Beet Discs Research Articles

Selection of a Suitable Disc Bioassay for the Screening of Anti-Tumor Molecules

The crown gall induced in potato discs by Agrobacterium tumefaciens is becoming largely utilised in screening anti-tumor agents. The present work is showing that beet discs are more adequate for the anti-tumor screening test. In fact, maximal tumor induction was observed on beet discs (87.5%), followed by carrot discs (75%) and potato discs (68.5%). Beet discs present the most sensibility to crown gall disease with a fast expression of symptoms and more visible galls without any staining need. The beet discs bioassay was carried out by using some synthesized organometallics known for their antitumor activity in mammalian cells. We found significant crown gall inhibition (20.7% to 40.55%) of the tested compounds. Overall results supported that beet bioassay might be a potential prescreen system of anti-tumor molecules in mammalian cells.

Changes in the level and activation state of the plasma membrane H(+)-ATPase during aging of red beet slices.

The effect of aging on the plasma membrane (PM) H(+)-ATPase of red beet (Beta vulgaris L.) parenchyma discs was analyzed in PM purified by aqueous two-phase partitioning. Aging increased both the activity in the amount of immunodetectable H(+)-ATPase in the PM. The activity assayed at slightly alkaline pH values increased earlier and more strongly than that assayed at acidic pH values, so that the pH curve of the enzyme from aged beet discs was shifted toward more alkaline values. Aging decreased the stimulation of the PM H(+)-ATPase activity by controlled trypsin treatments or by lysophosphatidylcholine. After trypsin treatment the pH dependence of H(+)-ATPase from dormant or aged beet discs became equal. These results indicate that aging not only increases the level of H(+)-ATPase in the PM, but also determines its activation, most likely by modifying the interaction between the autoinhibitory carboxyl-terminal domain and the catalytic site. When the PM H(+)-ATPase activity was assayed at a slightly alkaline pH, the tyrosine modifier N-acetylimidazole inhibited the H(+)-ATPase in the PM from dormant beet discs much less than in the PM from aged discs, suggesting that modification of a tyrosine residue may be involved in the activation of the PM H(+)-ATPase induced by aging. The results are discussed with regard to aging-induced development of transmembrane transport activities.

Membrane damage caused by exposure to t-butyl hydroperoxide

As well as red beet discs, turnip and sweet potato storage tissues exposed to t-butyl hydroperoxide ( t-BHP) showed leakage of several cellular components including potassium ions, reducing sugars and UV-absorbing materials. Even at the beginning of the eight hr-treatment period, leakage of potassium ions was accompanied by a decrease in respiratory activity. At the time when the respiration of turnip discs started to decrease, accumulation of lipid hydroperoxides was detected by four different methods. Just after t-BHP treatment started incorporation of t- BHP was observed and the amount increased linearly to a maximum after ca four hr and then decreased gradually. This disc experiment suggests that t-BHP, a model compound of oxygen stress, is incorporated into plant tissues and may cause directly or indirectly lipid peroxidation in various biomembranes which leads to a leakage of cellular components and a decrease in respiratory activity.

<i>tert</i>.-Butylhydroperoxide 処理による赤ビート切片からのベタシアニン リーケージに及ぼす各種の添加物質の影響

With the purpose of getting much information about the mechanism of leakage observed at the onset of plant senescence or other physiological disorders, a model system using red beet discs has been investigated in our study. The present paper describes the effects of various chemicals added to the incubation medium, in which red beet discs are exposed or pre-exposed to tert. -butylhydroperoxide (BHP), on the leakage of red betacyanin pigment from the cells. The mechanism of betacyanin leakage induced by the BHP treatment is suggested from these results.1. Under the same conditions reported in our previous paper, the treatment with BHP (10mM and 8hrs) evidently caused the abrupt increase of OD 540nm (leakage of red betacyanin pigment), and the optical density reached about 1.0 from 5hrs to 7hrs after the treatment.2. The addition of 5mM sodium diethyldithiocarba mate, 20mM thiourea, 5.0mM (+)-catechin and 1mM n-propyl gallate, separately, to the incubation medium effectively inhibited the increase in OD 540nm, when each chemical and BHP were added simultaneously.3. L-Cysteine (5mM) and D-ascorbic acid (5mM), which are well known as endogenous reducing agents, also showed the inhibitory effect on Betacyanin leakage of red beet discs.4. When (+)-catechin (5mM), cysteine (10mM) and thiourea (20mM) wereadded, separately, to the incubation medium in the absence of BHP after the BHP treatment (10mM and 8hrs), they also showed significant effects on inhibition of betacyanin leakage.5. The presence of 5mM EDTA or 2.5mM FeCl3 in the incubation medium containing 10mM BHP enhanced the increase in OD 540nm considerably.These results suggest that BHP taken into the discs of red beet, as indicated in the red cells of human blood, may be transformed by either free Fe3+, chelated Fe3+ or other endogenous unknown factors to the tert. -butoxyl radical, and consequently will attack various biomembranes in the cells to cause the lakage of red betacyanin pigment. Further biochemical investigation will be required to confirm the mechanism.

<i>tert</i>.-Butylhydroperoxide 処理による赤ビート切片からのベタシアニン リーケージの諸性質

In order to obtain information on the mechanism of leakage which is often observed at the onset of plant senescence or some physiological disorders, a model experimental system has been established, which examines the kinetics and properties of betacyanin leakage from red beet discs treated with tert.-butylhydroperoxide (t-BHP).1. No leakage of betacyanin was observed even after 4 days when red beet discs (φ13mm×1.5mm) were incubated in MES-NaOH (pH 5.8) buffer containing 0.2 M mannitol and streptomycin (0.5μg/ml) in the absence of t-BHP in the dark at 20°C.2. In this experimental system, changing the concentration (2-10mM) of t-BHP and the duration (2-8 hours) of the treatment enabled the time when the betacyanin leakage occurred to be easily controlled.3. As well as betacyanin, potassium ions, reducing sugars, UV-absorbing components and proteins also leaked out from red beet discs after the t-BHP treatment, but the extent of leakage appeared to be related to the molecular size.4. The leakage showed a remarkable pH dependence between pH 5.05 and 7.43; the leakage of betacyanin occurred much faster below pH 5.8 than near pH 7.5. Addition of calcium ion and spermine to the medium effectively prevented the leakage of betacyanin until t-BHP treatment caused leakage. Glutathione was effective only when it was used before treatment with t-BHP. Their mechanisms of prevention seem to differ.This experimental system, using red beet discs, appears to be very useful for investigating the mechanism of leakage.

Activity of the Ethylene-forming Enzyme Measured in vivo at Different Cell potentials

Summary Activity of the ethylene-forming enzyme (EFE) has been determined in hypocotyl segments of mung bean ( Vigna radiata L.) and in discs of red beet ( Beta vulgaris L.) at the same time as the plasma membrane potential difference, measured with a microelectrode, has been progressively reduced by titration with the protonophores 2,4-dinitrophenol and carbonyl cyanide m-chloropheny1hydrazone and with KCl. Treatment with the protonophores affected EFE activity in both the mung bean hypocotyls and in the beet discs, although in different ways in the two tissues, while the depolarisation resulting from KCl treatment did not affect the EFE activity of either tissue. The results obtained are not consistent with the proposal of John (1983, FEBS Letters 152, 141) that a potential-dependent EFE activity is located at the plasma membrane of plant cells.

The decolorization of betacyanin by polyamines

The pigment betacyanin, the efflux of which has been used as a measure of membrane integrity in red beet discs, has now been shown to react with di- and polyamines with consequent decolorization.

A Comparison of Methods for Measuring Turgor Pressures and Osmotic Pressures of Cells of Red Beet Storage Tissue

Two methods for measuring the turgor pressures of cells in discs of storage tissue of red beet (Beta vulgaris L.) were compared, and a centrifugation method for extracting sap from frozen and thawed tissue was evaluated. Turgor pressures were measured directly using a pressure probe, or indirectly using a vapour pressure osmometer. With the latter, discs were placed directly in the osmometer chamber and turgor was calculated as the difference in osmotic pressure before and after freezing and thawing. Turgor in freshly cut discs, measured with the pressure probe, was between 0 012 MPa and 0-118 MPa with a mean ±s.d. of 0 092 + 0-032 MPa (n = 24). That measured with the osmometer varied between 0-08 MPa and 0-12 MPa with a mean ±s.d. of 0-09 ±0-10 MPa(n = 54). After vacuum infiltration of discs with distilled water, the turgor measured with the pressure probe increased to 1-05 1-12 MPa. Turgor measured with the osmometer also increased after vacuum infiltration but was, on average, 12% lower than that measured with the pressure probe. Overall, the results suggest that for routine measurements, the osmometer can provide reasonable estimates of the turgor of cells in beet discs. This is because a number of factors that, potentially, could interfere with this method have only a small effect in this tissue. None of the measured turgors is indicative of that occurring in intact storage roots because both excision and vacuum infiltration of discs alter the concentrations of solutes in the extracellular space. The osmotic pressure of sap extracted by centrifugation from frozen and thawed discs was not significantly different from that measured by placing frozen and thawed discs directly in the osmometer. Solute concentrations in the sap were not significantly different from those measured by chemical extraction of discs.

Auxin-Induced Ethylene Production as Related to Auxin Metabolism in Leaf Discs of Tobacco and Sugar Beet

Exogenously supplied indole-3-acetic acid (IAA) stimulated ethylene production in tobacco (Nicotiana glauca) leaf discs but not in those of sugar beet (Beta vulgaris L.). The stimulatory effect of IAA in tobacco was relatively small during the first 24 hours of incubation but became greater during the next 24 hours. It was found that leaf discs of these two species metabolized [1-(14)C]IAA quite differently. The rate of decarboxylation in sugar beet discs was much higher than in tobacco. The latter contained much less free IAA but a markedly higher level of IAA conjugates. The major conjugate in the sugar beet extracts was indole-3-acetylaspartic acid, whereas tobacco extracts contained mainly three polar IAA conjugates which were not found in the sugar beet extracts. The accumulation of the unidentified conjugates corresponded with the rise of ethylene production in the tobacco leaf discs. Reapplication of all the extracted IAA conjugates resulted in a great stimulation of ethylene production by tobacco leaf discs which was accompanied by decarboxylation of the IAA conjugates. The results suggest that in tobacco IAA-treated leaf discs the IAA conjugates could stimulate ethylene production by a slow release of free IAA. The inability of the exogenously supplied IAA to stimulate ethylene production in the sugar beet leaf discs was not due to a deficiency of free IAA within the tissue but rather to the lack of responsiveness of this tissue to IAA, probably because of an autoinhibitory mechanism existing in the sugar beet leaf discs.

Effects of pH and Ethephon on Betacyanin Leakage from Beet Root Discs

Betacyanin leakage from beet root discs was found to increase with decreasing pH of the incubation medium. Although 10 millimolar Ca(2+) reduced pigment leakage at pH 3.5, it was ineffective at pH 2.3. Leakage was also stimulated by 100 micrograms per milliliter (2-chloroethyl)-phosphonic acid (pH 3.1), but when the solution was neutralized, this leakage was eliminated. Bubbling C(2)H(4) through a neutral medium containing beet discs had no effect on pigment leakage; it appears that the effect of Ethephon solutions on this process is a function of their low pH.

Effect of Glyphosate on Membrane Permeability in Red Beet (Beta vulgaris) Root Tissue

When discs of red beet (Beta vulgarisL.) root were immersed in various concentrations of glyphosate [N-(phosphonomethyl)glycine] either in phosphate (pH 6.8) or citrate (pH 6.2) buffer, no efflux of betacyanin occurred even after 1 week. This suggests that glyphosate had little or no effect on membrane permeability. When beet discs were immersed in distilled water containing glyphosate, the betacyanin efflux increased with time and concentration. Measurement of pH indicated that the solutions were strongly acidic at first, but tended to approach a pH of 6.0 over 8 h, after which time betacyanin efflux slowed down or stopped. The tissue was able to buffer the solutions sufficiently to prevent further efflux. When tissue discs were immersed in a series of citrate buffer ranging from pH 3 to 6.2, efflux occurred at pH 3 and slightly at pH 6.2. Efflux was not altered significantly with the addition of glyphosate.

Effect of humic acid on invertase synthesis in roots of higher plants

Humic acid (HA) stimulated the development of invertase activity in beetroot storage tissue discs washed in water and also in pea root segments, excised from the 2–4 mm region behind the root tip, during their most active growth phase when cultured in sucrose media. In contrast HA had no effect on the incorporation of [ 14C]leucine into proteins in either tissue. Inhibitors of protein synthesis ( d- threo-chloramphenicol, cyclohexamide and puromycin) and RNA synthesis (actinomycin d) inhibited invertase development and the incorporation of [ 14C]leucine into proteins in both tissues, while also abolishing the stimulation of invertase development in the presence of HA. Similar effects were produced by the amino acid analogues, p-fluorophenylalanine, cis-4-hydroxy- l-proline and azetidine-2-carboxylic acid but, with the exception of p-fluorophenylalanine in beet discs, the inhibitory effects of the analogues were prevented in the presence of the corresponding natural amino acid. The results show that HA stimulated the synthesis of de noro invertase during the period when it also enhanced cell elongation and the effect was dependent on continuous RNA and protein synthesis.

INDUCTION OF WILTING BY MANNOSE IN SPINACH BEET LEAVES

SUMMARYMannose, but not glucose, induces loss of turgor in whole plants, detached leaves and leaf discs of spinach beet. There is no comparable effect in leaves of tobacco and geranium. Supply of mannose to spinach beet discs causes an enhanced conductivity in the bathing medium after 8–10 h and this is associated with increased leakage of potassium ions over controls. After 24–36 h, water‐soaking is evidence of intercellular accumulation of water in the tissue. Previous work has established that mannose sequesters orthophosphate as mannose phosphate in leaves of spinach beet but not in those of tobacco and geranium, and the implied changes in cell membrane permeability are probably due to limited availability of Pi for ATP synthesis and turnover of phospholipid membrane components. The rate of cuticular transpiration may also be increased.

Free Space Characteristics of Barley Leaf Slices

Measurements are described of free space content of barley leaf slices. It is shown that the leaf slices contain a Donnan free space of about 3 µ-equiv/g fresh weight of tissue at a concentration of 300 mN, together with a water free space occupying 0.21 ml/g fresh weight. The Donnan free space is shown to be located in cell walls, as in other tissues (beet discs, barley roots) but the water free space is largely due to cut or damaged cells, injected intercellular spaces, and surface films of solution. The results are discussed in relation to free space of intact leaves.

Correlation between the Suppression of Glucose and Phosphate Uptake and the Release of Protein from Viable Carrot Root Cells Treated with Monovalent Cations

Treating carrot (Daucus carota L.) discs with ice-cold NaCl solutions for 30 minutes caused three effects that appear to be functionally related: the exchange of tissue Ca(2+) and Mg(2+) for Na(+), the release of protein, and the suppression of active uptake of glucose and orthophosphate. Cyclosis continued apparently unabated after treatment with NaCl at concentrations of up to 0.25 m, so the cells remained viable and energetically competent. The correlation between the release of Ca(2+) and Mg(2+) and release of protein, and between these effects and the suppression of glucose and orthophosphate uptake, supports the hypothesis that divalent cations maintain, and monovalent cations disrupt, linkages between the outer cell surface and proteins required for active solute uptake. Calcium preserved uptake activity only when it was added in time to prevent the release of protein. Cells gradually recovered some glucose uptake activity after it had been completely inactivated by treatment with 0.25 m NaCl. This recovery occurred in the absence of added Ca(2+). It was inhibited by puromycin and so appears to require some protein synthesis. Beet (Beta vulgaris L.) discs were more resistant than carrot discs to treatment with NaCl solutions, thus reflecting the difference in tolerance of the two species to sodicity.

Effects of Humic Acid on Protein Synthesis and Ion Uptake in Beet Discs

Humic acids stimulate the development of phosphate uptake-capacity in beet discs during ageing under aseptic conditions without affecting phosphate uptake per se. Chloride uptake is inhibited by humic acids as is the development of chloride uptake capacity. The uptake of proline and leucine is not influenced by ageing in humic acid. While measurements of the incorporation of these amino acids into the sub-cellular particles of discs indicate that humic acid does not affect protein synthesis in general, the stimulation of invertase development during ageing does show a definite effect on some aspect of protein synthesis.

The Plasmalemma: Seat of the Type 2 Mechanisms of Ion Absorption

According to the classical view, a plant cell is a metabolic microcosm separated from the medium without by a membrane, the plasmalemma, which acts as a permeability barrier to polar solutes including inorganic ions. Many such solutes nevertheless are taken into the cell. This phenomenon is commonly thought to be due, not to diffusion of the free ions across the plasmalemma, but to the operation of mechanisms of active transport (3). The resistance of this membrane to free diffusion of ions and many other solutes, coupled with its capacity for transport, is thought to fulfill the twin requirement for the maintenance of chemical homeostasis within the cytoplasm and for the regulated transport of nutrients and metabolites into and out of the cell. This, the classical view of the function of the plasmalemma, has been repeatedly challenged. It has been claimed, specifically, that inorganic ions readily diffuse across the plasmalemma, a claim which has been countered by cogent arguments on the part of defenders of the classical view )(1, 2, 3, 4,8,9). Nevertheless, Laties and his collaborators (11, and references cited there) have once again put forward the contention that ions readily negotiate the plasmalemma by diffusion. They confirmed for corn roots and beet discs the operation of dual mechanisms of absorption of a given ion, as described by Epstein (5). They went on to contend that the type 2 mechanisms-those that become apparent at concentrations above 1 mM-are located in the tonoplast, and are reached by the ions after they have diffused across the plasmalemma, considered by them to be permeable in this range o,f concentrations. We have shown elsewhere (16) that even in the range of high concentrations (> 1 mM), where the type 2 mechanisms are operative, the plasmalemma does not become permeable to monovalent cations, and that mechanisms 1 and 2 of alkali cation absorption operate in parallel across the plasmalemma. In the present paper we confirm this conclusion by means of independent evidence, and extend it to the absorption of Cl. In the experiments, duplicate or triplicate sets of 1.00-g samples of excised roots of barley, Hordeum vulgare, Arivat, enclosed in open-weave cheesecloth tea bags (7) were exposed to aerated experimental solutions containing 0.5 mM CaSO4 and KCl labeled with 86Rb or NaCl labeled with 36Cl, at pH 5.5 ± 0.3 and 30°. Unless otherwise indicated, the experimental period was terminated by procedure B of Epstein et al. (7) in which readily exchangeable and diffusible ions (those in the outer space and on charged sites of the cell wall) are eluted by a 30-min exposure of the tissue to a 0.5 mM CaSO4, 5 mM KCl or NaCl solution (unlabeled), in the cold (about 4°). In the experiment reported in table I, the root samples were exposed to the experimental solutions either for a continuous 10-min period, or for 5 1-min pulses interrupted by 1-min periods of rinsing in unlabeled solutions. The temperature remained 30° during both the periods of absorption and the interruptions when the tissue was being rinsed in unlabeled solutions. For the 0.1 mM run, the unlabeled rinsing solution was 0.5 mM CaSO4, since nonmetabolic exchange adsorption of the labeled K is insignificant at this K concentration, in the presence of 0.5 mM Ca l(6). At the end of the experiment the samples were rinsed with 0.5 mM CaSO4 followed by water. For the run at 5 mM, where exchange adsorption is appreciable (7), the roots were rinsed, during the 1-min interruptions of the 1-min absorption pulses, with a 0.5 mM CaSO4, 5 mM KCl solution (unlabeled). The very last rin,se, just before reimmersion in the labeled experimental solutions, was with 0.5 mM CaSO4 solution. Table I shows that at both 0.1 and 5 mM K the amounts of K absorbed in five 1-min pulses were half the amounts absorbed in continuous 10-min periods. Table II presents the results of an experiment in which the roots were exposed to the labeled expt rimental solutions for either 1 or 60 min. At both concentrations (0.5 and 25 mnI) the amounts absorbed in 1 min were one-sixtieth those absorbed in 60 min.

Compartmentation of Malate in Relation to Ion Absorption in Beet

Malate in beet discs treated in different salt solutions was labeled by a 30 min pulse of (14)CO(2), and subsequent changes in specific activity were followed for several hr. In treatments which resulted in net acid synthesis in response to excess cation absorption, malate specific activity fell slowly after removal of (14)CO(2). In solutions where no net acid synthesis occurred, and from which cation and anion were absorbed equally, malate specific activity fell rapidly when (14)CO(2) was removed. The foregoing suggests that the net synthesis of organic acids in response to excess cation absorption leads to the removal of organic anions from cytoplasmic metabolic pools as counter-ions in salt transport to the vacuole.The latter hypothesis was further examined by direct measurement of the distribution of labeled malate between cytoplasm and vacuole using the wash-exchange method of compartmental analysis, previously described for inorganic ions. The method satisfied the criterion of exchange specificity necessary for this purpose. Much higher retention of label in the cytoplasm was observed in KCl solutions (no net synthesis) than in K(2)SO(4) solutions (net synthesis) after 3 hr (14)CO(2) fixation and subsequent wash-exchange. The observed distribution is consistent with the rapid removal of organic anions to the vacuole during net acid synthesis. The significance of organic acid transport in relation to metabolism is discussed.

Interpretation of the Dual Isotherm for Ion Absorption in Beet Tissue

Beet discs aged in 0.5 mM CaSO(4) develop a capacity to absorb K(+) and Cl(-) from solutions of low concentration. The initial influx of these ions is described by a hyperbolic relationship with concentration in the range 0.01 to 0.5 mM KCl, which is identical with the system 1 absorption isotherm found in other tissues. A second hyperbolic isotherm, attributable to system 2, is found at higher concentrations (1-50 mM KCl).When the transport of labeled ion to the vacuole is studied by wash-exchanging the bulk of the cytoplasmic label following the absorption period, it is noted that in the range of system 1, isotope influx to the vacuole increases with time as the concentration of labeled ions in the cytoplasm increases, while in the range of system 2, influx to the vacuole is constant from the beginning. Diminution of the cytoplasmic specific activity during radio-isotope absorption by prefilling the cytoplasm with the analogous unlabeled salt, markedly reduces subsequent radioisotope uptake to the vacuole only in the range of system 1. These experiments suggest that the cytoplasm serves as a mixing chamber, and that the plasma membrane controls ion uptake to the tissue at low concentrations, indicating that the system 1 isotherm reflects ion movement into the cytoplasm through the plasma membrane. Flux experiments support this conclusion, showing that development with age of the system 1 isotherm corresponds to a quantitatively similar increase in plasma membrane influx in 0.2 mM KCl.At higher concentrations the outer membrane no longer rate-limits entry of ions to the vacuole. Isotope influx under these conditions, described by the system 2 isotherm, presumably reflects movement across the tonoplast.

The Effects of Aqueous Extracts of Red Beet Root on Salt Accumulation and Respiration of Discs of Red Beet Root

An account is given of the preparation of aqueous extracts of red beet root which are shown to stimulate potassium uptake in beet discs washed for a short period, but inhibit potassium uptake in discs washed for four days or more. Analysis of extracts showed them to contain organic anions (especially citrate and malate) which affect both the metabolic phase of potassium uptake and respiration of the tissue. The effects of extracts and organic acids on uptake of manganese by beet discs is described and compared with effects on potassium absorption. The results are discussed with respect to current theories of salt accumulation and in relation to the hypothesis relating an inhibitor of salt accumulation to the lag phase of ion uptake by beet discs.