Plasmatubules in the pollen tubes of Nicotiana sylvestris

Ultrastructural studies of the pollen tubes of Nicotiana sylvestris grown in the pistil revealed an extensive development of plasmatubules formed by evaginations of the plasma membrane. The plasmatubules occurred as twisted tubular structures in the periplasmic space along the tube wall and, in cross section, exhibited circular profiles with an outer diameter of 28±4 nm. They were also seen in deep, pocket-like invaginations of the plasma membrane and in this case the profiles had an outer diameter of 34±8 nm. In the pocket-like invaginations they were partially branched and often closely packed to form groups with obvious patterns. The enlargement of the plasma-membrane area resulting from plasmatubules formed along the tube wall was about six-to tenfold. Pollen tubes grown in vitro exhibited poorly developed plasmatubules. It is suggested that the large extension of the plasma membrane could enhance the uptake of nutrients, and thus might be responsible for the comparatively fast growth of pollen tubes in the pistil. Moreover, it is also assumed that the turnover rate of the Golgi apparatus must be higher in pollen tubes growing in vivo than in vitro, in order to provide a sufficient amount of membrane for the formation of the plasma membrane with its tubular modifications.     

Potential dependence of the admittance ofChara plasmalemma

Summary A computer-controlled apparatus is described, which combines the two powerful methods of voltage-clamping and admittance measurement. The 5-Hz admittance ofChara plasmalemma is obtained for transmembrane PD from −400 mV to 0. DC conductance is also measured by the bipolar staircase method. Both the DC and 5-Hz conductances at steady state display a central maximum at ≈−250 mV. This feature is attributed to the conductance/voltage characteristics of the H⁺ pump. The steady-state capacitance does not show any trend throughout the potential interval. At the time of the delay, before excitation commences, the 5-Hz conductance is smaller than after excitation. At the time of excitation the 5-Hz conductance echoes the time-course of the ionic current, while the capacitance undergoes a sharp decrease followed by an increase. A possible explanation of the capacitance behavior is attempted involving transport number effects and reactances associated with the Hodgkin-Huxley gating mechanism. At punchthrough the membrane becomes inductive.     

Plasmatubules in transfer cells of pea (Pisum sativum L.)

Plasmatubules are tubular evaginations of the plasmalemma. They have previously been found at sites where high solute flux between apoplast and symplast occurs for a short period and where wall proliferations of the transfer cell type have not been developed (Harris et al. 1982, Planta 156, 461–465). In this paper we describe the distribution of plasmatubules in transfer cells of the leaf minor veins of Pisum sativum L. Transfer cells are found in these veins associated both with phloem sieve elements and with xylem vessels. Plasmatubules were found, in both types of transfer cell and it is suggested that the specific distribution of the plasmatubules may reflect further membrane amplification within the transfer cell for uptake of solute from apoplast into symplast.     

Comparison of K,MgATPases in purified plasmalemma from wheat and oat. – Substrate specificities and effects of pH, temperature and inhibitors

Plasmalemma from 8-day old oat ( Avena sativa L. cv. Brighton) and spring wheat ( Triticum aestivum L. cv. Drabant), grown in the dark at 18°C, was prepared from the 10000 g (10 min) – 30 000 g (60 min) root homogenate by two-phase separation in three steps with 6.5% (w/w) Dextran T 500 and 6.5% (w/w) polyethylene glycol 4 000. Biochemically and with respect to activation by Mg²⁺ as well as by (Mg²⁺+ K⁺), the oat preparations clearly appeared as ATPase(s) in the pH range 5–8. They showed high specificity for ATP, temperature optima between 38 and 40°C, and were inhibited by vanadate, DCCD (dicyclohexylcarbodiimide) and SH-reagents, but not by oligomycin, ammonium molybdate or ouabain. In contrast, the preparations from wheat contained more than one type of MgATPase/ nucleotidase, as revealed by complex dependence on both pH and temperature as well as by comparatively low specificity towards nucleotides. However, no unspecific phosphatase was present, and the effect of K⁺ over and above that of Mg²⁺ was almost as specific as in oat by all criteria used. The data available from this and earlier investigations from our group would indicate that the complex reactions of preparations of wheat plasmalemma may not be due to contamination but, rather, expressions of the many biological functions that must be associated with the plasmalemma in vivo and which may be located in sub-units that are more firmly attached to wheat than to oat plasmalemma.     

Potassium ion channels in the plasmalemma

The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K⁺ transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K⁺ equilibrium potential (E_K) – membrane potential (V_m), or secondary active by coupling through a carrier to the inward driving force of H⁺ or Na⁺. Known K⁺ channels are permeable to monovalent cations, a permeability order being K⁺ > Rb⁺ > NH₄⁺ > Na⁺≥ Li⁺ > Cs⁺. The macroscopic K⁺ currents across a cell or protoplast surface commonly show rectification, i.e. a V^m-dependent conductance which in turn, may be controlled by the cytosolic activity of Ca²⁺, of K⁺, of H⁺, or by the K⁺ driving force. Analysis by the patch clamp technique reveals that plant K⁺ channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K⁺ channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.     

Microplicae: characteristic ridge-like folds of the plasmalemma

Scanning electron microscopy reveals that the free surfaces of stratified squamous epithelial cells lining the alimentary tract, cornea, and conjunctiva exhibit characteristic ridge-like folds of plasmalemma. These microplicae are approximately 0.1-0.2 micronm in width, of variable height (0.2-0.8 micronm) and length, may followstraight or winding paths, often branch, and exhibit a wide variety of patterns over the surfaces of cells. Transmission electron microscopy reveals that microplicae often have a fine (100-150 A) electron-dense zone subjacent to their plasmalemma and an intracellular matrix characterized by a disorderly arrary of fine filaments (40-60 A in diameter). Microplicae appear to arise from plasmalemmal fold which once provided for intercellular interdigitation and desmosome abhesion between adjacent cells. Ruthenium red staining demonstrates that microplicae and interplical grooves are covered with a polyanionic glycocalyx. Although free surface microplicae may merely represent the renmants of intercellular interdigitations or a modified expression of microvillous-like extensions, it is also possible that they serve another specific function. In this regard it is speculated that microplical and interplical grooves may function to hold a layer of lubricating and cushioning mucin designed to protect the underlying plasmalemma from abrasive abuse.     

Measurements of redox activity at the plasmalemma

Salt (NaCl) tolerance of 3 eucalypt species ( Eucalyptus alba Reinw. ex Bl., E. camaldulensis Dehnh and E. microtheca f.v. Muell.) was studied: three-month-old seedlings grown in a greenhouse were watered by a saline solution (up to 700 m M ) for 1 month. Mineral, water and sugar contents were highly affected by the saline stress. Sodium, K and Ca were accumulated in the leaves. No significant differences were found between E. camaldulensis and E. microtheca , the tolerant species, in water and mineral contents. Sugar contents were greater in E. microtheca . In E. microtheca Na was highly accumulated in roots [up to 910 μmol (g fresh weight)⁻¹], less in stems [up to 580 μmol (g fresh weight)-1] and leaves [up to 410 μmol (g fresh weight)^−r]. Chloride was also accumulated, its content was greater than the total content of Na and K, especially in the salt-tolerant provenance. Potassium and Ca contents were variously affected by the saline stress whereas soluble protein, amino acid and sugar contents were increased. Just after the saline stress, plants showed a large increase in the Na content of the leaf while the decrease in the K content of the stem and leaf continued. Plants were killed by the stress, probably because of too high accumulation of Na in leaves or roots according to the provenance. Osmoregulation and especially the participation of Na are discussed.     

Properties of a plasmalemma ATPase of the maize scutellum

Properties of a plasmalemma phosphatase of the maize scutellum, tentatively identified as an ATPase in a previous paper, were investigated. Fresh and frozen-thawed scutellum slices, that had been treated with 10 mM HCl to destroy acid phosphatases, were used as a source of enzyme. With the exceptions of the Na⁺, K⁺ and dinitrophenol experiments, the two kinds of slices gave similar results. ATP and CTP were the best substrates for the enzyme followed by TTP, UTP, CDP, ADP and GTP. UDP, nucleoside monophosphates, sugar phosphates, inorganic pyrophosphate and p-nitrophenyl phosphate were relatively ineffective as substrates. The K_m's for ATP and ADP were 0.65 and 5 mM, respectively, but the two substrates gave the same V_max (49.8 μmol P_i/hr/g slices). Previously, it was shown that the products of ATP hydrolysis are ADP, AMP and P_i. Using these previous results and from the time courses of ATP disappearance from the bathing solution and the appearance of P_i and ADP, it was concluded that ATP and ADP were hydrolysed by the same enzyme. The ATPase was not inhibited by oligomycin. N-N′-Dicyclohexylcarbodiimide (DCCD) was a poor inhibitor, and a water soluble analog of DCCD, 1-ethyl-3 (3 dimethyl-aminopropyl)-carbodiimide, gave only 33% inhibition. The relative effectiveness of divalent cations for stimulating ATPase activity was Mn²⁺ > Mg²⁺ ? Ca²⁺ > Co²⁺ · Na⁺ and K⁺ gave a small additional stimulation in the presence of Mg²⁺. However, Na⁺ and K⁺ gave a much greater stimulation when no divalent cation was added, and this occurred only when fresh slices were used. Dinitrophenol also increased ATPase activity only when fresh slices were used. Since it is likely that both the uptake of Na⁺ and K⁺ and the action of dinitrophenol would lower the electrochemical gradient of protons across the plasmalemma, the different results obtained with fresh slices indicate that the ATPase in these slices was under the constraint of a proton gradient.     

Isolation and properties of the plasmalemma in yeast

Summary A method is described for the isolation of fragments of the plasmalemma based on differential and density gradient centrifugation using cell free extracts from anaerobically grown Saccharomyces cerevisiae. Electron microscopically investigated frozen-etched specimens of isolated plasmalemma revealed the presence of globular particles attached to the outer surface of the membrane; these particles correspond to those observed in situ.In isolated plasmalemma a high specific activity of Mg⁺⁺-dependent ATPase, which is not sensitive to Oligomycin, is present. Yeast plasmalemma contains protein, lipids (including phospholipids) and an appreciable amount of polysaccharide. Hydrolysis of this polysacharide yields only mannose.The treatment of the isolated plasmalemma with detergents liberates the globular particles which can be isolated by density gradient centrifugation. Protein and polysaccharide occur in the respective fraction; therefore the globular particle represents a mannan-protein. It is concluded that the particles, which cover the plasma-membrane of plant cells, represent glycoproteins, that is, building stones to be incorporated into the fibrillar network of the cell walls.     

Current-voltage relationships of potassium channels in the plasmalemma ofAcetabularia

Summary The outer membrane of mechanically prepared protoplasmic droplets fromAcetabularia mediterranea has been investigated by patch-clamp techniques. These membranes are shown to consist of physiologically intact plasmalemma. With the Cl^– pump inhibited, microscopic currents through K⁺-selective channels were studied. These currents compare well with macroscopic K⁺ currents as previously determined by standard microelectrode techniques and tracer flux measurements. There is about one K⁺ channel per m² in the plasmalemma. The current-voltage relationship (I–V curve) of the main open channel (channel A) is sigmoid over a voltage range between about –100 and +100 mV with saturation currents of about ±10 pA. A second species (or different state of channel A) of K⁺-selective channels (channel B) differs from channel A by smaller saturation currents (about ±7 pA) and a much smaller open probability. The open probability of channel A increases from almost zero at large negative voltages to about 1/2 at large positive voltages. Taking the closed times into account, the mean steady-stateI–V curve of channel A displays outward rectification about the equilibrium voltage for K⁺ and negative slope conductance at larger negative voltages. The open channelI–V curve of the open channels A and B, the changes of theI–V curve of the open channel A upon variation of the external K⁺ concentration, as well as the mean steady-stateI–V curves of channel A are described by simple reaction kinetic models, the parameters of which are determined to fit the experimental data. The results are discussed with respect to data from other K⁺ channels in plants and with respect to regulation of the cytoplasmic K⁺ concentration inAcetabularia.     

Significance of plasmalemma aquaporins for water‐transport in Arabidopsis thaliana

The plant plasma membrane intrinsic protein, PIP1b, facilitates water transport. These features were characterized in Xenopus oocytes and it has asked whether aquaporins are relevant for water transport in plants. In order to elucidate this uncertainty Arabidopsis thaliana was transformed with an anti-sense construct targeted to the PIP1b gene. Molecular analysis revealed that the anti-sense lines have reduced steady-state levels of PIP1b and the highly homologous PIP1a mRNA. The cell membrane water permeability was analyzed by swelling of protoplasts, which had been transferred into hypotonic conditions. The results indicate that the reduced expression of the specific aquaporins decreases the cellular osmotic water permeability coefficient approximately three times. The morphology and development of the anti-sense lines resembles that of control plants, with the exception of the root system, which is five times as abundant as that of control plants. Xylem pressure measurement suggests that the increase of root mass compensates the reduced cellular water permeability in order to ensure a sufficient water supply to the plant. The results obtained by this study, therefore, clearly demonstrate that aquaporins are important for plant water transport.     

Calcium influx at the plasmalemma of Chara corallina

Influx of ⁴⁵Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La³⁺-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5–15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca²⁺ externally was 0.25–0.5 pmol·cm^-2·s^-1, but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K⁺. In cells in which the control flux was less than about 0.5 pmol·cm^-2·s^-1 there was no effect of 50 M nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm^-2·s^-1 (whether by natural variability, pretreatment, or by depolarisation in 20 mM K⁺), the flux was reduced by 50 M nifedipine to a value in the range 0.25–0.59 pmol·cm^-2·s^-1. It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 M BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm^-2 with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca²⁺ the measured influx is a reasonable estimate of the influx at the plasmalemma.Abbreviations 0.4K-APW6 artificial pondwater, pH 6, containing 0.4 mM KCl - 20 K-APW6 artificial pondwater, pH 6, containing 20 mM KCl - Ca_o external Ca²⁺     

Identification of albumin-binding proteins of thymocyte plasmalemma

In the present work we examined whether the interaction between albumin molecules and thymocytes involves albumin-binding proteins (ABP). Two plasmalemma-rich fractions obtained by differential centrifugation from rat thymus lymphocytes were characterized biochemically and morphologically. These fractions were examined by ligand-blotting and ligand affinity chromatography techniques. Plasmalemma proteins separated by SDS-PAGE were electrotransferred onto nitrocellulose membranes and incubated with¹²⁵I-albumin, in the presence or absence of excess native albumin. The autoradiogram revealed specific binding to two sets of polypeptides of 16–18 and 29–31 kDa, which could be blocked by native albumin. To elucidate whether albumin-binding proteins are exposed on the cell surface, intact lymphocytes were surface radioiodinated and membrane fractions prepared from them were subjected to affinity chromatography on albumin-agarose beads. The proteins thus purified had, like ABP, M_r of 16 and 31. These data indicate that ABP (i) are components of thymocyte plasma membrane, (ii) have apparent molecular mass of 16–18 and 29–31 kDa, and (iii) are exposed on the outer membrane surface.Abbreviations ABP albumin-binding proteins - Alb bovine serum albumin - Au gold - DAPI 4,6-diamidino-2-phenylindol - EM electron microscopy - NC nitrocellulose - PAGE polyacrylamidegel electrophoresis - PBS phosphate buffered saline - PEG polyethylene glycol - PMSF phenylmethylsulfonyl fluoride - WGA Wheat germ agglutinin     

Effect of cyanide on the plasmalemma potential of mnium

By centrifuging Mnium cuspidatum leaf cells, the cytoplasm can be distinguished from the vacuole and a microelectrode tip can be located unambiguously in the cytoplasm. The site of the electrogenic pump is clearly demonstrated to reside in the plasmalemma as shown by depolarization of the cell electropotential induced by CN⁻.