Tyramine Hydroxycinnamoyl Transferase Research Articles

Engineering Saccharomyces cerevisiae for production of the capsaicinoid nonivamide

BackgroundCapsaicinoids are produced by plants in the Capsicum genus and are the main reason for the pungency of chili pepper fruits. They are strong agonists of TRPV1 (the transient receptor potential cation channel subfamily V member 1) and used as active ingredients in pharmaceuticals for the treatment of pain. The use of bioengineered microorganisms in a fermentation process may be an efficient route for their preparation, as well as for the discovery of (bio-)synthetic capsaicinoids with improved or novel bioactivities.ResultsSaccharomyces cerevisiae was engineered to over-express a selection of amide-forming N-acyltransferase and CoA-ligase enzyme cascades using a combinatorial gene assembly method, and was screened for nonivamide production from supplemented vanillylamine and nonanoic acid. Data from this work demonstrate that Tyramine N-hydroxycinnamoyl transferase from Capsicum annuum (CaAT) was most efficient for nonivamide formation in yeast, outcompeting the other candidates including AT3 (Pun1) from Capsicum spp. The CoA-ligase partner with highest activity from the ones evaluated here were from Petunia hybrida (PhCL) and Spingomonas sp. Ibu-2 (IpfF). A yeast strain expressing CaAT and IpfF produced 10.6 mg L−1 nonivamide in a controlled bioreactor setup, demonstrating nonivamide biosynthesis by S. cerevisiae for the first time.ConclusionsBaker’s yeast was engineered for production of nonivamide as a model capsaicinoid, by expressing N-acyltransferases and CoA-ligases of plant and bacterial origin. The constructed yeast platform holds potential for in vivo biocatalytic formation of capsaicinoids and could be a useful tool for the discovery of novel drugs.

Hydroxycinnamic acid functional ingredients and their biosynthetic genes in tubers of Solanum tuberosum Group Phureja

AbstractPotato is an ideal candidate for the delivery of functional ingredients due to its high worldwide consumption. The metabolites in cooked tubers of eight diploid potato genotypes from Colombia were explored. Potato tubers were harvested, cooked，lyophilized, and then stored at −80°C. Metabolites were extracted from flesh samples and analyzed using liquid chromatography and high-resolution mass spectrometry. A total of 294 metabolites were putatively identified, of which 87 metabolites were associated with health-benefiting roles for humans, such as anticancer and anti-inflammatory properties. Two metabolites, chlorogenic acid and N-Feruloyltyramine were detected in high abundance and were mapped on to the potato metabolic pathways to predict the related biosynthetic enzymes: hydroxycinnamoyl-CoA quinate transferase (HQT) and tyramine hydroxycinnamoyl transferase (THT), respectively. The coding genes of these enzymes identified nonsynonymous single-nucleotide polymorphisms (nsSNPs) in AC09, AC64, and...

Transcription factor StWRKY1 regulates phenylpropanoid metabolites conferring late blight resistance in potato.

Quantitative resistance is polygenically controlled and durable, but the underlying molecular and biochemical mechanisms are poorly understood. Secondary cell wall thickening is a critical process in quantitative resistance, regulated by transcriptional networks. This paper provides compelling evidence on the functionality of StWRKY1 transcription factor, in a compatible interaction of potato-Phytophthora infestans, to extend our knowledge on the regulation of the metabolic pathway genes leading to strengthening the secondary cell wall. A metabolomics approach was used to identify resistance-related metabolites belonging to the phenylpropanoid pathway and their biosynthetic genes regulated by StWRKY1. The StWRKY1 gene in resistant potato was silenced to decipher its role in the regulation of phenylpropanoid pathway genes to strengthen the secondary cell wall. Sequencing of the promoter region of StWRKY1 in susceptible genotypes revealed the absence of heat shock elements (HSEs). Simultaneous induction of both the heat shock protein (sHSP17.8) and StWRKY1 following pathogen invasion enables functioning of the latter to interact with the HSE present in the resistant StWRKY1 promoter region. EMSA and luciferase transient expression assays further revealed direct binding of StWRKY1 to promoters of hydroxycinnamic acid amide (HCAA) biosynthetic genes encoding 4-coumarate:CoA ligase and tyramine hydroxycinnamoyl transferase. Silencing of the StWRKY1 gene was associated with signs of reduced late blight resistance by significantly increasing the pathogen biomass and decreasing the abundance of HCAAs. This study provides convincing evidence on the role of StWRKY1 in the regulation of downstream genes to biosynthesize HCAAs, which are deposited to reinforce secondary cell walls.

Metabolomics deciphers quantitative resistance mechanisms in diploid potato clones against late blight.

Resistance to late blight in potato is either qualitative or quantitative in nature. The quantitative resistance is durable, but the molecular and biochemical mechanisms underlying quantitative resistance are poorly understood, and are not efficiently utilised in potato breeding. A non-targeted metabolomics, using high resolution hybrid mass spectrometry, was applied to decipher the mechanisms of resistance in the advanced breeding diploid potato genotypes (Solanum tuberosum L. Group Phureja), with valuable sources of genetic diversity. The metabolomics profiles of resistant genotypes (AC04 and AC09) were compared with a susceptible commercial genotype (Criolla Colombia), following Phytophthora infestans or mock-inoculation, to identify the resistance related (RR) metabolites. Metabolites belonging to phenylpropanoids, flavonoid and alkaloid chemical groups were highly induced in resistant genotypes relative to susceptible. Concurrently, the biosynthetic genes, tyrosine decarboxylase (TyDC) and tyramine hydroxycinnamoyl transferase (THT), involved in the biosynthesis of hydroxycinnamic acid amides (HCAAs), and chalcone synthase (CHS) and flavonol synthase (FLS), involved in flavonoid biosynthesis, were also upregulated, as confirmed by quantitative real-time PCR. Probable genes coding for these enzymes were sequenced and nonsynonymous single-nucleotide polymorphisms (nsSNPs) were identified. The resistance to late blight observed in this study was mainly associated with cell wall thickening due to deposition of HCAAs, flavonoids and alkaloids.

Transgenic tomato plants overexpressing tyramine N-hydroxycinnamoyltransferase exhibit elevated hydroxycinnamic acid amide levels and enhanced resistance to Pseudomonas syringae.

Hydroxycinnamic acid amides (HCAA) are secondary metabolites involved in plant development and defense that have been widely reported throughout the plant kingdom. These phenolics show antioxidant, antiviral, antibacterial, and antifungal activities. Hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase (THT) is the key enzyme in HCAA synthesis and is induced in response to pathogen infection, wounding, or elicitor treatments, preceding HCAA accumulation. We have engineered transgenic tomato plants overexpressing tomato THT. These plants displayed an enhanced THT gene expression in leaves as compared with wild type (WT) plants. Consequently, leaves of THT-overexpressing plants showed a higher constitutive accumulation of the amide coumaroyltyramine (CT). Similar results were found in flowers and fruits. Moreover, feruloyltyramine (FT) also accumulated in these tissues, being present at higher levels in transgenic plants. Accumulation of CT, FT and octopamine, and noradrenaline HCAA in response to Pseudomonas syringae pv. tomato infection was higher in transgenic plants than in the WT plants. Transgenic plants showed an enhanced resistance to the bacterial infection. In addition, this HCAA accumulation was accompanied by an increase in salicylic acid levels and pathogenesis-related gene induction. Taken together, these results suggest that HCAA may play an important role in the defense of tomato plants against P. syringae infection.

Quantitative resistance in potato leaves to late blight associated with induced hydroxycinnamic acid amides

Late blight is a serious economic threat to potato crop, sometimes leading to complete crop loss. The resistance in potato to late blight can be qualitative or quantitative in nature. Qualitative resistance is not durable. Though quantitative resistance is durable, the breeding is challenging due to polygenic inheritance. Several quantitative trait loci (QTLs) have been identified, but the mechanisms of resistance are largely unknown. A nontargeted metabolomics approach was used to identify resistance-related (RR) metabolites in a resistant genotype (F06025), as compared to a susceptible (Shepody) genotype, mock- or pathogen-inoculated. The RR metabolites, which had high fold change in abundance, mainly belonged to phenylpropanoid, flavonoid, fatty acid, and alkaloid chemical groups. The most important phenylpropanoids identified were hydroxycinnamic acid amides, the polyaromatic domain of suberin that is known to be associated with cell wall reinforcement. These metabolites were mapped on to the potato metabolic pathways, and the candidate enzymes and their coding genes were identified. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay revealed a higher upregulation of 4-coumarate: CoA ligase (4-CL), tyrosine decarboxylase (TyDC), and tyramine hydroxycinnamoyl transferase (THT) in the pathogen-inoculated resistant genotype than in susceptible. These genes were sequenced in both resistant and susceptible genotypes, and nonsynonymous single-nucleotide polymorphisms (nsSNPs) were found. The application of these genes in potato resistance improvement, following validation, is discussed.

Identification of Late Blight Resistance-Related Metabolites and Genes in Potato through Nontargeted Metabolomics

Late blight of potato (Solanum tuberosum) caused by Phytophthora infestans significantly reduces the productivity of potato around the world. Resistance to late blight in potato is either qualitative or quantitative. Qualitative resistance governed by race-specific single R genes is well characterized and gives complete resistance, but is not durable. Quantitative resistance governed by polygenes gives partial resistance, but is durable in nature. However, the quantitative resistance mechanisms are poorly studied and are not efficiently exploited in potato breeding. A nontargeted metabolic profiling of resistant (F06037) and susceptible (Shepody) potato cultivars, using high-resolution liquid chromatography–mass spectrometry, was applied to elucidate the quantitative resistance mechanisms against P. infestans (US-8 genotype). The hydroxycinnamic acid amides (HCAAs) of the shunt phenylpropanoid pathway were highly induced following pathogen inoculation in F06037. In parallel, the transcript abundances of genes that catalyze the biosynthesis of these metabolites, such as 4-coumarate:CoA ligase, tyrosine decarboxylase, ornithine decarboxylase, tyramine hydroxycinnamoyl transferase, and putrescine hydroxycinnamoyl transferase, were also higher in the resistant genotype. Sequencing of the coding genes of these enzymes revealed single-nucleotide polymorphisms between resistant and susceptible genotypes, and the amino acid changes caused missense mutations altering protein functions. HCAAs deposited at host cell walls inhibit pathogen colonization, thus reducing lesion expansion. In addition, these also act as phytoalexins, leading to the reduced biomass of the pathogen. Following validation, the HCAAs can be used as biomarker metabolites for late blight resistance. The putative candidate genes can be either used to develop allele-specific markers for marker-assisted breeding programs or suitably stacked into elite cultivars through cisgenic approaches, following validation.

Production of plant-specific tyramine derivatives by dual expression of tyramine N-hydroxycinnamoyltransferase and 4-coumarate:coenzyme A ligase in Escherichia coli

Plant-specific bioactive compounds including feruloyltyramine (FT), 4-coumaroyltyramine (CT), and caffeoyltyramine (CaT) were simultaneously produced in Escherichia coli by heterologous expression of two biosynthetic genes encoding 4-coumarate:coenzyme A ligase and tyramine N-hydroxycinnamoyltransferase (THT) cloned from Arabidopsis thaliana and pepper, respectively. Simultaneous supplementation of substrates to the recombinant E. coli resulted in the secretion of multiple tyramine derivatives into the medium at high yield: CT (189 mg l(-1)), FT (135 mg l(-1)), CaT (40 mg l(-1)). In addition, the recombinant E. coli also produced, albeit at low concentration, a range of dopamine derivatives such as feruloyldopamine due to THT's ability to accept dopamine as a substrate.

Endosperm-specific expression of tyramine N-hydroxycinnamoyltransferase and tyrosine decarboxylase from a single self-processing polypeptide produces high levels of tyramine derivatives in rice seeds

The plant-specific tyramine derivatives, feruloyltyramine (FT) and 4-coumaroyltyramine (CT), represent bioactive compounds found at low levels in many plant species. We generated transgenic rice seeds that produce high levels of CT (14 microg g(-1) seeds) and FT (2.7 microg g(-1) seeds) through the dual expression of tyramine N-hydroxycinnamoyltransferase and tyrosine decarboxylase, using the self-processing foot-and-mouth disease virus 2A sequence and the endosperm-specific prolamin promoter.

Induction of p-Coumaroyldopamine and Feruloyldopamine, Two Novel Metabolites, in Tomato by the Bacterial Pathogen Pseudomonas syringae

Inoculation of tomato plants (Solanum lycopersicum cv. Rutgers) with Pseudomonas syringae pv. tomato led to the production of a hypersensitive-like response in this pathovar of tomato. Accumulation of hydroxycinnamic acid amides (HCAA) of tyramine (p-coumaroyltyramine and feruloyltyramine) and dopamine (p-coumaroyldopamine and feruloyldopamine) was detected after bacterial infection. Two of them, p-coumaroyldopamine and feruloyldopamine, are described for the first time. The accumulation of HCAA was preceded by an increment of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase (THT) gene expression. HCAA also accumulated in transgenic NahG tomato plants overexpressing a bacterial salicylic hydroxylase. However, treatment of plants with the ethylene biosynthesis inhibitor, aminoethoxyvinilglycine, led to a reduction in the accumulation of THT transcripts and HCAA. Together, the results suggest that pathogen-induced induction of ethylene is essential for HCAA synthesis, whereas salicylic acid is not required for this response. In addition, notable antibacterial and antioxidant activities were found for the new HCAA, thus indicating that they could play a role in the defense of tomato plants against bacterial infection.

Enhanced synthesis of feruloyltyramine and 4-coumaroyltyramine is associated with tyramine availability in transgenic rice expressing pepper tyramine N-hydroxycinnamoyltransferase

Tyramine N-hydroxycinnamoyltransferase (THT) is a 28-kDa soluble protein that catalyzes the condensation of hydroxycinnamates and tyramine via the thioesters. The representative products synthesized by THT include feruloyltyramine (FT) and 4-coumaroyltyramine (CT). The THT gene of Capsicum annuum was introduced into the rice genome using Agrobacterium-mediated transformation. Eight independent transgenic rice plants were selected and characterized. The transgenic lines had a single- or two-copy insertion of the THT transgene. Abundant THT mRNA with high THT enzyme activity was detected in transgenic leaves, but not in wild-type leaves. In young leaves, the level of CT + FT was very low, but differed significantly between transgenics and the wild type. However, in old and senesced leaves, a high level of CT + FT was detected in both transgenics and the wild type, at 32 times higher in the best transgenic line than in the wild type. Tyramine applied exogenously to roots was directly coupled with the increased synthesis of CT + FT in young leaves in a dose-dependent manner. Our data suggest that CT and FT synthesis is developmentally regulated and closely associated with the presence of tyramine substrate in rice plants.

Systemic Potato virus X infection induces defence gene expression and accumulation of β-phenylethylamine-alkaloids in potato.

A better understanding of defence responses elicited during compatible plant-virus interactions is a current goal in plant pathology. We analysed defence responses during infection of Solanum tuberosum L. cv. Desiree with Potato virus X (PVX) at the transcript and metabolite level. A mostly unchanged primary metabolism reflects the compatible nature of this plant-virus interaction. Salicylic acid biosynthesis and expression of several defence genes including PR-1 and glutathione-S-transferase, which are involved in ethylene and reactive oxygen species dependent signalling, were highly up-regulated in upper-uninoculated (systemic) leaves of PVX-infected potato plants compared with mock-inoculated controls. Moreover, the β-phenylethylamine-alkaloids tyramine, octopamine, dopamine and norepinephrine were highly induced upon infection. β-phenylethylamine-alkaloids can contribute to active plant defence responses by forming hydroxycinnamic acid amides (HCAA), which are thought to increase cell wall stability by extracellular peroxidative polymerisation. Expression of tyramine-hydroxycinnamoyl transferase (THT) and apoplastic peroxidase (POD) was highly induced upon PVX infection in systemic leaves, which suggests synthesis and extracellular polymerisation of HCAA. Since cell-wall-bound ion concentrations could contribute to this process, we measured cell-wall-bound and total ion concentrations in PVX-infected and mock-inoculated leaves. The observed metabolic and transcriptional changes might represent a systemic acquired resistance response against subsequent pathogen challenge.

Functional Analysis of the Amine Substrate Specificity Domain of Pepper Tyramine and Serotonin N-Hydroxycinnamoyltransferases

Pepper (Capsicum annuum) serotonin N-hydroxycinnamoyltransferase (SHT) catalyzes the synthesis of N-hydroxycinnamic acid amides of serotonin, including feruloylserotonin and p-coumaroylserotonin. To elucidate the domain or the key amino acid that determines the amine substrate specificity, we isolated a tyramine N-hydroxycinnamoyltransferase (THT) gene from pepper. Purified recombinant THT protein catalyzed the synthesis of N-hydroxycinnamic acid amides of tyramine, including feruloyltyramine and p-coumaroyltyramine, but did not accept serotonin as a substrate. Both the SHT and THT mRNAs were found to be expressed constitutively in all pepper organs. Pepper SHT and THT, which have primary sequences that are 78% identical, were used as models to investigate the structural determinants responsible for their distinct substrate specificities and other enzymatic properties. A series of chimeric genes was constructed by reciprocal exchange of DNA segments between the SHT and THT cDNAs. Functional characterization of the recombinant chimeric proteins revealed that the amino acid residues 129 to 165 of SHT and the corresponding residues 125 to 160 in THT are critical structural determinants for amine substrate specificity. Several amino acids are strongly implicated in the determination of amine substrate specificity, in which glycine-158 is involved in catalysis and amine substrate binding and tyrosine-149 plays a pivotal role in controlling amine substrate specificity between serotonin and tyramine in SHT. Furthermore, the indisputable role of tyrosine is corroborated by the THT-F145Y mutant that uses serotonin as the acyl acceptor. The results from the chimeras and the kinetic measurements will direct the creation of additional novel N-hydroxycinnamoyltransferases from the various N-hydroxycinnamoyltransferases found in nature.

Elevated tyrosine decarboxylase and tyramine hydroxycinnamoyltransferase levels increase wound-induced tyramine-derived hydroxycinnamic acid amide accumulation in transgenic tobacco leaves

Feruloyltyramine (FT) and 4-coumaroyltyramine (4CT) participate in the defense of plants against pathogens through their extracellular peroxidative polymerization, which is thought to reduce cell wall digestibility. Hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) and tyrosine decarboxylase (TYDC; EC 4.1.1.25) are purported to play key roles in the stress-induced regulation of tyramine-derived hydroxycinnamic acid amide (HCAAT) metabolism. Transgenic tobacco (Nicotiana tabacum cv. Xanthi) was engineered to constitutively express tobacco THT. A T1 plant over-expressing THT was crossbred with T1 tobacco expressing opium poppy TYDC2, to produce a T2 line with elevated THT and TYDC activities compared with wild type plants. The effects of an independent increase in TYDC or THT activity, or a dual increase in both TYDC and THT on the cellular pools of HCAAT pathway intermediates and the accumulation of soluble and cell wall-bound FT and 4CT were examined. Increased TYDC activity resulted in a larger cellular pool of tyramine and lower levels of L-phenylalanine in transgenic leaves. In contrast, elevated THT activity reduced tyramine levels. HCAAT levels were low in healthy leaves, but were induced in response to wounding and accumulated around wound sites. Similarly, endogenous THT and TYDC activities were wound-induced. The rate of wound-induced HCAAT accumulation was highest in transgenic plants with elevated THT and TYDC activities showing that both enzymes exert control over the flux of intermediates involved in HCAAT biosynthesis under some conditions.

Elicitation of primary and secondary metabolism during defense in the potato

Exposure of potato tuber tissues to Phytophthora infestans, the late blight pathogen, elicits multiple defense responses, including an oxidative burst, accumulation of phenylpropanoid compounds, and sesquiterpenoid phytoalexins (lubimin and rishitin), which are accompanied by de novo synthesis of the enzymes that produce them. These reactions are also induced in potato tubers treated with a crude elicitor (hyphal wall components, or HWC) prepared from the mycelia of the pathogen. Differential hybridization was used to identify elicitor-responsive cDNAs from potato tubers. Database searches revealed that these genes appear to encode tyramine hydroxycinnamoyl transferase, arginine decarboxylase, glucose 6-phosphate dehydrogenase, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, NADP-dependent malic enzyme, pyruvate decarboxylase, and other potential defense components. RNA gel blot analyses indicated that the accumulation of transcripts related to both secondary and primary metabolism was induced by the HWC elicitor, arachidonic acid or salicylic acid. This suggests that the signals from the pathogen activate not only secondary metabolism but also the primary metabolism that supports the rapid expression of defense responses in potato tubers.

Induction of hydroxycinnamoyl-tyramine conjugates in pepper by Xanthomonas campestris, a plant defense response activated by hrp gene-dependent and hrp gene-independent mechanisms.

Inoculation of pepper leaves, Capsicum annuum cv. Early Calwonder ECW 10R, with strains of Xanthomonas campestris led to an accumulation of the phenolic conjugates feruloyltyramine (FT) and p-coumaroyltyramine (CT) 24 h postinoculation in nonhost- and gene-for-gene-determined incompatible interactions with X. campestris pv. campestris and X. campestris pv. vesicatoria, respectively. In contrast, neither compound was detected in compatible interactions with X. campestris pv. vesicatoria. The accumulation of FT and CT was preceded by an increase in the extractable activity of tyrosine decarboxylase as well as increases in the transcription of genes encoding phenylalanine ammonia-lyase and tyramine hydroxycinnamoyl transferase. No such changes were detected in compatible interactions. Very rapid accumulation of FT and CT occurred (4 h postinoculation) in pepper in response to a X. campestris pv. campestris mutant carrying a deletion of the hrp gene cluster. In contrast, hrp mutants of X. campestris pv. vesicatoria failed to elicit the production of FT and CT. These observations suggest the existence of hrp gene-dependent and -independent activation mechanisms of a defense response involving hydroxycinnamoyltyramines.

Bacterial Lipopolysaccharides And Plant—pathogen Interactions

Lipopolysaccharides are amphipathic molecules forming the outermost layer of the cell surface of Gram-negative bacteria. They are essential for protecting the cell from hostile environments and, in the case of pathogens, they play a direct role in interactions with eukaryotic host cells. Mutants with altered lipopolysaccharide structure have been obtained with several plant pathogenic bacteria; such mutants generally show reduced virulence. Purified lipopolysaccharide has several effects on plants, notably suppression of the hypersensitive response to subsequently inoculated avirulent pathogens. The suppression is strictly localized and is observed within a time ‘window’ of, typically, 10–30 h. Although infiltration of lipopolysaccharide into leaves produces no macroscopic symptoms, characteristic changes in plant gene expression can be observed. One effect is to sensitize the plant tissue to subsequent bacterial inoculation so that the sensitized tissue responds more rapidly and intensely, giving partial inhibition of bacterial growth. The synthesis of antimicrobial hydroxycinnamoyl tyramine conjugates is one facet of the process which provides an excellent biochemical model for analysing the phenomenon. Lipopolysaccharide induces the synthesis of two enzymes involved in conjugate production (tyrosine decarboxylase and tyramine-hydroxycinnamoyl transferase), but the conjugates themselves are not produced until bacteria are subsequently inoculated. Using this and other examples we discuss the mechanisms of lipopolysaccharide action on plants in the context of plant disease.

Induction of N-hydroxycinnamoyltyramine synthesis and tyramine N-hydroxycinnamoyltransferase (THT) activity by wounding in maize leaves.

Both N-p-coumaroyl- and N-feruloyltyramine accumulated in response to wounding in leaf segments of maize. The amount of N-hydroxycinnamoyltyramines started to increase 3-6 h after wounding and peaked at 12 h. Thereafter, the amount of N-p-coumaroyltyramine decreased rapidly, while the N-feruloyltyramine content remained at a high level. The accumulation of N-hydroxycinnamoyltyramines was accompanied by an increase in the tyramine N-hydroxycinnamoyltransferase (THT) activity. This increase was initially detected 3 h after wounding and reached a maximum at 36 h, the level of activity being 40 and 11 times that in the leaves before wounding and in the control leaves, respectively. Partial purification of THT from wounded leaves by (NH4)2SO4 precipitation and subsequent two steps of anion-exchange chromatography resulted in a 12.5-fold increase in specific activity. Kinetic studies with this partially purified enzyme revealed that the best substrates were tyramine and feruloyl-CoA, although tryptamine and sinapoyl-CoA also efficiently served as substrates. The apparent native molecular weight of the enzyme was determined by gel filtration as 40 kDa.

Temporal Correlation of Tyramine Metabolism with Alkaloid and Amide Biosynthesis in Elicited Opium Poppy Cell Cultures : In Honour of Professor G. H. Neil Towers 75th Birthday

Aromatic amines are involved in the biosynthesis of many plant secondary metabolites, including benzylisoquinoline alkaloids and hydroxycinnamic acid amides. Incorporation of tyramine and dopamine into both the antibiotic alkaloid sanguinarine and hydroxycinnamic acid amides was shown to occur in elicitor-treated opium poppy cell cultures. A temporal correlation was demonstrated between the biosynthesis of alkaloids and amides, the accumulation of tyrosine\\dopa decarboxylase (TYDC) and berberine bridge enzyme (BBE) mRNAs, and the induction of tyramine hydroxycinnamoyl transferase (THT) activity. The induction of TYDC mRNAs occurred most rapidly, whereas the subsequent activation of BBE mRNAs and THT activity correlated with the synthesis and accumulation of sanguinarine and amides, respectively. In pulse-labeling experiments, a maximum of 1.2 and 0.6% of exogenous 14C-tyramine and 14C-dopamine, respectively, was incorporated into sanguinarine after elicitor treatment of cell cultures. In contrast, a maximum of 24 and 35% of exogenous 14C-tyramine and 14C-dopamine, respectively, was incorporated into an acid-insoluble fraction of the cell wall after elicitor treatment. Insolubilized radioactivity was released from cell walls after hydrolytic extraction and recovered mostly as hydroxycinnamic acid amides and free amines. The elicitor-induced incorporation of 14C-amines into both sanguinarine and cell walls decreased during the cell culture growth cycle. The prolonged induction of THT activity in elicited cultures, coupled with high and constitutive peroxidase activity, suggests that the observed decrease in cell wall labeling 5 h after the addition of elicitor was due to hydroxycinnamoyl-CoA or H2O2 limitation.

Elicitor-stimulated biosynthesis of hydroxycinnamoyltyramines in cell suspension cultures of Solanum tuberosum

Treatment of suspension-cultured potato cells (Solanum tuberosum L. cv. Desiree) with an elicitor from Phytophthora infestans induced increased incorporation of 4-hydroxybenzaldehyde, 4-hydroxybenzoate, and N-4-coumaroyl- and N-feruloyltyramine into the cell␣wall and secretion of N-4-coumaroyl- and N-feruloyltyramine into the culture medium. Induced metabolite accumulation was preceded by rapid and transient increases in activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25), exhibiting maximal activities 5–10 h after initiation of elicitor treatment. Activities of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (EC 2.3.1.110), catalyzing the formation of N-4-coumaroyl- and N-feruloyltyramine, increased later and remained at high levels. The phenolic defense compounds appear to be involved in cell wall reinforcement and may further directly affect fungal growth in the apoplastic space.