Chromatographic degradation of phloridzin

Phloridzin, the main phenolic glucoside in apple leaves, has been found to undergo transformation during chromatography. When chromatographed repeatedly in ammoniacal solvents, at least 2 new derivatives appeared. One of these was identified as phloretic acid. When bioassayed in the presence of indole-3-acetic acid this substance behaved as though it promoted the destruction of the auxin. Comparative bioassay with naphthaleneacetic acid suggested that phloretic acid acts on indoleacetic acid destruction via stimulation of indoleacetic acid oxidase. However, at low concentration and in presence of a small amount of phloridzin it also showed a synergistic effect with indoleacetic acid.

A substance with the same characteristics was obtained directly from apple leaves, which are known to contain phloridzin when the extracts were chromatographed only once in the same (alkaline) solvent. While not completely confirmed, this suggests that phloretic acid is normally present in apple leaves, where it may affect growth there by promoting indoleacetic acid oxidation.

     

Phloridzin and apple scab

Phloridzin, sieboldin, trilobatin, phloretin and 3-hydroxyphloretin can all be used as carbon sources by Venturia inaequalis in culture. Resistance to apple scab was not linked with inheritance of sieboldin or trilobatin in seedlings. There is no direct connection between phloridzin or its breakdown products and scab resistance.     

连作苹果土壤酚酸对平邑甜茶幼苗的影响

为探讨连作(重茬)苹果土壤中酚酸类物质的积累与苹果连作障碍的关系,在砂培条件下,取连作果园土壤中实际浓度的酚酸类物质处理平邑甜茶幼苗,探讨了连作2a的果园土壤中实测浓度的根皮苷、间苯三酚、根皮素、对羟基苯甲酸和肉桂酸对平邑甜茶幼苗根系线粒体指标、抗氧化酶活性、膜过氧化程度及活性氧(ROS)含量的影响。结果表明:连作土壤中实际浓度的5种酚酸类物质均使平邑甜茶幼苗生长受到抑制,根系受影响程度高于地上部分,表现为根冠比降低;线粒体膜通透性转换孔(MPTP)开放程度增大,线粒体膜电位降低,细胞色素Cyt c/a比值下降;降低了幼苗根系中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,增加了过氧化氢(H2O2)、超氧阴离子自由基(O·-2)以及丙二醛(MDA)的含量。土壤浓度的5种酚酸类物质中,以根皮苷处理抑制效果最显著,间苯三酚处理抑制力最小。因此,根皮苷是引起苹果连作障碍的主要酚酸,实践中应重点考虑对根皮苷的降解以缓解苹果连作障碍。     

Stimulation of indoleacetic acid production in a <Emphasis Type="Italic">Rhizobium</Emphasis> isolate of <Emphasis Type="Italic">Vigna mungo</Emphasis> by root nodule phenolic acids

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in l-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 μg ml⁻¹) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

The Effect of Some Inhibitory Sugars upon the Content of Adenosine Triphosphate in Wheat Roots

The content of adenosine triphosphate (ATP) in roots of -wheat (Triticum aestivum L.) was determined with the fire-fly-luciferase method. The content is decreased by D-mannose, which inhibits root growth, respiration and chloride uptake. In intact seedlings the inhibition of root growth is relieved by other sugars and also by the flavanone naringenin and by 2,4-dinitrophenol. This reversal is combined with an increased content of ATP. The inhibition of chloride uptake by mannose in excised roots is reversed by some other sugars (including D-galactose which is in itself inhibitory to root growth), and also in this case the ATP content is increased. Naringenin and dinitrophenol do not relieve the inhibition of chloride uptake caused by mannose. Nor do they increase the content of ATP in this case. The primary effect of mannose seems to be inhibition of glycolysis whereas the effect upon root growth is secondary. Galactose, which also inhibits root growth, does not inhibit respiration or reduce the ATP content and the primary effect of galactose (and also of 2-deoxy-D-glucose and 2-deoxy-D-galactose) seems to be on the synthesis of cell wall substances.     

The constitutive phenolic composition of two Malus × domestica genotypes is not responsible for their contrasted susceptibilities to fire blight

Apple (Malus × domestica) leaf phenolics, in particular the three major dihydrochalcones, phloridzin, trilobatin and sieboldin, were studied for their potential contribution to the resistance to Erwinia amylovora, the causative agent of fire blight. The constitutive phenolic compositions of part of a progeny resulting from a cross between two apple genotypes, displaying contrasted susceptibilities to fire blight, were investigated by RP–HPLC–DAD. With regards to the major dihydrochalcones, offsprings were found to display the parent profile: either phloridzin alone or a combination of phloridzin, trilobatin and sieboldin. Despite a strong antioxidant activity that could interfere with the oxidative burst triggered by the bacteria, sieboldin did not segregate with resistance even though it was strongly associated with a high constitutive antioxidant capacity of leaf extracts. Several hydroxycinnamic acids and flavonols were identified in the progeny but profiles were very similar. However, concentrations of several flavonols positively correlated with resistance in the genotypes that possessed trilobatin and sieboldin. The major dihydrochalcones that represent more than 200 mg/g of leaf DW are not directly responsible for resistance to fire blight in this system. In addition, a high constitutive antioxidant activity is unlikely to be sufficient to block bacterial spread.     

Biosynthesis of oleanolic acid glycosides in protoplasts isolated from Calendula officinalis L. roots

Many medicinal plants contain oleanane saponins in roots, however, only scarce data on their biosynthesis in this organ are available so far, including our previous results concerning Calendula officinalis plant. Thus, the purpose of the present work was to confirm the presumable biosynthetic pathway of oleanolic acid glycosides in roots of young C. officinalis plants. First of all, the effective method of isolation of protoplasts from C. officinalis roots was established. Then, isolated root protoplasts were supplied with radioactive precursors, [2-¹⁴C] mevalonate (MVA) and [3-³H] oleanolic acid (OL) and their transformations were studied with comparison to results obtained with excised roots. The penetration of both precursors into protoplasts was more rapid and effective than in the case of excised roots. The labeling of sterols and OL during the incubation with MVA showed that the isoprenoid pathway leading to triterpenoids was operative in excised roots as well as isolated root protoplasts. Moreover, the transformations of OL into two series of its glycosides, i.e. glucosides and glucuronides were investigated. It has been shown that both series of OL glycosides are synthesized in isolated root protoplasts in the same way as in excised roots of young marigold plants.     

The allelopathic potential of alfalfa root medicagenic acid glycosides and their fate in soil environments

Summary The allelopathic effect of alfalfa (Medicago media Pers.) and red clover (Trifolium pratense L.) root saponins on winter wheat seedling growth and the fate of these chemicals in soil environments were studied. Seed germination, seedling and test fungus growth were suppressed by water and by alcohol extracts of alfalfa roots, and by crude saponins of alfalfa roots, indicating that medicagenic acid glycosides are the inhibitor. Powdered alfalfa roots inhibited wheat seedling growth when added to sand. At concentrations as low as 0.25% (w/w) the root system was completely destroyed whereas seedling shoots suffered little damage. Red clover roots caused some wheat growth inhibition when incorporated to sand, but their effect was much lower than in the alfalfa root treatment. Soil textures had a significant influence on the inhibitory effect of alfalfa roots. The inhibition of seedling growth was more pronounced on light than on heavy soils. This was attribted to the higher sorption of inhibitors by heavy soils. Incubation of alfalfa roots mixed into loose sand, coarse sand, loamy sand and clay loam for a period of 0–8 days resulted in decreased toxicity to bothT. viride and wheat seedlings. This decrease occurred more quickly in heavier soils than in loose sand, due to the hydrolysis of glycosides by soil microorganisms. Soil microbes were capable of detoxifying medicagenic acid glycosides by partial hydrolysis of sugar chain to aglycone. These findings illustrate the importance of medicagenic acid glycosides as an inhibitor of wheat seedling growth, and of their fate in different soil environments.     

Differential IAA dose response relations of the axr1 and axr2 mutants of Arabidopsis

In comparison to wild type Arabidopsis thaliana, the auxin resistant mutants axr1 and axr2 exhibit reduced inhibition of root elongation in response to auxins. Several auxin-regulated physiological processes are also altered in the mutant plants. When wild-type, axr1 and axr2 seedlings were grown in darkness on media containing indoleacetic acid (IAA), promotion of root growth was observed at low concentrations of IAA (10^?11 to 10^?7M) in 5-day-old axr2 seedlings, but not in axr1 or wild-type seedlings. In axr1 there was little or no measurable root growth response over the same concentration range. In wild type, root growth was inhibited at concentrations greater than 10^?10M and no detectable root growth response was observed at lower concentrations. In addition, production of lateral roots in response to IAA increased in axr2 seedlings and decreased in axr1 seedlings relative to wild type. Promotion of root elongation and initiation of lateral roots in axr2 seedlings in response to auxin indicate that axr2 seedlings are able to perceive and respond to IAA.     

The Behaviour of Phloridzin in the Coleoptile Straight-growth Test

Phloridzin was identified as one of the two inhibitors of coleoptile-sectionextension in the acid fraction of extracts of apple shoots.The tips of the sections showed a characteristic flaccidityand staining at concentrations of 200 p.p.m. and above. Sectionswhich had been inhibited by phloridzin did not recover so wellafter washing and transfer to sodium ß-indolylacetateas did those which had been in contact with an inhibitor fromplum shoots. Phloridzin was more effective as an inhibitor than its breakdownproducts phloretin, phloroglucinol, p-hydroxyhydrocinnamic acid,or p-hydroxybenzoic acid. The scanty knowledge of the role of phloridzin in plant metabolismis briefly discussed.     

Indoleacetic Acid Oxidase: A Dual Catalytic Enzyme?

The isolation of a unique enzyme capable of oxidizing indoleacetic acid, but devoid of peroxidase activity, has been reported for preparations from tobacco roots and commercial horseradish peroxidase. Experiments were made to verify these results using enzyme obtained from Betula leaves and commercial horseradish peroxidase. Both indoleacetic acid oxidase and guaiacol peroxidase activity appeared at 2.5 elution volumes from sulfoethyl-Sephadex. These results were obtained with both sources of enzyme. In no case was a separate peak of indoleacetic acid oxidase activity obtained at 5.4 elution volumes as reported for the tobacco enzyme using the same chromatographic system. Both types of activity, from both sources of enzyme, also eluted together during gel filtration. Successful column chromatography of Betula enzyme was dependent upon previous purification by membrane ultrafiltration. These results indicate indoleacetic acid oxidase activity and guaiacol peroxidase activity are dual catalytic functions of a single enzyme.     

Influence of Nitrogen and Sucrose in the Medium and of Irradiance of the Stock Plants on Root Formation in Pelargonium Petioles Grown in vitro

In vitro root formation was studied in petiole explants from Pelargonium×hortorom Bailey cv. Radio exposed to different irradiance (2.5. 11.6 or 23.0 W/m²). Optimal root formation was found when stock plants were subjected to 2.5 W/m² and explants were grown on a medium with 20 g/I of sucrose and 5.0 mM of nitrogen. The number of roots per explant decreased with increasing nitrogen or sucrose concentrations in the growth medium and with increasing irradiance applied to the stock plants. Variation in temperature (17, 21 or 25°C) did not affect root formation. The number of roots per explant was higher in explants from young petioles than in explants from older petioles. The initial nitrogen content of the explants was highest at the lowest irradiance. The content of reducing sugars and sucrose increased with increasing irradiance. The endogenous levels of lysine, arginine and ornithine were highest at 2.5 W/m² and the levels of proline. alanine, γ-aminobutyric acid, glutamic acid, glutaminc, aspartic acid and asparagine were highest at 23.0 W/m². With rising nitrogen concentration in the medium, the final endogenous content of nitrogen in the explants increased, whereas the content of reducing sugars decreased. When the sucrose concentration in the medium increased, the endogenous content of reducing sugars rose, and the nitrogen level was lowered. Variations in the nitrogen concentration in the growth medium influenced the final level of free amino acids in the explants.     

Progressive Effects of Phloridzin on Melanogenesis in B16 Mouse Melanoma Cells

When we studied the effects of polyphenols from apple fruits on melanogenesis in B16 mouse melanoma cell lines, phloridzin had dose-dependent progressive effects on melanogenesis between 10 and 500 μg/ml without inhibiting cell growth. At a concentration of 500 μg/ml, phloridzin increased the melanin content in the cells to 181% of that in control cells. In contrast, phloretin, the aglycon of phloridzin, did not activate melanogenesis in the cells and was cytotoxic at a concentration of 5 μg/ml. Phloridzin increased the activity of tyrosinase to 223% of that in control cells. Furthermore, phloridzin inhibited the activity of protein kinase C (PKC), which is recognized to regulate tyrosinase activity. The inhibition of PKC activity continued for 120min from the addition of phloridzin. Therefore, we estimated that the activation of melanogenesis by phloridzin resulted from the increase of tyrosinase activity caused by the inhibition of PKC activity.     

Correlation between Root-Generated Ionic Currents, pH, Fusicoccin, Indoleacetic Acid, and Growth of the Primary Root of Zea mays

Correlations between root-generated ionic currents, extracellular pH, indoleacetic acid, fusicoccin, and growth were investigated. Current consistently entered the meristematic and elongating tissues of intact growing roots of Zea mays cv Golden Bantam. Mature root regions generated the outward limb of the current loop. Ion-substitution and pH-profile experiments suggested that the bulk of the ionic current was carried by H⁺. Calcium ions did not carry current, but calcium may regulate the proton circulation since the proton current density was slightly larger in calcium-depleted media. Increased root elongation at low pH was associated with increased current density and an extended zone of inward current. Conversely decreased elongation at high pH was associated with a reduced current density and a more restricted zone of inward current. The effect of the fungal toxin fusicoccin was to increase the current density of the inward limb of the ion current and to increase root extension. Concentrations of indoleacetic acid that reduced root growth, also reduced the density of the inward current and shortened the inward current zone. The results emphasize the point that roots are electrically contiguous over many millimeters and that the electrophysiology of root growth is best studied in intact root systems.     

Rapid effects of indoleacetic Acid and ethylene on the growth of intact pea roots

Root auxanometers were used to determine the growth rates of individual intact primary roots accurately and quickly. The growth of pea (Pisum sativum L.) roots was inhibited by both indoleacetic acid and ethylene within 20 minutes. A supramaximal concentration of ethylene inhibited root growth less than did 5 to 20 mum indoleacetic acid, indicating that inhibition of root growth by auxin was not due only to indoleacetic acid-induced ethylene production. Inhibition of root growth was largely relieved within 60 minutes of removal of both growth regulators.     

Growth and biochemical characterization of associations between cyanobionts and wheat seedlings in co-culturing experiments

N₂-fixing cyanobacteria are unique in their capacity to form symbiotic associations with a wide range of eukaryotic hosts belonging to different plant groups. The present study was undertaken to analyze the interactions of the cyanobiont PI 01 (from Azolla pinnata) and Nostoc PCC 9229 (from Gunnera monoika) with wheat seedlings, in co-culturing experiments. Each of the cyanobionts enhanced significantly the volume of root and shoot biomass in the experimental cultures. The transverse sections of roots in the co-cultured seedlings revealed the presence of aseriate packets of cyanobionts below the root epidermis. The investigated cyanobionts excreted amino acids (His, Met, Val) and sugars into the medium, while indoleacetic acid was detected when the cyanobionts were grown in a tryptophan containing medium. During the co-culturing, sugars and proline were detected in the extracellular filtrates. It can be hypothesized that these sugars and amino acids may serve as signal substances in the development of functional associations between the relevant cyanobionts and the wheat seedlings.     

Direct rhizogenesis and establishment of fast growing normal root organ culture of Withania somnifera Dunal

Direct rooting from leaf explants of Withania somnifera was achieved on half strength Murashige and Skoog’s medium supplemented with 15 g l⁻¹ sucrose, and different concentrations of growth regulators. Basal medium supplemented with 2.85 μM indoleacetic acid and 9.85 μM indolebutyric acid achieved maximum number of roots with 100% response. The roots were cultured on MS liquid medium for the establishment of root-organ culture with the same plant growth regulators and incubated on an orbital shaker at 80 rpm at 25 ± 2 °C. A root biomass of 6.15 ± 0.17 g was obtained after 5 weeks. When 1 g roots were inoculated to 2.5 l bubble column reactor, 47 g roots were obtained after 6 weeks. The concentration of alkaloids was increased as compared to field grown roots. The maximum concentration of withanolides (10 mg g⁻¹ dry weight) was obtained in the bioreactor.     

Dihydrochalcones: Implication in resistance to oxidative stress and bioactivities against advanced glycation end-products and vasoconstriction

Flavonoids are a group of polyphenol compounds with known antioxidant activities. Among them, dihydrochalcones are mainly found in apple leaves (Malus domestica). Glycosylated dihydrochalcones were previously found in large amounts in leaves of two genotypes of Malus with contrasting resistance to fire blight, a bacterial disease caused by Erwinia amylovora. In the present study we demonstrate that soluble polyphenol patterns comprised phloridzin alone or in combination with two additional dihydrochalcones, identified as sieboldin and trilobatin. Presence of sieboldin in young leaves correlated well with a high 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. Moreover, these leaves displayed enhanced tolerance to paraquat, a photooxidative-stress generating herbicide. Interestingly, phloridzin had a high activity in the oxygen radical absorbance capacity (ORAC) assay, but its presence alone in leaves did not correlate with tolerance to paraquat. In order to further characterise the activity of these compounds, we tested their ability to prevent oxidative-dependent formation of advanced glycation end-products (AGEs) and phenylephrine-induced contraction of isolated rat mesenteric arteries. The antioxidant capacity of sieboldin was clearly demonstrated by showing that this compound (i) prevented vasoconstriction and (ii) inhibited AGEs formation. Both assays provided interesting information concerning a potential use of sieboldin as a therapeutic. Hence, our results strongly argue for a bioactivity of dihydrochalcones as functional antioxidants in the resistance of Malus leaves to oxidative stress. In addition, we demonstrate for the first time that sieboldin is a powerful multipotent antioxidant, effective in preventing physiopathological processes. Further work should aim at demonstrating the potential use of this compound as a therapeutic in treating free radical-involving diseases.     

Effects of Pargyline on Mitochondrial Amine Oxidase Activity, Indoleacetic Acid Synthesis and Growth of Crown-Gall Tumor Callus

Vinca rosea L. crown-gall tumor callus tissue cultures treated with N-benzyl-N methyl propargylamine (pargyline) exhibited a decrease in the level of endogenous indoleacetic acid from 0.42 μg/mg of protein to less than 0.30 μg/mg of protein. A simultaneous decrease in the specific activity of mitochondrial amine oxidase from 3000 units to less than 250 units at 1.0 μM, 0.01 mM, 0.1 mM and 1.0 mM pargyline, suggested a relationship between amine oxidase function and indoleacetic acid synthesis. Tryptamine incorporation into indoleacetic acid was also decreased at these concentrations. Pargyline inhibited tumor callus growth significantly (based on fresh weight measurements) at the highest concentration, 1.0mM. These data support the hypothesis of a coordinate metabolic system linking mitochondrial amine oxidase activity and indole acetic acid synthesis. Inhibitory action of pargyline on the enzyme is reflected in reduced indoleacetic acid levels and, ultimately, in reduced callus growth rates.     

Electrophoretic study on peroxidase,indoleacetic acid oxidase,and o-diphenol oxidase fractions in extracts from different growth zones ofvicia faba L. roots

Using electrophoresis in acrylamide gel, fractions of peroxidase, indoleacetic acid oxidase, and o-diphenol oxidase were investigated in extracts from three growth zones ofVicia faba L. roots. Three peroxidase fractions (zones) moving towards the anode were revealed as well as four peroxidase fractions (zones) migrating towards the cathode. Three peroxidase fractions showed detectable indoleacetic acid oxidase activity. The o-diphenol oxidase activity was revealed in all peroxidase fractions moving towards the anode, in those moving towards the cathode the o-diphenol oxidase activity differred according to the substrate used. One fraction with both peroxidase and o-diphenol oxidase activity occurred only in electrophoreograms of extracts from the maturation zone; in this fraction no indoleacetic acid oxidase activity was demonstrable.