A Phytotoxic Glycopeptide from Cultures of Corynebacterium insidiosum

Cultures of Corynebacterium insidiosum produce an extra-cellular phytotoxic glycopeptide that possesses the ability to wilt plant cuttings. Wilt induced by this glycopeptide is directly dependent upon time and upon concentration with measureable wilt occurring in 40 nm solutions in 1 hour. The organism produces 1.3 grams toxin/liter of culture medium. The toxin was purified, and the physical, chemical, and biological properties were measured. The glycopeptide has an empirical formula of C₁₀₈H₂₂₆O₁₃₂N based on 1 atom of nitrogen. The molecular weight as estimated by light scattering and column gel chromatography indicated values approximating 5 × 10⁶. The toxin does not dissociate into small molecular weight subunits when treated with 8 m urea or 30% pyridine.     

The Active Site on the Phytotoxin of Corynebacterium sepedonicum

Corynebacterium sepedonicum produces an extracellular phytotoxic glycopeptide that possesses a capacity to wilt plant cuttings. It has been previously demonstrated that the integrity of some of the membranes of the host cells is destroyed, suggesting the possibility that a biologically active site is present on the toxin molecule. The toxin was chemically altered in the following ways and then tested for biological activity: (a) the NH₂-terminal group on the peptide portion of the toxin was blocked by the dansylation technique; (b) the OH groups on the sugar and amino acid residues as well as the NH groups on the amino acid residues were blocked by exhaustive methylation; (c) the COO⁻ groups were converted to their respective methyl esters; (d) the peptide moiety was removed by pronase digestion. Experimental results indicate that the carboxyl groups of the nonpeptide portion of the molecule are responsible for the biological activity of the toxin. Other experiments showed that the toxin does not affect the membranes of animal cells.     

Light Requirement for AgNO(3) Inhibition of Ethrel-Induced Leaf Abscission from Cuttings of Vigna radiata

To obtain information regarding the antiethylene properties and binding site of Ag⁺, studies were initiated to define conditions under which Ag⁺ does or does not inhibit ethylene action. AgNO₃, applied as a leaf spray, inhibited 2-chloroethylphosphonic acid (Ethrel)-induced leaf abscission from green cuttings of Vigna radiata in white light but lost considerable activity in the dark. In the absence of Ethrel, AgNO₃ stimulated abscission in the dark. When cuttings were dark-aged for 24 hours prior to treatment with AgNO₃ and aged for an additional 24 hours in the dark after treatment, good inhibition of subsequent Ethrel-induced abscission was restored by returning the cuttings to light. However, when dark aging was preceded by far-red irradiation, considerably less inhibition of Ethrel-induced abscission was restored in the light. AgNO₃ was completely inactive on cuttings aged in the dark and treated with Ethrel in the dark. Light is required for the antiethylene activity of AgNO₃ with regard to leaf abscission of Vigna.     

Changes in Alfalfa Stem Conductance Induced by Corynebacterium insidiosum Toxin

A toxin involved in bacterial wilt of alfalfa (Medicago sativa L.) has been isolated from cultures of the pathogen, Corynebacterium insidiosum, as well as from diseased plants (S. M. Ries and G. A. Strobel. 1972. Physiological Plant Pathology 2: 133-142). The influence of this toxin, a glycopeptide with a molecular weight of 5 × 10⁶, on the water relations of alfalfa was examined. It was found that very small amounts of the toxin (2 μg) significantly reduced stem conductance through 15-cm long stems. This decrease in stem conductance caused by the toxin best explains the rapid decrease in transpiration and stomatal conductance and the resultant wilting after alfalfa cuttings have been in 200 μg ml⁻¹ toxin for 2 hours. Membrane damage resulting in water leakage was ruled out as a factor in the wilting during the 2-hour period. It is postulated that the toxin acts by interfering with water movement through pit membranes.     

Purification and Properties of a Phytotoxic Polysaccharide Produced by Corynebacterium sepedonicum

A polysaccharide possessing a capacity to wilt plant cuttings has been isolated and purified from cultures of Corynebacterium sepedonicum. The molecular weight, based on the average of molecular weights determined by 3 physical methods, is 21,450. The empirical formula of the polysaccharide is C₄₈H₉₆O₄₈N. It is antigenic and the borate complex migrates in an electric field. It has an intrinsic viscosity of 0.125 and an S_20w of 0.76. A hydrolysate of the compound yields glucose, mannose, 2 unidentified reducing compounds and 1 unidentified non-reducing compound. The nitrogen in the toxin can be accounted for in 6 amino acids. Although the toxin is primarily polysaccharide it might more aptly be termed a glycopeptide.     

DNA damage in potato plants induced by cadmium,ethyl methanesulphonate and γ-rays

We have calibrated the alkaline protocol of the plant comet (Single Cell Gel Electrophoresis) assay as a method for detecting the extent of induced DNA damage in potato plants (Solanum tuberosum L. cultivar Korela). After 2 and 24 h treatments of the rooted cuttings with the heavy metal cadmium (Cd²⁺), a dose–response increase in DNA damage was noted versus controls in root nuclei. With a 24 h recovery period, the Cd²⁺-induced DNA damage in roots increased significantly. No significant increase in DNA damage was demonstrated in leaf nuclei after 24 h Cd²⁺ treatments, but continuous Cd²⁺ treatments for 2 weeks resulted in an increase in leaf DNA damage. This increase may be however associated with necrotic and apoptotic DNA fragmentation, as the affected plants had inhibited growth and distorted yellowish leaves. For comparison, the monofunctional alkylating agent ethyl methanesulphonate, and γ-rays were assessed for induced DNA damage. Analysis of the accumulation of cadmium by inductively coupled plasma optical emission spectrometry demonstrates that roots accumulate almost 9-fold more cadmium than aboveground parts of the rooted potato cuttings. This may explain the absence of Cd²⁺ genotoxicity in leaves after short-term treatments.     

Inhibition of Dark CO(2) Fixation and Photosynthesis in Leaf Discs of Corn Susceptible to the Host-specific Toxin Produced by Helminthosporium maydis, Race T

The host-specific toxin produced by Helminthosporium maydis, race T, causes 50% inhibition of dark fixation of ¹⁴CO₂ by leaf discs of susceptible (Texas male sterile) corn when it is diluted to approximately 1/10,000 of the volume of the original fungus culture filtrate. Dilutions of 1/10 or less are required for equivalent inhibition of discs prepared from resistant (N) corn. Root growth and photosynthesis were considerably less sensitive (dilution values 1/3000 and 1/1200, respectively), as was leakage of ¹⁴C induced by toxin from preloaded discs. Based on literature values for dilutions causing ion leakage or inhibition of mitochondrial oxidation, toxin dilutions several orders of magnitude greater bring about inhibition of dark CO₂ fixation. Preincubation of discs in light increased sensitivity of dark fixation to toxin and an effect of light on symptom development was shown. Phosphoenolypruvate carboxylase activity in extracts of roots or leaves was not affected by toxin nor was the enzyme level altered in excised leaves treated with toxin. Inhibition of dark fixation of CO₂ provides a bioassaay for race T toxin which is both reliable and rapid.     

Effects of Lead on Ultrastructure of Isoetes sinensis Palmer (Isoetaceae), a Critically Endangered Species in China

Isoetes sinensis Palmer (Isoetaceae) is a critically endangered fern that is a marsh plant (that is an aquatic or amphibious plant) in China. To evaluate damage or influence of lead (Pb) on cell ultrastructure in I. sinensis, we used 2000mg·L^-1 Pb(NO₃)₂ solution to treat I. sinensis for 35d, and used transmission electron microscope (TEM) to observe the cell ultrastructure of leaf blades and roots of the plant. Our results indicated that Pb induced distinct changes of the organelles including chloroplast, mitochondria, nucleolus and vacuole. The level of damage organ was lower leaf > upper leaf > root The typical performance of the damages caused by lead shown that part of the nucleolus cracked; the cristae dilated, matrix vacuolized and membrane structure blurred in mitochondria; the vacuole cracked; grana lamella decreased, stroma lamella loosed, starch grains decreased, and membrane structure was disrupted in chloroplasts; Pb deposits were present on cell wall. The damages to chloroplasts and mitochondria were relatively severe, while damage to the nucleus was relatively lighter. The damage to the cell ultrastructure of leaf blades with direct contact with Pb was more severe than that without direct contact with Pb.     

Interactions of the tomato with two formae speciales of Fusarium oxysporum

Tomato cuttings were inoculated with Fusarium oxysporum f.sp. lycopersici (FL) and F. oxysporum f.sp. pisi (FP) by standing the cuttings in suspensions of bud-cells of the fungi. FP never induced external symptoms although the fungus persisted in the lower parts of the cutting. FL at concentrations from 10³ to 10⁶ spores per ml induced typical wilt symptoms but there was subsequent recovery of some cuttings with the production of uninvaded side shoots. When the cuttings were inoculated with mixed suspensions of bud cells of the two fungi there was marked reduction of symptoms. The extent of this reduction was related to the proportion of FP/FL bud cells for a fixed inoculum of FL in the mixture and was moderate at a rate of 1/3 and complete at ratios from 4/1 to 9/1. Mixed suspensions of heat-killed bud cells of FP with live bud cells of FL in the ratio of 4/1 induced normal symptoms and it was concluded that the symptom mitigation induced by FP was related to the presence of living cells of the fungus. Root inoculations with mixed suspensions also gave less wilt than with FL alone. Symptom mitigation was apparently associated with a reduction of the extent of invasion of the cuttings but in vitro tests failed to demonstrate that exudates or extracts from normal or invaded tomato tissue induced any reduction of growth of the tomato pathogen.     

The lose of juvenility elicits adventitious rooting recalcitrance in apple rootstocks

For perennial woody plants, softwood cutting is an efficient technique for larger scale propagation and adventitious rooting of cuttings is one of the most crucial steps. To evaluate the significance of juvenility on adventitious rooting, rooting rates was compared between softwood cuttings collected from apomictic seedlings (juvenile), in vitro cultured plants (rejuvenated), suckers (juvenile like) and canopy shoots (adult) of reproductively mature trees in Malus xiaojinensis. After pre-treatment with indole-3-butytric acid (IBA) (3,000 mg L^?1) + H₂O₂ (50 mM), rooting rates in cutting from juvenile, juvenile like and rejuvenated donor plants were significantly higher (>90 %) than that from adult trees. The effects of IBA on adventitious rooting were enhanced significantly by exogenous H₂O₂. After 15 passages of in vitro subculture, the micro-shoots from adult phase explants were rejuvenated successfully, marked by the elevated expression of miR156 in the leaflets of the micro-shoots. But the rooting ability of rejuvenated micro-shoots was recovered delayed at the 18th or 21st passage of subculture. During the process of rejuvenation, the leaf indole-3-acetic acid contents and the expressions of rooting related genes CKI1, ARRO-1, ARF7 and ARF19 increased significantly. In contrary, the leaf abscisic acid contents decreased. A lack of juvenility is the most important limiting factor governing adventitious rooting of softwood cuttings in apple rootstocks.     

Neuroprotection of the leaf and stem of Vitis amurensis and their active compounds against ischemic brain damage in rats and excitotoxicity in cultured neurons

Vitis amurensis (Vitaceae) has been reported to have anti-oxidant and anti-inflammatory activities. The present study investigated a methanol extract from the leaf and stem of V. amurensis for neuroprotective effects on cerebral ischemic damage in rats and on excitotoxicity induced by glutamate in cultured rat cortical neurons. Transient focal cerebral ischemia was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion (MCAO/reperfusion) in rats. Orally administered V. amurensis (25-100 mg/kg) reduced MCAO/reperfusion-induced infarct and edema formation, neurological deficits, and neuronal death. Depletion of glutathione (GSH) level and lipid peroxidation induced by MCAO/reperfusion was inhibited by administration of V. amurensis. The increase of phosphorylated mitogen-activated protein kinases (MAPKs), cyclooxygenase-2 (COX-2), and pro-apoptotic proteins and the decrease of anti-apoptotic protein in MCAO/reperfusion rats were significantly inhibited by treatment with V. amurensis. Exposure of cultured cortical neurons to 500 μM glutamate for 12 h induced neuronal cell death. V. amurensis (1-50 μg/ml) and (+)-ampelopsin A, γ-2-viniferin, and trans-?-viniferin isolated from the leaf and stem of V. amurensis inhibited glutamate-induced neuronal death, the elevation of intracellular calcium ([Ca²⁺]_i), the generation of reactive oxygen species (ROS), and changes of apoptosis-related proteins in cultured cortical neurons, suggesting that the neuroprotective effect of V. amurensis may be partially attributed to these compounds. These results suggest that the neuroprotective effect of V. amurensis against focal cerebral ischemic injury might be due to its anti-apoptotic effect, resulting from anti-excitotoxic, anti-oxidative, and anti-inflammatory effects and that the leaf and stem of V. amurensis have possible therapeutic roles for preventing neurodegeneration in stroke.     

The influence of leaf area on the rooting physiology of leafy stem cuttings of Terminalia spinosa Engl.

Summary The effect of different leaf areas on the rooting of Terminalia spinosa Engl. cuttings in an non-mist propagation system in glasshouses at Edinburgh was investigated by trimming the leaves to 0, 7.5, 15 and 30 cm² before cuttings were severed from stockplants. Cuttings were taken to a standard length of 5 cm from the lateral shoots of previously pruned stockplants grown in a tropicalised glasshouse. During the rooting period, photosynthetic rate, stomatal conductance, water potential and relative water content of the cuttings were assessed at regular intervals. It was found that (i) removal of the entire leaf area prevented rooting; (ii) cuttings with a 7.5 cm², 15 cm² and 30 cm² leaf all achieved 80% rooting after 3 weeks; (iii) an increase in leaf area from 7.5 cm² to 30 cm² increased the rate of rooting and the length of the longest root after 2 weeks, but also increased the number of original leaves abscised after 6 weeks; and (iv) the greatest number of new leaves were produced by cuttings with 7.5 cm² and 15 cm² leaf area per cutting. All leafy cuttings actively photosynthesized during the propagation period, with a mean rate of 2 mol CO₂ m^-2 s^-1 with an irradiance of 100 mol m^-2 s^-1. Cuttings with 30 cm² leaf area had lower relative water contents, lower stomatal conductances and lower photosynthetic rates per unit leaf area than those with a 7.5 cm² and 15 cm² leaf. It was concluded that T. spinosa cuttings are easy to root, provided the cuttings have leaves to produce current assimilates.A member of the Edinburgh Centre for Tropical Forests     

Some phytotoxic glycopeptides from Ceratocystis ulmi, the Dutch Elm Disease pathogen

Ceratocystis ulmi, the causal agent of Dutch Elm Disease, produces phytotoxic glycopeptides in culture. A mixture of phytotoxic glycopeptides has been prepared by affinity chromatography on a concanavalin A-Sepharose column and collectively they have been termed the toxin. The polydisperse component that makes up the majority of the toxin (80%) by weight has a molecular weight of about 2.7·10⁵. The large molecular weight component (<5%) elutes at the void volume of a Bio-Gel A50 m column. The other component (15%) appears as a trailing peak on the edge of the major component and has an approximate molecular weight of 7 · 10⁴. The toxin is composed of 38% sugar residues, primarily rhamnose and mannose, and 7% amino acid residues. Methylation analysis coupled with mild acid hydrolysis indicates that the backbone of the polysaccharide portion of the toxin is composed of α-1,6-linked mannosyl residues with a 3-linked terminal rhamnosyl residue linked to C-3 of almost every mannosyl residue. The carbohydrate portion of the molecule is linked to the peptide via O-glycosidic linkages to both threonyl and seryl residues. All three components of the toxin are capable of causing wilt in stem cuttings of American elm.     

An explanation for the light requirement of AgNO3 to inhibit ethylene-induced abscission

The loss of the antiethylene activity of Ag⁺ on leaf abscission by incubation in the dark was investigated. When primary leaves were removed from cuttings of Vigna radiata previously sprayed with AgNO₃, dark-induced abscission of the petioles was inhibited, compared to untreated leafless controls, in the presence or absence of ethephon, an ethylene-releasing compound. Malformin did not negate inhibition of petiole abscission induced by Ag⁺. Although leaf removal restored the antiethylene activity of Ag⁺ in the dark, macerates of leaves from dark-aged cuttings did not negate the ability of Ag⁺ to inhibit petiole abscission in the dark. Abscisic acid completely abolished the ability of Ag⁺ to counteract ethephon-induced leaf abscission in the light, and almost completely abolished the Ag⁺-induced inhibition of petiole abscission from explants in the dark. It is proposed that the phytochrome requirement for the antiethylene activity of Ag⁺ on ethephon-induced leaf abscission involves prevention of the formation, accumulation, or transport of a substance in leaves in the dark which negates Ag⁺ activity. This substance may be abscisic acid or another substance with similar biological activity.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

A Novel Enolic beta-Ketoaldehyde Phytotoxin Produced by Stemphylium botryosum f. sp. lycopersici : PARTIAL CHEMICAL AND BIOLOGICAL CHARACTERIZATION

A new phytotoxin, stemphyloxin I, C₂₁H₃₂O₅, was isolated from cultures of the pathogenic fungus Stemphylium botryosum f. sp. lycopersici. The toxin is a tricyclic compound possessing a most unusual β-ketoaldehyde group. Injection of stemphyloxin I into a tomato leaflet caused unlimited necrotic spots and a loss of turgor, which at higher toxin concentration wilted the whole compound leaf. Visible symptoms could be observed at a toxin concentration as low as 2.7 micromolar. Stemphyloxin I is a nonspecific toxin. It exhibits a differential toxicity towards various plants, tomato and eggplant being the most sensitive. Incorporation of [¹⁴C]amino acids into proteins of exponentially growing tomato cell suspension was completely suppressed in the presence of 1 micromolar toxin. The toxin showed no significant difference in its inhibitory activity against green and white tomato cell cultures. The methoxy derivative of stemphyloxin I, in which the β-ketoaldehyde group is exclusively modified, showed a reduction of approximately 50 times in its inhibitory activity as compared to the toxin. The diacetate derivative conferred the same activity as stemphyloxin I.     

3,4-Dihydroxybenzaldehyde purified from the barley seeds (Hordeum vulgare) inhibits oxidative DNA damage and apoptosis via its antioxidant activity

Barley is a major crop worldwide. It has been reported that barley seeds have an effect on scavenging ROS. However, little has been known about the functional role of the barley on the inhibition of DNA damage and apoptosis by ROS. In this study, we purified 3,4-dihydroxybenzaldehyde from the barley with silica gel column chromatography and HPLC and then identified it by GC/MS. And we firstly investigated the inhibitory effects of 3,4-dihydroxybenzaldehyde purified from the barley on oxidative DNA damage and apoptosis induced by H₂O₂, the major mediator of oxidative stress and a potent mutagen. In antioxidant activity assay such as DPPH radical and hydroxyl radical scavenging assay, Fe²⁺ chelating assay, and intracellular ROS scavenging assay by DCF-DA, 3,4-dihydroxybenzaldehyde was found to scavenge DPPH radical, hydroxyl radical and intracellular ROS. Also it chelated Fe²⁺. In in vitro oxidative DNA damage assay and the expression level of phospho-H2A.X, it inhibited oxidative DNA damage and its treatment decreased the expression level of phospho-H2A.X. And in oxidative cell death and apoptosis assay via MTT assay and Hoechst 33342 staining, respectively, the treatment of 3,4-dihydroxybenzaldehyde attenuated H₂O₂-induced cell death and apoptosis. These results suggest that the barley may exert the inhibitory effect on H₂O₂-induced tumor development by blocking H₂O₂-induced oxidative DNA damage, cell death and apoptosis.     

Thermotolerance of Leaf Discs from Four Isoprene-Emitting Species Is Not Enhanced by Exposure to Exogenous Isoprene

The effects of exogenously supplied isoprene on chlorophyll fluorescence characteristics were examined in leaf discs of four isoprene-emitting plant species, kudzu (Pueraria lobata [Willd.] Ohwi.), velvet bean (Mucuna sp.), quaking aspen (Populus tremuloides Michx.), and pussy willow (Salix discolor Muhl). Isoprene, supplied to the leaves at either 18 μL L⁻¹ in compressed air or 21 μL L⁻¹ in N₂, had no effect on the temperature at which minimal fluorescence exhibited an upward inflection during controlled increases in leaf-disc temperature. During exposure to 1008 μmol photons m⁻² s⁻¹ in an N₂ atmosphere, 21 μL L⁻¹ isoprene had no effect on the thermally induced inflection of steady-state fluorescence. The maximum quantum efficiency of photosystem II photochemistry decreased sharply as leaf-disc temperature was increased; however, this decrease was unaffected by exposure of leaf discs to 21 μL L⁻¹ isoprene. Therefore, there were no discernible effects of isoprene on the occurrence of symptoms of high-temperature damage to thylakoid membranes. Our data do not support the hypothesis that isoprene enhances leaf thermotolerance.