Extraction,Antioxidant Activity and Identification of Flavonoids from Root Tubers of <i>Kosteletzkya virginica</i>

Kosteletzkya virginica (K. virginica) is used for revegetation of salt-affected coastal tidal flats and as a raw material of biodiesel. K. virginica root tuber, a biowaste with low economic value, is rich in bioactive compounds. This study aimed to extract and identify flavonoids from K. virginica root tubers. The optimal extraction conditions were 1/25 (w/v) solid/liquid ratio, 40% ethanol concentration at 40°C for 60 min. Under these conditions, 65.2 ± 3.7 mg/g total flavonoid content was extracted from the roots, which were collected from salinized soil in late autumn of the third year. Antioxidant activity was evaluated through 1,1-diphenyl-2-picrylhydrazyl, hydroxyl radical, and superoxide anion scavenging assays. The extracted flavonoids exhibited antioxidant activity in a dose-dependent manner. Five flavonoids, glucoliquiritin apioside, licoisoflavone B, 5-methoxy-7,8-diprenyl- flavone, 7,2′-dihydroxy-6,8-dimethyl-4′,5′-methylenedioxyflavan, and 5,7,4′-trihydroxy-3′-methoxy-6,8-di-Cmethylflavanone, were identified by ultra-performance liquid chromatography–tandem mass spectrometry. Our results suggest that the flavonoids of K. virginica root tubers might be potent antioxidants and can be effectively applied as an ingredient in food and natural medicine.     

Salt Tolerance in Cell Suspension Cultures of the Halophyte Kosteletzkya virginica

Tolerance to NaCl was studied in cell suspension cultures ofKosteletzkya virginica (L.) Presl. (Malvaceae), a dicotyledonoushalophyte that grows in tidal marshes of the eastern UnitedStates. Growth of salinized cultures was significantly inhibitedat high (255 mol m^–3 NaCl), but not at lower externalsalinities. Adjustment of cell suspensions to Nacl was rapid,with the duration of the normal growth cycle unaffected by salinity.Maximum biomass was attained when cultures were exposed to NaClduring early log growth. Patterns of inorganic ion accumulationreflected the utilization of both Na⁺ and K⁺ as osmotica, withNa⁺ content substantially increasing when cells were grown atan external salinity sufficient to reduce growth. K⁺ uptakeselectivity was high and Na⁺/K⁺ ratios were low in salt-treatedcultures even though K⁺ content was somewhat lower comparedto unsalinized cultures. Free proline and microsomal lipid contentincreased in salt-treated cell cultures. Key words: Kosteletzkya virginica, halophyte, salt tolerance, cell suspension culture     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Labeling of LH/hCG receptor in rat ovaries.

Human chorionic gonadotropin (hCG) was found to stimulate the incorporation of [¹⁴C] N-acetyl-D-glucosamine in rat ovary in vitro. Subcellular fractionation of the ovarian tissue revealed that the plasma membranes were stimulated maximally to the extent of 200 to 300% by the hormone indicating the stimulation of the synthesis of plasma membrane glycoproteins. In addition, and appreciable amount of the radioactivity was incorporated in the cell surface LH/hCG receptor. The evidence in support of the labeling of the receptor was derived from the behavior of the detergent solubilized receptor on Sepharose 6B column and on hCG-Sepharose affinity adsorbent. The labeled receptor thus purified showed binding affinity for [¹²⁵I] hCG. Thus, the hormone stimulates the synthesis of cell surface glycoproteins as well as the LH/hCG receptor.     

An assessment of the cultural capabilities of protoplasts of some wild species of linum

Protoplasts of several wildLinum species were isolated enzymatically from roots, hypocotyls and cotyledons of seedlings, and also from theirin vitro grown shoots and cell suspension cultures. When cultured all these protoplasts divided to produce callus but only good plant regeneration capability was evident in the case ofLinum lewissii and to a much lesser extent forL. strictum. Only rhizogenesis was observed withL. alpinum, L. narbonense, L. grandiflorum andL. altaicum. The high plant regeneration capacity ofL. lewissii from protoplast -derived tissues ofin vitro shoots and cell suspension cultures makes this species an attractive experimental system for somatic genetic manipulation.Abbreviations BAP benzylaminopurine - NAA -naphthaleneacetic acid - CPW cell and protoplast wash solution - gFW gram fresh weight On leave from Department of Crop Sciences University of Alexandria Egypt     

ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomyces cerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane and was found in the detergent-resistant membrane fraction. Compared to control cells, a pdr10∆ mutant was resistant to sorbate but hypersensitive to the chitin-binding agent Calcofluor White. Calcofluor sensitivity was attributable to a partial defect in endocytosis of the chitin synthase Chs3, while sorbate resistance was attributable to accumulation of a higher than normal level of the sorbate exporter Pdr12. Epistasis analysis indicated that Pdr10 function requires Pdr5, Pdr12, Lem3, and mature sphingolipids. Strikingly, Pdr12 was shifted to the detergent-resistant membrane fraction in pdr10∆ cells. Pdr10 therefore acts as a negative regulator for incorporation of Pdr12 into detergent-resistant membranes, a novel role for members of the ABC transporter superfamily.     

Plasma membrane ATPase and H+ transport activities in microsomal membranes from mycorrhizal tomato roots

ATPase activity, ATP-dependent H⁺ transport and the amount of antigenic tomato plasma membrane H⁺-APTase have been analysed in membrane vesicles isolated from Glomus mosseae- or Glomus intraradices-colonized roots and from non-mycorrhizal tomato roots. Microsomal protein content was higher in mycorrhizal than in control roots. The specific activity of the plasma membrane H⁺-ATPase was not affected by mycorrhizal colonization, although this activity increased in membranes isolated from mycorrhizal roots when expressed on a fresh weight basis. Western blot analysis of microsomal proteins using antibodies raised against the Arabidopsis thaliana plasma membrane H⁺ - ATPase showed that mycorrhizal colonization did not change the relative amount of tomato plasma membrane ATPase in the microsomes. However, on a fresh weight basis, there was a greater amount of this protein in roots of mycorrhizal plants. In addition, mycorrhizal membranes showed a higher specific activity of the vanadate-sensitive ATP-dependant H⁺ transport than membranes isolated from control roots. These results suggest that mycorrhiza might regulate the plasma membrane ATPase by increasing the coupling efficiency between H⁺ transport and ATP hydrolysis. The observed effects of mycorrhizal colonization on plasma membrane H⁺-ATPase were independent of the AM fungal species colonizing the root system.     

Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogenes

Hairy root culture of Ginseng (Panax ginseng) was established after roots were induced on callus following infection with Agrobacterium rhizogenes. The transformed cultures of ginseng could be subcultured as an axenic root culture in the absence of phytohormones, and grew with extensive lateral branches more rapidly than the ordinary cultured roots induced by hormonal control from ginseng callus. The hairy roots synthesized the same saponins, ginsenosides, as those of the native root, up to about 2.4 times in the quantity, and up to about 2 times in comparison with that of the ordinary cultured roots, on dry weight basis.Abbreviations Ms medium Murashige and Skoog's medium - 2,4-D 2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - K kinetin This paper is Part 52 in the series "Studies on Plant Tissue Culture". For Part 51, see Ayabe S, Udagawa A, Furuya T (1987).     

Isolated cell walls exhibit cation binding properties distinct from those of plant roots

Aims

The principal contributor to the cation binding properties of roots is currently considered to be the cell wall or, alternatively, the plasma membrane. The aim of this study was to highlight their respective contributions in the binding properties.

Methods

Cell walls of a dicotyledon (Solanum lycopersicum L.) and monocotyledon (Triticum aestivum L.) were isolated from roots and their binding properties were compared to those of their respective roots. Cell wall and root binding capacities were evaluated by potentiometric titrations and cation exchange capacity measurements, while their biochemical composition was analyzed by ¹³C-NMR spectroscopy.

Results

The lower binding capacity of isolated cell walls compared to roots revealed that cell plasma membranes had a higher binding site density than cell walls. The significant decrease in some NMR signals, i.e. carbonyl C, N alkyl/methoxyl C and alkyl C regions, suggested that carboxyl, amine and phosphate binding sites, borne by proteins and phospholipid plasma membranes, contribute to the binding capacity.

Conclusions

Cell walls and plasma membranes were found to be jointly involved in root binding properties and their respective contributions seemed vary between plants.     

Induction of phenylpropanoid compounds by UV-B irradiation in roots of seedlings and cell cultures from dill (Anethum graveolens L.)

In both cell cultures and seedling roots from dill (Anethum graveolens L.) UV irradiation selectively induced a quercetin arabinoside and two other phenylpropanoid compounds with caffeic acid as the aglycone. Only wavelengths below 360 nm were effective in this induction. Maximal effectiveness was observed below 320 nm.Supported by the Deutsche Forschungsgemeinschaft (SFB 46).     

Active cation transport and Na+K+Mg ATPase of the monotreme erythrocytes

The erythrocytes of the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus), which are practically devoid of intracellular ATP content (1), were examined for active Rb⁸⁶ influx and for the presence of Na+K+Mg ATPase. We found that intact erythrocytes of both species possess the ability to actively transport cations. Ouabain sensitive Rb⁸⁶ influx in the echidna was approximately 0.17 μmoles/ml cells × hr, whereas the platypus exhibited a higher value of 0.43 μmoles/ml cells × hr. Surprisingly, ouabain sensitive Na+K+Mg ATPase activity of isolated membranes was high amounting to some 15 to 25 fold higher than the human erythrocyte counterpart determined under identical conditions. These findings suggest that a trace amount of ATP is sufficient to maintain active cation transport across the monotreme cell membranes.     

High-yield production of tropane alkaloids by hairy-root cultures of aDatura candida hybrid

Hairy root cultures were obtained following inoculation of the stems of sterile plantlets of aDatura candida hybrid withAgrobacterium rhizogenes. The scopolamine and hyoscyamine content was quantified by HPLC and compared with the non-transformed plants. The alkaloid yield (0.68% dry weight) obtained with the hairy roots was 1.6 and 2.6 times the amount found in the aerial parts and in the roots of the parent plants, respectively. Only a small proportion of alkaloids was released into the growth medium. Scopclamine was the principal alkaloid and the scopolamine/hyoscyamine ratio of ca. 5:1 makes these hairy roct cultures worthy of consideration as a source of scopolamine.     

Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.)

In vitro propagation of trees using cell, tissue and organ culture is a fast emerging area. We report here the clonal propagation of Indian rosewood (Dalbergia latifolia Roxb.) from shoot callus cultures of 5 year old trees. Bud regeneration was obtained on MS media supplemented with BA and NAA. About 35% of the cultures showed organogenesis. Shoots measuring about 3–5 cm can be excised and rooted in White's medium supplemented with 1–2 mg/L IAA. Rooted plants were successfully established in soil.Abbreviations BA Benzyladenine - CM Coconut milk - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthaleneacetic acid - PVP-360 Polyvinyl pyrrolidone     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Cell cycle variations in HeLa 65 plasma membrane alkaline phosphatase

HeLa plasma membranes from M, G₁, and S phase cells were isolated from growing synchronous cell cultures. It was found that the specific activity of plasma membrane alkaline phosphatase was over three times higher in the M phase cell than in the G₁ and S phase cell. However, sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis showed that the S phase plasma membrane contained 5.5 times more alkaline phosphatase protein than did the plasma membrane from mitotic cells, and 11.0 times more than the G₁ phase plasma membrane. This would indicate that the high specific activity in mitosis was due to modification of the alkaline phosphatase protein resulting in increased enzymatic activity.     

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% ¹. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient ². Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K¹⁵NO₃ as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest ³. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control ⁴.     

Physiological factors for tolerance of <Emphasis Type="Italic">Kosteletzkya virginica</Emphasis> (L.) Presl to one-instar bollworms of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Hubner)

An experiment was designed to investigate the tolerance and response of the Kosteletzkya virginica (L.) Presl to one-instar bollworms of Helicoverpa armigera (Hubner). In this experiment, the insects obviously brought an enhancement in damaged rate of leaf area (DRLA), average diameter of bitten hole (ADBH) and average grade of leaf damage (AGLD) in K. virginica seedlings. When amount of insect increased to 4 per top leaf, the average DRLA, AABH, ADBH and AGLD were 8.6%, 9.2 mm, 2.7 mm and 2.0 mm, respectively, and the seedlings showed a potential insect-tolerance to this insect stress. Furthermore, with an increase of insect density, MDA, relative permeability of membrane (RPM), O₂^−· production and soluble sugar concentration all enhanced, whereas activities of SOD and POD increased and subsequently declined. By statistical analysis, it was drawn that bio-toxicity of O₂^−· was the first destroying factor, cell membrane decomposition ranked the second, and the destruction of membrane lipid peroxidation was the slightest to K. virginica seedlings. Moreover, under the insect stress, POD was the most significant factor in protecting the seedlings, soluble sugar acted as the subordinate one, and the protection of SOD was the slightest compared to two others. In addition, insect stress obviously affected chlorophyll-fluorescence characteristics, and resulted in a significant increase in Fo and qN, and a significant reduction in Fv/Fm, ETR and qP during experiment.     

Diacylglycerol kinase in plasma membranes from wheat

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg²⁺ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl₂. The Mg²⁺ requirement could be substituted only partially by Mn²⁺ and not at all by Ca²⁺. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

Activation of plasmalemmal NADPH oxidase in etiolated maize seedlings exposed to chilling temperatures

Five-day-old etiolated seedlings of maize (Zea mays L.) were used to study the kinetics of hydrogen peroxide formation upon lowering growth temperature from 25 to 6°C. The total content of hydrogen peroxide in root and shoot tissues increased by 30–40% after 2-h cooling compared to the control level but returned to the initial level or decreased even lower after 24-h cooling. In order to prove the involvement of plasma membrane NADPH oxidase in changes of hydrogen peroxide content upon cooling, isolated plasma membranes were obtained from untreated plants and from seedlings chilled at 6°C for 2 and 24 h. The NADPH-dependent generation of superoxide anion radical in isolated plasma membranes was quantified by measuring the rate of formazan production from the tetrazolium salt XTT. The activity of plasma membrane NADPH oxidase in shoots was 50 ± 9 nmol O₂/(mg protein min), which was 1.5 times higher than the activity in roots. The enzyme activity in plasma membranes was inhibited by low concentrations of diphenyleneiodonium. The effective concentration EC₅₀ was 5.10 μM for shoots and 9.05 μM for roots. The activity of plasma membrane NADPH oxidase increased after 2-h cooling of seedlings but reversed to the control level after 24-h cooling. This transient activation of NADPH oxidase upon cooling was similar to the pattern of hydrogen peroxide formation in shoots and roots. Analysis of NADPH oxidase activity of plasma membrane proteins after their separation in denaturing conditions followed by subsequent renaturation revealed four diphenyleneiodonium-sensitive bands with mol wt of 130, 88, 51, and 48 kD. Western blot analysis of the reaction with antibodies against the catalytic domain of phagocyte NADPH oxidase revealed the proteins with mol wt of only 88 and 48 kD. The properties of molecular organization of plasma membrane NADPH oxidase are discussed in terms of its role in cell signaling.