[Cloning and expression analysis of a tyrosine decarboxylase gene from Rehmannia glutinosa].

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of phenylethanoid glycosides. Based on the transcriptome data, we cloned the full-length cDNA (GenBank accession NO. KU640395) of RgTyDC gene from Rehmannia glutinosa, and then performed bioinformatic analysis of the sequence. Further, we detected the expression pattern in different organs and hair roots treated with four elicitors by qRT-PCR. The results showed that the full length of RgTyDC cDNA was 1 530 bp encoding 509 amino acids. The molecular weight of the putative RgTyDC protein was about 56.6 kDa and the theoretical isoelectric point was 6.25. The RgTyDC indicated the highest homology with Sesamum indicum SiTyDC and Erythranthe guttata EgTyDC, both of them were reached 88%. RgTyDC highly expressed in R. glutinosa leaf, especially in senescing leaf, and rarely expressed in tuberous root. After the treatment of SA and MeJA, the relative expression level of RgTyDC mRNA was substantially increased. The results provide a foundation for exploring the molecular function of RgTyDC involved in phenylethanoid glycosides biosynthesis.

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.

Expression of Lactobacillus brevis IOEB 9809 tyrosine decarboxylase and agmatine deiminase genes in wine correlates with substrate availability.

AIMS: Lactobacillus brevis IOEB 9809 is able to produce both tyramine and putrescine via tyrosine decarboxylase and agmatine deiminase enzymes, respectively, when cultured on synthetic media. The aims of this study were to assess the expression of L. brevis IOEB 9809 tdc and aguA1 genes, during wine fermentation and to evaluate the effect of substrate availability and pH on tdc and aguA1 expression, as well as on biogenic amine production and L. brevis viability. METHODS AND RESULTS: The relative expression of L. brevis IOEB 9809 tdc and aguA1 genes was analysed in wine by quantitative real-time RT-PCR (qRT-PCR) during a period of incubation of 30 days. Cell viability, pH values, putrescine and tyramine concentration were monitored throughout the experiments. CONCLUSIONS: The wine trials indicated that L. brevis IOEB 9809 is able to produce both tyramine and putrescine during wine fermentation. Increased cell viability was also observed in wine supplemented with tyrosine or agmatine. qRT-PCR analysis suggests a strong influence of substrate availability on the expression of genes coding for tyrosine decarboxylase and agmatine deiminase in L. brevis IOEB 9809. Less evident is the relationship between putrescine and tyramine production and tolerance to wine pH. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this study represents the first assessment of relative expression of L. brevis IOEB 9809 genes involved in biogenic amine production in wine. Furthermore, an effect of biogenic amine production on viability of L. brevis during wine fermentation was established.

Role of aromatic aldehyde synthase in wounding/herbivory response and flower scent production in different Arabidopsis ecotypes.

Aromatic L-amino acid decarboxylases (AADCs) are key enzymes operating at the interface between primary and secondary metabolism. The Arabidopsis thaliana genome contains two genes, At2g20340 and At4g28680, encoding pyridoxal 5'-phosphate-dependent AADCs with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in floral scent production. The At4g28680 gene product was recently biochemically characterized as an L-tyrosine decarboxylase (AtTYDC), whereas the function of the other gene product remains unknown. The biochemical and functional characterization of the At2g20340 gene product revealed that it is an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. AtAAS knock-down and transgenic AtAAS RNA interference (RNAi) lines show significant reduction in phenylacetaldehyde levels and an increase in phenylalanine, indicating that AtAAS is responsible for phenylacetaldehyde formation in planta. In A. thaliana ecotype Columbia (Col-0), AtAAS expression was highest in leaves, and was induced by methyl jasmonate treatment and wounding. Pieris rapae larvae feeding on Col-0 leaves resulted in increased phenylacetaldehyde emission, suggesting that the emitted aldehyde has a defensive activity against attacking herbivores. In the ecotypes Sei-0 and Di-G, which emit phenylacetaldehyde as a predominant flower volatile, the highest expression of AtAAS was found in flowers and RNAi AtAAS silencing led to a reduction of phenylacetaldehyde formation in this organ. In contrast to ecotype Col-0, no phenylacetaldehyde accumulation was observed in Sei-0 upon wounding, suggesting that AtAAS and subsequently phenylacetaldehyde contribute to pollinator attraction in this ecotype.

Wound-inducible biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine in tryptophan and tyrosine decarboxylase transgenic tobacco lines.

The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC xTYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine.

The catecholamine potentiates starch mobilization in transgenic potato tubers.

In human and animal cells, the catecholamines are involved in glycogen mobilization. Since the compounds are found in a potato, their function in starch mobilization was hypothesized. In order to verify this hypothesis, the transgenic potato plants Solanum tuberosum L. cv. Desiree overexpressing tyrosine decarboxylase (TD EC 4.1.1.25) cDNA from parsley has been generated. The cDNA expression was judged by the northern blot analysis and the enzyme activity measurements. Four independent transgenic lines with the highest TD mRNA expression were selected and analyzed. The expected substantial decrease in tyrosine content was followed by significant increase in tyramine and dramatic enhancement of norepinephrine synthesis was detected. The level of L-3,4-dihydroxyphenylalanin (L-Dopa) was only slightly increased and dopamine significantly decreased in most cases in these plants. The increase in norepinephrine was accompanied by changes in carbohydrate metabolism. The significant increase in glucose and sucrose and the decrease in starch content were characteristic features of TD overexpressed transgenic potato tubers. The features mentioned above indicate that catecholamines potentiate starch mobilization in potato plants in common with animal cells. The decrease in tyrosine content in transgenic plants is also compensated by significant increase in chlorogenic acid synthesis thus potentially increasing the antioxidant capacity of transgenic tubers. The glycoalkaloids content is changed in the transformants. This may originate from glucose accumulation and glycolysis activation. The obtained transgenic potato provides material for further detailed studies of the physiological function of catecholamines in plants.

Analysis of promoters from tyrosine/dihydroxyphenylalanine decarboxylase and berberine bridge enzyme genes involved in benzylisoquinoline alkaloid biosynthesis in opium poppy.

Tyrosine/dihydroxyphenylalanine decarboxylase (TYDC) and the berberine bridge enzyme (BBE) represent the entry point and a key branch point, respectively, in the biosynthesis of benzylisoquinoline alkaloids in select species of the Papaveraceae and Fumariaceae. Genomic clones for tydc7 and bbe1 from opium poppy (Papaver somniferum L.) were isolated. Deletion analysis of tydc7 and bbe1 5'-flanking regions revealed the location of putative regulatory domains necessary for expression of the beta-glucuronidase (gus) reporter gene in a transient assay system based on the microprojectile bombardment of cultured opium poppy cells. A 105-nucleotide region between -393 and -287 of the tydc7 5'-flanking region, and a 155-nucleotide region between -355 and -200 of the bbe1 5'-flanking region, were found to be essential for promoter activity. RNA gel blot analysis showed that tydc7 and bbe1 expression is induced in cultured opium poppy cells in response to wounding or treatment with a pathogen-derived elicitor. Time-courses for the induction of tydc7 and bbe1 mRNAs in wounded cells were nearly identical to those for GUS activity in cells bombarded with select promoter-gus constructs when the -393 to -287 region of tydc7, or the -355 to -200 region of bbe1, was present. Our data suggest that the wound signal caused by the entry of DNA-coated microcarriers into opium poppy cells was sufficient to induce tydc7 and bbe1 promoter activity, and that wound-responsive regulatory elements are located within domains identified by deletion analysis.

Expression patterns conferred by tyrosine/dihydroxyphenylalanine decarboxylase promoters from opium poppy are conserved in transgenic tobacco.

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the beta-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.

Molecular analysis of a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family.

An aromatic amino acid decarboxylase DNA fragment was generated from opium poppy (Papaver somniferum L.) genomic DNA by the PCR using primers designed from conserved amino acid sequences of other aromatic amino acid decarboxylase genes. Using this fragment as a probe, a genomic clone was isolated that encodes a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family (TyDC5). The predicted TyDC5 amino acid sequence shares extensive identity with other opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylases (84%), and when expressed in Escherichia coli, it is active against tyrosine and to a lesser extent against 3,4-dihydroxyphenylalanine. Ribonuclease protection assays indicate that TyDC5 is expressed primarily in the roots of mature poppy plants. A peak of TyDC5 expression was also observed during germination, coincident with the emergence of the radicle from the seed coat. Parallel results were obtained in transgenic tobacco using a TyDC5 promoter fragment (-2060) translationally fused to the beta-glucuronidase reporter gene (GUS). IN TyDC5::GUS tobacco, GUS activity transiently appeared in all parts of the seedling during germination, but was limited to the roots in older plants. These results indicate that TyDC5 expression is transcriptionally regulated and suggest that the TyDC5 enzyme may play an important role in providing precursors for alkaloid synthesis in the roots and germinating seedlings of opium poppy.

Expression in Escherichia coli and partial characterization of two tyrosine/dopa decarboxylases from opium poppy.

Two tyrosine/dopa decarboxylases (TYDC1 and TYDC2) from opium poppy (Papaver somniferum) were heterologously expressed in Escherichia coli and partially characterized. TYDC1 and TYDC2 are representative members of the two major isoform sub-classes of genes found in opium poppy which share less than 75% amino acid identity. Although both enzymes exhibit a marginal preference in vitro for L-dopa over L-tyrosine, the apparent Kms of both TYDC1 and TYDC2 in total protein extracts for either substrate were equal (Kms = 1 mM) at pH 7.2. Both TYDC1 and TYDC2 exhibited a similar broad pH optimum in the range 7.5-8.5, and their activity was enhanced in the presence of pyridoxal phosphate co-factor. The Vmax values for TYDC1 with either tyrosine or dopa as substrate were virtually identical (Vmax = 0.59 fkat mg-1 protein), whereas, the Vmax for TYDC2 was two-fold greater with dopa (Vmax = 0.21 fkat mg-1 protein) than with tyrosine (Vmax = 0.12 fkat mg-1 protein) as substrate. Bacterial cell cultures expressing the TYDC1 polypeptide accumulated up to 350 micrograms ml-1 tyramine and 360 micrograms ml-1 dopamine in the medium within 8 hr after the addition of exogenous tyrosine or dopa, respectively. In contrast, cultures expressing the TYDC2 polypeptide accumulated 160 micrograms ml-1 tyramine and 110 micrograms ml-1 dopamine 8 hr after adding tyrosine or dopa, respectively. The higher in vivo conversion rates by bacterial cultures expressing TYDC1 relative to bacteria expressing TYDC2 is consistent with the higher specific activity of TYDC1 measured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy.

Two early and potential rate-limiting steps in the biosynthesis of isoquinoline alkaloids, such as morphine and codeine, in opium poppy (Papaver somniferum) involve decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-dopa) to yield tyramine and dopamine, respectively. A DNA fragment was amplified by polymerase chain reaction (PCR) using degenerate primers designed to two highly conserved domains found in other aromatic amino acid decarboxylases. A poppy seedling cDNA library was screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dopa decarboxylase (TYDC/DODC) was isolated. Two other independent cDNAs (cTYDC2 and cTYDC3) encoding TYDC/DODC were isolated by heterologous screening with a plant tryptophan decarboxylase (TDC) cDNA as probe. A poppy genomic library was screened with cTYDC1 and two intronless genomic clones (gTYDC1 and gTYDC4) were isolated. The deduced amino acid sequences of all poppy clones share extensive identity with other reported pyridoxal phosphate-dependent decarboxylases from both plants and animals. Based on sequence homology, members of the gene family were divided into two subsets (cTYDC1 and gTYDC4; cTYDC2 and cTYDC3) of proteins with predicted M(r) = 56,983 and 59,323, respectively. Within each subset the clones exhibit greater than 90% identity, whereas clones between subsets share less than 75% identity. Expression of gTYDC1 and cTYDC2 as beta-galactosidase fusion proteins in Escherichia coli resulted in catalytically active enzymes immunodetectable with TDC-specific polyclonal antibodies. Each enzyme showed marginally higher substrate specificity for L-dopa over L-tyrosine, but did not accept L-tryptophan and L-phenylalanine as substrates. Genomic DNA blot-hybridization analysis revealed 6 to 8 genes homologous to cTYDC1 and 4 to 6 genes homologous to cTYDC2 in the tetraploid poppy genome. A premature translation stop codon was found in the gTYDC4 clone suggesting that it may not encode a functional protein. RNA blot-hybridization with probes specific to the gTYDC1- or cTYDC2-like subsets showed that members of the TYDC gene family are differentially expressed in various plant tissues.