Enterococcus-derived tyramine hijacks α2A-adrenergic receptor in intestinal stem cells to exacerbate colitis.

Inflammatory bowel disease (IBD) is characterized by dysbiosis of the gut microbiota and dysfunction of intestinal stem cells (ISCs). However, the direct interactions between IBD microbial factors and ISCs are undescribed. Here, we identify α2A-adrenergic receptor (ADRA2A) as a highly expressed GPCR in ISCs. Through PRESTO-Tango screening, we demonstrate that tyramine, primarily produced by Enterococcus via tyrosine decarboxylase (tyrDC), serves as a microbial ligand for ADRA2A. Using an engineered tyrDC-deficient Enterococcus faecalis strain and intestinal epithelial cell-specific Adra2a knockout mice, we show that Enterococcus-derived tyramine suppresses ISC proliferation, thereby impairing epithelial regeneration and exacerbating DSS-induced colitis through ADRA2A. Importantly, blocking the axis with an ADRA2A antagonist, yohimbine, disrupts tyramine-mediated suppression on ISCs and alleviates colitis. Our findings highlight a microbial ligand-GPCR pair in ISCs, revealing a causal link between microbial regulation of ISCs and colitis exacerbation and yielding a targeted therapeutic approach to restore ISC function in colitis.

Piperine improves levodopa availability in the 6-OHDA-lesioned rat model of Parkinson's disease by suppressing gut bacterial tyrosine decarboxylase.

AIM: Tyrosine decarboxylase (TDC) presented in the gut-associated strain Enterococcus faecalis can convert levodopa (L-dopa) into dopamine (DA), and its increased abundance would potentially minimize the availability and efficacy of L-dopa. However, the known human decarboxylase inhibitors are ineffective in this bacteria-mediated conversion. This study aims to investigate the inhibition of piperine (PIP) on L-dopa bacterial metabolism and evaluates the synergistic effect of PIP combined with L-dopa on Parkinson's disease (PD). METHODS: Metagenomics sequencing was adopted to determine the regulation of PIP on rat intestinal microbiota structure, especially on the relative abundance of E. faecalis. Then, the inhibitory effects of PIP on L-dopa conversion and TDC expression of E. faecalis were tested in vitro. We examined the synergetic effect of the combination of L-dopa and PIP on 6-hydroxydopamine (6-OHDA)-lesioned rats and tested the regulations of L-dopa bioavailability and brain DA level by pharmacokinetics study and MALDI-MS imaging. Finally, we evaluated the microbiota-dependent improvement effect of PIP on L-dopa availability using pseudo-germ-free and E. faecalis-transplanted rats. RESULTS: We found that PIP combined with L-dopa could better ameliorate the move disorders of 6-OHDA-lesioned rats by remarkably improving L-dopa availability and brain DA level than L-dopa alone, which was associated with the effect of PIP on suppressing the bacterial decarboxylation of L-dopa via effectively downregulating the abnormal high abundances of E. faecalis and TDC in 6-OHDA-lesioned rats. CONCLUSION: Oral administration of L-dopa combined with PIP can improve L-dopa availability and brain DA level in 6-OHDA-lesioned rats by suppressing intestinal bacterial TDC.

Optimization of tyrosol-producing pathway with tyrosine decarboxylase and tyramine oxidase in high-tyrosine-producing Escherichia coli.

Tyrosol (4-hydroxyphenylethanol) is a phenolic compound used in the pharmaceutical and chemical industries. However, current supply methods, such as extraction from natural resources and chemical synthesis, have disadvantages from the viewpoint of cost and environmental protection. Here, we developed a tyrosol-producing Escherichia coli cell factory from a high-tyrosine-producing strain by expressing selected tyrosine decarboxylase-, tyramine oxidase (TYO)-, and medium-chain dehydrogenase/reductase (YahK)-encoding genes. The genes were controlled by the strong T7 promoter and integrated into the chromosome because of the advantages over plasmid-based systems. The strain produced a melanin-like pigment as a by-product, which is suggested to be formed from 4-hydroxyphenylacetaldehyde (a TYO product/YahK substrate). By using a culture medium containing a high concentration of glycerol, which was reported to enhance NADH supply required for YahK activity, the final titer of tyrosol reached 2.42 g/L in test tube-scale cultivation with a concomitant decrease in the amount of pigment. These results indicate that chromosomally integrated and T7 promoter-controlled gene expression system in E. coli is useful for high production of heterologous enzymes and might be applied for industrial production of useful compounds including tyrosine and tyrosol.

Association Between Microbial Tyrosine Decarboxylase Gene and Levodopa Responsiveness in Patients With Parkinson Disease.

BACKGROUND AND OBJECTIVES: Interindividual variability in levodopa efficacy is a challenge for the personalized treatment of Parkinson disease (PD). Gut microbiota might represent a new approach for personalized medicine. Recently, a novel microbial levodopa metabolism pathway was identified, which is mediated by tyrosine decarboxylase mainly encoded by tyrosine decarboxylase gene (tyrDC) in Enterococcus faecalis. In this study, we aimed to identify whether the abundance of microbial tyrDC gene and E faecalis is associated with levodopa responsiveness and could predict the drug response. METHODS: This cross-sectional study enrolled patients with PD between December 2019 and January 2022 and evaluated levodopa responsiveness using a levodopa challenge test. Patients were stratified into moderate and good responders based on levodopa responsiveness. The tyrDC gene and E. faecalis abundance in fecal samples were measured using quantitative real-time PCR. Plasma levodopa concentrations were measured using liquid chromatography-tandem mass spectrometry analysis. The predictive models for levodopa responsiveness were constructed and verified through cross-validation and external validation. RESULTS: A total of 101 patients with PD were enrolled in the primary cohort and 43 were enrolled in the external validation cohort. Moderate responders had higher abundances of the tyrDC gene (3.6 [3.1-4.3] vs 2.6 [2.1-2.9], p < 0.001) and E faecalis (3.2 [2.5-4.4] vs 2.6 [2.1-3.6], p = 0.010) than good responders. The tyrDC gene abundance was independently associated with levodopa responsiveness (OR: 5.848; 95% CI: 2.664-12.838; p < 0.001). Notably, tyrDC gene abundance showed certain discriminative power for levodopa responsiveness in primary cohort (sensitivity: 80.0%; specificity: 84.3%; area under the curve [AUC]: 0.85; 95% CI: 0.77-0.93; p < 0.001) and external validation cohort (sensitivity: 85.0%; specificity: 95.7%; AUC: 0.95; 95% CI: 0.89-1.02; p < 0.001). The prediction of levodopa responsiveness based on tyrDC gene abundance had good calibration and discrimination in cross-validation (C-index in training and test sets: 0.856 and 0.851, respectively) and external validation (C-index: 0.952). DISCUSSION: The microbial tyrDC gene abundance could serve as a potential biomarker of levodopa responsiveness. Novel strategies targeting the tyrDC gene may provide new approaches for personalized levodopa treatment.

Enzymatic synthesis of benzylisoquinoline alkaloids using a parallel cascade strategy and tyrosinase variants.

Benzylisoquinoline alkaloid derived pharmaceuticals are widely applied in modern medicines. Recent studies on the microbial production of benzylisoquinolines have highlighted key biological syntheses towards these natural products. Routes to non-natural benzylisoquinolines have been less explored, particularly halogenated compounds which are more challenging. Here, we show the use of a tyrosinase, tyrosine decarboxylase, transaminase, and norcoclaurine synthase which are combined in a parallel cascade design, in order to generate halogenated benzylisoquinoline alkaloids in high enantiomeric excess. Notably, mutagenesis studies are applied to generate tyrosinase mutants, which enhance the acceptance of halogenated tyrosines for use in the biocatalytic cascades developed.

Functional characterization of tyrosine decarboxylase genes that contribute to acteoside biosynthesis in Rehmannia glutinosa.

MAIN CONCLUSION: The RgTyDCs possess typical decarboxylase functional activity in vitro and in vivo and participate in acteoside biosynthesis in R. glutinosa, positively controlling its production via activated acteoside/tyrosine-derived pathways. Acteoside is an important ingredient in Rehmannia glutinosa and an active natural component that contributes to human health. Tyrosine decarboxylase (TyDC) is thought to play an important role in acteoside biosynthesis. Several plant TyDC family genes have been functionally characterized and shown to play roles in some bioactive metabolites' biosynthesis by mediating the decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-DOPA); however, one TyDC (named RgTyDC1) in R. glutinosa has been identified to date, but the family genes that contribute to acteoside biosynthesis remain largely characterized. Here, by in silico and experimental analyses, we isolated and identified three RgTyDCs (RgTyDC2 to RgTyDC4) in this species; these genes' sequences showed 50.92-82.55% identity, included highly conserved domains with homologues in other plants, classified into two subsets, and encoded proteins that localized to the cytosol. Enzyme kinetic analyses of RgTyDC2 and RgTyDC4 indicated that they both efficiently catalysed L-tyrosine and L-dopa. The overexpression of RgTyDC2 and RgTyDC4 in R. glutinosa, which was associated with enhanced TyDC activity, significantly increased tyramine and dopamine contents, which was positively correlated with improved acteoside production; moreover, the overexpression of RgTyDCs led to upregulated expression of some other genes-related to acteoside biosynthesis. This result suggested that the overexpression of RgTyDCs can positively activate the molecular networks of acteoside pathways, enhancing the accumulation of tyramine and dopamine, and promoting end-product acteoside biosynthesis. Our findings provide an evidence that RgTyDCs play vital molecular roles in acteoside biosynthesis pathways, contributing to the increase in acteoside yield in R. glutinosa.

Parkinson's Disease Medication Alters Small Intestinal Motility and Microbiota Composition in Healthy Rats.

Parkinson's disease (PD) is known to be associated with altered gastrointestinal function and microbiota composition. To date, the effect of PD medication on the gastrointestinal function and microbiota, at the site of drug absorption, the small intestine, has not been studied, although it may represent an important confounder in reported microbiota alterations observed in PD patients. To this end, healthy (non-PD) wild-type Groningen rats were employed and treated with dopamine, pramipexole (in combination with levodopa-carbidopa), or ropinirole (in combination with levodopa-carbidopa) for 14 sequential days. Rats treated with dopamine agonists showed a significant reduction in small intestinal motility and an increase in bacterial overgrowth in the distal small intestine. Notably, significant alterations in microbial taxa were observed between the treated and vehicle groups; analogous to the changes previously reported in human PD versus healthy control microbiota studies. These microbial changes included an increase in Lactobacillus and Bifidobacterium and a decrease in Lachnospiraceae and Prevotellaceae. Markedly, certain Lactobacillus species correlated negatively with levodopa levels in the systemic circulation, potentially affecting the bioavailability of levodopa. Overall, the study highlights a significant effect of PD medication intrinsically on disease-associated comorbidities, including gastrointestinal dysfunction and small intestinal bacterial overgrowth, as well as the gut microbiota composition. The results urge future studies to take into account the influence of PD medication per se when seeking to identify microbiota-related biomarkers for PD. IMPORTANCE Parkinson's disease (PD) is the second most common neurodegenerative disorder and is known to be associated with altered gastrointestinal function and microbiota composition. We previously showed that the gut bacteria harboring tyrosine decarboxylase enzymes interfere with levodopa, the main treatment for PD (S. P. van Kessel, A. K. Frye, A. O. El-Gendy, M. Castejon, A. Keshavarzian, G. van Dijk, and S. El Aidy, Nat Commun 10:310, 2019). Although PD medication could be an important confounder in the reported alterations, its effect, apart from the disease itself, on the microbiota composition or the gastrointestinal function at the site of drug absorption, the small intestine, has not been studied. The findings presented here show a significant impact of commonly prescribed PD medication on the small intestinal motility, small intestinal bacterial overgrowth, and microbiota composition, irrespective of the PD. Remarkably, we observed negative associations between bacterial species harboring tyrosine decarboxylase activity and levodopa levels in the systemic circulation, potentially affecting the bioavailability of levodopa. Overall, this study shows that PD medication is an important factor in determining gastrointestinal motility and, in turn, microbiota composition and may, partly, explain the differential abundant taxa previously reported in the cross-sectional PD microbiota human studies. The results urge future studies to take into account the influence of PD medication on gut motility and microbiota composition when seeking to identify microbiota-related biomarkers for PD.

Overexpression of the tyrosine decarboxylase gene MdTyDC in apple enhances long-term moderate drought tolerance and WUE.

Drought stress affects the apple yield and quality. Tyrosine decarboxylase (TyDC) plays a fundamental role in many secondary metabolite reactions in higher plants (including those involving dopamine). Our aims of this study are: 1) to identify the role of TyDC in dopamine derivative biosynthesis and its function in long-term moderate drought conditions; and 2) to explore the role of MdTyDC in plant growth and development as well as the drought stress response. Wild type and three independently apple plants overexpression of MdTyDC were treated for long-term moderate drought stress. The growth and physiological parameters of apple plant, photosynthetic capacity, antioxidant enzymes activity, water use efficiency (WUE), stomatal behavior, amino acid content and dopamine content were detected under long-term moderate drought stress. Overexpression of MdTyDC (OE) in apple showed better growth performance, higher photosynthetic capacity and higher capacity for photochemical reactions than wild type lines (WT). Under long-term moderate drought stress, OE lines showed higher WUE, increased ABA content, decreased stomatal aperture, higher antioxidant activity, lower accumulation of ROS and increases in amino acids, such as proline, phenylalanine and lysine. In addition, qRT-PCR revealed higher gene expression of MdTyDC and dopamine content in OE compared with WT lines under long-term moderate drought stress. These results indicate that MdTyDC confers long-term moderate drought tolerance by improving photosynthetic capacity, WUE, antioxidant activity, dopamine content and changing the contents of amino acids (such as proline accumulation).

Cloning and characterization of tyrosine decarboxylase (TDC) from Litopenaeus vannamei, and its roles in biogenic amines synthesis, immune regulation, and resistance to Vibrio alginolyticus by RNA interference.

The biogenic amines, tyramine and octopamine, in the octopaminergic synthesis pathway play critical roles in regulating physiological and immunological homeostasis in Litopenaeus vannamei. Tyrosine decarboxylase (TDC) is an enzyme catalyzing the first decarboxylation step in the biosynthesis of tyramine and octopamine. The full-length gene sequence of TDC cloned from the brain of L. vannamei (LvTDC) was predicted to encode a 779-amino acid protein with a pyridoxal-dependent decarboxylase-conserved domain in close phylogenetic relationship with arthropod TDCs. LvTDC gene expression was found to be abundant in nervous thoracic ganglia. RNA interference was used to assess the immune and physiological function of LvTDC. The LvTDC knockdown shrimp revealed significant decreases in the total haemocyte count, hyaline cells, antimicrobial peptides, respiratory bursts, gene expression, respiratory bursts of haemocytes per unit of haemolymph, and phagocytic activity and clearance efficiency toward Vibrio alginolyticus. Furthermore, LvTDC knockdown was accompanied by decreases in octopamine deficiency. In the V. alginolyticus challenge test, the survival rate of LvTDC knockdown shrimp was lower than the shrimp injected with DEPC-water or GAPDH-dsRNA. In conclusion, the cloned LvTDC was responsible for octopaminergic synthesis, which then regulated physiological and immune responses in L. vannamei.

Tyramine synthesis, vesicular packaging, and the SNARE complex function coordinately in astrocytes to regulate Drosophila alcohol sedation.

Identifying mechanisms underlying alcohol-related behaviors could provide important insights regarding the etiology of alcohol use disorder. To date, most genetic studies on alcohol-related behavior in model organisms have focused on neurons, leaving the causal roles of glial mechanisms less comprehensively investigated. Here, we report our studies on the role of Tyrosine decarboxylase 2 (Tdc2), which converts tyrosine to the catecholamine tyramine, in glial cells in Drosophila alcohol sedation. Using genetic approaches that drove transgene expression constitutively in all glia, constitutively in astrocytes and conditionally in glia during adulthood, we found that knockdown and overexpression of Tdc2, respectively, increased and decreased the sensitivity to alcohol sedation in flies. Manipulation of the genes tyramine ß-hydroxylase and tyrosine hydroxylase, which respectively synthesize octopamine and dopamine from tyramine and tyrosine, had no discernable effect on alcohol sedation, suggesting that Tdc2 affects alcohol sedation by regulating tyramine production. We also found that knockdown of the vesicular monoamine transporter (VMAT) and disruption of the SNARE complex in all glia or selectively in astrocytes increased sensitivity to alcohol sedation and that both VMAT and the SNARE complex functioned downstream of Tdc2. Our studies support a model in which the synthesis of tyramine and vesicle-mediated release of tyramine from adult astrocytes regulates alcohol sedation in Drosophila. Considering that tyramine is functionally orthologous to norepinephrine in mammals, our results raise the possibility that gliotransmitter synthesis release could be a conserved mechanism influencing behavioral responses to alcohol as well as alcohol use disorder.

Crystallographic Snapshots of the Dunathan and Quinonoid Intermediates provide Insights into the Reaction Mechanism of Group II Decarboxylases.

PLP-dependent enzymes catalyze a plethora of chemical reactions affecting diverse physiological functions. Here we report the structural determinants of the reaction mechanism in a Group II PLP-dependent decarboxylase by assigning two early intermediates. The in-crystallo complexes of the PLP bound form, and the Dunathan and quinonoid intermediates, allowed direct observation of the active site interactions. The structures reveal that a subtle rearrangement of a conserved Arg residue in concert with a water-mediated interaction with the carboxylate of the Dunathan intermediate, appears to directly stabilize the alignment and facilitate the release of CO2 to yield the quinonoid. Modeling indicates that the conformational change of a dynamic catalytic loop to a closed form controls a conserved network of hydrogen bond interactions between catalytic residues to protonate the quinonoid. Our results provide a structural framework to elucidate mechanistic roles of residues that govern reaction specificity and catalysis in PLP-dependent decarboxylation.

A neurotransmitter produced by gut bacteria modulates host sensory behaviour.

Animals coexist in commensal, pathogenic or mutualistic relationships with complex communities of diverse organisms, including microorganisms1. Some bacteria produce bioactive neurotransmitters that have previously been proposed to modulate nervous system activity and behaviours of their hosts2,3. However, the mechanistic basis of this microbiota-brain signalling and its physiological relevance are largely unknown. Here we show that in Caenorhabditis elegans, the neuromodulator tyramine produced by commensal Providencia bacteria, which colonize the gut, bypasses the requirement for host tyramine biosynthesis and manipulates a host sensory decision. Bacterially produced tyramine is probably converted to octopamine by the host tyramine ß-hydroxylase enzyme. Octopamine, in turn, targets the OCTR-1 octopamine receptor on ASH nociceptive neurons to modulate an aversive olfactory response. We identify the genes that are required for tyramine biosynthesis in Providencia, and show that these genes are necessary for the modulation of host behaviour. We further find that C. elegans colonized by Providencia preferentially select these bacteria in food choice assays, and that this selection bias requires bacterially produced tyramine and host octopamine signalling. Our results demonstrate that a neurotransmitter produced by gut bacteria mimics the functions of the cognate host molecule to override host control of a sensory decision, and thereby promotes fitness of both the host and the microorganism.

Crystal structures clarify cofactor binding of plant tyrosine decarboxylase.

Plant tyrosine decarboxylase (TyrDC) is a group II pyridoxal 5'-phosphate (PLP)-dependent decarboxylase that mainly catalyzes the decarboxylation of tyrosine to tyramine. This is biologically important for diverting essential primary metabolites into secondary metabolic pathways. Intensive studies have characterized the effective of PLP-binding and the substrate specificity of mammalian 3,4-dihydroxyphenyl-l-alanine (Dopa) decarboxylases, a member of group II PLP-dependent decarboxylase. However, the characteristics of PLP binding and substrate specificity of plant TyrDCs remain unknown. In this study, we focus on the PLP binding manner, and determined the crystal structures of the apo and PLP binding form of type II TyrDC from Papaver somniferum (PsTyrDCII and PsTyrDCII-PLP). The structures showed that, unlike mammalian Dopa decarboxylase, the binding of PLP does not induce distinct conformational changes of PsTyrDCII regarding the overall structure, but the PLP binding pocket displays conformational changes at Phe124, His203 and Thr262. Combining structural comparation and the obtained biochemical findings, it is demonstrated that PsTyrDCII does not binds PLP tightly. Such characteristics of PLP binding may be required by its catalytic reaction and substrate binding. The activity of TyrDC probably regulated by the concentration of PLP in cells.

Discovery and inhibition of an interspecies gut bacterial pathway for Levodopa metabolism.

The human gut microbiota metabolizes the Parkinson's disease medication Levodopa (l-dopa), potentially reducing drug availability and causing side effects. However, the organisms, genes, and enzymes responsible for this activity in patients and their susceptibility to inhibition by host-targeted drugs are unknown. Here, we describe an interspecies pathway for gut bacterial l-dopa metabolism. Conversion of l-dopa to dopamine by a pyridoxal phosphate-dependent tyrosine decarboxylase from Enterococcus faecalis is followed by transformation of dopamine to m-tyramine by a molybdenum-dependent dehydroxylase from Eggerthella lenta These enzymes predict drug metabolism in complex human gut microbiotas. Although a drug that targets host aromatic amino acid decarboxylase does not prevent gut microbial l-dopa decarboxylation, we identified a compound that inhibits this activity in Parkinson's patient microbiotas and increases l-dopa bioavailability in mice.

A Neuronal Pathway that Commands Deceleration in Drosophila Larval Light-Avoidance.

When facing a sudden danger or aversive condition while engaged in on-going forward motion, animals transiently slow down and make a turn to escape. The neural mechanisms underlying stimulation-induced deceleration in avoidance behavior are largely unknown. Here, we report that in Drosophila larvae, light-induced deceleration was commanded by a continuous neural pathway that included prothoracicotropic hormone neurons, eclosion hormone neurons, and tyrosine decarboxylase 2 motor neurons (the PET pathway). Inhibiting neurons in the PET pathway led to defects in light-avoidance due to insufficient deceleration and head casting. On the other hand, activation of PET pathway neurons specifically caused immediate deceleration in larval locomotion. Our findings reveal a neural substrate for the emergent deceleration response and provide a new understanding of the relationship between behavioral modules in animal avoidance responses.

Gut bacterial tyrosine decarboxylases restrict levels of levodopa in the treatment of Parkinson's disease.

Human gut microbiota senses its environment and responds by releasing metabolites, some of which are key regulators of human health and disease. In this study, we characterize gut-associated bacteria in their ability to decarboxylate levodopa to dopamine via tyrosine decarboxylases. Bacterial tyrosine decarboxylases efficiently convert levodopa to dopamine, even in the presence of tyrosine, a competitive substrate, or inhibitors of human decarboxylase. In situ levels of levodopa are compromised by high abundance of gut bacterial tyrosine decarboxylase in patients with Parkinson's disease. Finally, the higher relative abundance of bacterial tyrosine decarboxylases at the site of levodopa absorption, proximal small intestine, had a significant impact on levels of levodopa in the plasma of rats. Our results highlight the role of microbial metabolism in drug availability, and specifically, that abundance of bacterial tyrosine decarboxylase in the proximal small intestine can explain the increased dosage regimen of levodopa treatment in Parkinson's disease patients.

Improving Soluble Expression of Tyrosine Decarboxylase from Lactobacillus brevis for Tyramine Synthesis with High Total Turnover Number.

The soluble expression of tyrosine decarboxylase (TDC) in heterologous host is often challenging. Here, acidic condition was found to be favorable for improving the soluble expression of TDC from Lactobacillus brevis in Escherichia coli, while addition of carbohydrates (such as glucose, arabinose, and fructose) was vital for decreasing the insoluble fraction. By simple pH control and addition of glucose, the specific activity of TDC in crude extract was enhanced to 46.3 U mg-1, 3.67-fold of that produced from LB medium. Optimization of the reaction conditions revealed that Tween-80 was effective in improving the tyramine production catalyzed by TDC, especially at high tyrosine loadings. As much as 400 mM tyrosine could be completely converted into tyramine with a substrate to catalyst ratio of 29.0 g g-1 and total turnover number of 23,300. This study provides efficient strategies for the highly soluble expression of TDC and biocatalytic production of tyramine.

Spatiotemporal oscillations of morphinan alkaloids in opium poppy.

Here, a comprehensive endeavor is made to simultaneously scrutinize spatiotemporal oscillations of three imperative morphinan alkaloids (i.e. thebaine, codeine, and morphine) alongside dynamic transcriptional patterns of TYDC, SalAT, COR, T6ODM, and CODM genes in different tissues of Papaver somniferum (i.e. root, bottom part of stem, upper part of stem, leaf, capsule wall, and capsule content) over five distinguished ontogenic stages (i.e. rosette, bud initiation, pendulous bud, flowering, and lancing). Apart from bottom stem and leaf, the maximum thebaine content occurred in lancing stage, while its minimum content did not follow a systematic rhythm, either among six tissues or five various sampling times. Regarding codeine, excepting upper stem, the highest ratios of codeine were observed at flowering and lacing stages, while negligible amounts were overall detected at early stages of plant growth like rosette. Considering morphine, apart from upper stem, it appears that late ontogeneic times including lancing and flowering are the most appropriate phases to achieve high amounts of morphine, while at early stages the aforesaid alkaloid possessed lower accumulation. Furthermore, all the five genes under study, overall, exhibited a variety of transcript levels either among six tissues or five various sampling times. Interestingly, a connection occurred between transcript ratio of SalAT and thebaine content, suggesting that thebaine biosynthesis is coordinated tightly by the enzymatic function of SalAT enzyme. Meanwhile, despite low magnitudes of T6ODM and CODM transcripts in the root-harvested samples at pendulous bud and flowering stages, both codeine and morphine were surprisingly in acceptable quantities, plausibly owing to the translocation of both alkaloids from the producing (source) tissues to the roots (sink), known as a phenomenon of 'source-to-sink transportation'. The results, altogether, could provide us enough information in acquiring new insights towards potential impacts of spatiotemporal oscillations on the magnitudes of all the above-mentioned alkaloids alongside transcription ratios of the key genes in opium poppy.