Sugar Beet Cell Research Articles

Extraction of Enzymes from Cell Walls of Sugar Beet Cells Grown in Suspension Culture

Extraction of Enzymes from Cell Walls of Sugar Beet Cells Grown in Suspension Culture

Acid and Alkaline Invertases in Suspension Cultures of Sugar Beet Cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K(m) values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K(m) values for sucrose.

Inhibition of Na+/H+ Antiport Activity in Sugar Beet Tonoplast by Analogs of Amiloride

The effects of amiloride and a series of amiloride analogs have been tested on the Na(+)/H(+) antiport activity in intact vacuoles and tonoplast vesicles isolated from sugar beet cell suspension cultures. There is a competitive interaction between amiloride analogs and sodium. Substitution of one or both H-atoms of the 5-amino group of amiloride (apparent K(i) about 150 micromolar) resulted in a 3- to 200-fold increase in inhibitory potency of the antiport activity.

Transient gene expression in electroporated protoplasts and intact cells of sugar beet

Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.

Structure and Function of Beta vulgaris Parenchyma Cells: Ultrastructure and Sugar Uptake Characteristics of Tissue and Cell in Suspension

Sugar-storing parenchyma cells of the fleshy sugarbeet (Beta vulgaris) root have (1) a very thin cytoplasm with many ribosomes and mitochondria and (2) a large sugar-storing vacuole. The walls are composed of loosely arranged microfibrils, and only a few branched plasmodesmata traverse the wall, indicating apoplastic transport. Numerous large, double-membrane-bound vesicles protrude into the vacuole, with little or no cytoplasm between the appressed membranes. The vesicles originate by simultaneous invaginations of the plasmalemma and tonoplast so that the inner membrane is derived from the plasmalemma and the outer one from the tonoplast. Vesicles do not pinch off into the vacuole, as during pinocytosis, but remain as invaginations of the original membranes. Uptake of membrane-impermeable mannitol and lead could not be detected with or without sugar, indicating that pinocytosis does not operate in sugar accumulation. The appressed double-membrane-bound vesicles enlarge the cell surface area. Water-washed...

Metabolism of demeton S-methyl sulfoxide (metasystox R) in cell suspension cultures of sugar-beet

The insecticide demeton S-methyl sufoxide (I) is oxidized by sugar-beet cell cultures to demeton S-methy sulfone (II). Cleavage of the thioester bond of I and II formed ethanethiols which were transformed to S-methylated compounds or S-glucosides. In addition another two metabolites were identified: ethyl vinyl sulfoxide (V), via ß-elimination of 0,0-dimethyl thiophospate from the parent insecticide, and [1-(ethylsulfinyl)-2-(methylthio)] -ethane (VII).

Callus formation from protoplasts of a sugarbeet cell suspension culture

Protoplasts of sugarbeet (Beta vulgaris L.) were isolated from cell suspension cultures and cultured in modified PGo medium. Conditions required for the efficient division of the protoplasts were investigated.The optimal combination of phytohormones was found to be 1 mg/l NAA, 0.2 mg/l 2,4-D, 0.5 mg/l zeatin. Protoplast division was also considerably stimulated by the addition of 250 mg/l casein hydrolysate, 200 mg/l yeast extract, and 20% v/v conditioned culture medium to the protoplast culture medium. The highest division rate (up to 35% of the protoplasts) was achieved at a density of 4×10(4)- 1×10(5) protoplasts/ml. From the colonies callus and suspension cultures were readily obtained.

Cytological identification of acrotrisomy in sugar beets

The analysis of sugar beet cells at three different somatic stages—prophase, early metaphase, and mid-metaphase—permits the identification and physical measurement of individual chromosomes and determines the break-point of some structural changes. This technique is successfully applied in the identification of an acrocentric chromosome 9S9L in the sugar beet.

Auxin-polyamine interaction with the calcium-controlled secretion of peroxidase by sugarbeet cells

Spermidine and ornithine given to normal auxin-requiring cell suspensions of sugarbeet inhibited peroxidase secretion in the absence of Ca(2+). Habituated (organogenic or not) cells did not respond. Both compounds counteracted the Ca(2+) - promoted enzyme secretion by three cell lines. Auxins (2,4-D and BSAA) did not modify the extracellular level of peroxidase activity in the absence of Ca(2+) When Ca(2+) was added, auxins increased its effect in normal cells and had practically no effect in habituated cells. The inhibitory effect of spermidine and ornithine was somewhat reduced by auxins in normal cells and increased in habituated cells. It was hypothesized that the effect of auxins did not involve the mediation of polyamines and that both types of compounds directly interacted with Ca(2+) at the membrane level.

Interaction of polyamines or their precursors with the calcium-controlled secretion of peroxidase by sugarbeet cells

Three polyamines tested (cadaverine, spermidine and spermine) and their 2 precursors (the amino acids arginine and ornithine) inhibit the Ca2+-mediated secretion of peroxidases by sugarbeet cells in suspension culture at concentrations ranging from 10−15 to 10−5M. In the absence of exogenous Ca2+, spermine added at higher concentrations mimics the activatory effect of Ca2+, the other polyamines being without effect.

Calcium-controlled peroxidase secretion by sugarbeet cells: Effect of ionophores in relation to organogenesis

Summary Peroxidase activity was studied in normal non-organogenic, habituated non-organogenic, and habituated organogenic calli of the same sugarbeet ( Beta vulgaris ) strain. Several subcellular fractions (soluble, membrane, ionic wall and covalent wall fractions) were prepared from these cells and assayed for peroxidase activity. All fractions from habituated non-organogenic cells were found less active than those from normal non-organogenic and habituated organogenic cells. Peroxidase release into the medium by the three types of cells was enhanced by 1 m M calcium chloride in proportion to their level of endogenous peroxidase activity. Calcium ionophores increased the effectiveness of calcium on peroxidase secretion only in habituated organogenic cells. Calcium-mediated peroxidase binding to membranes in vitro was also measured for the three cell lines. It was found that membranes from habituated organogenic cells were the most sensitive to calcium. It is proposed that the calcium-mediated binding of peroxidases to membranes could be related to the secretory process of these enzymes.

Auxin control of calcium-mediated peroxidase secretion by auxin-dependent and auxin-independent sugarbeet cells

Peroxidase secretion by three sugarbeet cell lines in the absence of Ca 2+ was unaffected by the addition of auxins of different chemical structures. The six auxins tested had no effect on the Ca 2+ -promoted peroxidase secretion by habituated non-organogenic cells (HNO), but they enhanced the secretion by normal non-organogenic cells (NNO) and three of them inhibited it in the case of the habituated organogenic line. A 5-week subculture of the auxinindependent HNO callus on auxin-containing media increased the sensitivity of cells to Ca 2+ and their responsiveness to auxins in suspension cultures. Conversely, subculture of the auxin-requiring NNO callus on auxin-deficient medium induced a decreased sensitivity to Ca 2+ and a complete loss of response to auxins. The possible regulation of peroxidase secretion by Ca 2+- and auxin-receptors is discussed.

The effect of ergosterol, ergocalciferol and cholecalciferol on calcium‐controlled peroxidase secretion by sugarbeet cells

Provitamin D2 (ergosterol), vitamins D2 and D3 reduced the calcium‐mediated peroxidase secretion in three types of sugarbeet cells in suspension cultures. Vitamin D2 was the most effective in habituated non‐organogenic cells which were the less sensitive to calcium; provitamin D2 was the most effective in habituated organogenic cells, while normal non‐organogenic cells were equally sensitive to the three types of vitamin D. The calcium ionophore A23187 slightly restricted peroxidase release in all cases, except in the habituated organogenic cells in the presence of calcium where it exerted a promotive effect. The inhibiting effect of vitamin D2 was not counteracted by the ionophore except in this habituated organogenic cell line.

Calcium-controlled peroxidase secretion by sugarbeet cell suspensions in relation to habituation

Sugarbeet cells from normal and habituated callus released peroxidases in liquid cultures, in proportion to their endogenous level. Calcium promoted this release more in the normal than in the habituated line. Treatment of the cells with sodium azide, sodium hydrogenarsenate or phenothiazine inhibited the calcium effect, which indicated a dependence on metabolic energy and on calmodulin regulation. The Ca ionophore Ro (bromolasalocid ethanolate) restricted peroxidase release.

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na(+) and K(+)) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.

Solute Distribution in Sugar Beet Leaves in Relation to Phloem Loading and Translocation

The distribution of solutes in the various cells of sugar beet (Beta vulgaris L.) source leaves, petioles, and sink leaves was studied in tissue prepared by freeze-substitution. The differences in degree of cryoprotection indicated that sieve elements and companion cells of the source leaf, petiole, and sink leaf contain a high concentration of solute. The osmotic pressure of various types of cells was measured by observing incipient plasmolysis in freeze-substituted tissues equilibrated with a series of mannitol solutions prior to rapid freezing. Analysis of source leaf tissue revealed osmotic pressure values of 13 bars for the mesophyll and 30 bars for the sieve elements and companion cells. The osmotic pressure of the mesophyll of sink leaves was somewhat higher.The sharp concentration increase at the membrane of the sieve element-companion cell complex of the source leaf indicates active phloem loading from the free space at this site. Active loading of the phloem is presumably needed to move the sugar from the chloroplasts of the mesophyll to the sieve tubes against the concentration gradient. The osmotic pressure of the mature sieve element-companion cell complex appears to be approximately the same in source leaf, path, and sink leaf tissue. There is a distinct difference in concentration between the mature sieve element-companion cell complex in the sink and the surrounding mesophyll. The solute distribution suggests that sugar is actively accumulated from the free space by the developing sink leaf tissue.The osmotic values observed in the various cells are consistent with the operation of a mass flow mechanism of translocation driven by active phloem loading and by active accumulation of sugar by sink tissues.

II. Lokalisation von Enzymen des reduktiven und dem oxydativen Pentosephosphat-Zyklus in den Chloroplasten und Permeabilität der Chloroplasten-Membran gegenüber Metaboliten

1. The interrelationship of metabolic activities in chloroplasts and cytoplasm of leaf cells of spinach, sugar beet and Elodea has been investigated. Different methods have been adopted to study the intracellular localization of enzymes and the flow of phosphorylated intermediates across the chloroplast membrane. The flow of substrates was investigated by determining the rates of the conversion of substrates added to aqueously isolated chloroplasts, prior to and after destruction of the outer chloroplast membrane. The observed differences yielded information as to whether a substrate could traverse the chloroplast membrane. Two methods mere used to investigate the localization of enzymes : a) The percentage distribution of photosynthetic and respiratory enzymes in chloroplasts and cytoplasm was calculated from data on enzyme activities in non-aqueous cell fractions. b) Low levels of enzymes in chloroplasts in the presence of high cytoplasmatic levels were detected by assaying enzyme activities in preparations of aqueously isolated chloroplasts prior to and after ultrasonic destruction of the outer chloroplast membrane. 2. If chloroplasts are isolated in aqueous sucrose buffer, their outer membranes act as an efficient barrier against the penetration of NADP, RuDP, GAP and, in some but not all experiments, of FMP and GMP. PGA, DHAP and, probably to a lesser extent, aspartate, ɑ-ketoglutarate, oxaloacetate and FDP can traverse this membrane. Chloroplast membranes are significantly altered when isolated in NaCI-buffer systems and do not correspond to the in vivo situation. 3. The conversion of Ri-5-P to RuDP occurs exclusively or nearly exclusively in the chloroplasts indicating that phosphoribulokinase and/or ribosephosphate isomerase are located only there. 4. The conversion of Ri-5-P to GAP and SuMP, which is catalyzed by the enzymes ribosephosphate isomerase, xylulosephosphate epimerase and transketolase, proceeds likewise only or at least predominantly in the chloroplasts and not, or only to a small extent, in the cytoplasm. 5. The major parts of glucose-6-phosphate dehydrogenase and of 6-phosphogluconate dehydrogenase reside in the cytoplasm. However, a small, but significant, level of these enzymes is to be found also in the chloroplasts. Hexokinase and transaldolase are also present there. Pyruvate kinase and phosphofructokinase appear to be absent from chloroplasts. 6. Since, with the presence of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, transaldolase and enzymes of the Calvin cycle, the enzymic machinery of the oxidative pentose phosphate pathway is complete in the chloroplasts, the results suggest that chloroplasts are engaged in the oxidative decomposition of carbohydrates. 7. In the dark the oxidative pentose phosphate pathway requires the control of NADPH formation and the transfer of hydrogen across the chloroplast membrane. 8. The available data on the intracellular localization of enzymes and on the kinetics of the distribution of labelled intermediates show that the photosynthetic carbon cycle operates exclusively within the chloroplasts. There is nothing to suggest that enzymes of chloroplasts and cytoplasm cooperate in the cyclic regeneration of the carbon acceptor molecule. However, the existence of phosphorylated transport metabolites suggests that secondary reactions of photosynthesis such as sucrose and amino acid synthesis, which proceed, at least in part, outside the chloroplasts, are directly linked with chloroplastic reactions by activated (phosphorylated) intermediates.