Cytoplasmic acidification with butyric acid does not alter the ionic conductivity of plasmodesmata.

The effect of lowering cytoplasmic pH on the ionic conductivity of higher-plant plasmodesmata was investigated with corn (Zea mays L. cv. Black Mexican Sweet) suspension culture cells. Exposure to butyric acid decreased the cytoplasmic pH by 0.8 units. Intercellular communication was monitored by electrophysiological techniques that allowed the measurement of membrane resistances of sister cells and the electrical resistance of the plasmodesmata connecting them. The decrease in cytoplasmic pH did not affect the resistance of plasmodesmata, despite the fact that the butyric acid treatment more than doubled the concentration of cytoplasmic calcium. This is discussed in light of previous findings that increases in cytoplasmic calcium increase the electrical resistance of plasmodesmata.

Effluxes of solutes from developing seed coats of Phaseolus vulgaris L. and Vicia faba l.: locating the effect of turgor in a coupled chemiosmotic system.

Cells lining the developing seed coats of legumes efflux photosynthates (mostly sucrose) and salts (mostly of potassium) into the apoplast for uptake by the developing embryo. These effluxes increase transiently in response to an increase in turgor in the effluxing cells. Detached coats of developing seed of P. haseolus vulgaris and Vicia faba were used to study the effects of turgor on the rates of efflux, on the membrane potential difference and on the membrane pH difference, using a number of inhibitors and agents which might affect signal cascades involving cytoplasmic calcium concentration. Effluxes were measured by measuring the concentrations of solutes of interest in solution samples placed in halves of detached seed coats, the paired halves serving as control and treated sample where appropriate. It is shown that a number of substances affect sucrose and potassium effluxes differently, and that hypo-osmotic shock depolarizes the efflux cells and acidifies the cytoplasm (in P. vulgaris). It is concluded that sucrose and potassium effluxes, although both are increased by an increase in turgor, are affected by different signal pathways. Further, it is also concluded that the signal that increases the rates of both sucrose efflux (via sucrose-proton antiport) and proton pump acts directly on the antiporter rather than on the pump. There are interesting parallels and contrasts between these processes and those in plants such as the charophyte Lamprothamnium after hypo-osmotic shock.

Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata.

The concentration of cytoplasmic free calcium ([Ca(2+)](cyt)) required to close higher plant plasmodesmata was investigated using corn (Zea mays L. cv. Black Mexican Sweet) suspension-culture cells. Physiological elevations of [Ca(2+)](cyt) were applied by cold treatment, and ion injection was also used to increase [Ca(2+)](cyt), by diffusion (for small increases) or by iontophoresis (for larger increases). The impact of such treatments on [Ca(2+)](cyt) was measured by ratiometric ion imaging. Intercellular communication during treatments was monitored using our recently developed electrophysiological technique that allows the electrical resistance of plasmodesmata and the plasma membranes of a sister-cell pair to be measured. A 4-fold increase in the calculated resistance of single plasmodesmata was observed in response to cold treatment that caused a 2-fold increase in average [Ca(2+)](cyt) (from 107 to 210 nM). In response to iontophoresis of Ca(2+), plasmodesmata were observed to go from "open" (low resistance) to "shut" (high resistance) and then back "open" within 10 s. Our results thus indicate that higher plant plasmodesmata respond quickly to physiological changes in [Ca(2+)](cyt).

Fast activation of a time-dependent outward current in protoplasts derived from coats of developing Phaseolus vulgaris seeds.

An outward current that appeared to activate instantaneously in response to depolarising voltage pulses at low sampling frequencies predominated in the plasma membrane of ground-parenchyma protoplasts derived from coats of developing Phaseolus vulgaris L. (cv. Redland Pioneer) seeds. However, the outward current showed time-dependent activation when higher sampling frequencies were used to measure the current. Activation of the current was best described as a double-exponential time course with the fast and slow time constants being 1 and 20 ms, respectively. The current also exhibited a rapid deactivation that followed a double-exponential time course with time constants of approximately 2 and 30 ms, respectively. "Tail-current" analysis allowed us to show that this current exhibited a low selectivity between K- and Cl- (PK:Cl = 1.8). Such a fast-activating current may account for some of the reports of time-independent, instantaneous currents that have been observed in plasma membranes of plant cells digitised at low sampling frequencies. Therefore, when "instantaneous" currents appear it is advisable to characterise these currents using higher sampling frequencies with correspondingly higher filtering frequency cut-offs.

The actions of calmodulin antagonists W-7 and TFP and of calcium on the gating kinetics of the calcium-activated large conductance potassium channel of the chara protoplasmic drop: a substate-sensitive analysis.

The effects of the calmodulin antagonists W-7 and trifluoperazine have been measured on the Ca2+-activated potassium channel in the membrane surrounding protoplasmic drops expressed from internodal cells of charophyte plants. The large-conductance (170 pS), voltage- and Ca2+-dependent gating, and prominent conductance substrate of this channel shows a strong kinetic resemblance to those of the Maxi-K channel from animal cells. This is the first study of the action of calmodulin antagonists which measures their effects on the most populated substates as well as the closed and main open states of Maxi-K channels. The substate analysis provides new evidence for different modes of action of- and different bindings sites for these calmodulin antagonists. Neither antagonist produces the simple closure of the channel reported previously as its effect on the Maxi-K channel, though both do induce flicker-block, reducing the mean current to near zero at high concentrations following an inverted Michaelis-Menten curve. W-7 reduces residence time in the fully open state, thus raising, in the same proportions, the probabilities of finding the channel in the closed state or a pre-existing substate. Its binding to the channel is voltage- and calcium-dependent. In contrast, trifluoperazine reduces residence in the open state and promotes an apparently new substate which overlaps the closed state at -50 mV but is distinguishable from it at voltages more negative than -100 mV. This substate may represent times that trifluoperazine is bound to the channel. Both antagonists have effects clearly distinguishable from that of withdrawing calcium from the channel, which does not affect open state residence time but increases closed state residence time. Thus neither antagonist reverses the activating effect of Ca2-. This is good kinetic evidence against the view that the channel is activated by Ca2+-calmodulin and that the effect of a calmodulin antagonist is to reverse this process by making Ca2--calmodulin less available.

Anion channel activity in the Chara plasma membrane: co-operative subunit phenomena and a model.

Anion channel activity in the plasma membrane of internodal cells of Chara australis has been studied using the patch clamp method and the records analysed with a program based on the Hidden Markov Model. The activity was variable in time, and often showed very noisy periods. At other times there were one or more of five types of channel-like activity. These are believed, like the noise, to arise from the same anion-conducting mechanism in the membrane. The channel-like activity shows many current levels, the actual levels varying on a time-scale of the order of seconds. The records show non-independent transitions between levels, and thus the activity is not the result of the patch containing many independent channels. The currents often move sequentially between adjacent levels. The current levels are at times more closely spaced as the magnitude of the current increases. Underlying this sequential transition scheme it is found that current and conductance frequently show levels that are integer multiples of a unit. Typically about 10 levels occur, a typical transition scheme being (in units) 0↔4↔7↔9. It is shown that it is possible to model such sequential transition schemes with underlying units by considering a new model based on a random walk on a semiregular fragmentary tessellation of a plane.

The Physiological Relevance of Na+-Coupled K+-Transport.

Plant roots utilize at least two distinct pathways with high and low affinities to accumulate K+. The system for high-affinity K+ uptake, which takes place against the electrochemical K+ gradient, requires direct energization. Energization of K+ uptake via Na+ coupling has been observed in algae and was recently proposed as a mechanism for K+ uptake in wheat (Triticum aestivum L.). To investigate whether Na+ coupling has general physiological relevance in energizing K+ transport, we screened a number of species, including Arabidopsis thaliana L. Heynh. ecotype Columbia, wheat, and barley (Hordeum vulgare L.), for the presence of Na+-coupled K+ uptake. Rb+-flux analysis and electrophysiological K+-transport assays were performed in the presence and absence of Na+ and provided evidence for a coupling between K+ and Na+ transport in several aquatic species. However, all investigated terrestrial species were able to sustain growth and K+ uptake in the absence of Na+. Furthermore, the addition of Na+ was either without effect or inhibited K+ absorption. The latter characteristic was independent of growth conditions with respect to Na+ status and pH. Our results suggest that in terrestrial species Na+-coupled K+ transport has no or limited physiological relevance, whereas in certain aquatic angiosperms and algae this type of secondary transport energization plays a significant role.

Modeling the current-voltage characteristics of Chara membranes: I. The effect of ATP removal and zero turgor.

We have obtained and modeled the electrical characteristics of the plasma membrane of Chara internodal cells: intact, without turgor and perfused with and without ATP. The cells were voltage and space-clamped to obtain the I/V (current-voltage) and G/V (conductance-voltage) profiles of the cell membrane. The intact cells yielded similar I/V characteristics with resting p.d.s. of -221 +/- 12 mV (cytoplasmic clamp, 5 cells) and -217 +/- 12 mV (vacuolar clamp, 5 cells). The cut unperfused cells were depolarized at -169 +/- 12 mV (7 cells) compared to the vacuole-clamped intact cells. The cells perfused with ATP fell into three groups: hyperpolarized group with resting p.d. -175 +/- 12 mV (4 cells) and I/V profile similar to the intact and cut unperfused cells; depolarized group with resting p.d. of -107 +/- 12 mV (6 cells) and I/V profiles close to linear; and excited cells with profiles showing a negative conductance region and resting p.d. at -59 +/- 12 mV (5 cells). The cells perfused with medium containing no ATP showed upwardly concave I/V characteristics and resting p.d. at -81 +/- 12 mV (6 cells). The I/V curves were modeled employing the "Two-state" model for the H+ pump (Hansen et al., 1981). The inward and outward rectifiers were fitted to exponential functions and combined with a linear background current. The excitation state in perfused cells was modeled by including an inward current, iexcit, with p.d.-dependence described by a combination of hyperbolic tangent functions. An inward current, ino-ATP, with a smaller amplitude, but very similar p.d.-dependence was also included in the simulation of the I/V curves from cells without ATP. This approach avoided I/V curve subtraction. The modeling of the total I/V and G/V characteristics provided more information about the parameters of the "Two-state" pump model, as well as more quantitative understanding of the interaction of the major transport systems in the plasmalemma in generation of the resting potential under a range of circumstances. ATP had little effect on nonpump currents except the excitation current; depolarization profoundly affected the pump characteristics.

Sodium-coupled symports in the plasma membranes of plant cells.

It has been a dogma since the success of chemiosmotic theory that plants use protons as a membrane-coupling mechanism--it has come to be implied that they use no other ion for the purpose. Following the finding that potassium can be taken up by sodium symport in Chara australis, we have established that urea is also taken up by sodium symport in the same organism. use of electrical and flux methods for finding sodium-driven symport are described; using these methods, sodium symport is also found for three solutes in Nitella translucens. Extending the search to higher plants has shown indications of sodium-driven uptake of potassium in Elodea canadensis roots, and Egeria sp. and Vallisneria leaves. Sugar and potassium uptake in Zea mays roots, and sugar uptake in Elodea canadensis showed no sodium requirement.

The uptake and metabolism of urea by Chara australis: IV. Symport with sodium--a slip model for the high and low affinity systems.

We have previously investigated the electrogenic influx of urea in Chara, and the urea- and sodium-dependent membrane current. We have shown that there is a sodium-stimulated component of urea influx and a urea-stimulated component of sodium influx, and that these are of the same size. We conclude that the electrogenic inward transport of urea, and of its analogues acetamide and acrylamide, is by sodium symport, with a stoichiometric ratio of 1:1. The kinetics of the fluxes and currents show two different KM values for sodium in different cells and two different kinds of kinetics for the effect of urea on membrane current, one of which fits the Michaelis-Menten equation, while the other shows a maximum and fits the difference of two Michaelis-Menten terms, suggesting a phenomenon like cis-inhibition. Similarities in kinetic characteristics between the inhibitory site and the electrically silent uptake site (System II) lead us to suggest that the same protein may be responsible for both the low-KM, electrogenic influx of urea (System I) and the high-KM, electrically silent influx by System II. We suggest a "slip" model for urea uptake in Chara.

Distribution of VA mycorrhizal entry points near the root apex: Is there an uninfectible zone at the root tip of leek or clover?

This paper re-examines the question of the susceptibility of plant roots to infection by vesicular-arbuscular mycorrhizal fungi, particularly near the root apex. The cumulative distributions of the observed distance between the root apex and the most apical mycorrhizal entry points are compared with expected distributions based on (i) a random distribution of successful encounters between hypha and root and (ii) an uninfectible apical region (of length Zo ). Data for both Allium porrum L. (leek) and Trifolium subterraneum L. (clover) are well fitted by the expected distributions and in the majority of cases (17/31) the best fit is obtained with zero Zo , with 8 additional cases being best fitted by values of Zo of 1 mm or less. Even where the best fits are obtained with values of Zo greater than zero, the distribution using Zo = 0 is still within the confidence limits for the data. These findings indicate that in neither species is there clear evidence for a subapical region that is immune to infection. The fact that there are short uninfected lengths immediately behind the apex is interpreted as representing a delay between an encounter and a detectable infection. Other consistent deviations of the data from the theoretical distributions are discussed. Fitting the distributions yields values for the frequency of infection (A). These are compared for the two host plants at different propagule densities. The values for leek are much lower than for clover. In both species there are increases in A with increases in propagule density which are consistent with earlier findings.

Whole-cell and single-channel currents across the plasmalemma of corn shoot suspension cells.

Whole-cell sealed-on pipettes have been used to measure electrical properties of the plasmalemma surrounding protoplasts isolated from Black Mexican sweet corn shoot cells from suspension culture. In these protoplasts the membrane resting potential (Vm) was found to be -59 +/- 23 mV (n = 23) in 1 mM Ko+. The mean Vm became more negative as [K+]o decreased, but was more positive than the K+ equilibrium potential. There was no evidence of electrogenic pump activity. We describe four features of the current-voltage characteristic of the plasmalemma of these protoplasts which show voltage-gated channel activity. Depolarization of the whole-cell membrane from the resting potential activates time- and voltage-dependent outward current through K(+)-selective channels. A local minimum in the outward current-voltage curve near Vm = 150 mV suggests that these currents are mediated by two populations of K(+)-selective channels. The absence of this minimum in the presence of verapamil suggests that the activation of one channel population depends on the influx of Ca2+ into the cytoplasm. We identify unitary currents from two K(+)-selective channel populations (40 and 125 pS) which open when the membrane is depolarized; it is possible that these mediate the outward whole-cell current. Hyperpolarization of the membrane from the resting potential produces time- and voltage-dependent inward whole-cell current. Current activation is fast and follows an exponential time course. The current saturates and in some cases decreases at membrane potentials more negative than -175 mV. This current is conducted by poorly selective K+ channels, where PCl/PK = 0.43 +/- 0.15. We describe a low conductance (20 pS) channel population of unknown selectivity which opens when the membrane is hyperpolarized. It is possible that these channels mediate inward whole-cell current. When the membrane is hyperpolarized to potentials more negative than -250 mV large, irregular inward current is activated. A third type of inward whole-cell current is briefly described. This activates slowly and with a U-shaped current-voltage curve over the range of membrane potentials -90 less than Vm less than 0 mV.

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops from Chara australis.

Patch-clamp studies of cytoplasmic drops from the charophyte Chara australis have previously revealed K+ channels combining high conductance (170 pS) with high selectivity for K+, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K+ greater than Rb+ greater than NH4+ much greater than Na+ and Cl-. Divalent cytosolic ions reduce the K+ conductance of this channel and alter its K+ gating in a voltage-dependent manner. The order of blocking potency is Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. The channel is activated by micromolar cytosolic Ca2+, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca2+ needed to open the channel. The possible role of the Ca(2+)-activated K+ channel in the tonoplast of Chara is discussed.

Sodium-coupled solute transport in charophyte algae: A general mechanism for transport energization in plant cells?

Ion-gradient-coupled transport systems in plants are normally electrophoretic and carry inward current. Rapid inward electrical currents elicited by K(+), by urea and by lysine in the freshwater acidophilic alga Nitella translucens Agh. are all very strongly dependent on the presence of Na(+) or (except in the case of K(+)) Li(+). These results indicate that Na(+)-coupled solute transport in plants, which had previously been demonstrated only in an alkalophilic species (Chara australis), did not evolve recently as an alternative to H(+)-coupled transport in high-pH environments, and might therefore be more widely distributed than has hitherto been recognised.

Transport of potassium in Chara australis: II. Kinetics of a symport with sodium.

An electrogenic K(+)-Na+ symport with a high affinity for K+ has been found in Chara (Smith & Walker, 1989). Under voltage-clamp conditions, the symport shows up as a change in membrane current upon adding either K+ or Na+ to the bathing medium in the presence of the other. Estimation of kinetic parameters for this transport has been difficult when using intact cells, since K(+)-Na+ current changes show a rapid falling off with time at K+ concentrations above 50 microM. Cytoplasm-enriched cell fragments are used to overcome this difficulty, since they do not show the rapid falling off of current change seen with intact cells. Current-voltage curves for the membrane in the absence or presence of either K+ or Na+ are obtained, yielding difference current-voltage curves which isolate the symport currents from other transport processes. The kinetic parameters describing this transport are found to be voltage dependent, with Km for K+ ranging from 30 down to 2 microM as membrane potential varies from -140 to -400 mV, and Km for Na+ ranging between 470 and 700 microM over a membrane potential range of -140 to -310 mV. Two different models for this transport system have been investigated. One of these involves the simultaneous transport of both the driver and substrate ions across the membrane, while the other allows for the possibility of the two ions being transported consecutively in two distinct reaction steps. The experimental results are shown to be consistent with either of these cotransport models, but they do suggest that binding of K+ occurs before that of Na+, and that movement of charge across the membrane (the voltage-dependent step) occurs when the transport protein has neither K+ nor Na+ bound to it.

THE APPARENT WIDTH OF THE RHIZOSPHERE OF TRIFOLIUM SUBTERRANEUM L. FOR VESICULAR-ARBUSCULAR MYCORRHIZAL NFECTION: EFFECTS OF TIME AND OTHER FACTORS.

We use the mathematical model developed by Ferriss (1981) for the study of plant pathogens to calculate the apparent width of the rhizosphere (W) of Trifolium subterraneum L. for infection by propagules of vesicular-arbuscular mycorrhizal fungi in soil, examining the effects of wetting the soil for different times before planting and of harvesting at different times. The reasons for choosing this model rather than that of Gilligan (1979) are given. Propagule densities were determined by the most probable number (MPN) method at 28 d using the same host species as the test plant, thus including a factor in the calculations which is the product of the efficiency of the propagules to infect and the susceptibility of the roots. Values for W at 10 to 12 d were between 2.5 and 6.5 mm for mixed soil inoculum and between 8.9 and 13.2 mm for Glomus mosseae (Nicol & Gerd.) Gerdeman & Trappe. W increased with time, associated with an increase in the number of infections formed. Factors contributing to this include progressive germination of propagules following wetting the soil, growth of hyphae from greater distances to the root and formation of secondary infections. Wetting the soil before planting had the greatest effect on W in 4 d old plants. By 10 d, there was no effect, supporting the idea that most propagules are likely to have germinated by that time. The contribution of secondary infections is harder to determine, but is likely to be important from about 10 d onwards, resulting in overestimation of W. By 12 to 16 d, in experiments in which six plants were grown in 210 or 222 cm3 soil, the apparent volume of the rhizosphere plus root exceeded the volume of soil. Values of the apparent width of the rhizosphere in our experiments are therefore most reliable before the plants are 10 d old. If the MPN method is used to determine propagule densities at the same harvest times and under the same conditions as those used to collect other data, then the equations can be used to compare different fungi and environmental conditions with respect to infection of young plants.