Cloning and Expression of a Potato cDNA Encoding Hydroxycinnamoyl-CoA:Tyramine N-(Hydroxycinnamoyl)transferase*

Axel SchmidtDagger , Rudi Grimm§, Jürgen Schmidt, Dierk Scheelparallel , Dieter StrackDagger **, and Sabine Rosahlparallel

From the Dagger  Abteilung Sekundärstoffwechsel,  Abteilung Naturstoffchemie and parallel  Abteilung Stress- und Entwicklungsbiologie of the Institut für Pflanzenbiochemie (IPB), Weinberg 3, D-06120 Halle (Saale), Germany, and § Waldbronn Analytical Division, Hewlett-Packard GmbH, Hewlett-Packard Str. 8, D-76337 Waldbronn, Germany

    ABSTRACT
Top
Abstract
Introduction
References

Hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) catalyzes the transfer of hydroxycinnamic acids from the respective CoA esters to tyramine and other amines in the formation of N-(hydroxycinnamoyl)amines. Expression of THT is induced by Phytophthora infestans, the causative agent of late blight disease in potato. The amino acid sequences of nine endopeptidase LysC-liberated peptides from purified potato THT were determined. Using degenerate primers, a THT-specific fragment was obtained by reverse transcription-polymerase chain reaction, and THT cDNA clones were isolated from a library constructed from RNA of elicitor-treated potato cells. The open reading frame encoding a protein of 248 amino acids was expressed in Escherichia coli. Recombinant THT exhibited a broad substrate specificity, similar to that of native potato THT, accepting cinnamoyl-, 4-coumaroyl-, caffeoyl-, feruloyl- and sinapoyl-CoA as acyl donors and tyramine, octopamine, and noradrenalin as acceptors tested. Elicitor-induced THT transcript accumulation in cultured potato cells peaked 5 h after initiation of treatment, whereas enzyme activity was highest from 5 to 30 h after elicitation. In soil-grown potato plants, THT mRNA was most abundant in roots. Genomic Southern analyses indicate that, in potato, THT is encoded by a multigene family.

    INTRODUCTION
Top
Abstract
Introduction
References

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 right-arrow 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.

    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 lambda 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. lambda  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).

    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.

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.

A cDNA library was constructed in lambda 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.

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.

                              
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Table I
Substrate specificity of the recombinant THT

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; black-square, elicited cultures; and triangle , 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).

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).

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.

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 (lambda max, nm) of compounds 1-5

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). open circle , nonelicited cultures (application of water); bullet , 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).


    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.

    ACKNOWLEDGEMENTS

We gratefully acknowledge Martina Rauscher and Dr. Claus Wasternack for critical reading of the manuscript. We thank Dagmar Knöfel for preparation of the hydroxycinnamoyl-CoAs and Birgit Kemmerling for providing the potato rRNA probe.

    FOOTNOTES

* This work was supported by the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (to D. Strack).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 correspondence should be addressed. Tel: 49-345-5582-205; Fax: 49-345-5582-106; E-mail: dstrack{at}ipb.uni-halle.de.

The abbreviations used are: THT, hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase; HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase; ES, electrospray; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; SSAT, spermidine/spermine N-acetyltransferase.
    REFERENCES
Top
Abstract
Introduction
References

  1. Fry, E. F., and Goodwin, S. B. (1997) Plant Dis. 81, 1349-1357
  2. Dixon, R. A., and Lamb, C. J. (1990) Annu. Rev. Plant Physiol. 41, 339-367[CrossRef]
  3. Kuc, J. (1982) in Phytoalexins (Bailey, J., and Mansfield, J., eds), pp. 81-105, Blackie Academic & Professional, Glasgow, Scotland
  4. Kuc, J., and Rush, J. S. (1985) Arch. Biochem. Biophys. 236, 455-472[Medline] [Order article via Infotrieve]
  5. Rohwer, F., Fritzemeier, K.-H., Scheel, D., and Hahlbrock, K. (1987) Planta 170, 556-561
  6. Fritzemeier, K. H., Cretin, C., Kombrink, E., Rohwer, F., Taylor, J., Scheel, D., and Hahlbrock, K. (1987) Plant Physiol. 85, 34-41
  7. Becker-André, M., Schulze-Lefert, P., and Hahlbrock, K. (1991) J. Biol. Chem. 266, 8551-8559[Abstract/Free Full Text]
  8. Joos, H.-J., and Hahlbrock, K. (1992) Eur. J. Biochem. 204, 621-629[Abstract]
  9. Keller, H., Hohlfeld, H., Wray, V., Hahlbrock, K., Scheel, D., and Strack, D. (1996) Phytochemistry 42, 389-396[CrossRef]
  10. Schmidt, A., Scheel, D., and Strack, D. (1998) Planta 205, 51-55[CrossRef]
  11. Clarke, D. D. (1982) in Active Defense Mechanisms in Plants (Wood, R. K. S., ed), pp. 321-322, Plenum Press, New York
  12. Beimen, A., Witte, L., and Barz, W. (1992) Bot. Acta 105, 152-160
  13. Negrel, J., Javelle, F., and Paynot, M. (1993) J. Plant Physiol. 142, 518-524
  14. Negrel, J., Pollet, B., and Lapierre, C. (1996) Phytochemistry 43, 1195-1199[CrossRef]
  15. Pearce, G., Marchand, P. A., Griswold, J., Lewis, N. G., and Ryan, C. A. (1998) Phytochemistry 47, 659-664[CrossRef]
  16. Fry, S. C. (1986) Annu. Rev. Plant Physiol. 73, 165-186
  17. Nicholson, R. L., and Hammerschmidt, R. (1992) Annu. Rev. Phytopathol. 30, 369-389[CrossRef]
  18. Matern, U., Grimmig, B., and Kneusel, R. E. (1995) Can. J. Bot. 73, 511-517
  19. Villegas, M., and Brodelius, P. E. (1990) Physiol. Plant. 78, 414-420[CrossRef]
  20. Negrel, J., and Javelle, F. (1995) Physiol. Plant. 95, 569-574[CrossRef]
  21. Negrel, J., and Martin, C. (1984) Phytochemistry 23, 2797-2801[CrossRef]
  22. Negrel, J., and Javelle, F. (1997) Eur. J. Biochem. 247, 1127-1135[Abstract]
  23. Hohlfeld, H., Schürmann, W., Scheel, D., and Strack, D. (1995) Plant Physiol 107, 545-552[Abstract/Free Full Text]
  24. Hohlfeld, H., Scheel, D., and Strack, D. (1996) Planta 199, 166-168
  25. Eckerskorn, E., and Grimm, R. (1996) Electrophoresis 17, 899-906[Medline] [Order article via Infotrieve]
  26. Stöckigt, J., and Zenk, M. H. (1975) Z. Naturforsch. 30c, 352-358
  27. Strack, D., Keller, H., and Weissenböck, G. (1987) J. Plant Physiol. 131, 61-73
  28. Gross, G. G., and Zenk, M. H. (1966) Z. Naturforsch. 21b, 683-690
  29. Hrazdina, G., Kreuzaler, F., Hahlbrock, K., and Grisebach, H. (1976) Arch. Biochem. Biophys. 175, 392-399[Medline] [Order article via Infotrieve]
  30. Dunsmuir, P., Bond, D., Lee, K., Gidoni, D., and Townsend, J. (1988) Plant Mol. Biol. Manual C 1, 1-17
  31. Geerts, A., Feltkamp, D., and Rosahl, S. (1994) Plant Physiol. 105, 269-277[Abstract/Free Full Text]
  32. Miersch, O., Knöfel, H. D., Schmidt, J., Kramell, R., and Parthier, B. (1998) Phytochemistry 47, 327-329[CrossRef]
  33. Lu, L., Berkey, K. A., and Casero, R. A. (1996) J. Biol. Chem. 271, 18920-18924[Abstract/Free Full Text]
  34. Coleman, C. S., Huang, H., and Pegg, A. E. (1995) Biochemistry 34, 13423-13430[Medline] [Order article via Infotrieve]
  35. Coleman, C. S., and Pegg, A. E. (1997) J. Biol. Chem. 272, 12164-12169[Abstract/Free Full Text]
  36. Grandmaison, J., Olah, G. M., Van Calsteren, M.-R., and Furlan, V. (1993) Mycorrhiza 3, 155-164


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