Epidermal Strips Research Articles

Observation of the Various Growth Stages of Rice Blast Fungus (<i>Pyricularia oryzae</i> Cavara) by Onion Epidermal Strip Method

薬剤試験を目的として病原菌の生育過程を同一実験系で観察しうる方法を検討した. その一例としてタマネギりん片上にイネいもち病菌の胞子懸濁液を点滴し, 25℃の湿室状態に保って, 本菌の生育過程を観察したところ, タマネギりん片上で生活史の各生育過程を演じることを知った. すなわち, 分生胞子発芽は3時間前後からはじまり, 5時間で90～100%に達した. 付着器形成は約3時間半後からはじまり, 5時間で70～89%, 20時間ではほぼ100%に近かった. 顆粒化は22時間後からおこり, 組織内への侵入は24時間後から見られ, 27時間後では約80～90%観察された. 菌糸は24時間で著しく伸長した. 胞子形成は2日目から4日目にかけて増加した. また各生育過程の途中, 水滴を除き, 約15分後に再び水滴を同一場所に点滴した. この操作によって本菌の発育は抑制されず, むしろ良好となった. 薬剤による阻害実験に本法を適用しうるので, 本法を“タマネギ剥離表皮検定法”としたい.

Potassium Involvement in Stomatal Movements ofPaphiopedilum

There are conflicting reports about whether guard cells of Paphiopedilum spp. accumulate K during stomatal opening. In this study X-ray microprobe analysis and histochemical staining for K indicated that K accumulated in guard cells in leaves of Paphiopedilum spp. when stomata opened. Additionally, stomatal opening in epidermal strips of P. harrisseanum could only be induced in an MES buffered KC1 medium. Element analysis off. harrisseanum epidermis also indicated substantial levels of K, Na and Ca within the tissue. We conclude that K is involved in the stomatal movements of Paphiopedilum

Effect of abscisic acid pretreatments on stomatal reopening in Vicia faba

After a pretreatment of 2 h exposure to a solution containing 2 × 10−4M ABA, reopening of stomata occurred in epidermal strips of Vicia faba L. cv. Cavalier on an ABA‐free incubation solution. After pretreatment with exogenous ABA stomatal apertures were greater when higher levels of KCl were incorporated into the solution used for reopening. Prolonged exposure to exogenous ABA (14 h) did not prevent stomatal reopening upon transfer to ABA‐free solutions. However, for both ABA and ABA‐free pretreatments, prolonged incubation (1 day after removal of epidermis) resulted in enhanced stomatal apertures when the epidermal strips were exposed to light. This effect was lost 2 days after removal of the epidermis and opening did not occur after 3 days. Epidermal strips containing endogenous ABA were obtained from wilted leaves. Reopening was greatly reduced by the endogenous ABA treatment, and variation of KCl concentration in the incubation solution had little effect on stomatal aperture. It is postulated that during wilting endogenous ABA becomes reversibly bound without loss of activity for a longer period than is obtained using exogenous ABA. The presence of other unidentified compounds may be involved in this process.

Microspectrophotometric analysis of the location of ] avenalumin accumulation in oat leaves in response to fungal infection

Cellular localization of avenalumins accumulated in oat leaves infected by either crown rust through stomata or rice blast fungus through the cuticular layer was demonstrated by microspectrophotometry using the fluorescence emission and u.v. absorption spectra characteristic of avenalumins. Thin sections of incompatible rust-infected leaves revealed collapsed mesophyll cells which emitted autofluorescence and absorbed u.v. light with spectral characteristics close to those of the avenalumins. Autofluorescent and u.v.-absorbing cells were detected only in incompatible rust race-oat combinations. Epidermal cells accumulated quite high levels of avenalumins in response to cuticular infection by the facultative pathogen. Spectrophotometry of the freshly stripped epidermis showed the presence of autofluorescent materials, with spectra characteristic of avenalumins, in infected and the directly adjacent cells. U.v.-absorption spectra close to those of avenalumins were also detected in fluorescent cells. Extraction of epidermal strips with methanol reduced fluorescence emission and u.v.-absorption in fluorescent cells by c. 40% of the original, thus suggesting that avenalumins could be partially eluted out of the fluorescent cells. Epidermal cells which were injected with aqueous solution of avenalumin I became fluorescent. Results suggest that avenalumins accumulate in the fluorescent mesophyll and epidermal cells formed in oat leaves at infection sites of fungal pathogens.

Cell Potentials and Turgor Pressures in Epidermal Cells of Tradescantia and Commelina

Cell Potentials and Turgor Pressures in Epidermal Cells of Tradescantia and Commelina

STIMULATION AND INHIBITION BY BICARBONATE OF STOMATAL OPENING IN EPIDERMAL STRIPS OF COMMELINA BENGHALENSIS

SUMMARYThe effect of 0 to 100μM bicarbonate on stomatal opening in epidermal strips of Commelina benghalensis was examined in the presence or absence of fusicoccin in light or darkness. Low concentrations of bicarbonate (up to 10 μM in the absence and 25 μM in presence of fusicoccin) stimulated stomatal opening while higher concentrations inhibited. The enhancement of opening by low concentrations of bicarbonate and phosphoenol pyruvate (PEP), and prevention of bicarbonate stimulation by malate or oxaloacetate suggested PEP carboxylase as a CO2 sensor in the guard cells. However, the inhibition of PEP carboxylase did not completely suppress the opening caused by fusicoccin. The action of fusicoccin therefore appears to involve a site other than CO2 fixation, presumably through the stimulation of proton excretion.

Effect of Ethephon on stomatal opening in detached epidermal strips of tobacco leaves (Nicotiana tabacum L. cv. Samsun)

The effect of Ethephon preparations upon stomatal openings in detached epidermal strips of tobacco leaves floated on 0.1 M KC1 was measured microscopically after different times of exposure. Addition of 0.01 v/v % or 0.1 v/v % Camposan, Ethrel, or E-BOH resulted in the opening or closing of the stomata depending upon the time of exposure and the concentration of Ethephon. It is evident from the results obtained in these studies that an increase in stomatal opening was induced by the acidity of the preparations and the released ethylene too. Prolonged times of action resulted in decreased stomatal openings or in stomata being closed, respectively. High concentrations of Ethephon also resulted in major damage being caused to guard cells after having been permitted to act upon them for an extended period of time.

Effects of Cations and Abscisic Acid on Chlorophyll a Fluorescence in Guard Cells of Vicia faba

The effects of cations and abscisic acid on chloroplast activity in guard cells of Vicia faba were investigated by analysis of the transient of chlorophyll a fluorescence. When epidermal strips containing guard cells as the only living cells were incubated in water and illuminated with strong light, chlorophyll a fluorescence rose rapidly to a high intensity and then declined slowly to a stationary level. The rate of this decline was enhanced by K(+) or Na(+), and the effect of these cations was greater when added with phosphate than with chloride as the anion. Ca(2+) suppressed the enhancement by Na(+) and, to a lesser extent, that by K(+). Abscisic acid also suppressed the enhancement by K(+) and Na(+). Since the fluorescence decline reflects the increase of intrathylakoid H(+) concentration necessary for photophosphorylation, the acceleration of the decline by K(+) (or Na(+) in the absence of Ca(2+)) implicates chloroplast activity in ion accumulation by guard cells in the light. The differential effects of phosphate and chloride suggest that chloroplast activity may be involved in malate formation in guard cells in the light.

Stomatal responses towards neutral red uptake and particulate movement in some arid zone plants

The stomata and their behaviour towards neutral red uptake in a few arid zone plant species under stress conditions have been inve stigated. Variable patterns of accumulation or non-accumulation and retention of this vital dye, by various cells of leaf epidermis have been observed. The neutral red uptake by the cells of the epidermal strip, coalescence of particles and their movement appear to be connected with the open/closed condition of the stomata.

The genetic relationship between the tomato mutants, flacca and lateral suppressor, with reference to abscisic acid accumulation

Two tomato mutants, Lycopersicon esculentum flacca and lateral suppressor, are assigned to map position 59 of chromosome 7. The tight linkage between these two gene loci was detected as a result of attempts to establish whether they would exhibit phenotypic interaction. The possibility that both mutants result in abnormalities of abscisic acid (ABA) accumulation is considered. ABA analysis supports the suggestion that plants homozygous for flacca have a substantially lower concentration but indicates that lateral suppressor homozygotes do not differ from normal in ABA content. An attempt is made to reconcile the results with those of Tucker (1976, New. Phytol. 77, 561-568) by suggesting that lateral suppressor plants may accumulate high levels of an ABA metabolite which is indistinguishable from ABA using the Commelina epidermal strip bioassay.

Kaempferol 3-O-Galactoside, 7-O-Rhamnoside is the Major Green Fluorescing Compound in the Epidermis of Vicia faba

The vacuoles of lower epidermal strips from Vicia faba exhibit an intrinsic green fluorescence when incubated in alkaline buffers. Using an alkaline-induced absorbance change as a spectrophotometric assay, the major pigment responsible for this fluorescence was isolated and identified as the flavonoid: kaempferol 3-O-galactoside, 7-O-rhamnoside. The aqueous absorption maxima were 394 and 341 nanometers at pH 10.0 and 6.0, respectively, with a pKa of 8.3 and the fluorescence emission maximum was 494 nanometers at pH 10.0. The in vivo concentration was estimated to be between 3 and 10 micromolar. The absorption spectrum of this flavonoid is different from the action spectrum for stomatal opening indicating that this compound is not the photoreceptor pigment for the blue light response of Vicia faba guard cells.

The mechanism of proton-induced increase of cell wall extensibility

Acid-induced elongation of Helianthus hypocotyl epidermal strips was investigated. We found no indications for the existence of an acid activated cell wall loosening enzyme, as the elongation of living or frozen — thawed epidermal strips show no pH optimum. Pretreatment with formaldehyde is without influence on acid-induced extension. The effect of hydrogen ions can be substituted by Ca 2+-chelating of EDTA with the same range of effectiveness. Other monovalent ions like potassium or sodium are much less effective than protons.

Lignification as a mechanism for induced systemic resistance in cucumber

Infection of the first true leaf of cucumber with Colletotrichum lagenarium induces systemic resistance (protection) against subsequent challenge by C. lagenarium or Cladosporium cucumerinum Conidia of C. lagenarium germinated and formed appressoria equally well on isolated epidermal strips from protected and control plants; however, penetration occurred less frequently into epidermal strips from protected plants. Inhibition of penetration was associated with host epidermal cell wall lignification directly under appressoria. Lignification occurred more rapidly and to a greater extent in protected than in control plants. Infection of etiolated seedlings resistant to scab with C. cucumerinum made the seedlings resistant to C. lagenarium, and elicited a rapid lignification of penetrated and adjacent host cells. This lignification reaction in the resistant cultivar was histologically indistinguishable from that at sites of penetration in a systemically protected “susceptible” cultivar challenged with C. cucumerinum. Germination of conidia and frequencies of penetration of C. cucumerinum were similar in protected and control plants, but development of the fungus in lignified tissue was markedly restricted. Coniferyl alcohol, a lignin precursor, was toxic to C. lagenarium and C. cucumerinum when only 0·3 hg were applied to thin-layer bioassay plates. This suggests that it might function as a phytoalexin. Mycelia of C. lagenarium and C. cucumerinum were “lignified” in the presence of coniferyl alcohol, peroxidase and hydrogen peroxide.

Mutual Antagonism of Sulfur Dioxide and Abscisic Acid in Their Effect on Stomatal Aperture in Broad Bean (Vicia faba L.) Epidermal Strips

Abscisic acid (ABA) was found to counteract the stomatal opening in Vicia faba L. caused by SO(2). The antagonism between SO(2) and ABA was mutual, and their combined effect depended upon which compound was in the greatest concentration. Stomatal apertures were monitored in detached epidermal strips floated in the light on aqueous solutions of SO(2) (sulfurous acid) and/or ABA in 0.01 molar sodium citrate buffer (pH 5.8). Low concentrations of sulfurous acid (10(-10) to 10(-7) molar) increased stomatal aperture, but concentrations greater than 10(-5) molar decreased it. A progressive decrease in aperture size occurred as ABA was increased from 10(-10) to 10(-5) molar.No evidence was found for a direct chemical reaction between the buffered sulfurous acid and ABA (exogenous or endogenous). Extractable, endogenous ABA in the strips remained relatively constant after exposure to several different concentrations of sulfurous acid. A technique for quantitating ABA from methanolic extracts of small samples of epidermis (20 milligrams dry weight) using reverse phase high performance liquid chromatography is described.

Stomatal responses in isolated epidermis of the crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr.

The optimal conditions for opening of stomata in detached epidermis of the Crassulacean Acid Metabolism (CAM) plant Kalanchoe daigremontiana were determined. Stomatal opening in CO2-free air was unaffected by light so subsequently all epidermal strips were incubated in the dark and in CO2-free air. Apertures were maximal after 3 h incubation and were significantly greater at 15° C than 25° C. Thus stomata in isolated epidermis of this species can respond directly to temperature. Stomatal opening was greatest when the incubating buffer contained 17.6 mol m(-3) K(+), but decreased linearly with increasing K(+) concentrations between 17.6 and 300 mol m(-3); the decrease in aperture was shown to be associated with increasing osmotic potentials of the solutions. Reasons for this behaviour, which differs from that of many C3 and C4 species, are discussed. Stomatal apertures declined linearly upon incubation of epidermis on buffer solutions containing between 10(-11) and 10(-5) mol m(-3) abscisic acid (ABA). Hence stomata on isolated epidermis of K. daigremontiana respond to lower concentrations of ABA than those of any species reported previously.

Low Proton Conductance of Plant Cuticles and Its Relevance to the Acid-Growth Theory

Evidence obtained on the relation between the pH of the medium and the growth of intact stem sections is compatible with the acid-growth theory only if the proton conductance of the cuticle is so low that the cuticle is an effective barrier to the entry or exit of protons from the tissue. By measuring the rate at which protons cross frozen-thawed epidermal strips of sunflower (Helianthus annuus L.) and soybean hypocotyls (Glycine max Morr.) and enzymically isolated cuticles of Berberis aquifolium Persh. and tomato (Lycopersicum esculentum Mill.) fruit, we have now demonstrated the low proton conductance of the cuticular layer. Unless the conductance is enhanced by abrasion of the cuticle or by removal of the cuticular waxes, proton movement into and out of a tissue across the cuticle will be significant only over long time periods.

Soluble carbohydrates in leaf epidermis of Allium cepa: a potential role in stomatal function?

Fructan polymers were found in the 80% ethanol extract of epidermal strips of Allium cepa L. These carbohydrate polymers were detectable in epidermis held in darkness in the presence of abscisic acid, but virtually disappeared in the presence of KCl in the light. The fructans in onion leaf epidermal strips constitute a very soluble polymeric carbon source which could be of importance in stomatal function.

Effect of boron on stomatal opening in epidermal strips of Commelina communis

Boron enhances stomalal opening in epidermal strips of Commelina communis L. under conditions conductive to opening, i.e. light and KCI. This effect is obtained when boron is given together or before KCI application. Since boron increases 86Rb net uptake in both illuminated epidermal strips and illuminated isolated guard cells, it is suggested that boron acts either by reducing potassium leakage or by increasing its uptake, thus leading to increased osmotic potential and, as a result, to stomatal opening.

Induced stomatal responses in epidermal strips ofVicia fabaL.

Induced stomatal responses in epidermal strips of<i>Vicia faba</i>L.

Energy Supply for Stomatal Opening in Epidermal Strips of Commelina benghalensis

The influence of light or darkness on stomatal opening in epidermal strips of Commelina benghalensis was evaluated in the presence or absence of O(2) and/or metabolic inhibitors. Opening was restricted in nitrogen and was promoted by NADH and acids of the tricarboxylic acid cycle (succinate and alpha-ketoglutarate) in CO(2)-free air in light as well as in darkness. The enhancement by light of stomatal opening was prevalent under nitrogen or in the presence of the respiratory inhibitors (sodium azide and oligomycin). Respiratory inhibitors decreased the opening in light or darkness under CO(2)-free air but exhibited no effect under nitrogen, whereas phosphorylation uncouplers were inhibitory in light or darkness under both CO(2)-free air and nitrogen. The results suggest that oxidative phosphorylation is a basic source of energy for stomatal opening, although photophosphorylation could be an energy source.