Ethylene-induced Isocoumarin Formation in Carrot Root Tissue

The concentrations of 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin (MMHD) formed in carrot roots inoculated with certain fungi or treated with indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), were related to the amount of ethylene produced by the root tissue. Ethylene applied exogenously in concentrations above 0.3 ppm induced the formation of MMHD in carrot root discs. Continued production of MMHD required the continued presence of ethylene. The amounts of MMHD in the discs were reduced by CO₂, an inhibitor of ethylene action, and by reduction of the partial pressure of ethylene in fungus-inoculated or 2,4,5-T-treated carrot root discs. The results indicate that ethylene is required for the induction of MMHD formation by carrot root tissue.     

Effect of Ethylene on the Qualitative and Quantitative Composition of the Phenol Content of Carrot Roots

Carrot (Daucus carota L.) roots stored at 3 ± 1°C in an atmosphere containing 100 μ1/I of ethylene had their total phenol content increased markedly as compared to control samples kept in air. The increase was two-fold: i. higher level of pre-existing phenols, particularly isochlorogenic acid, ii. de novo synthesis of (at least) four compounds, two of which were identified as 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin, and 5-hydroxy-7-methoxy-2-methylchromone, which are not normally present in carrot tissues.     

Phenolic metabolism in root slices of selected carrot cultivars

Carrot root slices, stored for 4 days at 20 °C reacted with a strong accumulation of total phenols, especially chlorogenic acid. A significant accumulation of isocoumarin content within the peel was observed in stored slices. Synthesis of phenols was accompanied by an increase in phenylalanine ammonia lyase activity, wound induced respiration and ethylene production. The great variability among the studied four cultivars was found concerning isocoumarin synthesis, PAL activity, respiration rate and ethylene evolution, but less distinct in the case of chlorogenic acid accumulation. The carrot slices obtained from freshly harvested roots were more sensitive to mechanical damage and short-term storage than those prepared from roots previously stored.     

Biosynthesis of ethylene in higher plants: the metabolic site of inhibition by phosphate*

Abstract. Phosphate inhibited endogenous as well as 1-aminocyclopropane-1-carboxylic acid (ACC)-stimulated ethylene synthesis in slices of tomato fruit, segments of carrot root and pea hypocotyls. ACC concentrations of up to 10 mol m^?3 did not overcome this inhibition. Phosphate inhibited the conversion of ¹⁴C ACC to ethylene in tomato fruit and vegetative tissue. Enzymatic conversion of ACC to ethylene by pea seedling homogenate was also inhibited by phosphate with a linear concentration dependency. The formation of ACC from S-adenosylmethionine (SAM) by extracts of pink tomatd fruit was slightly, but not significantly, affected by phosphate. However, the SAM to ACC conversion was greater when extracts from tomato fruit were made in phosphate rather than in HEPES-KOH buffer. Non-enzymatic ethylene synthesis from ACC in a model system was stimulated by phosphate. We suggest that phosphate is an inhibitor of ethylene biosynthesis in higher plants and that one site of its control is the conversion of ACC to ethylene.     

Energy dependence of O-antigen synthesis in Salmonella typhimurium.

The uncoupler 2,4-dinitrophenol prevents in vivo synthesis of O antigen in Salmonella typhimurium by inhibiting the first reaction of the pathway, formation of galactosyl-pyrophosphoryl-undecaprenol. Inhibition was observed only in intact cells; dinitrophenol had no effect on activity of the synthase enzyme in isolated membrane fractions. In vivo inhibition could not be explained by changes in intracellular nucleotide pools or a shift in the equilibrium of the reaction and appeared to be specific for the first step in the pathway. Neither the subsequent mannosyl transferase, which catalyzes formation of the trisaccharide-lipid intermediate, mannosyl-rhamnosyl-galactosyl-pyrophosphoryl-undecaprenol, nor O-antigen polymerase was inhibited. In addition, incorporation of galactose into core lipopolysaccharide was only modestly inhibited under conditions in which O-antigen synthesis was abolished. The results suggest that maintenance of proton motive force is required for access of substrate, UDP-galactose and/or undecaprenyl phosphate, to the active site of the galactosyl-pyrophosphoryl-undecaprenol synthase enzyme.     

Cytokinins: new apoptotic inducers in plants

High concentrations of cytokinins block cell proliferation and induce programmed cell death (PCD) in both carrot ( Daucus carota L.) and Arabidopsis thaliana (L.) Heynh. cell cultures [13 and 27 micro M N(6)-benzylaminopurine (BAP), respectively]. In the present work, cell death was scored by Evan's blue staining and was also demonstrated to be programmed by various parameters, including chromatin condensation, oligonucleosomal DNA degradation (laddering), and release of cytochrome c from mitochondria. In carrot cells, this induction takes approximately 24 h, with proliferating cells being more sensitive than quiescent ones. Two hormones, namely abscisic acid and 2,4-dichlorophenoxyacetic acid (2,4-D), protect cells against the cytokinin-induced death. PCD is not merely a consequence of the inability of the culture to proliferate, since high levels of 2,4-D block carrot cell proliferation without promoting PCD. Increased ethylene production was also observed in BAP-treated cultures, although this increase was not responsible for PCD because inhibitors of ethylene synthesis and action did not block PCD in BAP-treated cultures. Programmed cell death in the form of DNA laddering was also seen in plants treated with cytokinins. This process was accompanied by accelerated senescence in the form of leaf yellowing.     

Polygonolide,an isocoumarin from Polygonum hydropiper possessing anti-inflammatory activity

A new isocoumarin polygonolide which inhibits the reversed passive Arthus reaction has been isolated from the methanol extract of the root of Polygonum hydropiper. The structure of polygonolide has been elucidated on the basis of spectroscopic data and confirmed to be 3,4-dimethyl-6-methoxy-8-hydroxyisocoumarin by total synthesis.     

Naturally produced isocoumarins: inhibitors of calmodulinsensitive cyclic guanosine 3′,5′-monophosphate phosphodiesterase

Summary Three isocoumarins have been isolated from a strain ofStreptoverticillium sp. and all inhibit the calmodulin-sensitive cyclic guanosine 3,5-monophosphate phosphodiesterase (EC 3.1.4.17, Boehringer Mannheim). Two of the compounds, 6,8-dihydroxy-7-methoxy-3-methyl isocoumarin and 6,7,8-trihydroxy-3-methyl isocoumarin have previously been isolated fromStreptomyces. The third fermentation product, 6,8-dihydroxy-3-methyl isocoumarin, was also found as a metabolite ofCeratocystis minor, a fungal species associated with the blue stain disease of pine [2,3].     

Discovery of an isocoumarin analogue that modulates neuronal functions via neurotrophin receptor TrkB

Isocoumarins are lactone ring-containing natural products, are quite abundant in microbes and higher plants, and have been shown to exhibit a broad range of pharmacological properties. However, the molecular mechanism or target of this class of molecules is not known. In this study, we have synthesized 14 isocoumarin derivatives and evaluated for their activity at TrkB receptor in transiently transfected HEK293T cells. We identified 8-hydroxy-3-aryl isocoumarin (1) as a high-affinity agonist at the TrkB receptor. We also demonstrated that isocoumarin 1 activated endogenously TrkB receptor in primary cortical neurons and modulated various markers of synaptic plasticity, and increased dendritic arborization. These results indicate therapeutic potential and molecular target of 8-hydroxy-3-aryl isocoumarin 1 for the treatment of various CNS disorders.     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

Biosynthesis of phytoalexin in carrot root requires ethylene action

The role of ethylene in phytoalexin production by carrot ( Daucus carota L.) roots was investigated using the ethylene action inhibitor 1-methylcyclopropene (MCP). Exposure of carrot roots to ethylene, UV-B irradiation, inoculation with fungal pathogens, treatment with 2,4-D or methyl jasmonate induced accumulation of the phytoalexin 6-methoxymellin (6-ME). Exposure to MCP for 4–12 h prior to the treatments completely inhibited 6-ME accumulation, indicating that 6-ME synthesis by carrot roots requires ethylene action.     

A direct, highly sensitive fluorometric assay for a microsomal cytochrome P450-mediated O-demethylation using a novel coumarin analog as substrate

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (lambdaA = 380 nm, lambdaF = 480 nm) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 380 nm and a fluorescence/emission wavelength of 480 nm, the fluorescence of this substance (lambdaA = 338 nm, lambdaF = 422 nm) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrome P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.     

Redirection of pigment biosynthesis to isocoumarins in Fusarium

Colonies of Fusarium species often appear red due to production of pigments, such as aurofusarin or bikaverin. The primary compounds in these biosynthetic pathways are YWA1 and pre-bikaverin, respectively, catalyzed by two multidomain polyketide synthases (PKSs), which both have a claisen-type cyclase domain (CLC) in their N terminal. Disruption of the CLC domains has been shown to result in formation of the lactones citreoisocoumarin and SMA93 instead of YWA1 and pre-bikaverin. In the present study we have discovered a medium with low nitrogen content which partially redirects the aurofusarin and bikaverin pathways to produce citreoisocoumarin and SMA93, respectively. This is first time that SMA93 is identified in a fungus and we suggest that it is renamed bikisocoumarin, as it is derived from the bikaverin pathway. The redirection of the aurofusarin and bikaverin biosynthetic pathways was reverted by adding inorganic nitrogen to the medium, whereas organic nitrogen in form of arginine or glutamine stimulated isocoumarin production. This suggests that nitrogen source can influence isocoumarin production. Production of isocoumarin was also repressed by alkaline conditions, which suggests that nitrogen supply is not the sole regulatory factor in the pathway. The redirection was observed in all producers of aurofusarin (6) and bikaverin (2), suggesting the presence of a conserved regulatory mechanism.     

The Biosynthesis of 8-Hydroxy-6-methoxy-3-methyl-3,4-dihydroisocoumarin and 5-Hydroxy-7-methoxy-2-methylchromone in Carrot Root Tissues Treated with Ethylene

Biosynthesis of ethylene-induced isocoumarin and eugenin in carrots (Daucus carota L.) was studied. Radioactive isocoumarin eugenin was isolated from these carrots fed with 1-¹⁴C-acetate and was isolated from ethylene treated carrots fed with 1-¹⁴C-acetate, 2-¹⁴C-malonate, and 3-¹⁴C-acetoacetate. Also, radioactive G-³H-5,7-dihydroxy-2-methylchromone. A trapping experiment with 5,7-dihydroxy-2-methyl-chromone as trapping agent indicated that this compound was synthesized in carrots from acetate. From the results obtained, it was inferred that the biosynthesis of isocoumarin and eugenin must proceed via the acetate pathway.     

Pterocarpanoid constituents of Swartzia leiocalycina

The proposed structure for leiocalycin from Swartzia leiocalycina has been confirmed by degradation and partial synthesis. Co-occurring with the pterocarp-6a-en are two new coumestones, 6-hydroxy-7-methoxy-11,12-methylenedioxycoumestone and 6-hydroxy-5,7-dimethoxy-11,12-methylenedioxycoumestone and the known 6aR,11aR-2-hydroxy-3-methoxy-8,9-methylenedioxypterocarpan.     

Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L.) cell suspension cultures

Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsibl for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.     

Effects of inhibitors of RNA and protein synthesis on bean hypocotyl hook opening and their implications regarding phytochrome action

Summary Inhibitors of protein and RNA synthesis (cycloheximide, puromycin, chloramphenicol, and actinomycin D), as well as Co⁺⁺, induce opening of the hypocotyl hook of bean seedlings during the early stage of the opening period both in the darkness and red light. The response is transitory, however, complete straightening of a hook can not be achieved in the presence of these agents. These agents abolish the response of hooks to red illumination. They also block the suppression of hook opening caused by IAA and ethylene. The response and sensitivity to GA are not affected by the inhibitors. Inhibitors of DNA synthesis (FUDR and mitomycin C) have no effect on hook opening. It appears that in this growth response RNA and protein synthesis are more immediately involved in ethylene action than they are in the cell elongation process or the action of GA thereon.The results indicate that phytochrome does not induce hook opening simply by activating genes whose products directly promote growth. It is suggested that the regulation of ethylene formation by light and auxins may be exerted by way of influences on tissue levels of phenolic inhibitors of ethylene biosynthesis.     

Synthesis and antifungal properties of alpha-methoxy and alpha-hydroxyl substituted 4-thiatetradecanoic acids

4-thiatetradecanoic acid exhibited weak antifungal activities against Candida albicans (ATCC 60193), Cryptococcus neoformans (ATCC 66031), and Aspergillus niger (ATCC 16404) (MIC=4.8-12.7 mM). It has been demonstrated that alpha-methoxylation efficiently blocks beta-oxidation and significantly improve the antifungal activities of fatty acids. We examined whether antifungal activity of 4-thiatetradecanoic acid can be improved by alpha-substitution. The unprecedented (+/-)-2-hydroxy-4-thiatetradecanoic acid was synthesized in four steps (20% overall yield), while the (+/-)-2-methoxy-4-thiatetradecanoic acid was synthesized in five steps (14% overall yield) starting from 1-decanethiol. The key step in the synthesis was the hydrolysis of a trimethylsilyloxynitrile. In general, the novel (+/-)-2-methoxy-4-thiatetradecanoic acid displayed significantly higher antifungal activities against C. albicans (ATCC 60193), C. neoformans (ATCC 66031), and A. niger (ATCC 16404) (MIC=0.8-1.2 mM), when compared with 4-thiatetradecanoic acid. In the case of C. neoformans the (+/-)-2-hydroxy-4-thiatetradecanoic acid was more fungitoxic (MIC=0.17 mM) than the alpha-methoxylated analog, but not as effective against A. niger (MIC=5.5 mM). The enhanced fungitoxicity of the (+/-)-2-methoxy-4-thiatetradecanoic acid, as compared to decylthiopropionic acid, might be the result of a longer half-life in the cells due to a blocked beta-oxidation pathway which results in more time to exert its toxic effects. Thus, these novel fatty acids may have applications as probes to study fatty acid metabolic routes in human cells.     

Regulation of root hair initiation and expansin gene expression in Arabidopsis

The expression of two Arabidopsis expansin genes (AtEXP7 and AtEXP18) is tightly linked to root hair initiation; thus, the regulation of these genes was studied to elucidate how developmental, hormonal, and environmental factors orchestrate root hair formation. Exogenous ethylene and auxin, as well as separation of the root from the medium, stimulated root hair formation and the expression of these expansin genes. The effects of exogenous auxin and root separation on root hair formation required the ethylene signaling pathway. By contrast, blocking the endogenous ethylene pathway, either by genetic mutations or by a chemical inhibitor, did not affect normal root hair formation and expansin gene expression. These results indicate that the normal developmental pathway for root hair formation (i.e., not induced by external stimuli) is independent of the ethylene pathway. Promoter analyses of the expansin genes show that the same promoter elements that determine cell specificity also determine inducibility by ethylene, auxin, and root separation. Our study suggests that two distinctive signaling pathways, one developmental and the other environmental/hormonal, converge to modulate the initiation of the root hair and the expression of its specific expansin gene set.     

Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana

Lateral root branching is a genetically defined and environmentally regulated process. Auxin is required for lateral root formation, and mutants that are altered in auxin synthesis, transport or signaling often have lateral root defects. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in the regulation of Arabidopsis lateral root formation are not well characterized. This study utilized Arabidopsis mutants altered in ethylene signaling and synthesis to explore the role of ethylene in lateral root formation. We find that enhanced ethylene synthesis or signaling, through the eto1-1 and ctr1-1 mutations, or through the application of 1-aminocyclopropane-1-carboxylic acid (ACC), negatively impacts lateral root formation, and is reversible by treatment with the ethylene antagonist, silver nitrate. In contrast, mutations that block ethylene responses, etr1-3 and ein2-5 , enhance root formation and render it insensitive to the effect of ACC, even though these mutants have reduced root elongation at high ACC doses. ACC treatments or the eto1-1 mutation significantly enhance radiolabeled indole-3-acetic acid (IAA) transport in both the acropetal and the basipetal directions. ein2-5 and etr1-3 have less acropetal IAA transport, and transport is no longer regulated by ACC. DR5-GUS reporter expression is also altered by ACC treatment, which is consistent with transport differences. The aux1-7 mutant, which has a defect in an IAA influx protein, is insensitive to the ethylene inhibition of root formation. aux1-7 also has ACC-insensitive acropetal and basipetal IAA transport, as well as altered DR5-GUS expression, which is consistent with ethylene altering AUX1-mediated IAA uptake, and thereby blocking lateral root formation.