Ecologocenotical characteristics of the herbaceous cover along the highways of Nizhny Novgorod

A total of 86 species of vascular plants have been found in the studied territories along the highways of Nizhny Novgorod. Asteraceae, Poaceae, and Fabaceae play a great role in the taxonomic structure of herbs. Cyperaceae and Rosaceae are less important. Among the ecologocenotical groups, meadow species form the basis of the herbaceous cover, but their participation diminishes at distances of 1–3 m from the highways. Herbaceous plants include also flowering meadow species, which play an indispensable aesthetic role.     

Osmotic Water Permeability of Vacuolar and Plasma Membranes Isolated from Maize Roots

The method of stopped flow was used to follow the changes in light scattering by the vesicles of plasmalemma and tonoplast isolated from maize (Zea maysL.) roots and treated by osmotic pressure. In both membrane preparations, the rate of the process depended on the osmotic gradient and was described with the simple exponential function. The rate constants derived from these functions were the following: the coefficient of water permeability in the tonoplast (P= 165 ± 7 m/s) exceeded by an order of magnitude the corresponding index for plasmalemma (11 ± 2 m/s). The presence of HgCl₂(1.6 nmol/g membrane protein) decreased the tonoplast water permeability by 80%. Microviscosity studies of the hydrocarbon zone in the isolated membranes by using a fluorescent diphenylhexatriene probe demonstrated that the two membranes do not differ in the phase state of their lipid bilayer. The authors conclude that the observed difference in water permeability does not depend on the state of the lipid phase and probably reflects the dissimilar functional activity of plasmalemma and tonoplast aquaporins.     

Osmotic water permeability of cell membranes from Mesembryanthemum crystallinum leaves: effects of age and salinity

The osmotic water permeability ( P _os) of cell membranes isolated from leaves of 40-, 50- and 60-day-old Mesembryanthemum crystallinum plants was estimated by measuring light-scattering kinetics using stopped-flow spectrophotometry. The measurements were performed on the plasma membrane (PM), purified tonoplast (TP), and TP-enriched vesicles. The PM and TP-enriched vesicles were obtained by partitioning the microsomal fraction in an aqueous polymer two-phase system, whereas the purified TP vesicles were prepared by microsomal vesicle flotation on a sucrose cushion. The P _os of isolated membranes declined with plant age. The kinetic experiments showed that there was no difference between the P _os of the PM and TP isolated from plants of all ages. A 24-h exposure of plants to 400 m M NaCl caused a decline in the P _os as well. These findings suggest that, during M. crystallinum transition to CAM, which was induced by plant ageing or salinity, plant osmoregulatory responses included changes in the P _os of the leaf-cell membranes. These variations in the P _os are discussed in the context of adaptive mechanisms responsible for the maintenance of the water balance in the common ice plant.     

Affinity of PIP-aquaporins to sterol-enriched domains in plasma membrane of the cells of etiolated pea seedlings

The hypothesis that sterol-enriched domains represent sites of preferred localization of PIP-aquaporins was tested in experiments on plasma membranes isolated from cells of etiolated pea (Pisum sativum L.) seedlings. Plasma membranes were isolated from microsomes by the partition in the aqueous two-phase polymer system and separated into vesicle fractions of different buoyant density by flotation in discontinuous OptiPrep gradient. Two types of plasma membrane preparations were used: one was treated with cold 1% Triton X-100 and the other was not. In untreated preparations, three populations of plasma membrane vesicles were obtained, while in the case of treated preparations, fractions of detergent-resistant membranes (DRM) and solubilized membrane proteins were obtained. In all membrane fractions collected after OptiPrep flotation, the amounts of proteins, sterols, and PIP-aquaporins were determined. The highest sterol content was detected in the membrane fraction with buoyant density 1.098 g/cm³ and in the DRM fraction (1.146 g/cm³). These fractions contained much more PIP-aquaporins than the other ones. Phase state of the lipid bilayer was determined by measuring generalized polarization excitation of fluorescence (GPEX) of laurdan incorporated into the membranes of different fractions. It was revealed that the lipid bilayer of the membranes with density of 1.098 g/cm³ had a higher extent of ordering than that of the fractions with density of ∼1.146 g/cm³. The results indicated that uppermost local concentrations of PIP-aquaporins were associated with tightly packed sterol-enriched domains. Moreover, upon solubilization of plasma membrane with Triton X-100, PIP-aquaporins mainly resided in DRM, thus exhibiting a high affinity to sterols.     

Effect of chilling temperatures on osmotic water permeability and aquaporin activity in the plasma membranes from pea roots

The osmotic water permeability of plasma membrane vesicles was examined after isolation from the roots of 7-day-old etiolated pea ( Pisum sativum, cv. Orlovchanin) seedlings grown at optimal temperature and those exposed to 1-day chilling at 8°C in the end of the growth period. The homogenization medium for obtaining plasma membranes was supplemented with either SH-reagents or protein phosphatase inhibitors. The plasmalemma vesicles were purified from the microsome fraction by means of two-phase polymer system. The osmotic water permeability of membrane vesicles was evaluated from the rate of their osmotically induced shrinkage. The lowering of growth temperature was accompanied by the increase in osmotic water permeability of plasmalemma. These changes occurred without the corresponding increase in aquaporin content or permeability of membrane lipid matrix. The membranes from cooled seedlings were markedly depleted in the content of SH-groups. Furthermore, the treatment of membrane samples with a thiol-reducing agent, tributylphosphine did not raise the SH-group content in membranes from chilled plants, unlike such changes in membranes from warm-grown plants. When the homogenization medium contained dithiothreitol and phenylarsine oxide (an inhibitor of tyrosine protein phosphatases), the osmotic permeability of plasmalemma in preparations from warm-grown seedlings also increased. Based on these results, it is supposed that aquaporin-mediated water permeability of membranes is regulated through different pathways under optimal and adverse conditions for plant growth. Direct action of endogenous SH redox regulators on aquaporin activity is likely under optimal growth conditions, while protein phosphatase might mediate changes in aquaporin activity under unfavorable growth conditions.     

PIP1 aquaporins,sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm³) and “heavy” (≥1.146 g/cm³) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.     

Identification and activity of superoxide-producing protein complexes of the plasma membrane of etiolated maize seedlings subjected to low positive temperature

Kinetics of superoxide anion generation by the isolated plasma membrane was determined by the rate of formazan formation from XTT in the presence of NADPH or NADH. The plasma membrane was prepared from (control) etiolated maize seedlings grown at 25°C and from (cooled) seedlings incubated at 6°C for the last day. Membrane vesicles from the control plants possessed superoxide-producing activity, and the rate of NADH oxidation was markedly higher than that of NADPH. The low-temperature incubation of the seedlings suppressed the NADPH-dependent activity, whereas the NADH-dependent one slightly increased. The solubilized by dodecyl maltoside (DDM) plasma membranes were separated into multiprotein complexes by high-resolution clear native electrophoresis (hrCN-PAGE). The aim was to find complexes exhibiting the superoxide-producing activity sensitive to inhibition by diphenylene iodonium. Several protein complexes from the plasma membrane capable of superoxide producion in the presence of NADPH or NADH were found. The maximum diphenylene iodonium-sensitive activity was found in the high-molecular weight complex, in which proteins reacting with antibodies against C-terminal peptide of phagocytic oxidase (gp91phox) were detectable. The activity of this complex was lower in the cooled than in the control seedlings and displayed higher affinity to NADPH than to NADH. To search for the cooling-induced changes in the polypeptide content of protein complexes, the two-dimensional difference gel electrophoresis (hrCN/SDS-PAGE) was used. Control and cooled samples, whose lysine had been labeled with fluorescent dyes Cy2 and Cy3, respectively, were separated by this method in one gel. Decrease in a temperature of plant growing affected the protein content of the complex so that some new proteins appeared and several polypeptides disappeared as compared with the control. There were no significant differences between the cooled and control counterparts in the content of proteins detectable with gp91phox antibodies. Therefore, the high-molecular complex containing NADPH oxidase looses proteins under low temperature that may decrease its superoxide-producing activity.     

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.     

Aquaporin Content in Cell Membranes of Mesembryanthemum crystallinum as Affected by Plant Transition from C3 to CAM Type of Photosynthesis

Western-blot analysis was used to determine the contents of aquaporin isoforms MIP A, MIP B, and MIP C in cell membranes isolated from roots and leaves of Mesembryanthemum crystallinum plants with C₃ and Crassulacean acid metabolism (CAM) types of photosynthesis. These membrane preparations were also used to assess osmotic water permeability; to this end, the rate of osmotic vesicle shrinking was registered as the light scattering intensity by the method of stopped flow. The cell membranes represented by the plasmalemma and the tonoplast-enriched fraction were obtained by separating the microsomes in a two-phase polymer system. Plant transition from C₃ to CAM-photosynthesis occurred in the course of plant development or was induced by salinization. All three isoforms under study were found in the plasma membranes of roots and leaves of the C₃ plants, whereas in the CAM plants, independent of the transition-inducing factor, the aquaporin contents notably decreased in the leaf membranes and remained unchanged in the roots. In the membranes isolated from roots and leaves of the C₃ plants, the values of osmotic water permeability exceeded two–threefold the corresponding indices characteristic of the CAM plants. The authors believe that aquaporin isoforms in M. crystallinum are under the organ- and tissue-specific control.     

Redox modulation of osmotic water permeability in plasma membranes isolated from roots and shoots of pea seedlings

Partitioning in a biphasic polymer system was used to isolate plasmalemma (PM) from roots and shoots of etiolated pea seedlings. The membrane preparations were used to assess the osmotic water permeability (P _os) with the stopped-flow method. The Western-blot technique was employed to determine the membrane content of the PIP-family of aquaporins, and their activity was estimated by measuring the rate of osmotic vesicle shrinking in the presence of inhibitors, HgCl₂ and AgNO₃. Monobromobimane fluorescent dye was used to determine the quantity of sulfhydryl groups in cell membranes and follow the effect of SH-oxidizing (diamide) and SH-reducing (dithiothreitol and tributylphosphine) agents on P _os of the root PM and oligomerization of aquaporins. The shoot PM was shown to combine high P _os with low aquaporin content. In the root PM, P _os was lower and the aquaporin content greatly exceeded that in the shoots. HgCl₂ and AgNO₃ did not decrease the rate of osmotic shrinking in root membrane vesicles, whereas considerably (by 40–50%) inhibited this index in the shoot membranes. Root and shoot PM preparations dramatically differed in their SH-group contents: the former exceeded the latter sixfold. When added to the homogenization medium, diamide and tributylphosphine affected the content of SH-groups and P _os in the root PM. In the roots, diamide decreased the quantity of SH-groups almost twofold and increased P _os fourfold, and the introduction of tributylphosphine produced a twofold increase in the quantity of SH-groups with only slight decrease in the P _os. Immunological analysis of membranes isolated in the presence of diamide showed that the ratio between the monomer and dimer forms of aquaporins in two membrane preparations depended on the presence of dithiothreitol in the denaturing buffer apparently because dithiothreitol exposed and reduced disulfide bonds essential for monomer interactions and inaccessible for interaction with redox modifiers of SH-groups in the membrane. Because of their inaccessibility, these modifiers could not cause the changes of P _os in the root PM produced by oxidation and reduction of SH-groups. This phenomenon is probably related to the change in the status of SH-groups in two cysteine residues at the N-end of the aquaporin loop C oriented outward into the apoplast.