Effect of Preparation Method on the Induction of (Sodium + Potassium) –Activated Adenosine Triphosphalase from Sugar Beet Root and its Lipid Composition

The (Na⁺+K⁺)–stimulation of the ATPase activity in a paniculate fraction from sugar beet roots was compared after inducing treatments with dithiothreitol (DTT), cystein or deoxycholate (DOC). Protection of SH groups by cystein or DTT increased the specific activity at the optimal Na: K ratio (25 :25 mM) with a factor of about 5 as compared to the untreated fraction, and with a factor of about 3 as compared to particles treated with DOC. DTT acted chiefly at the optimum ratio of Na : K (25 :25 mM), whereas a less specific stimulation at any proportion of the couple Na : K was observed after treatment with DOC or cystein. A combined treatment (DOC + DTT) led to inactivation of the DTT effect. Treatment with DTT resulted in a loss of lipids, which were replaced by water, but otherwise little swelling took place. The loss of lipids was almost exclusively due to loss of phospholipids, and among these the zwitterkonic phos–phatidyl choline and phosphatidyl ethanolamine were affected more than the acidic phosphatidyl glycerol or phosphatidyl inositol. The sulfolipid was maintained within the particles. The combined treatment “with (DOC + DTT) led to further losses of lipid material at the same time as strong swelling took place. As compared to the treatment with DTT alone, the additional loss of lipids occurred mainly from the (sterol glycoside + cerebroside + digalactosyl diglyceride –fraction) and from the solfolipid, with some losses also from the (pigments + neutral), phosphatidyl choline and phosphatidyl glycerol fractions. The results are in agreement with our earlier data, which also show that lipid fractionation is important for the revealing of (Na⁺ 4– K⁺)–stimulated ATPase activity, possibly because of the negative electric charge of the sulfolipid, which is characteristic for the induced preparations and which may be connected with the specific site of stimulation by the cations.     

Lipids from Bean, Barley and Sugar Beet in Relation to Salt Resistance

A comparison was made between the lipid and fatty acid composition of the salt-sensitive bean (Phaseolus vulgaris L. cv. Saxa), the less salt-sensitive barley (Hordeum vulgaris L. cv. Wisa) and the salt-tolerant sugar beet (Beta vulgaris L. cv. Kawemono). Sugar beet roots showed a higher content of sterol components and sulfolipid as compared with bean and barley roots. The lipids of sugar beet roots contained more linoleic acid and less linolenic acid than those of bean and barley roots. For barley and sugar beet roots a higher amount of extra-long chain fatty acids was observed than for bean roots. It was concluded that differences in membrane structure are correlated with differences in membrane permeability to sodium and chloride and in salt-resistance of the studied species.     

Properties of Mg2+-Stimulated and (Na++K+)-Activated Adenosine-5'-Triphosphatase from Sugar Beet Cotyledons

Sugar beet leaf homogenate contains Mg²⁺-stimulated ATPase activity with the highest specific activity in the 25,000–30,000 ×g-fraction. This fraction also has (Na⁺+ K⁺)-activated ATPase activity. Both activities have two pH optima, one stable at pH 7.9 and one variable at lower pH. When optimal conditions of Na⁺ and K⁺ were tested with 64 combinations of these ions, at least two mountains of activity were revealed. The (Na⁺+ K⁺)-ATPase had a high specificity for ATP. It had lost about 50% of its original activity after 56 days of storage at ?85°C. The activity drop was most pronounced at high ionic concentrations in the test medium. The (Na⁺+ K⁺)-ATPase shows four peaks of activity when tested at constant ionic strength. The idea is put forward that the four peaks reflect two ATPases, one in the tonoplast and one in the plasmalemma, which undergo conformational changes in relation to the ionic milieu.     

Gallus Regeneration from Protoplasts of Sugar Beet(Beta vulgaris)

Conditions appropriate for isolation and culture of protoplasts from cell suspension cultures of sugar beet (Beta vulgaris L. ) were investigated. Protoplasts with high yields and high quality were obtained by treating cells with a mixture of cellulase, macerozyme R-10 and driselase, or other enzyme combinations. Protoplasts were cultured in MS liquid medium or solid agar medium. Callus was obtained from the cultured protoplasts.     

Effects of Potassium Deficiency on the Photosynthesis and Respiration of Leaves of Sugar Beet under Conditions of Low Sodium Supply

Sugar beet plants (Beta vulgaris L. cv. F58-554H1) were germinated and cultured under standardized environmental conditions. The effects of K deficiency on photosynthetic and respiratory CO₂ exchange rates of attached leaves were studied under conditions of low Na supply by withholding both Na and K from the culture medium at cut-off (28 days after planting). Potassium and Na concentrations in the leaf blade and petiole decreased rapidly during the 8 days after cut-off, then more slowly.     

Effects of Ca2+ and Cd2+ on the Carbohydrate Metabolism in Sugar Beet (Beta vulgaris)

Sugar beet (Beta vulgaris L. cv. Monohill) were cultivated ina nutrient solution with different combinations of Ca²⁺ (36,180, 720 or 3560µM) and Cd²⁺ (0, 1, 5 or 20µM).The dry and fresh weights, the content of Ca²⁺ and Cd²⁺ , sucrose,fructose, glucose and starch in 5-week-old plants was analysedas well as the rate of [¹⁴C]-sucrose uptake in discs from 3-month-oldstorage roots. The carbohydrate metabolism was indirectly affectedby the presence of calcium or cadmium. Cadmium caused a diminisheddry weight and carbohydrate concentration. The dry weight wasunaffected by the Ca²⁺ level but the carbohydrate distributionbetween storage and growth processes was affected; at low Ca²⁺in the tissue, the growth was retarded and the level of storagecarbohydrate increased, while at high Ca²⁺ the opposite wasfound. The [¹⁴C]-sucrose uptake decreased in tap roots cultivatedat low Ca²⁺ . Long term exposure to Cd²⁺ also decreased thesucrose uptake in tap roots. Direct Cd²⁺ addition to the assaymedium, however, increased the sucrose uptake, probably at thetonoplast, while Ca²⁺ had no transient effect on the uptake.Cadmium increased the Ca²⁺ concentration in the plant, but Ca²⁺did not affect the net-uptake of Cd²⁺. Key words: Sugar beet, cadmium uptake, calcium uptake, carbohydrate formation, growth     

Bolting Susceptibility of Sugar Beet (Beta vulgaris) in Relation to Contents of Sulfhydryls and Disulfides and to Protein Composition of Membranes

The aim of the investigation was to correlate the sensitivity to low temperature that leads to bolting of sugar beet with a property of seedlings or seeds, analyzable without cold treatment. Populations or inbred lines were investigated, the bolting percentages of which had been determined in field trials. Sulfhydryls (—SH) and disulfide sulphur (-S-) were analyzed by amperometric titration with silver nitrate. Disulfides were broken with sulphite, and proteins were unfolded with urea. Negative correlations were found between the bolting percentage and (-SH + -S-) per leaf fresh or dry weight unit and per leaf protein nitrogen, with urea at the titrations. Positive correlations were obtained between the bolting percentage and the ratio of (-SH + -S-) titratable in the absence of urea to that titratable in the presence of urea, when leaf homogenates or leaf or seed protein precipitates were examined. Centrifugation experiments showed that membrane proteins were responsible for the correlation. By polyacrylamide gel electrophoresis of the membrane proteins it was found that the proportion between some of these proteins was correlated with the bolting percentage. From these results and such from analyses of beet plants during and after cold treatment a hypothesis was constructed: Low bolting susceptibility, i.e., low sensitivity to low temperature, is caused by a high proportion of a hydrophobic membrane protein, rich in -SH or -S-. This protein interferes with the reactivity of the plants to gibberellins resulting from low temperatures or long days.     

Adenosine Triphosphatases Bound to Plasmalemma, Mitochondria, and Microsomes or Present in the Cytoplasmic Phase from Potato Tubers

Between pH 4–10, basal ATPase activity, measured in the absence of mineral ions, was 10 to 100 times higher in the final cytoplasmic supernatant from potato tuber homogenates than in the membraneous fractions (purified plasmalemma, purified mitochondria and microsomes). The soluble ATPase was slightly inhibited, whereas the membrane-bound ATPases were all stimulated by Mg²⁺ ions. A further stimulation by Na⁺ or K⁺ ions was only observed in purified plasmalemma or mitochondria, at alkaline pH (7.5–9.5). At a fixed (Na⁺+ K⁺) concentrations (80 mM), this last stimulation was much greater in purified mitochondria (350%) than in plasmalemma (33%); it also increased with (Na⁺+ K⁺) concentrations up to 200 mM in mitochondria whereas, in plasmalemma, it was roughly constant for monovalent ion concentrations between 20 and 200 mM. General properties of the plasma membrane-bound ATPase have been determined, i.e. substrate specificity, activity variations with quantity of substrate, temperature, pH, etc. Divalent cations stimulated strongly the ATPase in the following order: Mn²⁺ > Mg²⁺ > Ca²⁺. The maximum ATP hydrolysis velocity for that part of ATPase activity which is strictly dependent on Mg²⁺ ions was 3.85 μmol × mg^?1 protein × h^?1. This plasma membrane ATPase was not sensitive to ouabaïn or to oligomycin.     

Kinetics Analysis of the Plasma Membrane Sucrose-H+ Symporter from Sugar Beet (Beta vulgaris L.) Leaves

The kinetics behavior of the H+-sucrose (Suc) symporter was investigated in plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves by analyzing the effect of external and internal pH (pHo and pHi, respectively) on Suc uptake. The apparent Km for Suc uptake increased 18-fold as the pHo increased from 5.5 to 7.5. Over this same pHo range, the apparent Vmax for Suc uptake remained constant. The effects of pHi in the presence or absence of internal Suc were exclusively restricted to changes in Vmax. Thus, proton concentration on the inside of the membrane vesicles ([H+]i) behaved as a noncompetitive inhibitor of Suc uptake. The Km for the proton concentration on the outside of the membrane vesicles was estimated to be pH 6.3, which would indicate that at physiological apoplastic pH Suc transport might be sensitive to changes in pHo. On the other hand, the [H+]i for half-maximal inhibition of Suc uptake was approximately pH 5.4, making regulation of Suc transport through changes in [H+]i unlikely. These results were interpreted in the framework of the kinetics models for co-transport systems developed by D. Sanders, U.-P. Hansen, D. Gradmann, and C. L. Slayman (J Membr Biol [1984] 77: 123-152). Based on their analysis, the behavior of the Suc symporter with respect to the [H+]i is interpreted as an ordered binding mechanism by which the binding of Suc on the apoplastic side of the membrane and its release on the symplastic side precedes that of H+ (i.e. a first-on, first-off model).     

Properties of (Sodium Potassium)-Stimulated ATPases in English Ryegrass Roots and Implications in Ion Transport

Maximum ATPase activities in the cell wall fraction of English ryegrass (Lolium perenne L.) roots were stimulated by foru discrete millimole ratios of (Na⁺+ K⁺); 40:0, 35:5, 5:35, and 0:40. The optimal pH for stimlation was found to be 6.5. Contrary to data in the literature, Mg²⁺ inhibited all stimulatory ratios of (Na⁺+ K⁺) when plants were cultured on an adequate nutrient solution. When grown on a dilute solution, Mg²⁺ enhanced (Na⁺+ K⁺)-stimulated ATPase activity in this membrane preparation. The single optimal combined concentration of (Na⁺+ K⁺) for all stimulatory ratios was 40 MM. The ratios of (Na⁺+ K⁺) which stimulated ATPase activity in the cell wall fraction varied with position along the root axis such that all rarely existed simultaneously nor did any exist in the terminal millimetre of the root. Both cell wall and microsomal fractions showed stimulation by (Na⁺+ K⁺) at all the above ratios indicating the possible presence of plasma membrane fragments in both fractions. Only the 35:5 ratio was stimulations were found in the supernatant. Implications of ion-stimulated ATPase involvement in ion transport were drawn from the appearance of ATPase activity at a 40:0 ratio of (Na⁺+ K⁺) and the disappearance of stimulations at 35:5, 5:35, and 0:40 ratios when plants were moved from a strong (35 mM total concentration) to a dilute (0.75 mM) nutrient solution.     

Uptake and Physiological Effects of Cadmium in Sugar Beet (Beta vulgaris) Related to Mineral Provision

Sugar beet seedlings (Beta vulgaris L. cv. Monohill) were grownfor 14 d on a nutrient solution based on the nutrient proportionsin healthy plants. Nutrients were supplied either once at relativelyhigh concentrations, or in small amounts with a daily incrementalincrease of 0?15 or 0?20 in accordance with an exponential growthrate. Cadmium (0, 0?6, 2?3, 50 or 20?0 µmol) was introducedeither by a single addition or in daily increments of 0?15 or0?20. Cadmium uptake, expressed as a percentage of total Cd²⁺supplied, decreased with increasing total C^d2+ content and withdecreasing availability of nutrients. With a daily supply ofcadmium, net uptake, transport and content per unit of dry weightin roots and shoots were related to the total Cd²⁺ supplied.Cadmium caused growth retardation, increased root/whole-plantratio, and decreased root-tip respiration and photosynthesis.At high initial nutrient concentrations, Cd²⁺ decreased thecontents of sucrose, glucose, fructose, and starch per unitof dry weight. The opposite was found if nutrients were addeddaily. In the latter case, the dry weight/fresh weight ratioalso increased. The effects of cadmium were related to [Cd²⁺]in proportion both to the root absorption area and to the nutrientconcentration. Key words: Sugar beet, mineral provision, cadmium uptake, sugar formation, growth     

Membrane Mg-(Ca)-Activated Adenosine Triphosphatase of Escherichia coli: Characterization in the Membrane-Bound and Solubilized States

The membrane-associated Mg(2+)-activated and Ca(2+)-activated adenosine 5'-triphosphatase (EC 3.6.1.3; ATPase) activities of Escherichia coli were further characterized. The degree of inhibition of membrane-bound Mg(2+)-(Ca(2+))-ATPase by a series of anions (i.e., sodium salts of nitrate, iodide, chloride, and acetate) was found to correlate with the relative chaotropic, or solubilizing, effectiveness of these anions. The enzyme was solubilized from washed membrane ghosts by treatment with 0.04% sodium lauryl sulfate at pH 9.0 and 37 C. Solubilized Mg(2+)-(Ca(2+))-ATPase exhibited an initial increase in activity, followed by fairly rapid inactivation, both ATPase activities being particularly cold-labile. The combined stabilizing effects of lauryl mercaptan (1-dodecanethiol), 0.01 m tris(hydroxymethyl)amino-methane-hydrochloride buffer (pH 9.0), 0.2 mm MgCl(2), and ambient temperature facilitated partial purification of the enzyme, the molecular weight of which was estimated to be approximately 100,000 by the gel filtration technique. In general, the membrane-associated Mg(2+)-(Ca(2+))-ATPase of E. coli resembles both mitochondrial membrane ATPase and the well-characterized membrane ATPases of Bacillus megaterium and Microcococcus lysodeikticus. It is of particular interest that N,N'-dicyclohexylcarbodiimide (DCCD), a known inhibitor of mitochondrial ATPase, of mitochondrial oxidative phosphorylation, and of the membrane-bound Mg(2+)-ATPase of Streptococcus faecalis was found to inhibit both the membrane-bound and the solubilized forms of E. coli Mg(2+)-(Ca(2+))-ATPase. The sensitivity of the membrane-associated Mg(2+)-(Ca(2+))-ATPase of E. coli to both anions and cations, its allotopic behavior, and its susceptibility to inhibition by DCCD favor the idea that this enzyme plays a key, probably polyfunctional, role in such biological activities of the membrane as oxidative phosphorylation and ion transport.     

Antibiotic and Biosurfactant Properties of Cyclic Lipopeptides Produced by Fluorescent Pseudomonas spp. from the Sugar Beet Rhizosphere

Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLP-producing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.     

Nectar Sugar Composition in Relation to Pollination Syndromes in Sinningieae (Gesneriaceae)

A putative correlation between nectar sugar composition andpollination syndrome was evaluated in the tribe Sinningieae(Neotropical Gesneriaceae). Sucrose, fructose and glucose werequantified in the nectar of 45 species using high performanceanion-exchange chromatography. Representative species of thehummingbird, bee, bat and moth pollination syndromes were sampledin relation to their numeric importance in the tribe. In hummingbirdand bee flowers, which represent 95% of the species in Sinningieae,nectar was sucrose-dominant (ratio [sucrose]/[hexose] > 1).Sugar ratios below one were only found in the nectar of threespecies with moth and bat syndromes. Sugar concentration averaged23.9 ± 10.6% (wt/total wt) in hummingbird flowers and28.7 ± 10.6% in bee flowers, whereas diluted nectar (7.1± 3.4%) was restricted to bat flowers. Similarities inthe nectar of hummingbird and bee flowers contrast with thepresence of specific morphological traits associated with thesetwo syndromes, indicating that plant-pollinator relationshipsrely on flower display rather than on nectar characteristics.By contrast, distinct nectar chemistry is correlated with thebat syndrome in which a particularly low sucrose productionis responsible for hexose dominance. Copyright 2001 Annals ofBotany Company Nectar sugar composition, pollination syndrome, Sinningia, Gesneriaceae, Brazil     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization

A (1→3)-β-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1→3)-β-glucan by methylation analysis and by glucanase digestion. (1→3)-β-Glucan synthase activity is markedly stimulated by Ca²⁺; activation is half-maximal at about 50 micromolar Ca²⁺ and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg²⁺, Mn²⁺, and Sr²⁺, also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by β-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein.     

Purification and Some Properties of Cell Wall-bound Invertases from Sugar Beet Seedlings and Aged Slices of Mature Roots

Cell wall-bound invertases (EC 3.2.1.26) from both sugar beet seedlings and aged slices of mature roots were purified to homogeneity separately with CM-cellulose chromatography and Bio-Gel P-150 gel filtrations. The enzymes behaved similarly throughout the purification procedures. The purified enzymes are identical as characterized by specific activity, gel electrophoretic mobility, K_m for sucrose and raffinose (1.33 and 4.0 millimolar, respectively), mobility on Bio-Gel P-150 (molecular weight 28,000), optimum pH (4.6 to 5.0), optimum temperature, and dependence on NaCl concentration for insolubilization by DNA. The results suggest that the enzymes may be encoded for by the same structural gene.     

Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris)

The present study compares the exogenous NAD(P)H oxidation and the membrane potential ([delta][psi]) generated in mitochondria isolated from different tissues of an important agricultural crop, sugar beet (Beta vulgaris}. We observed that mitochondria from taproots, cold-stored taproots, and in vitro-grown tissue cultures contain a functional NADH dehydrogenase, whereas only those isolated from tissue cultures displayed a functional NAD(P)H dehydrogenase. It is interesting that the NADH-dependent [delta][psi] of mitochondria from cold-stored taproots and from tissue cultures was not affected by free Ca2+ ions, whereas free Ca2+ was required for the mitochondrial NADPH oxidation by in vitro-grown cells and cytosolic NADH oxidation by mitochondria from fresh taproots. A tentative model accounting for the different response to Ca2+ ions of the NADH dehydrogenase in mitochondria from cold-stored taproots and tissue cultures of B. vulgaris is discussed.     

栽培甜菜卵细胞、合子及二细胞原胚的超微结构

为丰富被子植物生殖生物学资料, 并为甜菜相关研究提供参考, 应用透射电镜技术研究栽培甜菜（Beta vulgaris）卵细胞、合子和二细胞原胚的超微结构特征。结果如下：在成熟卵细胞中多聚核糖体数量不多, 且细胞代谢活性较弱; 初期合子内, 核仁大量合成核糖体前体物质, 胞质中多聚核糖体数目众多, 细胞代谢活性较强; 休眠期合子的核仁变小, 胞质中核糖体数量急剧减少, 仅有少量多聚核糖体, 细胞代谢活性较弱; 合子分裂前期和二细胞原胚期, 核仁显著, 胞质中核糖体的密度增加, 出现大量多聚核糖体, 细胞代谢活性较强。根据上述结果可以得出, 栽培甜菜从卵细胞成熟→合子初期→合子休眠期→合子分裂前期→二细胞原胚的超微结构变化中多聚核糖体的变化最为显著, 表现为“少→多→少→多”的数量变化过程, 反映出细胞代谢状态也经历了“弱→强→弱→强”的变化过程, 这种变化趋势与配子体世代向孢子体世代转变有关。     

Adenosine Triphosphatase Activities in Leaves of the Mangrove Avicennia nitida Jacq: Influence of Sodium to Potassium Ratios and Salt Concentrations

Homogenates from the salt-excreting leaves of the mangrove Avicennia nitida were subjected to differential centrifugation and investigated for adenosine triphosphatase activities. At pH 6.75 a salt stimulation with peaks at three different sodium to potassium ratios could be demonstrated above the activity due to Mg²⁺ ions. The stimulation by sodium and potassium depends on the ionic strength of the test medium, higher salt concentrations being inhibitory. The plant system seems thus more complicated than the animal activities. Technically, this means that a search for (Na⁺ + K⁺)-activated ATPases in plants should be performed with a close spacing of Na:K ratios at several constant levels of salt. Literature data on the transport of Na⁺ and K⁺ indicate that the physiological situation is rather complex in plants.