Biosynthesis Of Amides Research Articles

Assessing the impact of Chlorantraniliprole (CAP) pesticide stress on oilseed rape (Brassia campestris L.): Residue dynamics, enzyme activities, and metabolite profiling

This study investigates the effects of chlorantraniliprole (CAP) pesticide stress on oilseed rape through comprehensive pot experiments. Assessing CAP residue variations in soil and oilseed rape (Brassia campestris L.), enzyme activities (POD, CPR, GST), and differential metabolites, we unveil significant findings. The average CAP residue levels were 18.38–13.70 mg/kg in unplanted soil, 9.94–6.30 mg/kg in planted soil, and 0–4.18 mg/kg in oilseed rape samples, respectively. Soil microbial influences and systemic pesticide translocation into oilseed rape contribute to CAP residue variations. Under the influence of CAP stress, oilseed rape displays escalated enzyme activities (POD, CPR, GST) and manifests 57 differential metabolites. Among these, 32 demonstrate considerable downregulation, mainly impacting amino acids and phenolic compounds, while 25 exhibit noteworthy overexpression, primarily affecting flavonoid compounds. This impact extends to 24 metabolic pathways, notably influencing amide biosynthesis, as well as arginine and proline metabolism. These findings underscore the discernible effects of CAP pesticide stress on oilseed rape.

Profiling Circular RNA Expression Across 11 Tissues in the Marine Gastropod Rapana venosa

The carnivorous marine gastropod, Rapana venosa, also known as the veined rapa whelk, has recently gained considerable attention due to its dual significance as both a valuable commercial seafood resource and a biological invader with widespread ecological implications. Significant efforts have been made to investigate the molecular pathways governing the specific adaptations in R. venosa. However, the regulatory mechanisms of these pathways remain largely unclear. Circular RNA (circRNA), a newly‐recognized class of non‐coding molecules, plays crucial roles in post‐transcriptional regulation, impacting various fundamental bioprocesses including development, cell cycle, immunity and disease pathogenesis. In this study, we conducted a comprehensive genome wide profiling of circRNAs across 11 tissues of R. venosa. We identified a total of 1214 circRNA genes across tissues, with 640 regarded as high‐confidence candidates. circRNAs displayed overall low expression levels, diverse isoform types and dynamic expression patterns across various tissues. Our comparative analyses revealed a few circRNA genes with a great diversity of isoforms and abundant expression in the salivary gland, suggesting potential roles in the feeding process of R. venosa. Furthermore, Gene Ontology (GO) enrichment analysis indicated the potential involvement of commonly expressed circRNAs in fundamental cellular processes, including cell division, amide biosynthesis and cellular response to hormone stimuli. Our study describes a preliminary examination of circRNAs across various tissues of R. venosa, offering a foundation for future research into the molecular mechanisms that influence the biology, ecology and behaviour of marine gastropods.

Characterization of Chromatin Accessibility in Fetal Bovine Chondrocytes.

Despite significant advances of the bovine epigenome investigation, new evidence for the epigenetic basis of fetal cartilage development remains lacking. In this study, the chondrocytes were isolated from long bone tissues of bovine fetuses at 90 days. The Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq) and transcriptome sequencing (RNA-seq) were used to characterize gene expression and chromatin accessibility profile in bovine chondrocytes. A total of 9686 open chromatin regions in bovine fetal chondrocytes were identified and 45% of the peaks were enriched in the promoter regions. Then, all peaks were annotated to the nearest gene for Gene Ontology (GO) and Kyoto Encylopaedia of Genes and Genomes (KEGG) analysis. Growth and development-related processes such as amide biosynthesis process (GO: 0043604) and translation regulation (GO: 006417) were enriched in the GO analysis. The KEGG analysis enriched endoplasmic reticulum protein processing signal pathway, TGF-β signaling pathway and cell cycle pathway, which are closely related to protein synthesis and processing during cell proliferation. Active transcription factors (TFs) were enriched by ATAC-seq, and were fully verified with gene expression levels obtained by RNA-seq. Among the top50 TFs from footprint analysis, known or potential cartilage development-related transcription factors FOS, FOSL2 and NFY were found. Overall, our data provide a theoretical basis for further determining the regulatory mechanism of cartilage development in bovine.

Analysis of reproduction-related transcriptomes on pineal-hypothalamic-pituitary-ovarian tissues during estrus and anestrus in Tan sheep.

Seasonal estrus is an important factor limiting the fertility of some animals such as sheep. Promoting estrus in the anestrus season is one of the major ways in improving the fecundity of seasonally breeding animals. The pineal-hypothalamus-pituitary-ovary (PHPO) axis plays a decisive role in regulating animal reproduction. However, the molecular mechanisms by which the PHPO axis regulates seasonal reproduction in animals are not well understood, especially in Tan sheep. To this end, we collected pineal, hypothalamus, pituitary and ovary tissues from Tan sheep during estrus and anestrus for RNA-Sequencing, and performed bioinformatics analysis on the entire regulatory axis of the pineal-hypothalamic-pituitary-ovary (PHPO). The results showed that 940, 1,638, 750, and 971 DEGs (differentially expressed genes, DEGs) were identified in pineal, hypothalamus, pituitary and ovary, respectively. GO analysis showed that DEGs from PHPO axis-related tissues were mainly enriched in "biological processes" such as transmembrane transport, peptide and amide biosynthesis and DNA synthesis. Meanwhile, KEGG enrichment analysis showed that the bile acid secretion pathway and the neuroactive ligand-receptor interaction pathway were significantly enriched. Additionally, four potential candidate genes related to seasonal reproduction (VEGFA, CDC20, ASPM, and PLCG2) were identified by gene expression profiling and protein-protein interaction (PPI) analysis. These findings will contribute to be better understanding of seasonal reproduction regulation in Tan sheep and will serve as a useful reference for molecular breeding of high fertility Tan sheep.

Ferrous sulfate efficiently kills Vibrio parahaemolyticus and protects salmon sashimi from its contamination

The primary seafood-borne pathogen Vibrio parahaemolyticus seriously threats the health of consumers preferring raw-fish products, becoming a global concern in food safety. In the present study, we found ferrous sulfate (FeSO4), a nutritional iron supplement, could efficiently induce the death of V. parahaemolyticus. Further, the bactericidal mechanisms of FeSO4 were explored. With a fluorescent probe of Fe2+, a significant influx of Fe2+ was determined in V. parahaemolyticus exposed to FeSO4, and the addition of an intracellular Fe2+ chelator was able to block the cell death. This suggested that cell death in V. parahaemolyticus induced by FeSO4 was dependent on the influx of Fe2+. It was intriguing that we did not observe the eruption of reactive oxygen species (ROS) and lipid hydroperoxides by Fe2+, but the application of liproxstatin-1 (a ferroptosis inhibitor) significantly modified the occurrence of cell death in V. parahaemolyticus. These results suggested FeSO4-induced cell death in V. parahaemolyticus be a ferroptosis differing from that in mammalian cells. Through transcriptome analysis, it was discovered that the exposure of FeSO4 disturbed considerable amounts of gene expression in V. parahaemolyticus including those involved in protein metabolism, amide biosynthesis, two-component system, amino acid degradation, carbon metabolism, citrate cycle, pyruvate metabolism, oxidative phosphorylation, and so on. These data suggested that FeSO4 was a pleiotropic antimicrobial agent against V. parahaemolyticus. Notably, FeSO4 was able to eliminate V. parahaemolyticus in salmon sashimi as well, without affecting the color, texture, shearing force, and sensory characteristics of salmon sashimi. Taken together, our results deciphered a unique ferroptosis in V. parahaemolyticus by FeSO4, and highlighted its potential in raw-fish products to control V. parahaemolyticus.

Screening and characterization of a nitrilase with significant nitrile hydratase activity.

We screened nitrilases with significant nitrile hydratase activity to exploit their potential in benzylic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on benzylic amide production by nitrilase. A sequence-based screening method using previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as "amide-formation hotspots") as molecular probes to identify putative amide-forming nitrilases. Based on the previously reported "amide-formation hotspots," we identified a nitrilase NitPG from Paraburkholderia graminis DSM 17151 that could produce a significant amount of mandelamide toward mandelonitrile and exhibited general hydration activity toward various benzylic nitriles. The time-course experiment with NitPG demonstrated that amide was also a true reaction product of nitrilase, suggesting that the nitrile catalysis by amide-forming nitrilase could be a post-transition state bifurcation-mediated enzymatic reaction. Further research demonstrated that low temperature, metal ion addition, and specific substrate structure could profoundly improve the amide formation capability of nitrilase. NitPG with broad hydration activity is a potential candidate for the enzymatic synthesis of benzylic amides for biotechnological applications. Studying the effect of nitrilase hydration activity could promote our understanding of the factors that influence amide and acid distribution.

Transcriptome Analysis Reveals Key Gene Expression Changes in Blue Catfish Sperm in Response to Cryopreservation.

The hybrids of female channel catfish (Ictalurus punctatus) and male blue catfish (I. furcatus) account for >50% of US catfish production due to superior growth, feed conversion, and disease resistance compared to both parental species. However, these hybrids can rarely be naturally spawned. Sperm collection is a lethal procedure, and sperm samples are now cryopreserved for fertilization needs. Previous studies showed that variation in sperm quality causes variable embryo hatch rates, which is the limiting factor in hybrid catfish breeding. Biomarkers as indicators for sperm quality and reproductive success are currently lacking. To address this, we investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm. Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. The present RNA-seq study identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage: MnSOD was induced to control ROS production; chaperones and ubiquitin ligases were upregulated to correct misfolded proteins or direct them to degradation; negative regulators of apoptosis, amide biosynthesis, and cilium-related functions were also enriched. Our study provides insight into underlying molecular mechanisms of sperm cryoinjury and lays a foundation to further explore molecular biomarkers on cryo-survival and gamete quality.

Abstract 143: Il-1β Antagonism Promotes SMC Differentiation And Accumulation In Fibrous Caps In Jak2 V617f Clonal Hematopoiesis

Patients with JAK2 V617F (JAK2 VF ) clonal hematopoiesis (CH) have increased JAK/STATsignaling that imparts an increased risk of premature coronary heart disease. We have shown that mice with Jak2 VF mutations modeling CH have increased AIM2 inflammasome activation that promotes atherosclerosis. Genetic inhibition of the inflammasome through deletion of Caspase1/11 or Gasdermin D in hematopoietic stem and progenitor cells (HSPC) harboring Jak2 VF mutations increased fibrous caps thickness. Therefore, we hypothesized that inhibition of IL-1β derived from Jak2 VF macrophages promotes smooth muscle cell (SMC) reprograming and accumulation in fibrous caps. To test this hypothesis, we transplanted bone marrow (BM) with Jak2 VF expression in all HSPCs into Ldlr -/- mice and then administered the IL-1 receptor antagonist Anakinra. After 12 weeks of Western type diet (WTD) feeding, mice treated with Anakinra had thicker fibrous caps and increased ACTA2 + SMCs within the cap region. In atheromas scRNA-Seq has identified SMC-derived populations including modified SMCs and fibroblasts which are enriched for Prg4 . Consistently, Anakinra treatment increased Prg4 -expressing cells within plaques as shown by RNA scope in situ hybridization. We then modeled Jak2 VF CH by transplanting mixtures of 20% Jak2 VF BM (or 20% littermate control BM) with 80% WT BM into Ldlr -/- mice followed by administration of antibodies to IL-1β. After WTD feeding, IL-1β antibody treatment led to increased cap thickness specifically in mice modeling Jak2 VF CH. To identify how IL-1β may alter cap thickness we isolated CD11b Negative cells from aortas and conducted bulk-cell RNA-Seq. Gene ontology analysis revealed that IL-1β antagonism specifically in Jak2 VF CH mice led to the significant enrichment of 6 biological processes all of which were related to biosynthetic processes including translation, peptide biosynthetic processes, peptide metabolism and amide biosynthesis. These changes were not seen in control mice indicating that inhibition of IL-1β derived from Jak2 VF hematopoietic cells leads to increased translation and biosynthetic processes in non-myeloid cells potentially supporting SMC differentiation and migration and resulting in increased fibrous cap formation.

Characterization of the Propham Biodegradation Pathway in Starkeya sp. Strain YW6 and Cloning of a Novel Amidase Gene mmH

We previously isolated a monocrotophos-degrading strain Starkeya sp. YW6, which could also degrade propham. Here, we show that strain YW6 metabolizes propham via a pathway in which propham is initially hydrolyzed to aniline and then converted to catechol, which is then oxidized via an ortho-cleavage pathway. The novel amidase gene mmH was cloned from strain YW6 and expressed in Escherichia coli BL21(DE3). MmH, which exhibits aryl acylamidase activity, was purified for enzymatic analysis. Bioinformatic analysis confirmed that MmH belongs to the amidase signature (AS) enzyme family and shares 26-50% identity with several AS family members. MmH (molecular mass of 53 kDa) was most active at 40 °C and pH 8.0 and showed high activity toward propham, with Kcat and Km values of 33.4 s-1 and 16.9 μM, respectively. These characteristics make MmH suitable for novel amide biosynthesis and environmental remediation.

Bm-iAANAT and its potential role in fatty acid amide biosynthesis in Bombyx mori

The purpose of this research is to unravel the substrate specificity and kinetic properties of an insect arylalkylamine N-acyltransferase from Bombyx mori (Bm-iAANAT) and to determine if this enzyme will catalyze the formation of long chain N-acylarylalkylamides in vitro. However, the determination of substrates and products for Bm-iAANAT in vitro is no guarantee that these same molecules are substrates and products for the enzyme in the organism. Therefore, RT-PCR was performed to detect the Bm-iAANAT transcripts and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis was performed on purified lipid extracts from B. mori larvae (fourth instar, Bmi4) to determine if long chain fatty acid amides are produced in B. mori. Ultimately, we found that recombinant Bm-iAANAT will utilize long-chain acyl-CoA thioesters as substrates and identified Bm-iAANAT transcripts and long-chain fatty acid amides in Bmi4. Together, these data show Bm-iAANAT will catalyze the formation of long-chain N-acylarylalkylamides in vitro and provide evidence demonstrating that Bm-iAANAT has a role in fatty acid amide biosynthesis in B. mori, as well.

Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi\u2013SlPH tomato line with reduced fruit acidity and higher pH

BackgroundThe destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi–SlPH tomato lines was sequenced and gene-expression patterns were compared.ResultsC. gloeosporioides inoculation of the RNAi–SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi–SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi–SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi–SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi–SlPH tomato, it mainly upregulates the nitrate metabolic process.ConclusionsModulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi–SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi–SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.

Mechanistic and Structural Analysis of a Drosophila melanogaster Enzyme, Arylalkylamine N-Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of N-Acetylarylalkylamides and N-Acetylhistamine.

Arylalkylamine N-acetyltransferase like 7 (AANATL7) catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related N-acetyltransferases, many members being linked to important roles in both human metabolism and disease. Drosophila melanogaster utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified D. melanogaster AANATL7 in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because D. melanogaster has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH-activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a pKa of 7.1 that is assigned to Glu-26 as a general base and a second pKa of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis.

Biosynthesis of bioactive piperamides in Piper Tuberculatum (Piperaceae)

Piper tuberculatum species belongs to the Piperaceae family and is an herb that typically grows in tropical areas of Brazil1. Previous chemical studies of the leaves and seeds from P. tuberculatum described the occurrence of isobutyl, piperidine and piperidone amides, which showed a potent trypanocidal, antifungal and insecticide activity. In general plant alkaloids comprise an important group of secondary metabolites because of their potent biological activity. Nevertheless, the biosynthesis of amides at the enzyme and gene levels is virtually unknown. In order to unravel this biosynthetic pathway, we have investigated the participation of malonic acid and L-valine and L-lysine amino acids as precursors. Additionally, the leaves and seeds protein profile of P. tuberculatum were investigated aiming to understand the amides formation as well as the regulatory process at the enzymatic level. The proteomic analysis indicated the presence of enzymes involved in primary and secondary metabolism, including a variety of defense proteins. All of these information will contribute significantly for further biosynthetic studies at gene levels.

Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells

Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.

Thematic review series: Lipid Posttranslational Modifications. Lysosomal metabolism of lipid-modified proteins

Much is now understood concerning the synthesis of prenylated and palmitoylated proteins, but what is known of their metabolic fate? This review details metabolic pathways for the lysosomal degradation of S-fatty acylated and prenylated proteins. Central to these pathways are two lysosomal enzymes, palmitoyl-protein thioesterase (PPT1) and prenylcysteine lyase (PCL). PPT1 is a soluble lipase that cleaves fatty acids from cysteine residues in proteins during lysosomal protein degradation. Notably, deficiency in the enzyme causes a neurodegenerative lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis. PCL is a membrane-associated flavin-containing lysosomal monooxygenase that metabolizes prenylcysteine to prenyl aldehyde through a completely novel mechanism. The eventual metabolic fates of other lipidated proteins (such as glycosylphosphatidylinositol-anchored and N-myristoylated proteins) are poorly understood, suggesting directions for future research.

A Tailoring Activity Is Responsible for Generating Polyene Amide Derivatives in Streptomyces diastaticus var. 108

We recently characterized rimocidin B (3b) and CE-108B (4b) as two polyene amides with improved pharmacological properties, produced by genetically modified Streptomyces diastaticus var. 108. In this work, genetic and biochemical analysis of the producer strain show that the two amides are derived from the parental polyenes rimocidin (3a) and CE-108 (4a) by a post-PKS modification of the free side chain carboxylic acid. This modification is mediated by an amidotransferase activity operating after the biosynthesis of rimocidin (3a) and CE-108 (4a) are completed. Two polyenes, intermediates of the biosynthetic pathway of rimocidin (3a) and CE-108 (4a), were also isolated and shown to have some improved pharmacological properties compared with the final products.

Oleic acid derived metabolites in mouse neuroblastoma N18TG2 cells.

Oleamide is an endogenous sleep-inducing lipid that has been isolated from the cerebrospinal fluid of sleep-deprived mammals. Oleamide is the best-understood member of the primary fatty acid amide family. One key unanswered question regarding oleamide and all other primary acid amides is the pathway by which these molecules are produced. One proposed pathway involves oleoyl-CoA and N-oleoylglycine as intermediates: oleic acid --> oleoyl-CoA --> N-oleoylglycine --> oleamide. The first and third reactions are known reactions, catalyzed by acyl-CoA synthetase and peptidylglycine alpha-amidating monooxygenase (PAM). Oleoyl-CoA formation from oleic acid has been demonstrated in vitro and in vivo while, to date, N-oleoylglycine cleavage to oleamide has been established only in vitro. PAM catalyzes the final step in alpha-amidated peptide biosynthesis, and its proposed role in primary fatty acid amide biosynthesis has been controversial. Mouse neuroblastoma N(18)TG(2) cells are an excellent model system for the study of oleamide biosynthesis because these cells convert [(14)C]-oleic acid to [(14)C]-oleamide and express PAM in a regulated fashion. We report herein that growth of the N(18)TG(2) cells in the presence of [(14)C]-oleic acid under conditions known to stimulate PAM expression generates an increase in [(14)C]-oleamide or in the presence of a PAM inhibitor generates [(14)C]-N-oleoylglycine. This represents the first identification of N-oleoylglycine from a biological source. In addition, N(18)TG(2) cell growth in the presence of N-oleoylglycine yields oleamide. These results strongly indicate that N-oleoylglycine is an intermediate in oleamide biosynthesis and provide further evidence that PAM does have a role in primary fatty acid amide production in vivo.

P-Coumaroylnoradrenaline, a Novel Plant Metabolite Implicated in Tomato Defense against Pathogens

The Avr9 peptide elicitor from the fungus Cladosporium fulvum, the bacterial pathogen Pseudomonas syringae pathovar tomato carrying the avirulence gene avrPto (Pst (avrPto)), and the organophosphorous insecticide fenitrothion induce resistance-related responses in tomato lines carrying the Cf-9, Pto, and Fen genes, respectively. These responses were associated with synthesis of p-coumaroyloctopamine and p-coumaroylnoradrenaline, a novel compound for plants. In susceptible near isogenic tomato lines (Cf-0, pto, fen) and wounded tomato leaves, the levels of these compounds were reduced or undetectable. The elevated levels of p-coumaroyloctopamine and p-coumaroylnoradrenaline were accompanied by elevated mRNA levels of genes encoding phenylalanine ammonia lyase, p-coumarate CoA ligase, and hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT), enzymes that are involved in the hydroxycinnamic acid amide biosynthesis. Southern hybridization indicated that THT is encoded by a multigene family in tomato. Four different THT full-length cDNAs were derived by reverse transcriptase-PCR using degenerate primers based on potato and tobacco THT sequences. Transcripts for all four homologs were present in unchallenged tomato leaves, but only tomTHT1-3 was highly expressed following challenge with Pst (avrPto). Furthermore, tomTHT1-3 showed a more substantial and rapid induction in the incompatible interaction than in the compatible interaction. The cDNAs tomTHT1-3, tomTHT7-1, and tomTHT7-8 encoded proteins with a high degree of amino acid sequence homology, although the recombinant proteins had different preferences for octopamine and noradrenaline. The fourth cDNA, tomTHT1-4, directed synthesis of a truncated enzymatically inactive protein due to the presence of a premature stop codon.

Hydroxycinnamic acid amide metabolism: physiology and biochemistry

Hydroxycinnamic acid amides (HCAAs) are a widely distributed group of plant secondary metabolites purported to function in several growth and developmental processes including floral induction, flower formation, sexual differentiation, tuberization, cell division, and cytomorphogenesis. Although most of these putative physiological roles for HCAAs remain controversial, the biosynthesis of amides and their subsequent polymerization in the plant cell wall are generally accepted as integral components of plant defense responses to pathogen challenge and wounding. Tyramine-derived HCAAs are commonly associated with the cell wall of tissues near pathogen-infected or wound healing regions. Moreover, feruloyltyramine and feruloyloctapamine are covalent cell wall constituents of both natural and wound periderms of potato (Solanum tuberosum) tubers, and are putative components of the aromatic domain of suberin. The deposition of HCAAs is thought to create a barrier against pathogens by reducing cell wall digestibility. HCAAs are formed by the condensation of hydroxycinnamoyl-CoA thioesters with phenylethylamines such as tyramine, or polyamines such as putrescine. The ultimate step in tyramine-derived HCAA biosynthesis is catalyzed by hydro xycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; E.C. 2.3.1.110). The enzyme has been isolated and purified from a variety of plants, and the corresponding cDNAs cloned from potato, tobacco (Nicotiana tabacum), and pepper (Capsicum annuum). THT exhibits homology with mammalian spermidine-spermine acetyl transferases and putative N-acetyltransferases from microorganisms. In this review, recent advances in our understanding of the physiology and biochemistry of HCAA biosynthesis in plants are discussed.Key words: hydroxycinnamic acid amides, hydroxycinnamoyl-CoA thioesters, metabolic engineering, phenylethylamines, plant cell wall, polyamines, secondary metabolism, tyramine.

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

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