Melatonin enhances stem strength by increasing lignin content and secondary cell wall thickness in herbaceous peony.

Cut flower quality is severely restrained by stem bending due to low stem strength. Melatonin has been shown to function in many aspects of plant growth and development, yet whether it can enhance stem strength, and the corresponding underlying mechanisms remain unclear. We investigated the role of melatonin in enhancement of stem strength in herbaceous peony (Paeonia lactiflora Pall.) by applying exogenous melatonin and changing endogenous melatonin biosynthesis. Endogenous melatonin content positively correlated with lignin content and stem strength in various P. lactiflora cultivars. Supplementation with exogenous melatonin significantly enhanced stem strength by increasing lignin content and the S/G lignin compositional ratio, up-regulating lignin biosynthetic gene expression. Moreover, overexpression of TRYPTOPHAN DECARBOXYLASE GENE (TDC) responsible for the first committed step of melatonin biosynthesis in tobacco, significantly increased endogenous melatonin, which further increased the S/G ratio and stem strength. In contrast, silencing PlTDC in P. lactiflora decreased endogenous melatonin, the S/G ratio and stem strength. Finally, manipulating the expression of CAFFEIC ACID O-METHYLTRANSFERASE GENE (COMT1), which is involved in both melatonin and lignin biosynthesis, showed even greater effects on melatonin, the S/G ratio and stem strength. Our results suggest that melatonin has a positive regulatory effect on P. lactiflora stem strength.

WRKY1-mediated regulation of tryptophan decarboxylase in tryptamine generation for withanamide production in Withania somnifera (Ashwagandha).

KEY MESSAGE: WsWRKY1-mediated transcriptional modulation of Withania somnifera tryptophan decarboxylase gene (WsTDC) helps to regulate fruit-specific tryptamine generation for production of withanamides. Withania somnifera is a highly valued medicinal plant. Recent demonstration of novel indolyl metabolites called withanamides in its fruits (berries) prompted us to investigate its tryptophan decarboxylase (TDC), as tryptophan is invariably a precursor for indole moiety. TDC catalyzes conversion of tryptophan into tryptamine, and the catalytic reaction constitutes a committed metabolic step for synthesis of an array of indolyl metabolites. The TDC gene (WsTDC) was cloned from berries of the plant and expressed in E. coli. The recombinant enzyme was purified and characterized for its catalytic attributes. Catalytic and structural aspects of the enzyme indicated its regulatory/rate-limiting significance in generation of the indolyl metabolites. Novel tissue-wise and developmentally differential abundance of WsTDC transcripts reflected its preeminent role in withanamide biogenesis in the fruits. Transgenic lines overexpressing WsTDC gene showed accumulation of tryptamine at significantly higher levels, while lines silenced for WsTDC exhibited considerably depleted levels of tryptamine. Cloning and sequence analysis of promoter of WsTDC revealed the presence of W-box in it. Follow-up studies on isolation of WsWRKY1 transcription factor and its overexpression in W. somnifera revealed that WsTDC expression was substantially induced by WsWRKY1 resulting in overproduction of tryptamine. The study invokes a key role of TDC in regulating the indolyl secondary metabolites through enabling elevated flux/supply of tryptamine at multiple levels from gene expression to catalytic attributes overall coordinated by WsWRKY1. This is the first biochemical, molecular, structural, physiological and regulatory description of a fruit-functional TDC.

Microbial Synthesis of Human-Hormone Melatonin at Gram Scales.

Melatonin is a commercially attractive tryptophan-derived hormone. Here we describe a bioprocess for the production of melatonin using Escherichia coli to high titers. The first engineered strain produced 0.13 g/L of melatonin from tryptophan under fed-batch fermentation conditions. A 4-fold improvement on melatonin titer was further achieved by (1) protein engineering of rate-limiting tryptophan hydroxylase to improve 5-hydroxytryptophan biosynthesis and (2) chromosomal integration of aromatic-amino-acid decarboxylase to limit byproduct formation and to minimize gene toxicity to the host cell. Fermentation optimization improved melatonin titer by an additional 2-fold. Deletion of yddG, a tryptophan exporter, exhibited an additive beneficial effect. The final engineered strain produced ∼2.0 g/L of melatonin with tryptophan supplemented externally and ∼1.0 g/L with glucose as the sole carbon source for tryptophan supply. This study lays the foundation for further developing a commercial melatonin-producing E. coli strain.

Clinical Metabolomics to Segregate Aromatic Amino Acid Decarboxylase Deficiency From Drug-Induced Metabolite Elevations.

BACKGROUND: Phenotyping technologies featured in the diagnosis of inborn errors of metabolism, such as organic acid, amino acid, and acylcarnitine analyses, recently have been supplemented by broad-scale untargeted metabolomic phenotyping. We investigated the analyte changes associated with aromatic amino acid decarboxylase (AADC) deficiency and dopamine medication treatment. METHODS: Using an untargeted metabolomics platform, we analyzed ethylenediaminetetraacetic acid plasma specimens, and biomarkers were identified by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. RESULTS: Elevated 3-methoxytyrosine (average z score 5.88) accompanied by significant decreases of dopamine 3-O-sulfate (-2.77), vanillylmandelate (-2.87), and 3-methoxytyramine sulfate (-1.44) were associated with AADC deficiency in three samples from two patients. In five non-AADC patients treated with carbidopa-levodopa, levels of 3-methoxytyrosine were elevated (7.65); however, the samples from non-AADC patients treated with DOPA-elevating drugs had normal or elevated levels of metabolites downstream of aromatic l-amino acid decarboxylase, including dopamine 3-O-sulfate (2.92), vanillylmandelate (0.33), and 3-methoxytyramine sulfate (5.07). In one example, a plasma metabolomic phenotype pointed to a probable AADC deficiency and prompted the evaluation of whole exome sequencing data, identifying homozygosity for a known pathogenic variant, whereas whole exome analysis in a second patient revealed compound heterozygosity for two variants of unknown significance. CONCLUSIONS: These data demonstrate the power of combining broad-scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and suggest that metabolic phenotyping of plasma can be used to identify AADC deficiency and to distinguish it from non-AADC patients with elevated 3-methoxytyrosine caused by DOPA-raising medications.

The central anorexigenic mechanism of amylin in Japanese quail (Coturnix japonica) involves pro-opiomelanocortin, calcitonin receptor, and the arcuate nucleus of the hypothalamus.

Amylin is a 37-amino acid peptide hormone that exerts anorexigenic effects in humans and animals. We demonstrated that central injection of amylin into chicks affected feeding and related behaviors via the hypothalamus and brainstem, although the molecular mechanisms remained elusive. Thus, the objective of this study was to investigate the molecular mechanisms underlying anorexigenic effects of amylin in 7 day-old Japanese quail. Food but not water intake was reduced after intracerebroventricular amylin injection, and the behavior analysis indicated that this was associated with decreased food pecks and preening. Whole hypothalamus and hypothalamic nuclei including the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), dorsomedial nucleus (DMN) and lateral hypothalamic area (LH) were extracted from quail at 1h post-injection for total RNA isolation. Real time PCR was performed to quantify mRNA abundance of amylin receptors, appetite-associated neuropeptides and monoamine-synthesis-related enzymes. Central amylin injection increased the mRNA abundance of calcitonin receptor (CALCR), receptor activity modifying protein 1 (RAMP1), pro-opiomelanocortin (POMC), and aromatic l-amino acid decarboxylase (AADC) in the hypothalamus and individual hypothalamic nuclei. Relative quantities of CALCR and POMC mRNA were greater in the ARC of the amylin- than vehicle-treated group. Thus, amylin-mediated effects on food intake may involve POMC, monoamine synthesis, and amylin receptor 1 (a complex of CALCR and RAMP1) in the ARC. Together, these data provide novel insights on the hypothalamic-specific molecular mechanisms of amylin-induced food intake.

Structural and functional analogies and differences between histidine decarboxylase and aromatic l-amino acid decarboxylase molecular networks: Biomedical implications.

Human histidine decarboxylase (HDC) and dopa decarboxilase (DDC) are highly homologous enzymes responsible for the synthesis of biogenic amines (BA) like histamine, and serotonin and dopamine, respectively. The enzymes share many structural and functional analogies, while their product metabolisms also follow similar patterns that are confluent in some metabolic steps. They are involved in common physiological functions, such as neurotransmission, gastrointestinal track function, immunity, cell growth and cell differentiation. As a consequence, metabolic elements of both BA subfamilies are also co-participants in a long list of human diseases. This review summarizes the analogies and differences in their origin (HDC and DDC) as well as their common pathophysiological scenarios. The major gaps of information are also underlined, as they delay the possibility of holistic approaches that would help personalized medicine and pharmacological initiatives for prevalent and rare diseases.

Missing proof of cooperative synthesis of dopamine by non-dopaminergic neurons.

L-DOPA accumulation in the extracellular medium was detected when the transfer of L-DOPA from the neurons containing tyrosine hydroxylase to the neurons containing aromatic L-amino acid decarboxylase was blocked, under conditions of inhibition of the L-DOPA degradation enzyme. Thus, the missing proof confirming the existence of cooperative synthesis of dopamine by neurons non-dopaminergic was obtained.

Characterization of A11 neurons projecting to the spinal cord of mice.

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons.

Integration of ¹H NMR and UPLC-Q-TOF/MS for a comprehensive urinary metabonomics study on a rat model of depression induced by chronic unpredictable mild stress.

Depression is a type of complex psychiatric disorder with long-term, recurrent bouts, and its etiology remains largely unknown. Here, an integrated approach utilizing (1)H NMR and UPLC-Q-TOF/MS together was firstly used for a comprehensive urinary metabonomics study on chronic unpredictable mild stress (CUMS) treated rats. More than twenty-nine metabolic pathways were disturbed after CUMS treatment and thirty-six potential biomarkers were identified by using two complementary analytical technologies. Among the identified biomarkers, nineteen (10, 11, 16, 17, 21-25, and 27-36) were firstly reported as potential biomarkers of CUMS-induced depression. Obviously, this paper presented a comprehensive map of the metabolic pathways perturbed by CUMS and expanded on the multitude of potential biomarkers that have been previously reported in the CUMS model. Four metabolic pathways, including valine, leucine and isoleucine biosynthesis; phenylalanine, tyrosine and tryptophan biosynthesis; tryptophan metabolism; synthesis and degradation of ketone bodies had the deepest influence in the pathophysiologic process of depression. Fifteen potential biomarkers (1-2, 4-6, 15, 18, 20-23, 27, 32, 35-36) involved in the above four metabolic pathways might become the screening criteria in clinical diagnosis and predict the development of depression. Moreover, the results of Western blot analysis of aromatic L-amino acid decarboxylase (DDC) and indoleamine 2, 3-dioxygenase (IDO) in the hippocampus of CUMS-treated rats indicated that depletion of 5-HT and tryptophan, production of 5-MT and altered expression of DDC and IDO together played a key role in the initiation and progression of depression. In addition, none of the potential biomarkers were detected by NMR and LC-MS simultaneously which indicated the complementary of the two kinds of detection technologies. Therefore, the integration of (1)H NMR and UPLC-Q-TOF/MS in metabonomics study provided an approach to identify the comprehensive potential depression-related biomarkers and helpful in further understanding the underlying molecular mechanisms of depression through the disturbance of metabolic pathways.

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.

Integrating carbon-halogen bond formation into medicinal plant metabolism.

Halogenation, which was once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways. However, only a small fraction of halogenated compounds have been isolated from terrestrial plants. Given the impact that halogenation can have on the biological activity of natural products, we reasoned that the introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group-a halide-is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biology efforts.

Neuroprotective effect of Rosmarinus officinalis extract on human dopaminergic cell line, SH-SY5Y.

Hydrogen peroxide (H2O2) is a major Reactive Oxygen Species (ROS), which has been implicated in many neurodegenerative conditions including Parkinson's disease (PD). Rosmarinus officinalis (R. officinalis) has been reported to have various pharmacological properties including anti-oxidant activity. In this study, we investigated the neuroprotective effects of R. officinalis extract on H2O2-induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that H2O2-induced cytotoxicity in SH-SY5Y cells was suppressed by treatment with R. officinalis. Moreover, R. officinalis was very effective in attenuating the disruption of mitochondrial membrane potential and apoptotic cell death induced by H2O2. R. officinalis extract effectively suppressed the up-regulation of Bax, Bak, Caspase-3 and -9, and down-regulation of Bcl-2. Pretreatment with R. officinalis significantly attenuated the down-regulation of tyrosine hydroxylase (TH), and aromatic amino acid decarboxylase (AADC) gene in SH-SY5Y cells. These findings indicate that R. officinalis is able to protect the neuronal cells against H2O2-induced injury and suggest that R. officinalis might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.

[Synthesis of monoamines by non-monoaminergic neurons: illusion or reality?]. / Synthèse de monoamines par des neurones non-monoaminergiques. Illusion ou réalité ?

In contrast to monoaminergic (MA-ergic) neurons possessing the whole set of the enzymes for MA synthesis from the precursor amino-acid, some, mostly peptidergic, neurons co-express only one of the enzymes of monoamine synthesis. They are widely distributed in the brain, being particularly numerous in ontogenesis and, in adulthood, under certain physiological conditions. Most monoenzymatic neurons possess one of the enzymes for dopamine (DA) synthesis, tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC). TH and AADC are enzymatically active in a substantial number of monoenzymatic neurons, where they are capable of converting L-tyrosine to L-3,4-dihydroxy-phenylalanine (L-DOPA) and L-DOPA to dopamine (DA) (or 5-hydroxy-tryptophan, 5-HTP to serotonin), respectively. According to our data L-DOPA synthesized in monoenzymatic TH-neurons is released and taken up by monoenzymatic AADC-neurons for DA synthesis. Moreover, L-DOPA captured by dopaminergic neurons and serotoninergic neurons serves to stimulate dopamine synthesis in the former and to start DA synthesis in the latter. Cooperative synthesis of MAs is considered as a compensatory reaction under a failure of MA-ergic neurons, e.g. in neurodegenerative diseases like hyperprolactinemia and Parkinson's disease, which are developed primarily because of degeneration of DA-ergic neurons of the tuberoinfundibular system and the nigrostriatal system, respectively. Noteworthy, the neurotoxin-induced increase of prolactin secretion returns with time to a normal level due to the stimulation of DA synthesis by the tuberoinfundibular most probably monoenzymatic neurons. The same compensatory mechanism is supposed to be used under the failure of the nigrostriatal DA-ergic system that is manifested by an increased number of monoenzymatic neurons in the striatum of animals with neurotoxin-induced parkinsonism and in humans with Parkinson's disease. Expression of the enzymes of MA synthesis in non-monoaminergic neurons is controlled by intercellular signals such as classical neurotransmitters (catecholamines), etc. Thus, a substantial number of brain neurons express partly the monoaminergic phenotype, namely individual complementary enzymes of MA synthesis, serving to produce MAs in cooperation, which is considered as a compensatory reaction under the failure of MA-ergic neurons.

Real-time MR imaging of adeno-associated viral vector delivery to the primate brain.

We are developing a method for real-time magnetic resonance imaging (MRI) visualization of convection-enhanced delivery (CED) of adeno-associated viral vectors (AAV) to the primate brain. By including gadolinium-loaded liposomes (GDL) with AAV, we can track the convective movement of viral particles by continuous monitoring of distribution of surrogate GDL. In order to validate this approach, we infused two AAV (AAV1-GFP and AAV2-hAADC) into three different regions of non-human primate brain (corona radiata, putamen, and thalamus). The procedure was tolerated well by all three animals in the study. The distribution of GFP determined by immunohistochemistry in both brain regions correlated closely with distribution of GDL determined by MRI. Co-distribution was weaker with AAV2-hAADC, although in vivo PET scanning with FMT for AADC activity correlated well with immunohistochemistry of AADC. Although this is a relatively small study, it appears that AAV1 correlates better with MRI-monitored delivery than does AAV2. It seems likely that the difference in distribution may be due to differences in tissue specificity of the two serotypes.

[Effects of nitrogen forms on camptothecin content and its metabolism-related enzymes activities in Camptotheca acuminata seedlings].

OBJECTIVE: To investigate the effects of nitrogen forms on the camptothecin (CPT) content, tryptophan synthase (TSB) and tryptophan decarboxylase (TDC) activities in Camptotheca acuminata seedlings. METHOD: The seedlings of C. acuminata with 6 pairs of leaves were subjected to 5 different NH4(+) -N/NO3(-) -N ratio (0 : 100, 75 : 25, 50 : 50, 25 : 75, 100 : 0) treatments by sand culture in a greenhouse. The CPT content, TSB activity in the young leaves and TDC in the stem barks of the seedlings were determined by HPLC on the 15th, 30th, 45th, 60th and 75th day, respectively. RESULT: The obvious relationship between CPT content and nitrogen forms was observed. When NH4(+) - N /NO3(-) -N ratio was 25 : 75, CPT accumulation in young leaves displayed the best advantages (the highest value is 5.69 per thousand) and increased in the early 30 days of treatment and then declined. There was no obvious relationship between TSB activity in the young leaves and nitrogen forms. TDC activity in the stem bark was the highest when NH4(+) -N /NO3(-) -N ratio was 25 : 75, and the change of TDC activity paralleled to CPT content in the young leaves. CONCLUSION: A short-term treatment that NH4(+) -N /NO3(-) -N ratio was 25:75 may gain high CPT content in the young leaves through enhancing the TDC activity in the stem bark of C. acuminata seedlings.

Humoral phototransduction: light transportation in the blood, and possible biological effects.

In our measurements plasma and, especially, the main plasma protein, albumin, exhibits a long-lasting light-induced luminescence, which should be capable of transporting light along the blood circulation. Moreover, albumin shows intense fluorescence, with emission at 337 nm, which is controlled by bilirubin. Furthermore, it is known that tryptophan decarboxylase, the last step of serotonin formation, is directly activated by light, with a maximum at 337 nm. As a hypothesis, we propose that light-induced luminescence of plasma components, such as albumin and free radicals, transports ambient light along the blood vessels. This emission could have photochemical and photobiological effects, e.g., photomodulation of enzymes. Albumin fluorescence emission could stimulate serotonin formation at 337 nm, modulated by bilirubin. Such mechanisms could be involved in the action of light therapy on serotonin formation, melatonin suppression and circadian rhythms, both in the pathophysiology of seasonal affective disorder and major depression, and in blood pressure regulation via photovasorelaxation. The proposed model can be called humoral phototransduction.

Effect of the beta3 adrenoceptor agonist CL 316243 on hypothalamic 5-HT synthesis and suppression of REM sleep in the rat.

Beta3 adrenoceptor agonists show an antidepressant-like profile in preclinical rodent assays and improve mood in clinically-obese patients. These observations suggest a possible antidepressant utility for beta3 adrenoceptor agonists. The present study examined the effects of acute and chronic administration of the beta3 adrenoceptor agonist CL 316243 on two physiological indicators of antidepressant activity in the rat: hypothalamic 5-HT synthesis and suppression of REM sleep. 5-HT synthesis was estimated by the accumulation of 5-hydroxytryptophan (5-HTP) after treatment with the L-aromatic acid decarboxylase inhibitor NSD 1015. Sleep-wake patterns were monitored using electroencephalogram and electromyogram signals collected by radiotelemetry. Rats were administered CL 316243 acutely or once daily for 11 days. Acute administration of CL 316243 significantly increased hypothalamic 5-HT synthesis, as indicated by increased levels of 5-HTP, and reduced the amount of REM sleep. However, chronic administration produced no changes in 5-HTP or REM compared with vehicle treatment. The present observations suggest that acute administration of CL 316243 causes antidepressant-like effects on REM sleep, possibly mediated by increased central 5-HT synthesis. However, these effects are not maintained with repeated dosing.

Dopamine synthesis by non-dopaminergic neurons in the arcuate nucleus of rat fetuses.

The aim of the present work was to verify the hypothesis that non-dopaminergic neurons expressing individual complementary dopamine synthesis enzymes can perform the co-located synthesis of dopamine. According to this hypothesis, neurons expressing tyrosine hydroxylase use L-tyrosine for the synthesis of L-dihydroxyphenylalanine (L-DOPA), which then enters neurons expressing aromatic amino acid decarboxylase, which converts L-DOPA to dopamine. Experiments were performed using the mediobasal hypothalamus of rat fetuses, which mostly contains single-enzyme neurons (>99%) and occasional double-enzyme neurons (<1%). Controls were obtained from the fetal substantia nigra, which is enriched with dopaminergic neurons. High-performance liquid chromatography was used to measure levels of dopamine and L-DOPA in cell extracts and the incubation medium after incubation in the presence and absence of exogenous L-tyrosine. Addition of L-tyrosine to the medium led to increases in the level of synthesis and release of L-DOPA in the mediobasal hypothalamus and substantia nigra. In addition, L-tyrosine increased dopamine synthesis in the substantia nigra and decreased dopamine synthesis in the mediobasal hypothalamus. This regional difference in levels of dopamine synthesis is probably due to inhibition of the uptake of L-DOPA from the intercellular medium by neurons in the mediobasal hypothalamus containing aromatic amino acid decarboxylase, due to the competitive binding of the L-DOPA transporter by L-tyrosine. Thus, these results provide the first evidence for the co-located synthesis of dopamine by non-dopaminergic neurons expressing single complementary enzymes involved in the synthesis of this neurotransmitter.

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.