Nicotinic Pathway Research Articles

Maternal choline supplementation modulates cognition and induces anti-inflammatory signaling in the prefrontal cortices of adolescent rats exposed to maternal immune activation

Maternal infection has long been described as a risk factor for neurodevelopmental disorders, especially autism spectrum disorders (ASD) and schizophrenia. Although many pathogens do not cross the placenta and infect the developing fetus directly, the maternal immune response to them is sufficient to alter fetal neurodevelopment, a phenomenon termed maternal immune activation (MIA). Low maternal choline is also a risk factor for neurodevelopmental disorders, and most pregnant people do not receive enough of it. In addition to its role in neurodevelopment, choline is capable of inducing anti-inflammatory signaling through a nicotinic pathway. Therefore, it was hypothesized that maternal choline supplementation would blunt the neurodevelopmental impact of MIA in offspring through long-term instigation of cholinergic anti-inflammatory signaling.To model MIA in rats, the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)) was used to elicit a maternal antiviral innate immune response in dams both with and without choline supplementation. Offspring were reared to both early and late adolescent stages (postnatal days 28 and 50, respectively), where anxiety-related behaviors and cognition were examined. After behavioral testing, animals were euthanized, and their prefrontal cortices (PFCs) were collected for analysis. MIA offspring demonstrated sex-specific patterns of altered cognition and repetitive behaviors, which were modulated by maternal choline supplementation. Choline supplementation also bolstered anti-inflammatory signaling in the PFCs of MIA animals at both early and late adolescent stages. These findings suggest that maternal choline supplementation may be sufficient to blunt some of the behavioral and neurobiological impacts of inflammatory exposures in utero, indicating that it may be a cheap, safe, and effective intervention for neurodevelopmental disorders.

Partial desensitization of MYC2 transcription factor alters the interaction with jasmonate signaling components and affects specialized metabolism

The activity of bHLH transcription factor MYC2, a key regulator in jasmonate signaling and plant specialized metabolism, is sensitive to repression by JASMONATE-ZIM-domain (JAZ) proteins and co-activation by the mediator subunit MED25. The substitution of a conserved aspartic acid (D) to asparagine (N) in the JAZ-interacting domain (JID) of Arabidopsis MYC2 affects interaction with JAZ, although the mechanism remained unclear. The effects of the conserved residue MYC2D128 on interaction with MED25 have not been investigated. Using tobacco as a model, we generated all possible substitutions of aspartic acid 128 (D128) in NtMYC2a. NtMYC2aD128N partially desensitized the repression by JAZ proteins, while strongly interacting with MED25, resulting in increased expression of nicotine pathway genes and nicotine accumulation in tobacco hairy roots overexpressing NtMYC2aD128N compared to those overexpressing NtMYC2a. The proline substitution, NtMYC2aD128P, negatively affected transactivation and abolished the interaction with JAZ proteins and MED25. Structural modeling and simulation suggest that the overall stability of the JID binding pocket is a predominant cause for the observed effects of substitutions at D128. The D128N substitution has an overall stabilizing effect on the binding pocket, which is destabilized by D128P. Our study offers an innovative tool to increase the production of plant natural products.

Investigating nicotine pathway-related long non-coding RNAs in tobacco.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200bp with low or no protein-coding ability, which play essential roles in various biological processes in plants. Tobacco is an ideal model plant for studying nicotine biosynthesis and metabolism, and there is little research on lncRNAs in this field. Therefore, how to take advantage of the mature tobacco system to profoundly investigate the lncRNAs involved in the nicotine pathway is intriguing. By exploiting 549 public RNA-Seq datasets of tobacco, 30,212 lncRNA candidates were identified, including 24,084 large intervening non-coding RNAs (lincRNAs), 5,778 natural antisense transcripts (NATs) and 350 intronic non-coding RNAs (incRNAs). Compared with protein-coding genes, lncRNAs have distinct properties in terms of exon number, sequence length, A/U content, and tissue-specific expression pattern. lincRNAs showed an asymmetric evolutionary pattern, with a higher proportion (68.71%) expressed from the Nicotiana sylvestris (S) subgenome. We predicted the potential cis/trans-regulatory effects on protein-coding genes. One hundred four lncRNAs were detected as precursors of 30 known microRNA (miRNA) family members, and 110 lncRNAs were expected to be the potential endogenous target mimics for 39 miRNAs. By combining the results of weighted gene co-expression network analysis with the differentially expressed gene analysis of topping RNA-seq data, we constructed a sub-network containing eight lncRNAs and 25 nicotine-related coding genes. We confirmed that the expression of seven lncRNAs could be affected by MeJA treatment and may be controlled by the transcription factor NtMYC2 using a quantitative PCR assay and gene editing. The results suggested that lncRNAs are involved in the nicotine pathway. Our findings further deepened the understanding of the features and functions of lncRNAs and provided new candidates for regulating nicotine biosynthesis in tobacco.

Clearing the air: clarifying the causal role of smoking in mental illness.

Clearing the air: clarifying the causal role of smoking in mental illness.

Memantine has a nicotinic neuroprotective pathway in acute hippocampal slices after an NMDA insult

Memantine is a non-competitive antagonist with a moderate affinity to the N-methyl-d-Aspartate (NMDA) receptor. The present study assessed memantine's neuroprotective activity using electrophysiology of ex-vivo hippocampal slices. Interestingly, a nicotinic component was necessary for memantine's neuroprotection (NP). Memantine demonstrated a bell-shaped dose-response curve of NP against NMDA. Memantine was neuroprotective at concentrations below 3 μM, but the NP declined at higher concentrations (>3 μM) when memantine inhibits the NMDA receptor. Additional evidence that memantine NP is mediated by an alternate mechanism independent of the inhibition of the NMDA receptor is supported by its ability to protect neurons when applied before or after the NMDA insult and in the presence of D(−)-2-Amino-5-phosphonopentanoic acid (APV), the standard NMDA receptor inhibitor. We found several similarities between the memantine NP mechanism and the neuroprotective nicotinic drug, the 4R cembranoid. Memantine's NP requires the release of acetylcholine, the activation of α4β2, and is independent of MEK/MAPK signaling. Both 4R and memantine require the activation of PI3K/AKT for NP against NMDA-mediated excitotoxicity, although at different concentrations. In conclusion, our studies show memantine is neuroprotective through a nicotinic pathway, similar to the nicotinic drug 4R. This information leads to a better understanding of memantine's mechanisms of action and explains its dose-dependent effectiveness in Alzheimer's and other neurological disorders.

Abstract PD6-06: Comprehensive characterization of neurotransmitters and neuronal signaling gene alterations in invasive breast cancers

Abstract BACKGROUNDIt has been reported that the sympathetic nervous system and associated neurotransmitters (NTs) play a pivotal role in driving breast cancer (BC) tumorigenesis and metastasis, however, comprehensive characterization of these pathways in BC is lacking. The purpose of this study was to retrospectively characterize NTs and neuronal signaling (NTNS) gene alterations in a large real-world BC cohort. METHODS A total of 6464 BC tumors were analyzed by next generation sequencing (NextSeq, 592 genes and WES, NovaSEQ, 720 genes) and whole transcriptome sequencing (WTS, NovaSeq) at Caris Life Sciences. Gene set variation analysis (GSVA) scores were calculated (positive: higher gene expressions in a selected gene set compared to genes outside that gene set in each tumor specimen, vice versa for negative) to assess expression of major NTNS genes, including GABA, nicotinic (NIC), muscarinic (MUS), dopamine (DA), reelin (RELN), and glial cell line-derived neurotrophic factor (GDNF). GSVA scores were compared by histologic subtype, primary or metastatic site, and hormone receptor (HR) and HER2 status with corrected Wilcoxon-Mann-Whitney testing. All significance levels were p<0.01. RESULTS The 6464 BC specimens in this cohort included 2520 primary sites and 3944 metastasis (mets) (liver: 1012; lymph node: 714; bone: 575; lung: 420; brain: 196). Predictive biomarker status in this cohort was HR+/HER2-: 3705; HR+/HER2+: 238; HR-/HER2+: 189; TNBC: 1654. Invasive ductal carcinomas (IDC) were the most common histologic subtype and demonstrated significantly higher GSVA scores for RELN and NIC pathways with respect to invasive lobular carcinomas (ILC) (Table). TNBC tumors had significantly higher enrichment overall (GABA, -0.04 vs -0.14; RELN, -0.05 vs -0.31; DA, -0.03 vs -0.08; MUS, 0.13 vs -0.16; NIC, 0.01 vs -0.12; and GDNF, 0.04 vs -0.04). HR-/HER2+ had significantly higher scores in GABA, -0.04 vs -0.14; RELN, -0.03 vs -0.31; MUS, 0.12 vs -0.16; and NIC, -0.01 vs -0.12 genes. Brain mets had significantly enriched pathway scores for GABA, 0.30 vs -0.13; MUS, 0.15 vs -0.08; and NIC, 0.13 vs -0.09 compared to primary tumors. Similarly, GABA,0.09 vs -0.13; DA, 0.07 vs -0.05; MUS, 0.17 vs -0.08; and NIC, 0.02 vs -0.09 pathways were enriched in bone mets compared to those from primary tumors. CONCLUSION Our results demonstrate that NTNS pathways are significantly enriched in IDC, TNBC tumors, and particularly in brain and bone mets. Our data advance the current understanding of the role of NTNS pathways in BC tumorigenesis and metastasis. Further investigation on genetic. determinants and signaling alternations associated with the observed NTNS pathway deregulation is warranted and could inform the development of novel therapeutic strategies. Significant comparisons with Bonferroni corrected p values are shown with an asterisk. Citation Format: Irene Kang, Krutika Deshpande, Sarah Persing, Jun Yin, Joanne Xiu, Wolfgang Michael Korn, Jia Zeng, Evanthia T Roussos-Torres, Janice Lu, Darcy Spicer, Stephen F Sener, Antoinette R Tan, Ashley Sumrall, David SB Hoon, Cynthia X Ma, Carey K Anders, Heather L McArthur, Reva Basho, Heinz-Josef Lenz, Josh Neman. Comprehensive characterization of neurotransmitters and neuronal signaling gene alterations in invasive breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-06.

NtMYB305a binds to the jasmonate-responsive GAG region of NtPMT1a promoter to regulate nicotine biosynthesis.

MYB transcription factors play essential roles in regulating plant secondary metabolism and jasmonate (JA) signaling. Putrescine N-methyltransferase is a key JA-regulated step in the biosynthesis of nicotine, an alkaloidal compound highly accumulated in Nicotiana spp. Here we report the identification of NtMYB305a in tobacco (Nicotiana tabacum) as a regulatory component of nicotine biosynthesis and demonstrate that it binds to the JA-responsive GAG region, which comprises a G-box, an AT-rich motif, and a GCC-box-like element, in the NtPMT1a promoter. Yeast one-hybrid analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed that NtMYB305a binds to the GAG region in vitro and in vivo. Binding specifically occurs at the ∼30-bp AT-rich motif in a G/C-base-independent manner, thus defining the AT-rich motif as previously unknown MYB-binding element. NtMYB305a localized in the nucleus of tobacco cells where it is capable of activating the expression of a 4×GAG-driven GUS reporter in an AT-rich motif-dependent manner. NtMYB305a positively regulates nicotine biosynthesis and the expression of NtPMT and other nicotine pathway genes. NtMYB305a acts synergistically with NtMYC2a to regulate nicotine biosynthesis, but no interaction between these two proteins was detected. This identification of NtMYB305a provides insights into the regulation of nicotine biosynthesis and extends the roles played by MYB transcription factors in plant secondary metabolism.

Transcriptomic analysis provides insights into the AUXIN RESPONSE FACTOR 6-mediated repression of nicotine biosynthesis in tobacco (Nicotiana tabacum L.).

NtARF6 overexpression represses nicotine biosynthesis in tobacco. Transcriptome analysis suggests that NtARF6 acts as a regulatory hub that connect different phytohormone signaling pathways to antagonize the jasmonic acid-induced nicotine biosynthesis. Plant specialized metabolic pathways are regulated by a plethora of molecular regulators that form complex networks. In Nicotiana tabacum, nicotine biosynthesis is regulated by transcriptional activators, such as NtMYC2 and the NIC2-locus ERFs. However, the underlying molecular mechanism of the regulatory feedback is largely unknown. Previous research has shown that NbARF1, a nicotine synthesis repressor, reduces nicotine accumulation in N. benthamiana. In this study, we demonstrated that overexpression of NtARF6, an ortholog of NbARF1, was able to reduce pyridine alkaloid accumulation in tobacco. We found that NtARF6 could not directly repress the transcriptional activities of the key nicotine pathway structural gene promoters. Transcriptomic analysis suggested that this NtARF6-induced deactivation of alkaloid biosynthesis might be achieved by the antagonistic effect between jasmonic acid (JA) and other plant hormone signaling pathways, such as ethylene (ETH), salicylic acid (SA), abscisic acid (ABA). The repression of JA biosynthesis is accompanied by the induction of ETH, ABA, and SA signaling and pathogenic infection defensive responses, resulting in counteracting JA-induced metabolic reprogramming and decreasing the expression of nicotine biosynthetic genes in vivo. This study provides transcriptomic evidence for the regulatory mechanism of the NtARF6-mediated repression of alkaloid biosynthesis and indicates that this ARF transcription factor might act as a regulatory hub to connect different hormone signaling pathways in tobacco.

A de novo regulation design shows an effectiveness in altering plant secondary metabolism

IntroductionTranscription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. ObjectivesWe selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. MethodsG-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. ResultsPAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. ConclusionG-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Time-Dependent Analysis of Paenarthrobacter nicotinovorans pAO1 Nicotine-Related Proteome.

Paenarthrobacter nicotinovorans is a soil Gram-positive nicotine-degrading microorganism (NDM) that harbors a 165 kb pAO1 catabolic megaplasmid. The nicotine catabolic genes on pAO1 have been sequenced, but not all the details on the regulation and interplay of this pathway with the general metabolism of the cell are available. To address this issue at the protein level, a time-based shotgun proteomics study was performed. P. nicotinovorans was grown in the presence or absence of nicotine, and the cells were harvested at three different time intervals: 7, 10, and 24 h after inoculation. The cells were lysed, separated on SDS-PAGE, and digested by in-gel digestion using trypsin, and the resulting peptide mixture was analyzed using nanoliquid chromatography tandem mass spectrometry. We found an extensive number of proteins that are both plasmidal- and chromosomal-encoded and that work together in the energetic metabolism via the Krebs cycle and nicotine pathway. These data provide insight into the adaptation of the bacterial cells to the nicotine metabolic intermediates and could serve as a basis for future attempts to genetically engineer the pAO1-encoded catabolic pathway for increased bioremediation efficiency or for the production of valuable chemicals. The mass-spectrometry-based proteomics data have been deposited to the PRIDE partner repository with the data set identifier PXD012577.

Impact of nicotine pathway downregulation on polyamine biosynthesis and leaf ripening in tobacco.

Traditional breeding and molecular approaches have been used to develop tobacco varieties with reduced nicotine and secondary alkaloid levels. However, available low‐alkaloid tobacco varieties have impaired leaf quality likely due to the metabolic consequences of nicotine biosynthesis downregulation. Recently, we found evidence that the unbalanced crosstalk between nicotine and polyamine pathways is involved in impaired leaf ripening of a low‐alkaloid (LA) Burley 21 line having a mutation at the Nic1 and Nic2 loci, key biosynthetic regulators of nicotine biosynthesis. Since the Nic1 and Nic2 loci are comprised of several genes, all phenotypic changes seen in LA Burley 21 could be due to a mixture of genetics‐based responses. Here, we investigated the commercial burley variety TN90 LC and its transgenic versions with only one downregulated gene, either putrescine methyl transferase (PMT‐RNAi) or PR50‐protein (PR50‐RNAi). Nicotine levels of cured lamina of TN90 LC, TN90 PMT‐RNAi and TN90 PR50‐RNAi, were 70.5 ± 3.8, 2.4 ± 0.5, and 6.0 ± 1.1 mg/g dry weight, respectively. Low‐alkaloid transgenic lines showed delayed leaf maturation and impaired leaf quality. We analyzed polyamine contents and ripening markers in wild‐type TN90 control plants (WT) and the two transgenic lines. The ripening markers revealed that the PMT‐RNAi line showed the most pronounced impaired leaf maturation phenotype at harvest, characterized by higher chlorophyll (19%) and glucose (173%) contents and more leaf mesophyll cells per area (25%), while the ripening markers revealed that maturation of PR50‐RNAi plants was intermediate between PMT‐RNAi and WT lines. Comparative polyamine analyses showed an increase in free and conjugated polyamines in roots of both transgenic lines, this being most pronounced in the PMT‐RNAi plants. For PMT‐RNAi plants, there were further perturbations of polyamine content in the leaves, which mirrored the general phenotype, as PR50‐RNAi transgenic plants looked more similar to the WT than PMT‐RNAi transgenic plants. Activity of ornithine decarboxylase, the enzyme that catalyzes the committing step of polyamine biosynthesis, was significantly higher in roots and mature leaves of PMT‐RNAi plants in comparison to WT, while there was no increase observed for arginine decarboxylase. Treatment of both transgenic lines with polyamine biosynthesis inhibitors decreased the polyamine content and ameliorated the phenotype, confirming the intricate interplay of polyamine and nicotine biosynthesis in tobacco and the influence of this interplay on leaf ripening.

Protein phosphatase NtPP2C2b and MAP kinase NtMPK4 act in concert to modulate nicotine biosynthesis.

Protein phosphatases (PPs) and protein kinases (PKs) regulate numerous developmental, defense, and phytohormone signaling processes in plants. However, the underlying regulatory mechanism governing biosynthesis of specialized metabolites, such as alkaloids, by the combined effects of PPs and PKs, is insufficiently understood. Here, we report the characterization of a group B protein phosphatase type 2C, NtPP2C2b, that likely acts upstream of the NICOTINE2 locus APETALA 2/Ethylene Response Factors (AP2/ERFs), to regulate nicotine biosynthesis in tobacco. Similar to the nicotine pathway genes, NtPP2C2b is highly expressed in roots and induced by jasmonic acid (JA). Overexpression of NtPP2C2b in transgenic hairy roots or stable transgenic tobacco plants repressed nicotine pathway gene expression and reduced nicotine accumulation. Additionally, transient overexpression of NtPP2C2b, together with the NtERF221, repressed transactivation of the quinolinate phosphoribosyltransferase promoter in tobacco cells. We further demonstrate that the JA-responsive tobacco mitogen-activated protein kinase (MAPK) 4 interacts with NtPP2C2b in yeast and plant cells. Conditional overexpression of NtMPK4 in tobacco hairy roots up-regulated nicotine pathway gene expression and increased nicotine accumulation. Our findings suggest that a previously uncharacterized PP-PK module acts to modulate alkaloid biosynthesis, highlighting the importance of post-translational control in the biosynthesis of specialized plant metabolites.

Degradome, small RNAs and transcriptome sequencing of a high-nicotine cultivated tobacco uncovers miRNA\u2019s function in nicotine biosynthesis

Tobacco (Nicotiana tabacum) is considered as the model plant for alkaloid research, of which nicotine accounts for 90%. Many nicotine biosynthetic genes have been identified and were known to be regulated by jasmonate-responsive transcription factors. As an important regulator in plant physiological processes, whether small RNAs are involved in nicotine biosynthesis is largely unknown. Here, we combine transcriptome, small RNAs and degradome analysis of two native tobacco germplasms YJ1 and ZY100 to investigate small RNA’s function. YJ1 leaves accumulate twofold higher nicotine than ZY100. Transcriptome analysis revealed 3,865 genes which were differently expressed in leaf and root of two germplasms, including some known nicotine and jasmonate pathway genes. By small RNA sequencing, 193 miRNAs were identified to be differentially expressed between YJ1 and ZY100. Using in silico and degradome sequencing approaches, six nicotine biosynthetic genes and seven jasmonate pathway genes were predicted to be targeted by 77 miRNA loci. Three pairs among them were validated by transient expression in vivo. Combined analysis of degradome and transcriptome datasets revealed 51 novel miRNA-mRNA interactions that may regulate nicotine biosynthesis. The comprehensive analysis of our study may provide new insights into the regulatory network of nicotine biosynthesis.

Cholinergic-induced anion secretion in murine jejunal enteroids involves synergy between muscarinic and nicotinic pathways.

Acetylcholine induces robust electrogenic anion secretion in mammalian intestine and it has long been hypothesized that it mediates the epithelial response through the M3 and, to a lesser extent, the M1 muscarinic receptors in the mouse. However, nicotinic receptors have recently been identified in intestinal enterocytes by quantitative real-time (qRT)-PCR/RNAseq, although any direct influence on intestinal transport has not been identified. We tested the hypothesis that cholinergic-induced anion secretion in the intestine is a result of both muscarinic and nicotinic pathways that are intrinsic to the intestinal epithelia. We developed a method to generate mouse jejunal enteroid monolayers which were used to measure active electrogenic anion secretion by the Ussing chamber/voltage-clamp technique. Here, we show that the cholinergic agonist carbachol (CCh) and the muscarinic agonist bethanechol (BCh) stimulate short-lived, concentration-dependent anion secretion in the epithelial cell-only enteroid monolayers. The muscarinic antagonist atropine completely inhibited CCh- and BCh-induced secretion, while the nicotinic antagonist hexamethonium reduced the CCh response by ~45%. While nicotine alone did not alter anion secretion, it increased the BCh-induced increase in short-circuit current in a concentration-dependent manner; this synergy was prevented by pretreatment with hexamethonium. In addition to being sensitive to hexamethonium, monolayers express both classes of cholinergic receptor by qRT-PCR, including 13 of 16 nicotinic receptor subunits. Our findings indicate that an interaction between muscarinic and nicotinic agonists synergistically stimulates anion secretion in mouse jejunal epithelial cells and identify a role for epithelial nicotinic receptors in anion secretion.

Genetic Manipulation of Transcriptional Regulators Alters Nicotine Biosynthesis in Tobacco.

The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.

Nicotine promotes activation of human pancreatic stellate cells through inducing autophagy via α7nAChR-mediated JAK2/STAT3 signaling pathway

AimPancreatic stellate cells (PSCs) are the main functional cells leading to pancreatic fibrosis. Nicotine is widely considered as an independent risk factor of pancreatic fibrosis, but the mechanism is still unclear. Our study was aimed to explore the effects of nicotine on human pancreatic stellate cells (hPSCs) and involved pathways. Materials and methodsPrimary human PSCs were cultured and treated with nicotine (0.1 μM and 1 μM). The proliferation, apoptosis, α-SMA expression, extracellular matrix metabolism and autophagy of hPSCs were detected by CCK-8 assay, flow cytometry, real-time PCR and Western blotting analysis. The α7nAChR-mediated JAK2/STAT3 signaling pathway was also examined, and an α7nAChR antagonist α-bungarotoxin (α-BTX) was used to perform inhibition experiments. Key findingsThe proliferation, α-SMA expression and autophagy of hPSCs were significantly promoted by 1 μM nicotine. Meanwhile, the apoptosis of hPSCs was significantly reduced. The extracellular matrix metabolism of hPSCs was also regulated by nicotine. Moreover, the α7nAChR-mediated JAK2/STAT3 signaling pathway was activated by nicotine, this pathway and effects of nicotine can be blocked by α-BTX. SignificanceOur finding suggests that nicotine can promote activation of human pancreatic stellate cells (hPSCs) through inducing autophagy via α7nAChR-mediated JAK2/STAT3 signaling pathway, providing a new insight into the mechanisms by which nicotine affects pancreatic fibrosis.

Associations of CYP2A6 Gene Polymorphism with Smoking Status Among Jordanians: Gender-Related Differences.

Cytochrome P450 2A6 enzyme (CYP2A6), an essential hepatic enzyme involved in the metabolism of drugs, is responsible for a major metabolic pathway of nicotine. Variation in the activity of polymorphic CYP2A6 alleles has been implicated in inter-individual differences in nicotine metabolism. The objective of the current study was to assess the association between the smoking status and the cytochrome P450 2A6 enzyme (CYP2A6) genotype in Jordanians. In the current study, 218 (117 Male and 101 female) healthy unrelated Jordanian volunteers were recruited. CYP2A6*1B, CYP2A6*4 and CYP2A6*9 were determined and correlated with subject smoking status. *1A/*1A was the most common genetic polymorphism in the overall study population, with no significant frequency differences between smokers and non-smokers. When the population was divided according to gender, only male smokers showed a significant correlation between genotype and smoking status. Considering the CYP2A6*9 genotype, the results showed differences in distribution between smokers and non-smokers, but only women showed a significant association between CYP2A6*9 allele genotype and smoking status. The results of this study show that there is a significant association between CYP2A6*9 genotype and smoking status. They also show that CYP2A6 genotype is significantly influenced by gender.

Exploration of Nicotine Metabolism in Paenarthrobacter nicotinovorans pAO1 by Microbial Proteomics.

Proteomics, or the large-scale study of proteins, is a post-genomics field that, together with transcriptomics and metabolomics, has moved the study of bacteria to a new era based on system-wide understanding of bacterial metabolic and regulatory networks. The study of bacterial proteins or microbial proteomics has found a wide array of applications in many fields of microbiology, from food, clinical, and industrial microbiology to microbial ecology and physiology. The current chapter makes a brief technical introduction into the available approaches for the large-scale study of bacterial proteins using mass-spectrometry. Furthermore, the advantages and disadvantages of using bacteria for proteomics studies are indicated as well as several example studies where MS-based bacterial proteomics had a fundamental role in deciphering the scientific question. Finally, the proteomics study of nicotine catabolism in Paenarthrobacter nicotinovorans pAO1 using nanoLC-MS/MS is given as an in-depth example for possible applications of microbial proteomics.The nicotine degradation pathway functioning in Paenarthrobacter nicotinovorans is encoded by the catabolic megaplasmid pAO1 that contains about 40 nicotine-related genes making out the nic-gens cluster. Despite the promising biotechnological potential for the production of green-chemicals, only half of the nic-genes have been experimentally linked to nicotine. In an attempt to systematically identify all the proteins involved in nicotine degradation, a gel-based proteomics approach was used to identify a total of 801 proteins when Paenarthrobacter nicotinovorans was grown on three carbon sources: citrate, nicotine and nicotine and citrate. The differences in protein abundance showed that the bacterium is able to switch between deamination and demethylation in the lower nicotine pathway based on the available C source. Several pAO1 putative genes including a hypothetical polyketide cyclase have been shown to have a nicotine-dependent expression and we hypothesize that the polyketide cyclase would hydrolyze the N1-C6 bond from the pyridine ring with the formation of alpha-keto-glutaramate. Two chromosomal proteins, a malate dehydrogenase, and a D-3-phosphoglycerate dehydrogenase were shown to be strongly upregulated when nicotine was the sole carbon source and could be related to the production of the alpha-keto-glutaramate by the polyketide cyclase.

Cloning and functional characterization of quinolinic acid phosphoribosyl transferase (QPT) gene of Nicotiana tabacum.

The quinolinate phosphoribosyl transferase (QPT) is a key enzyme that converts quinolinic acid into nicotinic acid mononucleotide. The QPT gene plays an essential role in the pyridine nucleotide cycle as well as in the biosynthetic pathway of the alkaloid nicotine. However, a clear role for QPT is yet to be characterized to validate the actual function of this gene in planta. In this study, an RNA interference (RNAi) approach was used to reveal the functional role of QPT. Transformation and analysis of the hairy roots (HRs) of the Nicotiana leaf explants was used, followed by plant regeneration and analysis. High-performance liquid chromatography (HPLC) analysis of the HRs and of the regenerated plants both revealed altered alkaloid biosynthetic cycle, with a substantially reduced content of nicotine and anabasine. The transgenic plants exhibited a significantly altered phenotype and growth pattern. Also, silencing of QPT led to a decrease in chlorophyll content, maximum quantum efficiency of PSII, net CO2 assimilation and starch content. Results clearly demonstrated that QPT was not only involved in the biosynthetic pathway of the alkaloids but also affected plant growth and development. Our results provide information to be considered when trying to engineer the secondary metabolite quality and quantity.

Genomic Insights into the Evolution of the Nicotine Biosynthesis Pathway in Tobacco.

In tobacco (Nicotiana tabacum), nicotine is the predominant alkaloid. It is produced in the roots and accumulated mainly in the leaves. Jasmonates play a central signaling role in damage-induced nicotine formation. The genome sequence of tobacco provides us an almost complete inventory of structural and regulatory genes involved in nicotine pathway. Phylogenetic and expression analyses revealed a series of structural genes of the nicotine pathway, forming a regulon, under the control of jasmonate-responsive ETHYLENE RESPONSE FACTOR (ERF) transcription factors. The duplication of NAD and polyamine metabolic pathways and the subsequent recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon were suggested to be the drivers for pyridine and pyrrolidine ring formation steps early in the pathway. Transcriptional regulation by ERF and cooperatively acting MYC2 transcription factors are corroborated by the frequent occurrence of cognate cis-regulatory elements of the factors in the promoter regions of the downstream structural genes. The allotetraploid tobacco has homologous clusters of ERF genes on different chromosomes, which are possibly derived from two ancestral diploids and include either nicotine-controlling ERF189 or ERF199 A large chromosomal deletion was found within one allele of the nicotine-controlling NICOTINE2 locus, which is part of one of the ERF gene clusters, and which has been used to breed tobacco cultivars with a low-nicotine content.