p-Coumaroylnoradrenaline, a Novel Plant Metabolite Implicated in Tomato Defense against Pathogens*

The Avr9 peptide elicitor from the fungus Cladosporium fulvum, the bacterial pathogen Pseudomonas syringae pathovar tomato carrying the avirulence gene avrPto (Pst (avrPto)), and the organophosphorous insecticide fenitrothion induce resistance-related responses in tomato lines carrying the Cf-9, Pto, and Fen genes, respectively. These responses were associated with synthesis of p-coumaroyloctopamine and p-coumaroylnoradrenaline, a novel compound for plants. In susceptible near isogenic tomato lines (Cf-0, pto, fen) and wounded tomato leaves, the levels of these compounds were reduced or undetectable. The elevated levels of p-coumaroyloctopamine and p-coumaroylnoradrenaline were accompanied by elevated mRNA levels of genes encoding phenylalanine ammonia lyase, p-coumarate CoA ligase, and hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT), enzymes that are involved in the hydroxycinnamic acid amide biosynthesis. Southern hybridization indicated that THT is encoded by a multigene family in tomato. Four different THT full-length cDNAs were derived by reverse transcriptase-PCR using degenerate primers based on potato and tobacco THT sequences. Transcripts for all four homologs were present in unchallenged tomato leaves, but only tomTHT1-3 was highly expressed following challenge with Pst (avrPto). Furthermore, tomTHT1-3 showed a more substantial and rapid induction in the incompatible interaction than in the compatible interaction. The cDNAs tomTHT1-3, tomTHT7-1, and tomTHT7-8 encoded proteins with a high degree of amino acid sequence homology, although the recombinant proteins had different preferences for octopamine and noradrenaline. The fourth cDNA, tomTHT1-4, directed synthesis of a truncated enzymatically inactive protein due to the presence of a premature stop codon.

Plant defense against microbial attack is associated with activation of plant responses that serve to restrict the growth of the pathogen and/or to eliminate it. Considerable progress has been made in elucidating many of these responses and how they are triggered following recognition of molecules of pathogen origin by the plant (reviewed in Refs. [1][2][3]. It is widely observed that plant resistance to pathogen challenge is correlated with alterations in phenolic metabolism and a rapid increase in the transcriptional activity of genes involved in the phenylpropanoid pathway. A number of products synthesized by this pathway have established roles in disease resistance in different plants; these include the signal molecule salicylic acid, antimicrobial phytoalexins, and lignin that can reinforce the plant cell wall (reviewed in Refs. 4 and 5). The work described in this paper had the initial aim of identifying further metabolites, originating from the phenylpropanoid pathway, that might play roles in disease resistance in tomato. We have compared the synthesis of soluble phenolics in response to both biotic (fungal elicitors, pathogenic bacteria) and abiotic agents (the organophosphorous insecticide fenitrothion) using nearly isogenic tomato lines that differ in their response to these agents. The resistance of tomato (Lycopersicon esculentum L.) to the fungus Cladosporium fulvum (C. fulvum) is conditioned by the presence of Cf resistance gene(s) in the plant together with the cognate avirulence (avr) gene(s) in the pathogen (6). The products of a number of avr genes are peptides that are found in intercellular fluids derived from C. fulvum-infected tomato leaves. These intercellular fluids induce chlorosis and necrosis in tomato in a Cf gene-dependent fashion (7).These responses are associated with increases in the levels of salicylic acid, indicating activation of the phenylpropanoid pathway (8). Tomato lines carrying the Pto gene are resistant to strains of Pseudomonas syringae pathovar tomato that carry the avirulence gene avrPto (Pst (avrPto)) (9). These strains cause a programmed cell death reaction, the hypersensitive response (HR), 1 when infiltrated into leaves. In contrast, tomato lines carrying the recessive pto allele do not show HR and are susceptible to infection by Pst (avrPto). The Fen gene, which is closely linked to Pto and belongs to the Pto gene family of serine threonine protein kinases, confers sensitivity to the organophosphorous insecticide fenthion (10). Treatment of Fen plants with fenthion or its analog fenitrothion results in a confluent necrotic reaction that resembles the HR, whereas fen plants show only sporadic appearance of necrotic spots (10,11). Both the Fen and Pto signaling pathways to cell death require the Prf gene (12); mutant lines carrying nonfunctional alleles (prf) show much lower sensitivity to fenthion and no HR in response to Pst (avrPto). By monitoring the profile of soluble phenolics, we have shown preferential induction of the synthesis of two phenolic compounds in those interactions that are associated with resistance. This is seen with both biotic elicitation and fenitrothion treatment. We identified these two metabolites as p-coumaroyloctopamine (p-CO) and p-coumaroylnoradrenaline (p-CN). Conjugates of hydroxycinnamic acids with tyramine and derivatives of tyramine, such as dopamine, methoxytyramine, and octopamine, are found in a wide range of plants. Synthesis of these amides has been shown previously to be activated by treatment with fungal elicitors, by attempted infection by fungi, viruses, and bacteria, and in some cases by wounding (13)(14)(15)(16)(17)(18)(19). However, as far as we are aware, this is the first description of the occurrence of p-CN in plants and consequently of its association with plant defense. The biosynthesis of p-CO and p-CN occurs by the condensation of the CoA derivative of p-coumaric acid with octopamine and noradrenaline, catalyzed by the enzyme hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110). The enzymes phenylalanine ammonia lyase (PAL; EC 4.3.1.5), and 4-coumarate CoA ligase (4CL; EC 6.2.1.12) are involved in the synthesis of p-coumaroyl-CoA; octopamine and noradrenaline are synthesized in a number of steps from tyrosine. The biosynthetic pathway for p-coumaroyltyramine is shown in Fig. 1. THT has been purified from potato, tobacco, and opium poppy, and the specificity for different acceptors has been established (20 -22). All enzymes have wide substrate specificity, although in the case of tobacco and potato, noradrenaline is the least favored acceptor (no experiments were performed with THT from opium poppy using noradrenaline as acceptor). In potato and tobacco, it has been shown that THT is encoded by a multigene family (23,24). Nevertheless, it is not known whether all genes encode active proteins with similar substrate preferences. So far, three THT cDNAs have been cloned from tobacco, one from potato as well as one from pepper (23)(24)(25). We have taken several approaches to investigate possible mechanisms underlying the enhanced synthesis of p-CO and p-CN during defense reactions in tomato. First, we have examined the transcriptional activity of several genes (PAL, 4CL, and THT) whose products are implicated in p-CO and p-CN biosynthesis. Second, we have used the sequences of the potato and tobacco THT genes to isolate four different tomato THT homologs and have examined the expression of these different THT genes after bacterial challenge. Finally, we have compared the acceptor preferences for the different recombinant THT enzymes. We report that the increased p-CO and p-CN synthesis is associated with the accumulation of transcripts for PAL, 4CL, and THT, although gene-specific analysis indicates that the different THT genes are differentially regulated upon bacterial challenge of plants. We also report that the different THT isoforms in tomato have different acceptor preferences and that the isoform that is most highly expressed upon bacterial challenge has a significantly higher affinity for noradrenaline than either the potato or tobacco enzymes.

EXPERIMENTAL PROCEDURES
Plant Material and Growth Conditions-For experiments with C. fulvum elicitors, the tomato cultivar Moneymaker (Cf-0) and two nearly isogenic lines homozygous for the resistance genes Cf-9 and Cf-2 were used. Plants were grown either to the sixth leaf stage (6 weeks) or to the cotyledon stage (3 weeks) in a growth room in Levington's M3 compost (Levington Horticulture Ltd., Fisons, Ipswich, UK) with a photoperiod of 16 h (light intensity of 100 microeinsteins). Growth room temperatures were 20°C in the light and 16°C in the dark. For the bacterial infection experiments, the tomato cultivar Rio Grande 76R carrying a functional Pto and Fen gene and a nearly isogenic line 76S (pto and fen) were grown to the sixth leaf stage. In addition, the nearly isogenic fast neutron-induced mutant lines cv. Rio Grande 76R prf3, prf9, and prf2 lacking a functional Prf gene were included in the FIG. 1. Biosynthetic pathway of p-coumaroyltyramine. THT catalyzes the synthesis of p-coumaroyltyramine from the thioester pcoumaroyl-CoA and the amine tyramine. The enzymes PAL and 4CL of the phenylpropanoid pathway are involved in the synthesis of p-coumaroyl-CoA. Tyramine is derived from tyrosine by the action of tyrosine decarboxylase (TDC). experiments (12). Plants to be challenged by bacteria or the insecticide fenitrothion were grown under the same light conditions as stated above but at a constant temperature of 22°C.
Fungal, Bacterial, and Chemical Elicitation and Wounding-The source of C. fulvum peptide elicitors Avr2 and Avr9 were intercellular washing fluids originating from tomato plants heavily infected by C. fulvum race 5 (referred to as IFAvr9) (26). For controls, intercellular fluids from healthy, noninfected plants (referred to as IF0) were used. For the time course analyses, the IF preparations were infiltrated with a blunt syringe in the intercellular space of tomato cotyledons previously nicked by a razor blade. For all other experiments, adult leaves were infiltrated.
P. syringae pathovar tomato carrying the avrPto gene (Pst (avrPto)) (27) and Pst (avrPto) ⌬hrp (K-R) (28) were grown overnight in King's B broth (29) containing the respective antibiotics. After centrifugation, the bacteria were suspended in 10 mM MgCl 2 to the appropriate concentration and infiltrated in the leaves as described above. For the chemical elicitation, the fenthion analog, fenitrothion, was used. The insecticide is available in the United Kingdom under the product name Dicofen (kindly provided by PBI Agrochemicals Ltd., Waltham Abbey, UK). Mature tomato leaflets were immersed for 30 s in a solution of 1% (w/v) fenitrothion containing 0.05% L-77-Silwett (Union Carbide). For wounding, plants were subjected to mechanical injury by gently rubbing the leaves with carborundum (320 grit powder; Fisher). The abrasive was then removed by careful rinsing with tap water.
Analysis of Free Phenolics-Extraction and analysis of the phenolics were performed essentially as described previously (30). In short, 3-4 mg of freeze-dried plant material was extracted with 70% methanol and analyzed via reverse phase HPLC using a Prodigy 5 ODS-2 column (25 cm ϫ 4.6 mm; Phenomenex Ltd., Macclesfield, UK). The following binary gradient, with a flow rate of 1 ml min Ϫ1 , was applied for elution: t ϭ 0 min, 10% A; t ϭ 20 min, 16% A; t ϭ 40 min, 50% A; t ϭ 45 min, 75% A; t ϭ 50 min, 95% A; t ϭ 55 min, 10% A). Solvent A was methanol/acetonitrile (1:1, v/v), solvent B was 10 mM ammonium formate, pH 3. Absorbences were measured with a diode array-scanning UV detector (Gilson, Middleton, WI). Quantification was accomplished using the standards derived from the enzyme assays (see "THT Enzyme Assay"). The molecular weight (M r ) and structures of peaks 6 and 8 were determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS). The conditions for LC/ESI MS have been stated before (30).
Test for Antimicrobial Activity-Cultures of Pst (avrPto) were grown overnight at 28°C in a rotary shaker in M9 minimal medium supplemented with 0.4% citrate, 25 mM MgSO 4 , and rifampicin (50 g/ml) (31). The bacterial concentrations were adjusted to 1 ϫ 10 4 colonyforming units (cfu)/ml by dilution into new broth. Samples of p-CN and p-CO derived by HPLC fractionation were lyophilized and redissolved in 35% MeOH to give a stock solution of 1 mM. To exclude inhibitory activity of putative impurities in the volatile HPLC fractions, a blank HPLC run was subjected to the same collection procedure as applied for p-CN and p-CO. The two blank fractions were combined, lyophilized, and redissolved in methanol in the same way. The phenolics and the blank control were added to the diluted bacterial cultures, which were incubated as above for a further 24 h. Bacterial viable counts were then determined by plating serial dilutions onto Kings B agar plates containing rifampicin. The experiment was performed on triplicate cultures and repeated three times.
RNA Isolation and Characterization for Northern Blot Analysis-Total RNA was extracted from frozen leaf material at various time points after inoculation with Pst (avrPto) (10 7 cfu/ml) or from control tissue using the Tripure Isolation Reagent (Roche Applied Science). For Northern blots, RNA samples (10 g of total RNA) were separated on formaldehyde-agarose gels and transferred to Hybond-N nylon membranes (Amersham Biosciences), according to the manufacturer's protocol. The integrity of the RNA was assessed by visualization of ribosomal RNA by ethidium bromide staining. The cDNA probes for PAL (35), THT (23), and 4CL (36) were labeled with 32 P, hybridized, washed, and exposed as described previously (37).
Cloning of the Tomato THT Genes-Two degenerate primer pairs were designed based on the consensus sequence of potato and tobacco THT (23,24): t1f, 5Ј-CATGAATATCATAATTATACTC-3Ј; t2f, 5Ј-GAA-GTTTCICCAACCCC-3Ј; t3r, 5Ј-CCATTITTTGCAGCAATAGAIGCAA-C-3Ј; t4r, 5Ј-CCATACCTAAAITCATCAAAIATTTCAACTCCC-3Ј. Reverse transcriptase-PCR (Titan TM One Tube reverse transcriptase-PCR system; Roche Applied Science) was carried out by using the primer combinations t1f/t3r and t2f/t4r on total RNA extracted from 76R plants 0, 4, 10, and 16 h postinoculation (hpi). The PCR profile was designed according to the manufacturer's instructions with slight alterations (incubation temperature, 55°C; denaturation time, 10 s; annealing temperature, 50°C; 10 ϩ 39 cycles). The amplification products were loaded on 1% agarose gels and separated by electrophoresis. Amplified DNA was extracted from gels and subcloned into the vector pGEM-T (Promega). Ten clones from each primer combination per given time point were sequenced. For the isolation of the tomato THT full-length cDNAs, mRNA was purified by using oligo(dT)-cellulose spin columns (Amersham Biosciences). The starting material consisted of a pool of total RNA samples of various time points after bacterial infection. The 5Ј-and 3Ј-rapid amplification of cDNA ends was carried out with gene-specific primers designed for each of the four candidate clones (Marathon TM cDNA amplification kit; Clontech; details for primer sequences and PCR profile available on request). Finally, full-length clones were amplified using primers containing BamHI (5Ј-end) and HindIII (3Ј-end) restriction sites to facilitate subsequent transfer into an expression vector. Candidate clones were sequenced to check for congruent results of full-length amplification and 5Ј-3Ј-rapid amplification of cDNA ends nucleotide analysis. The sequences of the four cDNAs were analyzed in silico by using the programs BlastP2.2.3 and ProtParamtool (available on the World Wide Web at www.expasy.ch). The alignment of the various sequences was performed applying the clustal method (gap penalty, 30; gap length penalty, 20; weight table, Pam 250).
Real Time PCR Analysis of the Tomato THT Homologs-The tomato cultivars Rio Grande 76R and 76S were infiltrated with Pst (avrPto) at a concentration of 10 7 cfu/ml. Single leaf discs (1 cm diameter) were cut out at 0, 4, 10, 16, and 24 hpi and frozen in liquid nitrogen. RNA was extracted using the RNAeasy plant miniprep kit following the manufacturer's instructions (Qiagen, Hilden, Germany). Subsequently, 2.5 g of cDNA were synthesized by reverse transcription using the Revert Aid First Strand Synthesis Kit as described in the manual (Fermentas, St. Leon-Rot, Germany). Real time PCR was carried out on an iCycler (Bio-Rad) featuring a reaction mixture with Sybr Green as fluorescent dye and 1 l of template cDNA in dilutions of 1:5, 1:10, 1:20, and 1:50 (38); details for primer sequences and PCR profile are available on request. Amplicons were subjected to melting curve analysis (60 -95°C, 0.5°C increments) and digested with HinfI, resulting in sequencespecific restriction patterns to assure amplification specificity. Message levels were determined as octuplicates at different dilutions (duplicates of each dilution; see above). Tomato Ef1␣ (accession number X53043) was used as constitutive standard. The level of THT cDNAs were normalized using the level of Ef1␣ cDNA as described in the ABI user bulletin 2.
Expression of Tomato THT cDNAs in Escherichia coli-The plasmids containing the coding region of the THT clones were digested with BamHI and HindIII, and the insert DNA was ligated into the expression vector pQUE-30 (Qiagen, Hilden, Germany).The construct was transformed into the E. coli strain M15(pREP4). Growth and induction of positive clones were performed as described by the manufacturer. The bacterial cultures were diluted to OD 0.3. After centrifugation of a 250-l aliquot, the pellet formed was resuspended in 25 l of SDSpolyacrylamide gel electrophoresis loading buffer, sonicated, and boiled for 5 min. These samples were spun down, and the supernatants were separated on 10 or 15% SDS-polyacrylamide gels (39), followed by staining with Colloidal Brilliant Blue (Sigma). For determination of THT activity, bacterial pellets were resuspended in 10 mM Tris-HCl, pH 7.5, and were broken by three passages through the French press. After centrifugation to remove cell debris, the supernatant was stored at Ϫ80°C.
THT Enzyme Assay-The substrate p-coumaroyl-CoA was enzymatically synthesized essentially as described by Meng and Campbell (40) using a recombinant tobacco 4CL (36). The reaction mixture was applied to a C18 Varian Bond Elut Extraction Cartridge (Phenomenex), and the CoA-conjugate was selectively eluted with acetonitrile/water (1:3, v/v). The material was lyophilized, and its purity was checked by HPLC. For the THT assay, 50 l of THT enzyme solution (4 g/l) were mixed with 200 l of 10 mM Tris-HCl (pH 7.5), 10 l of p-coumaroyl-CoA (1 mM), and 5 l of tyramine, dopamine, octopamine, and noradrenaline to a final concentration ranging from 0.01 to 0.5 mM. After 10 min at 25°C, the reaction was stopped by the addition of 50 l of 50% (w/v) trichloroacetic acid. The assay mixture was spun down, and 200 l of the supernatant were subjected to reverse phase HPLC analysis on a C18 column, exactly as described earlier for the phenolic analysis. The amine conjugates were identified by retention time and UV spectra. The K m values for the different acceptor amines were calculated from Lineweaver-Burk plots from double measurements at each concentration.

Differential Accumulation of Phenolics in Response to Fungal
Elicitors-Intercellular fluid containing the C. fulvum Avr9 and Avr2 peptides (IFAvr9) was used for infiltration of resistant (Cf-9) and susceptible (Cf-0) tomato plants. At 12-16 hpi, a graying necrosis could be seen in the incompatible interaction ( Fig. 2A, Cf-9), whereas no macroscopic symptoms could be detected for the Cf-0 plants ( Fig. 2A, Cf-0). The production of soluble phenolics during these interactions was monitored by HPLC analysis of extracts from the infiltrated leaf areas. Of 13 detectable compounds, only two (referred to as peaks 6 and 8) showed a reliable alteration over three independent time course experiments (Figs. 2A and 3A). Tomato Cf-9 cotyledons infiltrated with IFAvr9 showed an accumulation of the peak 6 compound starting at 15 hpi and the peak 8 compound starting at 6 hpi. The maximum level of the two substances induced in Cf-9 plants were achieved at 21 hpi; thereafter, the levels declined rapidly. The levels at the maximum values were ϳ20and 80-fold higher, respectively, than in Cf-0 plants. In Cf-9 cotyledons infiltrated with IF0 (containing no Avr peptides), an accumulation of the peak 8 compound was observed beginning at 6 hpi and reaching a maximum value at 9 hpi (Fig. 3A). The maximum level was ϳ10-fold lower than that seen with IFAvr9; thereafter, the level declined slowly. There was no accumulation of the peak 6 compound in this interaction. To show that the induction of the two phenolics was not just specific for interactions involving Cf-9, a comparable experiment using Cf-2 plants was carried out. At 24 h after IFAvr9 inoculation, chlorosis could be seen in the incompatible Cf-2 interaction, correlating with an induction of peaks 6 and 8. The accumulation profiles of these two compounds were comparable with the Cf-9 interaction (peak ratio P8/P6 ϭ ϳ3:1), although the maximum levels were approximately 10 times lower than in the Cf-9 interaction (data not shown). The triggering of the synthesis of the two phenolic compounds in these experiments could be a specific response of tomato to fungal elicitors rather than a broad defense mechanism capable of being activated by a range of pathogens. To test this, we monitored the levels of phenolics in tomato leaves challenged with an avirulent bacterial pathogen and with a derivative of fenthion that triggered programmed cell death in tomato.
Synthesis of Phenolics after Bacterial Challenge-In the tomato cultivar 76R, the Pto locus confers resistance to Pst strains expressing the avirulence gene avrPto (9). Infiltration of Pst (avrPto) into 76R at concentrations of 10 7 cfu/ml lead to an HR, indicated by dry necrosis, at about 16 hpi (Fig. 2B, Pto). Infiltration of the same inoculum in the nearly isogenic-susceptible relative 76S, carrying the pto allele, resulted in watersoaked necrotic lesions (Fig. 2B, pto). Strong induction of peaks 6 and 8 was only seen in the incompatible (HR) interaction with 76R (Fig. 2B). Time course analysis revealed that the peak 8 compound started to accumulate at 4 hpi, and maximum accumulation was reached at 16 hpi. The peak 6 compound started to accumulate at 10 hpi but again showed a maximum accumulation at 16 hpi (Fig. 3B). Maximum levels of peak 8 were 10 times higher than that of peak 6. By contrast, in the compatible interaction with 76S, levels of both compounds were about 30 times lower than the corresponding values with 76R. Control plants infiltrated with water showed no induction of the peak 6 and 8 compounds (data not shown).
The cultivar 76R was also inoculated with the Pst (avrPto) ⌬ hrp (K-R) mutant, which carries a deletion within the hrp gene cluster (28). Genes within this cluster encode components of a Type III secretion system believed to deliver the AvrPto protein to its site of recognition within the plant cell. The deletion mutant strain does not trigger HR and showed no induction of the phenolics in the infiltrated leaf area (Fig.  3B). Pst (avrPto) was also inoculated into the prf2, prf3, and prf9 mutant lines derived from 76R (12). These lines have mutations in the Prf gene, which is involved in the signal transduction pathway leading to HR, and hence are susceptible to Pst (avrPto). All of these lines gave very low levels of peak 6 and peak 8 compounds, comparable with that seen in the susceptible cultivar 76S (data not shown). Overall, these results showed that the greatly enhanced synthesis of the two phenolics in tomato/Pst interactions is dependent on the presence of avrPto, Pto, and the signal transduction component Prf and hence is correlated with the triggering of HR and the expression of resistance.
Synthesis of Phenolics after Chemical Elicitation-The tomato cultivar 76R carries the Fen gene, which is tightly linked to Pto and conditions sensitivity to the organophosphorous insecticide fenthion, leading to a necrotic reaction comparable with the HR (10,11). The near isogenic tomato cultivar 76S (fen) shows a high degree of insensitivity to fenthion. We challenged these plants with the fenthion analog fenitrothion. After 48 h, the 76R plants developed dark, large, necrotic lesions covering the whole leaflet (Fig. 2C, Fen). In contrast, challenge of 76S and the mutant line prf3 derived from 76R resulted only in sporadic appearance of necrotic spots (Fig. 2C, fen). At the given time point, the level of the peak 6 and peak 8 compounds were 10 and 3 times higher in the Fen plants compared with the fen or prf3 plants. Interestingly, after fenitrothion treatment, peak 6 is prevalent within the spectrum of phenolics, whereas with fungal elicitors or bacteria peak 8 is predominant.
Synthesis of Phenolics after Wounding-Tomato leaves were gently rubbed with carborundum and carefully washed to remove the abrasive. After 24 h, the whole leaf area showed dry, dark green, necrotic lesions, which were not accompanied by the induction of the phenolics in question (Fig. 2D). This result established that the synthesis of the peak 6 and peak 8 phenolics was not a general response to tissue necrosis caused by any agent but was specific to the programmed cell death responses associated with disease resistance.
Structural Characterization of the Peak 6 and 8 Compounds-Comparison of the absorbance spectra of peak 6 and 8 ( max 289 and 305 nm) with the UV spectrum of the standard p-coumaroyltyramine (Fig. 4, B and E) showed that they belong to the same family of phenolic amines. The identity of peak 6 was revealed by LC/ESI MS to be p-CN (Fig. 4, A and C). This was indicated by a (M ϩ H) Ϫ peak at m/z 316.2 and fragment ions at m/z 147.0 (p-coumaroyl moiety; fragmentation occurred between C-10 and the nitrogen atom) and (M ϩ H-H 2 O) ϩ at m/z 298.2 (water loss at the carbon skeleton of the noradrenaline residue). Generally, in the amine conjugates with a hydroxy function at C-7Ј, the ESI mass spectra display a prominent (M ϩ H-H 2 O) ϩ. These ions can be used to differentiate between 7Ј-hydroxy compounds and those with hydroxyl substitution on the benzene ring of the amine moiety (23). To our knowledge, this is the first description of this conjugate in plants. The mass spectrometry analysis of peak 8 revealed a 16-Da lower (M ϩ H) Ϫ peak at m/z 300.2, indicating the presence of a compound with one hydroxy function less then p-coumaroylnoradrenaline (Fig. 4, D and F). The fragment ions at m/z 147.1 and 282.1 (water loss at the carbon skeleton of the octopamine residue) verified it to be p-CO. This metabolite has already been identified in cell cultures of Solanum khasianum and S. tuberosum (23,41). In addition to the MS analysis comparison of the retention time values of peaks 6 and 8 with the retention times of synthesized products derived from the THT enzyme, assays using noradrenaline and octopamine as substrates (see "Enzymatic Properties of Recombinant THT Enzymes") gave further Antimicrobial Activity of p-CN against Pst (avrPto)-The phenolic conjugates p-CN and p-CO were purified by HPLC from extracts of challenged leaves and were added to liquid cultures of Pst (avrPto) to various final concentrations. Effects on bacterial growth were monitored after a 24-h culture period. Control cultures received either water or lyophilized HPLC buffer (blank control), which had a small but reproducible growth-promoting activity compared with water (Fig. 5). Preliminary experiments showed that at a range of concentrations up to 100 M, p-CO had no significant effect on bacterial growth or viability. At 100 M, p-CN reduced the growth of Pst (avrPto) 10-fold in comparison with the blank control. At 10 M, the effect was smaller, and a 2-fold reduction was observed. The inhibitory activity of p-CN appeared to be lower when calculated using the water control; 100 M p-CN reduced bacterial growth by 7-fold, and 10 M p-CN had no significant inhibitory effect.
Association of p-CN and p-CO Accumulation with Transcript Alterations of THT, PAL, and 4CL-As outlined in the Introduction, the last step in the biosynthesis of p-CN and p-CO in tomato is catalyzed by the enzyme THT. This protein conjugates p-coumaroyl-CoA (synthesized through the action of PAL and 4CL) and a variety of amines (e.g. tyramine, octopamine, or noradrenaline). We tested for the presence of THT genes in tomato and other plants by performing a botanical garden blot using a potato THT cDNA as probe. Although only very weak signs of hybridization could be observed with the monocot plant barley, the four solanaceous species (potato, pepper, tobacco, and tomato) showed multiple strong hybridization signals (data not shown). This indicated the presence of a multigene family in each of these four species. The transcript levels for THT, PAL, and 4CL were measured in both incompatible and compatible interactions of Pst (avrPto) with tomato cultivars 76R and 76S using the potato THT cDNA and tomato PAL and 4CL cDNAs as probes (Fig. 6). Untreated tomato leaves showed a moderate, constitutive expression of THT, but no transcripts for PAL or 4CL could be detected. In the incompatible interaction, a strong accumulation of THT transcripts in response to Pst (avrPto) could be observed as early as 4 hpi, with high transcript levels being maintained up to 24 hpi. In contrast, transcripts for PAL and 4CL revealed increased levels at 4 hpi but declined slowly within the next 20h (Fig. 6, Pto ϩ Pst (avrPto)). In the compatible interaction, THT transcript levels showed an increase at 4 hpi, although to a lower level than in the incompatible interaction. Subsequently, the THT transcript levels dropped at 10 hpi before a second phase of accumulation at 16 and 24 hpi, although again to a lower level than that seen in the incompatible interaction. Transcripts for PAL and 4CL only accumulated at 16 hpi and were reduced by 24 hpi (Fig. 6, pto ϩ Pst (avrPto)). Infiltration of water into the plants also triggered the accumulation of transcripts for THT at 4 hpi and for PAL at 16 hpi, although no accumulation of transcripts for 4CL was detected at any time point (Fig. 6, water). The patterns of THT and PAL transcript accumulation in compatible interactions with Pst (avrPto) could thus be mimicked by water inoculation alone, whereas the accumulation of 4CL transcripts required the presence of bacteria.
Isolation and Characterization of Tomato THT cDNA Clones-RNA was isolated from the leaves of tomato cultivar 76R responding to Pst (avrPto) and used to isolate THT clones by degenerative reverse transcriptase-PCR followed by 5Ј-3Јrapid amplification of cDNA ends. The degenerate primers were designed on the consensus of THT genes from tobacco (accession number AJ005062) and potato (23). Four different tomato THT clones could be identified. The clones tomTHT1-3 and tomTHT1-4 (accession numbers AY081905 and AY081908, respectively) could be amplified with the primer combination t1f/t3r. The predicted coding regions were 724 or 729 nucleotides, respectively. With the primer pair t2f/t4r, two additional THT genes, tomTHT7-1 and tomTHT7-8, could be isolated with coding regions of 741 and 723 nucleotides, respectively (accession numbers AY081906 and AY081907).
The predicted proteins encoded by the four cDNAs showed a high degree of amino acid sequence similarity (Fig. 7); tomTHT1-3, tomTHT7-1, and tomTHT7-8 all exhibited 93% sequence similarity to each other, and all were 73% similar to tomTHT1-4. The major difference in tomTHT1-4 compared with the other predicted tomato proteins was in the amino acid stretch 98 -122, which ended in a premature stop signal. In terms of amino acid sequence, the THTs from tomato were more closely related to the THT of S. tuberosum than to THT from tobacco. The similarity between sequences of potato and tomTHT1-3/7-1/7-8/1-4 was 95, 89, 93, and 76%, respectively; the similarity between sequences of tobacco and tomTHT1-3/7-1/7-8/1-4 was 86, 83, 84, and 70%, respectively.
Gene-specific Transcript Analysis of the Four Tomato THT cDNA Clones-The accumulation of transcripts for the four tomato THT cDNAs was analyzed by real time PCR in both the incompatible and compatible interaction of Pst (avrPto) with the tomato cultivar 76R and 76S, respectively. In the incompatible interaction, levels of transcripts corresponding to tomTHT1-3 showed a substantial and rapid increase upon in- FIG. 4. ESI MS spectra, absorbance spectra and chemical structure of peak 6 and 8 compounds. The compounds in peak 6 and peak 8 were identified by mass spectrometry as p-coumaroylnoradrenaline (A) and p-coumaroyloctopamine (D), and the structures (C and F) together with UV-absorbance spectra (B and E) are shown. oculation and were 63-fold higher at 4 h after inoculation than in unchallenged plants. This induction was followed by a decline of transcript levels to ϳ50% of the maximum level at 16 hpi (Fig. 8A). Elevated levels of tomTHT1-3 transcripts could also be seen in the compatible interaction. In this case, however, maximum induction occurred at a much later time point (16 hpi) and to a lesser extent (about 16-fold less) than in the incompatible interaction (Fig. 8B). Furthermore, the constitutive levels of tomTHT1-3 transcripts were 6 times lower in susceptible compared with resistant plants. The accumulation profiles of transcripts for tomTHT7-8, tomTHT1-4, and tomTHT7-1 were clearly different from that of tomTHT1-3. Although in unchallenged plants tomTHT7-8, tomTHT1-4, and tomTHT7-1 had approximately the same transcript level as tomTHT1-3, after challenge the levels of transcripts of all three genes declined in both compatible and incompatible interactions.
Enzymatic Properties of Recombinant THT Enzymes-The tomato THTs were expressed in E. coli using the vector pQE30. Analysis of the extracts from the induced cultures by SDSpolyacrylamide gel electrophoresis confirmed the slightly different sizes of tomTHT1-3, tomTHT7-8, and tomTHT7-1 predicted from the translated DNA sequence of the cDNA clones. The expression of tomTHT1-4 could also be induced, but this (truncated) protein was only detectable on a 15% SDS gel (data not shown).
Crude bacterial extracts containing the recombinant tomato THTs were tested for their enzymatic activity and substrate specificity with p-coumaroyl-CoA as acyl donor and various amines as possible acceptors (Table I). Extracts from bacteria carrying the empty expression vector pQE30 or expressing the truncated protein tomTHT1-4 showed no activity with any of the acceptor substrates. The other three recombinant tomTHTs (1-3, 7-1, and 7-8) were all enzymatically active. All three exhibited the highest affinity for tyramine, although the K m value of tomTHT7-8 was about 10-fold higher than that of tomTHT1-3 and tomTHT7-1. For tomTHT1-3 and tomTHT7-1, the affinities for the different acceptor decreased in the order tyramine Ͼ octopamine Ͼ dopamine Ͼ noradrenaline and tyramine Ͼ dopamine Ͼ octopamine Ͼ noradrenaline, respectively. In contrast, the affinity of tomTHT7-8 for noradrenaline was higher than for both dopamine and octopamine.

DISCUSSION
Conjugates of hydroxycinnamic acids with tyramine or with derivatives of tyramine, such as dopamine, methoxytyramine, and octopamine, are found in a wide range of plants including onion, potato, pepper, and tobacco (13)(14)(15)(16)37). It has been shown that their synthesis is activated in response to fungal elicitors as well as in response to attempted infection by fungi, viruses, and bacteria and, in some cases, wounding. Increased synthesis of feruloyl-and p-coumaroyl-derivatives of tyramine (FT and p-CT) and octopamine (FO and p-CO) is associated with Phytophthora infestans elicitor stimulation of potato cell suspension cultures (23,44). Increased synthesis of FT and CT is also associated with the resistance of potato plants to P. infestans (15) and with both the nonhost-and gene-for-genedetermined resistance reactions of pepper to Xanthomonas campestris (37). Biosynthesis of FT is induced in tobacco by tobacco mosaic virus infection (14). In tomato, potato, and tobacco, accumulation of FT occurs after wounding with a hemostat or a pressure-applying tool, although in wounded pepper and nightshade no FT can be detected (19). Here we have shown that markedly enhanced synthesis of p-CO and p-CN (a novel compound for plants) is specifically associated with resistance reactions triggered in tomato by a range of agents (fungal elicitors, avirulent bacteria, and the chemical fenitrothion). However, wounding with the abrasive carborundum causing necrotic cell death did not lead to the induction of these two compounds.
Two major roles for hydroxycinnamoyl tyramines in plant defense have been proposed. They can be incorporated into the plant cell wall to strengthen it against microbial degradation, or potentially they can act as direct antimicrobial agents. We have not addressed the issue of incorporation of p-CN and p-CO into the plant cell wall, but it could account for the reduction of the levels of soluble p-CO and p-CN at later time points after treatment with C. fulvum elicitors and Pst (avrPto). There are only a few reports of antimicrobial activity of hydroxycinnamoyl tyramines. Grandmaison et al. (42) showed that FT induced hyphal branching but reduced total growth of mycorhizal fungi. Newman et al. (37) demonstrated antibacterial activity of both FT and CT against X. campestris. In contrast, McLusky et al. (16) failed to detect any antimicrobial activity of hydroxycinnamoyl tyramines from onion induced by attempted Botrytis infection against Botrytis spp. We have shown that p-CN has a relatively weak activity against Pst (avrPto) (a 10-fold growth reduction at 100 M; a 2-fold growth reduction at 10 M). The maximum value of p-CN accumulating in resistant tomato leaves after challenge with Pst (avrPto) was 21 nmol/100 mg dry weight leaf tissue (Fig. 3B). This is equivalent to a concentration of 24 M p-CN in the tomato leaf, assuming the compound was distributed throughout the cellular water. This concentration lies within the range in which p-CN has antimicrobial activity against Pst (avrPto). In addition, as discussed by Newman et al. (37), the localization of p-CN to the apoplast could increase its effective concentration in the immediate bacterial environment, so that we do not exclude the possibility that p-CN contributes to resistance through its antimicrobial action. It is also possible that hydroxycinnamoyl conjugates including p-CN and p-CO have further roles in plant-pathogen interactions that are as yet undefined but which could include activity as signal molecules.
The increased synthesis and accumulation of p-CN and p-CO in tomato during incompatible interactions with Pst (avrPto) is associated with elevated mRNA levels of the genes encoding PAL, 4CL, and THT at 4 h after bacterial inoculation. In compatible interactions, by contrast, the amount of PAL and 4CL transcripts increased at a later time point (at 16 h after  bacterial inoculation), although there was still an increase in THT mRNA levels at 4 hpi. The induction of PAL and THT seen in compatible interactions may be a response to the inoculation procedure, since inoculation with water produced very similar effects. Induction of 4CL, however, was specific to bacterial interactions and was not induced by water. The induction of THT alone at 4 h in compatible interactions and after water inoculation was not sufficient to allow synthesis of p-CO and p-CN. Elevated THT mRNA level accompanies the elicitorinduced accumulation of p-CO, p-CT, FT, and FO in potato suspension culture (23). Similar differential induction of transcript levels of PAL and THT has been described in pepper in response to avirulent and virulent strains of X. campestris and is associated with differential synthesis of FT and p-CT (43).
Southern analysis indicated that, as in potato and tobacco, THT in tomato is encoded by a multigene family. Four different cDNAs were cloned. One of these cDNAs, tomTHT1-4, encoded a truncated inactive THT because of the presence of a premature stop codon. Examination of the tomato expressed sequence tag (EST) data base (available on the World Wide Web at tigrblast.tigr.org/tgi/) indicates the presence of two further highly homologous THT isoforms, defined by ESTs TC98928 and TC98925 (92 and 74% homology to tomTHT1-3 on the DNA level). We did not detect these two homologs, most likely due to the design of the degenerate primers and subsequent preference of the other clones. The three cDNAs tomTHT1-3, tomTHT7-1, and tomTHT7-8 encode proteins that are overall highly similar to each other but show divergence particularly in the N terminus and extreme C terminus. The tobacco THT isoforms defined through cDNA cloning are much less divergent (24); two predicted proteins are identical, and a third is altered in only two amino acid positions. It is not known whether these alterations give rise to alterations in substrate preference. The two tobacco cDNAs encoding proteins with identical amino acid sequence differ in their 3Ј-and 5Ј-untranslated region sequences, which may reflect differences in their regulation.
Using real time PCR we have shown differential expression of the genes encoding the tomato THT homologs during incompatible and compatible interactions with Pst (avrPto). The transcript analysis suggests that transcripts for all four homologs are present in unchallenged tomato leaves but that only tomTHT1-3 seems to be highly expressed following bacterial challenge in whole leaf tissue. Furthermore, tomTHT1-3 shows a more substantial and rapid induction in resistant compared with susceptible plants. These results reflect those of the Northern blot analysis, where all THT transcripts would be measured together. Differential expression of the tomato THT family members is also indicated by their EST distribution profile (available on the World Wide Web at tigrblast.tigr.org/ tgi/). All of the THT gene family members show alterations in their expression patterns in different plant tissues (roots, shoots, or mature green fruit) under a variety of developmental stages or in leaves under elicitation conditions. Transcripts for tomTHT1-3 (EST TC98926) are the most abundant THT gene transcripts among ESTs derived from bacterially challenged leaf tissue as well as callus tissue. The expression level is comparable with "housekeeping" genes such as those encoding ␣-tubulin or glyceraldehyde-3-phosphate dehydrogenase.
By comparison with mammalian spermine/spermidine Nacetyl transferases (SSATs), Schmidt et al. (23) have proposed that the amino acid sequence RKLGMGS in potato THT (residues 176 -182) is responsible for acyl-CoA binding and that the residues underlined within this sequence are essential for catalytic activity. The three active tomato THTs have the amino acid sequence RKLGMG(S/K) at the homologous position, with conservation of all three essential residues. Tyramine is the preferred substrate for all five THT proteins discussed. However, all three tomato isoforms have a lower K m for noradrenaline than either the potato and tobacco enzymes (measured with feruloyl-CoA as acyl donor) (20,23). Other differences between the tomato isoforms and potato and tobacco enzymes are seen in their relative affinities for dopamine and octopamine. For THT1-3, the affinity for octopamine is higher than for dopamine. For THT7-1 and THT7-8, the affinity for octopamine is lower than for dopamine, whereas for the tobacco and potato enzymes the reverse is true and the affinity for dopamine is at least 10 times lower than for octopamine. In addition, THT7-8 seems to have apart from tyramine the highest affinity for noradrenaline.
It is evident from Fig. 2A that although p-CO and p-CN synthesis is induced in tomato by fungal elicitor, bacterial inoculation, and fenitrothion treatment, the relative levels of the two products differ in each case. Other hydroxycinnamic amides such as p-CT were also detected at lower concentrations; however, no reproducibly significant difference between resistant and susceptible responses were observed. The pattern of the hydroxycinnamoyl conjugates seen will depend on the relative level and substrate specificity of the THT isoforms present in the tissue but presumably more importantly on the availability of the different acceptors and of the acyl donors. Differential induction of THT isoforms with different specificity in tomato represents one possible mechanism to alter the pattern of products in response to different stimuli and perhaps also in plant development. The mechanisms that determine the relative availability of tyramine and its hydroxylation derivatives octopamine and noradrenaline under different elicitation conditions are not known. The work described here provides correlative evidence for a role for p-CN and p-CO in tomato resistance against microbial attack. Future studies involving overexpression and gene silencing of the THT family members will increase our insight into the interrelation of the biosyn- thesis of the phenolic amine conjugates and the outcome of plant-microbe interactions.