Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Expression of an mRNA with sequence similarity to pea dehydrin (Psdhn 1) in guard cells of Vicia faba in response to exogenous abscisic acid

The function and location of guard cells uniquely subject them to stress. First, stomatal movements require large fluctuations in the concentration of potassium salts. Second, guard cell inner walls are the first surfaces exposed to evaporation and apoplastic solutes may accumulate there as a result. We have therefore investigated whether guard cells exhibit atypical expression of dehydrin genes because dehydrins accumulate in vegetative tissues in response to water stress. We have also assayed for osmotin mRNA, which is up-regulated in leaves in response to various stresses. mRNA probes for several representative genes were used with RNA extracts from control and water-stressed Vicia faba leaflets. Correlatively, these probes were used with RNA extracts from "isolated' guard cells that had been incubated with combinations of abscisic acid, mannitol and Ca²⁺. (Isolated guard cells are epidermal strips sonicated to destroy cells other than guard cells.) Hybridization with the probe prepared for a dehydrin from Pisum sativum (Psdhn 1) was detected in leaf extracts only if the leaf had been stressed. Similarly, after 1- and 6-h incubations with abscisic acid, isolated guard cells contained an mRNA that hybridized with the probe for Psdhn 1. Appearance of this abscisic acid-dependent mRNA required neither mannitol nor exogenous Ca²⁺. Regardless of the conditions or tissue, no hybridization was detected with the probe against osmotin, but our interpretation of this result is qualified. The simplest conclusion is that atypical expression of dehydrin is not the mechanism by which guard cells cope with their peculiar function and location.     

Development and accumulation of ABA in fluridone-treated and drought-stressed Vicia faba plants under different light conditions

The effects of fluridone on guard cell morphology, chloroplast ultrastructure and accumulation of drought stress-induced abscisic acid (ABA) were studied in Vicia faba L. plants grown under different light conditions. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. The appearance of defective and undeveloped stomata, and chloroplasts with a destroyed thylakoid membrane system was found in fluridone-treated plants grown at a photosynthetic photon flux (PPF) of 600 μmol m^-2 s^-1. Plants grown at a PPF of 40 μmol m^-2 s^-1 had diminished levels of ABA after imposition of dehydration. Fluridone treatment reduced the level of ABA in both unstressed and dehydrated leaves. Accumulation of ABA in the control plants was considerably reduced when they were exposed to dark periods of 24, 48 and 72 h just before imposition of the stress. Twenty-four hours after the dark treatment dehydration of the leaves resulted in a 3-fold decrease in the level of stress-induced ABA, and 72 h after dark treatment the amount of stress-induced ABA approximated the prestressed values. Fluridone-treated plants failed to accumulate ABA under water stress. In addition to functionally active chloroplasts, well-developed and functional stomata are required for drought stress to elicit a rise in ABA.     

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.     

ATP-driven Ca2+ transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelina communis

Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U₃+U₃′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven ⁴⁵Ca²⁺ transport [? 15 pkat (mg protein)⁻¹]. Ca²⁺ uptake into the vesicles had a pH optimum of 7.2 and apparent K_m values for Ca²⁺ of 4.4 μM and for Mg-ATP of 300 μM. Ca²⁺ uptake, K⁺, Mg²⁺-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm⁻³) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca²⁺ transport into the vesicles, excluding a Ca²⁺/H⁺ exchange driven by a proton gradient. ATP-dependent Ca²⁺ uptake was inhibited by erythrosin B (I₅₀= 0.1 μM), ruthenium red (I₅₀= 30 μM), La³⁺ (I₅₀= 10 μM) and vanadate (I₅₀= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I₅₀= 70 μM) and W-7 (I₅₀= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca²⁺ efflux from the vesicles rather than inhibit Ca²⁺ uptake. Our results demonstrate the presence of a Ca²⁺-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca²⁺ out of the cell. Its high affinity for Ca²⁺ makes it a likely component involved in adjusting low cytoplasmic Ca²⁺ levels. No indications for a secondary active Ca²⁺/H⁺ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca²⁺-ATPase from the H⁺-translocating K⁺. Mg²⁺-ATPase in C. communis plasma membranes.     

Changes in the Light Scattering by Symbiosomes from Vicia faba Root Nodules as Indicator of Ion and Metabolite Transport across the Peribacteroid Membrane

Passive transport of ions and metabolites across the peribacteroid membrane (PBM) was investigated on symbiosome preparations isolated from the broad bean (Vicia faba L.) root nodules and suspended in a potassium-free medium. Optical density of the symbiosome suspension at 546 nm was monitored as an indicator of light-scattering changes. Depolarization of the PBM with tetraphenylphosphonium cation (TPP⁺) caused an increase in light scattering of symbiosome suspension. This effect was enhanced after adding a K⁺ ionophore valinomycin to the incubation medium. A similar effect was observed after supplementing the symbiosome suspension with nigericin, a K⁺/H⁺ antiporter. Similar experiments on bacteroid suspensions prepared from isolated symbiosomes did not reveal any appreciable changes in light scattering in the presence of the same membrane-active substances. The light scattering by symbiosome suspensions decreased after adding malate or succinate, while the subsequent addition of centimolar concentrations of K⁺ substantially accelerated this process. Light scattering by the symbiosome suspension was insensitive to the addition of glutamate, a substance normally impermeant through the PBM of legume root nodules. These results suggest that the changes in light scattering by symbiosomes reflect the osmotically induced changes of symbiosome volume. These volume changes were assigned to alteration of the peribacteroid space (PBS). The incubation of symbiosomes in a potassium-free medium acidified their the PBS; this acidification was accelerated by valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and nigericin, and it was abolished in the presence of comparatively high concentrations of K⁺ in the incubation medium. The results indicate a relatively high permeability of the PBM to K⁺ ions.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

On the presence of inside-out plasma membrane vesicles and vanadate-inhibited K+,Mg2+-ATPase in microsomal fractions from wheat and maize roots

We have estimated the amount of inside-out plasma membrane (PM) vesicles in microsomal fractions from wheat (Triticum aestivum L. cv. Drabant) and maize (Zea mays L.) roots; non-latent activities of the PM markers vanadate-inhibited K⁺, Mg²⁺-ATPase (ΔVO₄-ATPase) and glucan synthase II (GS II, EC 2.4.1.34) were used as markers for inside-out PM vesicles, latent activities as markers for right-side-out PM vesicles, and specific staining with silicotungstic acid (STA) as a general marker for the PM. Separation of presumptive inside-out PM vesicles from right-side-out ones was achieved by counter-current-distribution (CCD) in an aqueous polymer two-phase system. Most of the GS II activity was latent and was found in material partitioning into the upper phase; a distribution which correlated well with that of STA-stained vesicles. Thus, most of the PM vesicles had a right-side-out orientation. ΔVO₄-ATPase, on the other hand, had a dual distribution (particularly pronounced in wheat) and was recovered both in material partitioning into the lower phase and into the upper phase. This indicates that ΔVO₄-ATPase activity was present also in membranes other than the PM. Additional evidence for this interpretation came from sucrose gradient centrifugation of wheat root material. This produced two peaks of ΔVO₄-ATPase activity with the membranes partitioning into the lower phase, none of which coincided with the peak obtained with right-side-out PM vesicles. Taken together, these results indicate that only very few inside-out PM vesicles are present in the microsomal fraction, and that ΔVO₄-ATPase as a marker for the PM, in contrast to GS II, may give quite misleading results with some plant materials. This stresses the need to use well-defined preparations of scaled, inside-out PM vesicles in solute uptake studies. The distribution of Ca²⁺-inhibited ATPase, on the other hand, agreed well with those of GS II and STA-stained vesicles both after CCD and sucrose gradient centrifugation, which suggests that Ca²⁺ inhibition may be a more specific property of the PM H⁺-ATPase than vanadate inhibition.