millikatal/kg.
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Enzymatic properties of recombinant THT--
The substrate
specificity of the recombinant THT activity tested with various
hydroxycinnamoyl-CoAs and amines as possible donors and acceptors,
respectively, is summarized in Table I. In general, the data resemble those obtained with the native THT from
potato cells (23) exhibiting a broad donor and acceptor specificity.
The enzyme had a high affinity toward cinnamoyl- and feruloyl-CoA with
apparent Km values of 0.06 and 0.10 mM,
respectively. Cinnamoyl-CoA gave the highest specificity (Vmax/Km), followed by
feruloyl-CoA (47%) and caffeoyl-CoA (20%); the specificities for
other CoA esters were in the range of 4-8%. As found with native THT,
there was a pronounced specificity for tyramine as acceptor in the
presence of cinnamoyl- and feruloyl-CoA as acyl donors. However, using
caffeoyl-, 4-coumaroyl-, and sinapoyl-CoAs as donors, the specificity
decreased to 2-4% of that measured with the former two. Octopamine
showed highest specificity, i.e. 530% compared with
tyramine with feruloyl-CoA. These results suggest that the formation of
various hydroxycinnamate amides accumulating in the cell wall and
secreted into the medium of suspension-cultured potato cells (Fig. 8)
is catalyzed by a single enzyme.
Expression of THT in Potato--
THT enzyme activity increases in
potato cells after elicitation with crude P. infestans
elicitor (10). Using the cDNA clone pTHT3, steady state levels of
THT mRNA after elicitation were determined. Total RNA was isolated
at different time points after the onset of elicitation and subjected
to Northern analyses. Fig. 4 shows that
THT transcripts of about 1-kb size start to accumulate 1 h after
elicitation and that maximal transcript levels are detected after
5 h. Background levels are reached after 40-60 h. Nonelicited control cells do not show an increase in THT transcript levels. Determination of THT activity in the same samples reveals that there is
a shift in the onset of enzyme activation compared with transcript
accumulation. In contrast to transcript levels, enzyme activity remains
high up to 30 h after initiation of elicitor treatment.

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Fig. 4.
Elicitor inducibility of THT expression in
cultured potato cells. Relative amount of THT-mRNA and THT
enzyme activity (100 = 7.4 millikatal/kg protein) measured in
extracts of samples taken at various times after elicitation (0 h = 5-day-old cultures). , THT activities of nonelicited cultures;
, elicited cultures; and , relative mRNA steady state levels.
Inset, Northern analysis with 15 µg of total RNA isolated
from potato cells (e, application of P. infestans
culture filtrate; c, application of water) at the times
indicated. Filters were hybridized against a radioactively labeled
probe derived from the 0.95-kb EcoRI fragment of pTHT3
(THT) and against a potato ribosomal probe
(rRNA).
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RNA was extracted from different tissues of potato plants grown in the
greenhouse and used in Northern analyses with the THT cDNA insert
as a probe (Fig. 5). Highest levels of
THT mRNA were present in roots, whereas low levels of THT
transcripts were detectable in leaves, petioles, stems, and tubers.
Flowers did not contain RNA hybridizing to the THT cDNA probe.

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Fig. 5.
Tissue-specific expression of THT in potato
plants. RNA was isolated from young (yl) and old leaves
(ol), petioles (p), stems (s), roots
(r), tubers (t), and flowers (f) of
6-week-old potato plants grown in the greenhouse. 15 µg of total RNA
were subjected to Northern analysis and hybridized against a
radioactively labeled probe derived from the 0.95-kb EcoRI
fragment of pTHT3 (THT) and against a potato ribosomal probe
(rRNA).
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Genomic Southern Analysis--
To analyze the structure of the
potato genes encoding THT, DNA was isolated from young leaves of potato
plants grown in the greenhouse, digested with restriction enzymes and
subjected to Southern analyses. Fig. 6
shows the presence of multiple bands hybridizing to the THT cDNA as
a probe indicating that THT is encoded by a multigene family in
potato.

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Fig. 6.
Genomic Southern analysis of THT genes.
DNA was isolated from young leaves of potato plants, digested with the
restriction enzymes as indicated, and subjected to Southern analysis.
The filter was hybridized against a radioactively labeled probe derived
from the 0.95-kb EcoRI fragment of pTHT3.
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Product Identification--
Because the analysis of enzymatic
properties of the recombinant THT revealed a high affinity for
octopamine as a substrate, we re-examined unidentified compounds,
accumulation of which was induced in elicitor-treated potato cells.
Using liquid chromatography/mass spectrometry (positive and negative
ion ES mass spectrometry) and HPLC/diode array detector, the amine
conjugates feruloyloctopamine (1), feruloyl-3'-methoxyoctopamine (2),
4-coumaroyltyramine (3), 4-coumaroyloctopamine (4), and
feruloyltyramine (5) could be identified. The molecular weights of the
feruloyl/4-coumaroyl amines 1-5 (Fig. 7)
were indicated in their positive and negative electrospray mass spectra
by [M+H]+/[M+2H]+ and [M-H]
ions, respectively. The main mass spectral fragmentation of compounds 1-5 under positive ion electrospray conditions is the formation of an
ion of type a indicating the feruloyl (m/z
177) and 4-coumaroyl moiety (m/z 147),
respectively (Table II). Generally, in
the amine conjugates with a hydroxy function at C-7' (1, 2, and 4) the
negative ES mass spectra display a prominent
[M
H
H2O]
ion. Additionally, in these
cases a weak [M+H]+/[M+2H]+ ion and a
stronger [M+H
H2O]+ ion appear in the
positive ion ES mass spectra. These ions can be used to differentiate
between 7'-hydroxy compounds and those being unsubstituted at the
benzylic position of the amine moiety. In the positive ion ES mass
spectra of tyramine conjugates, 3 and 5 show the ion at
m/z 121 characteristic for ES spectra of tyramine
conjugates (32). Therefore, LC/ES mass spectroscopy represents a very
useful tool for the identification of feruloyl/4-coumaroyl amines even
in trace amounts.

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Fig. 7.
Structures of the hydroxycinnamic acid
amides, accumulating in cell walls and medium of suspension-cultured
potato cells. 1, feruloyloctopamine; 2,
feruloyl-3'-methoxyoctopamine; 3, 4-coumaroyltyramine;
4, 4-coumaroyloctopamine; 5,
feruloyltyramine.
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Table II
Positive and negative ion ES mass spectra (m/z, relative intensities),
HPLC retention times (RT, min) and UV absorbance maxima
( max, nm) of compounds 1-5
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As shown in Fig. 8, apart from the
tyramine amides both the 4-coumaroyl and feruloyloctopamine accumulate
as cell wall-bound amides. However, only 4-coumaroyloctopamine, along
with the tyramine amides, is being secreted into the medium.
Feruloyl-3'-methoxyoctopamine was identified as a compound whose levels
were reduced in elicitor-treated potato cells.

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Fig. 8.
Level of hydroxycinnamic acid amides in cell
walls and medium of suspension-cultured potato cells at the time of
elicitor application (0, 5-day-old cultures).
, nonelicited cultures (application of water); , elicited
cultures (application of P. infestans culture filtrate).
Values are means ± SD from three independent extractions.
Panel A, cell wall-bound compounds; panel B,
compounds secreted into the culture medium. To be complete, the
accumulation patterns of the tyramine amides were taken from our
previous publication (10).
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DISCUSSION |
By isolating a THT cDNA clone from suspension-cultured potato
cells we were able to characterize enzymatic properties and expression
patterns of THT. The cDNA clone pTHT3 encodes a protein with a
calculated molecular mass of 28.4 kDa, which is slightly more than the
apparent mass of about 25 kDa that was estimated for THT from
SDS-polyacrylamide gel electrophoresis of proteins purified from
elicitor-treated potato cells (23). A molecular mass of 24 kDa was
determined for THT from cultured tobacco cells treated with Pronase as
an elicitor (22). The potato cDNA clone pTHT3 contains the
complete protein-encoding region, because the first ATG, at
position 53, is preceded by an in-frame stop codon 42-bp upstream. As
there are no potential intron acceptor splice sites within this region,
it is unlikely that intron sequences are present. It is therefore
concluded that the ATG at position 53 represents the start codon for THT.
THT belongs to the class of acyltransferases (EC 2.3.1) and indeed, the
highest homology of the deduced amino acid sequence is found with other
enzymes of this class, namely spermidine/spermine N-acetyltransferases (SSATs) from mice, pigs, and humans.
Two conserved domains have been identified in
N-acetyltransferases from microbes and animals that are also
present in potato THT. Domain I contains a stretch of seven amino acids
(RGFGIGS), that was shown to be required for SSAT activity and binding
of acetyl-CoA (33), and that corresponds to RKLGMGS (amino acids 173 to
179) in potato THT. Mutation of the arginine or either of the two
C-terminal glycine residues in RGFGIGS resulted in complete loss of
SSAT activity (33). Interestingly, all three residues are conserved in
the sequence of potato THT. In addition to its homology to domain I,
potato THT also exhibits similarity to the conserved domain II of
N-acetyltransferases (33). Furthermore, the spacing between
domain I and II as well as the location of both domains in the
C-terminal part of the protein are conserved as well. On the other
hand, potato THT lacks the C-terminal motif MATEE, which is critical
for polyamine binding in SSATs (34) and has a longer N terminus.
Some enzymatic properties of the purified potato THT (23) could not be
found with the recombinant THT. For example, we were unable to
demonstrate stimulation of THT activity by Ca2+ or
Mg2+. It was also impossible to determine a definite pH
optimum. Depending on the buffer used, we determined pH optima between
6.5 and 6.8 for the native enzyme with 50% of these activities at pH
levels of about 6.0 and 9.1 (23). In contrast, recombinant THT
exhibited highest activities between pH 9.0 and 10.0 with half-maximal
activities at pH levels of about 5.3 and 12.4 (data not shown). The
broad substrate specificity of the recombinant THT coincides with that obtained with the potato THT (23). Thus, the various amides accumulating in cultured potato cell walls and those secreted into the
medium (Fig. 8) could be formed by a single enzyme. This was also found
with the purified tobacco THT (22). Consequently, control of the
in vivo formation of the different amides appears to be
achieved by supply of the respective hydroxycinnamoyl-CoAs and the amines.
For human SSAT, dimerization is necessary to form the active enzyme
(35). The apparent native molecular mass of potato THT was determined
as 49 kDa (23) with subunits of about 25 kDa (24) suggesting that THT
is also active as a dimer. Expression of THT cDNA sequences in
E. coli and subsequent gel filtration analyses on Superdex
G-75 showed that the active enzyme had a relative molecular mass of
about 63 kDa (data not shown). Because the calculated mass of the
recombinant THT expressed in the His-tag vector is 30 kDa, THT appears
to be active as a homodimer, as supposed by Negrel and Javelle (22) for
the tobacco THT. Whether heterodimers are also active, as suggested
(24), remains to be elucidated.
A comparison of the properties of the recombinant THT from potato to
those of the enzyme purified from suspension-cultured tobacco cells
treated with Pronase as an elicitor (22) reveals several similarities.
For example, the calculated pI for potato THT (5.1) and the one
described for tobacco THT (5.2) are nearly identical. Furthermore, the
activity of neither enzyme is affected by the addition of
CaCl2 or MgCl2. For both enzymes, tyramine and
feruloyl-CoA are among the best substrates. As differences we noted
that the Km for cinnamoyl-CoA is low for the recombinant potato THT, whereas a relatively low specificity for cinnamoyl-CoA derivatives was described for the tobacco enzyme. Caffeoyl-CoA, which is an efficient substrate for recombinant THT from
potato, was used as a competitive inhibitor for tobacco THT (22). Two
of the peptide sequences reported for the tobacco enzyme (ATESVLXDLLFK
and LFYQIHEYHNYTHLYK) fit well with the deduced amino acid sequence of
the potato cDNA clone. However, the third peptide (FYGPSHEYHN) is
not present as a contiguous sequence.
THT activity also increases in potato tubers in response to wounding
(13). This induction and the synthesis of hydroxycinnamoyltyramines are
considered to constitute an early response of tuber disks to wounding
and might indicate an involvement of THT in wound healing. The low
levels of THT transcripts detected in tubers correspond to the
observation that untreated tubers contain barely detectable THT
activity (13). We are presently analyzing whether the increase in THT
activity in wounded tubers correlates with an increase in THT mRNA.
Analyses of the tissue-specific expression of THT revealed the highest
transcript levels in roots. Because products of the THT reaction have
been shown to be active against mycorrhizal fungi (36) it is tempting
to speculate on a function of THT in defense against root pathogens.
The determination of pathogen-inducibility of THT gene expression
in planta and the functional analysis in transgenic plants
will show whether THT plays a role in the plant's response to pathogen attack.