Potassium Transport in Roots

Potassium Transport in Roots

LEON V . KOCHIANa and WILLIAM J . LUCASb a

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U.S.Plant. Soil and Nutrition Laboratory. USDA.ARS. Cornell University. Ithaca. New York. USA Department of Botany. University of California. Davis. California. USA

Introduction

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Plasma Membrane Transport of K+ in Roots . . . . . . . A . Early Work: the Carrier-Kinetic A proach . . . . . . B . Are Root K+ Fluxes Coupled to H ? . . . . . . . . C . Uptake at High K+ Concentrations: the Linear Component D . Summary . . . . . . . . . . . . . . . .

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Redox-coupledPlasmalemmaTransportof K+ . . A . Influence of Exogenous N A D H on K+Influx . B Membrane Transport and the Wound Response C. DevelopmentofanIntegratedNADHModel .

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Regulation of K+ Fluxes within the Plant . . . . . . . . . A . Allosteric Regulation of K+ Transport . . . . . . . . B . K+ Cycling within the Plant: an Integration of Regulatory . . . . . . . . . . . . . . . . Mechanisms

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Radial K+ Transport to the Xylem . . A . Site of K+ Entry into the Symplasm B . Radial Pathway . . . . . . C . Lag Phase in Xylem Loading . . D . K+ Transport into the Xylem . .

Future Research and Prospects

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Advances in Botanical Research Vol . 15

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Copyright 0 1988 Academic Press Limited All rights of reproduction in any form reserved .

ISBN 0-12-005915-0

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I. INTRODUCTION The involvement of Kf in a number of plant functions (enzyme activation, maintenance of turgor, protein synthesis, etc.), along with the relatively high permeability of plant cell membranes to K’, has been the basis for the study of K+ transport as a model system for plant ion transport. Numerous investigations have been conducted over the past 40 years, aimed at furthering our understanding of the mechanism(s), energetics, and cellular location of K+ transport systems in roots. Despite the surfeit of literature in these areas, it can be said with some confidence that the processes by which K+ ions are transported into and across the root are still far from being fully resolved. This is due, in part, to the complex nature of the root, with its various cell types and tissues. Additional complications arise from the fact that the individual cells are coupled (both electrically and physiologically) via plasmodesmata, and also simply from the complex nature of individual cells, with their multiple compartments and subcompartments. In the first part of this chapter, we will outline and summarize what is known (or at least thought to be known) concerning mechanistic aspects of K+ transport in roots. Once this background has been established, the regulation of these transport processes can be discussed in the context of the integration of these cellular processes at the organ and whole plant level.

11. PLASMA MEMBRANE TRANSPORT OF K+ IN ROOTS A. EARLY WORK: THE CARRIER-KINETIC APPROACH

Over the past half century, most of the studies that have been conducted concerning ion absorption by plants have generally utilized three basic types of plant material: the giant algae such as Chum, Nitellu, and Vuloniu, slices cut from storage tissue of beet, potato, and carrot, and either intact roots or roots excised from seedlings. The use of excised roots as research material can be traced to the classical paper of Hoagland and Broyer (1936). They found that the roots of barley seedlings grown in dilute salt solutions exhibited extremely high initial rates of ion accumulation. Because radioisotopes had not yet been introduced, the ability of these roots to maintain high rates of accumulation made them very useful experimental material. Hence, the now well-known “low-salt roots” characterized by low salt content, high sugar content, and a large capacity for ion transport, became widely used in research. It was during this era that many of the basic concepts of membrane transport were developed. Much of the work conducted with low-salt roots concerned the absorption of K+ and other alkali cations (for the reasons discussed above). Although the concept of the lipid bilayer nature of the plasmalemma had not yet been fully developed, many researchers were

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beginning to realize that plant (and animal) cells were bounded by a non-aqueous layer that was relatively impermeable to electrolytes (Osterhout, 1931). Furthermore, the rapid absorption of Kf by plant cells led a number of investigators to hypothesize that special structures must exist that would facilitate K+ uptake across this outer boundary (Jacobsen and Overstreet, 1947; Jacobsen et al. , 1950; Osterhout, 1950,1952). The concept that, in the plasmalemma, specific carriers were involved in K+ uptake was developed further by Epstein and co-workers. When K+(86Rb+)uptake from dilute solutions into excised barley roots was studied, the kinetic profile approximated a rectangular hyperbola. This response was quite similar to that found in classical enzyme kinetic studies [Fig. 1 (see low concentration range)]. Epstein and Hagen (1952) were the first to apply Michaelis-Menten enzyme kinetics to ion transport. They postulated that specific alkali cation transport systems operated in a fashion analogous to substrate-specific enzymes. They went on to show, for the uptake of K+ and other alkali cations, that at higher external concentrations their observed kinetics deviated from classical Michaelis-Menten form (Leggett and Epstein, 1956; Epstein et af., 1963). Saturation was attained at low external K+ concentrations, but then at higher concentrations the curves appeared to reach a second level of saturation (Fig. 1). This biphasic pattern was labelled the “dual isotherm of uptake” by Epstein, and was hypothesized to be due to the operation of two separate classes of carriers in the plasmalemma. In the low K+ concentration range (<0.5 mM), mechanism I was hypothesized to be a K+ transport system with a high affinity for K+ 25 I

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K+concentration ( mM )

Fig. 1 . Rate of K+ absorption in barley roots as a function of K+concentration. Note that the concentration scale has been changed after 0.2 mM K’. Data from Epstein et al. (1963).

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(particularly with respect to Na’). In the higher concentration range, mechanism I1 was proposed to operate as a low-affinity, high-velocity transport mechanism that was less specific for K’. Further studies in the mechanism I1 range suggested that the kinetic profile was not a smooth curve, but was characterized by multiple inflections (Elzam et al., 1964; Epstein and Rains, 1965). This pioneering work by Epstein and his colleagues had a significant influence on the field of plant membrane transport. Subsequently, publications emerged from many laboratories indicating that the uptake of a number of inorganic and organic solutes in most plant tissues followed complex kinetics. These complex kinetics were, for the most part, interpreted to be the result of two or more transport systems. Subsequent kinetic analyses suggested that each transport system (for a particular solute) exhibited distinctly different Michaelis-Menten kinetics. The great volume of published work reflected the interest and importance associated with this field. A number of controversies arose concerning the kinetic aspects of K+ absorption (and the uptake of other solutes). Conflicting hypotheses were presented concerning such aspects as the number of phases and the cellular and subcellular localization of each phase (for a review see Laties, 1969; Epstein, 1976). The most direct challenge to Epstein’s “dual isotherm” hypothesis was introduced in the early 1970s by Nissen, who presented an alternative interpretation for multiphasic uptake kinetics (Nissen, 1971, 1973, 1974). Nissen’s approach was to analyse both his own and other researcher’s kinetic data for solute uptake (uptake versus solute concentration) by performing various kinetic transformations of the primary data. These transformations (usually Lineweaver-Burk reciprocal plots) yield linear relationships for uptake kinetics that followed Michaelis-Menten relationships. Because the kinetics for solute uptake in plants are almost always complex, the reciprocal plots, quite naturally, yielded non-linear transformations. Nissen has fitted the transformed data by means of a statistical computer program and has found that the best fit, generally, for uptake of Kf and any other solutes that he and others have studied, is a series of adjacent linear segments (e.g. Nissen and Nissen, 1983; Nissen, 1987). Nissen claims that this type of analysis is strong evidence for the operation of a single complex transport system located in the plasmalemma. As external K+ concentration is increased, this putative transport system is thought to undergo abrupt transitions at discrete external K+ concentrations. Within each phase, the transport system is thought to obey Michaelis-Menten kinetics, with the kinetic constants for each phase usually increasing in a fairly regular manner. This approach is somewhat controversial and has been subjected to some fairly strong criticisms (Walker, 1974; Wyn Jones, 1975; Borstlap, 1981a,b, 1983; Kochian and Lucas, 1982b).

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K+concentration ( mM) Fig. 2. Potassium (=Rb') influx as a function of Kf concentration in the experimental medium, obtained on corn root segments that were grown in 5 mM KCI + 0.2 mM CaS04 (high-salt status), Flux determinations were performed using the apparatus developed by Kochian and Lucas (1982a). Data points represent the mean k SE (points which lack error bars do so because the standard errors were smaller than symbols used). Linear regression performed on data points for K+ concentrations from 1.0 to 10 mM gave a value of 0.334 wmol (g fresh weight)-' h-' for the first-order rate coefficient of the linear component (the regression coefficient was 0.999).

Some of these criticisms, and an overall critique of the carrier-kinetic approach to Kf transport in roots, will be discussed in a later section. Most of the work of this era centred around the deduction of ion transport mechanisms from either the shapes of kinetic curves (influx versus concentration) or from the shapes of curves resulting from various mathematical transformations of the kinetic data. Many of these published data are of limited value because, in many cases, the kinetic profiles lack a sufficient number of data points for the concentration ranges tested. Also, data replication was often lacking. Kochian and Lucas (1982a) addressed this problem by developing an experimental apparatus which enabled them to generate large numbers of data points on the [ S ] axis, with enough replicates to obtain sufficiently precise values. As illustrated in Fig. 2, no abrupt discontinuities were observed for K+ transport into corn roots. Kinetic curves for Kf influx into roots of both low- and high-salt-grown corn seedlings were found to be smooth and nonsaturating; the curves approached linearity at concentrations above 1 mM and exhibited no tendency towards saturation at concentrations up to 50 mM. The kinetics for K+ transport could be resolved into saturable and nonsaturating, or first-order kinetic components. The saturable component could be specifically inhibited by application of sulphhydryl reagents (Fig. 3), whereas the nonsaturating component could be specifically inhibited by either the application of K+ channel-blocking

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Fig. 3. Kinetic curves for 86Rb+influx into high-salt-grown corn root segments. In (a), the control curve (0)has been separated into its saturable and linear components. In (b), the influence of N-ethyl maleimide (NEM) exposures on %Rb+ influx is depicted. Corn root segments were pretreated with 0.3 mM NEM for 10 or 30 s, then washed for 10 min in 1 mM dithioerythritol (DTE) prior to %Rb+uptake. Data from Kochian and Lucas (1982a).

agents, or by substituting C1- in the uptake solution with other anions (Kochian et al., 1985).The discrepancy between Epstein's mechanism I1 and the first-order kinetic component observed in corn roots, will be addressed in Section 1I.C. Although the carrier-kinetic approach has provided important information on the mechanisms of ion transport, clearly there are limitations associated with this approach. In studying root ion transport, one is dealing with a system consisting of many cell types, each potentially in a different physiological state, with the problems of unstirred layers and diffusion limitation further confounding data interpretation. In studying the concentration dependence of uptake, the bulk solution ion concentration is generally taken as representing the substrate level available for transport, despite the knowledge that the cation and anion concentrations at the surface of the plasmalemma are probably considerably different from those in the bulk solution. This point is highlighted by the recent work of Newman et al. (1987), in which ion-selective microelectrodes were utilized to analyse the

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electrochemical gradients for H+ and K+ in the unstirred layer near the surface of corn roots. Newman etal. (1987) found that in the presence of low levels of K+ (less than 50 p ~ )the , steady-state concentration at the root surface was about one-half that of the bulk solution, due to depletion arising from net K+ uptake into the root. This considerable difference may be magnified further within the cell walls and at the surface of the plasmalemma, where additional diffusion limitation and ionic effects may have a dramatic influence on cation and anion activities. These points are of particular significance when mechanistic models, such as those presented by Nissen, are based solely on small differences between and within uptake kinetic curves. ' ? B. ARE ROOT K+ FLUXES COUPLED TO H

It has long been accepted that K+ influx into roots is either coupled to the transport of H+ or, at the very least, influenced by H+ activities (or activity gradients) associated with the K+ transport system. Like most areas of plant membrane transport, the concept that K+ and H+ fluxes are coupled has been controversial. Over the past 40 years, various models have been presented to explain the mechanism(s) by which K+ uptake is coupled to H+ efflux. The earliest models proposed that H+ efflux was dependent upon K+ uptake, or more specifically, depended upon cation uptake that was in excess of anion absorption. The H+ excreted by the root had a compensatory role; that is, they were involved in maintaining charge balance across the plasmalemma. Organic acid metabolism and cytoplasmic pH regulation were also involved in this model (Ulrich, 1941;Jacobsen et al., 1950;Jackson and Adams, 1963;Torii and Laties, 1966; Hiatt, 1967a,b). More recent models have reversed the dependency between K+ and H+ fluxes. The influence of Mitchell's chemiosmotic hypothesis (Mitchell, 1970) has led some researchers to hypothesize that active H+ efflux is the primary event (and driving force) for the subsequent (and dependent) K+ uptake. Although most plant transport physiologists now generally accept that K+ uptake is dependent on H+ efflux, controversy still exists concerning the degree of this dependence. There appear to be two groups of adherents, with some researchers subscribing to a direct chemical coupling (Poole, 1974; Hanson, 1977; Leonard and Hanson, 1972; Lin and Hanson, 1976; Cheeseman and Hanson, 1979a,b; Cheeseman et al., 1980), and others arguing that the two fluxes are indirectly coupled, through the electrical component of the protonmotive force generated by the electrogenic, plasmalemma H+ATPase (e.g. Pitman et al., 1975; Marrk, 1977, 1979; Bellando et al., 1979; Bellando and Trotta, 1980). Glass and co-workers have severely questioned the existence of either a direct or an electrical coupling of the two fluxes, and have again suggested that the maintenance of charge balance may be the primary role for H+ fluxes in relation to K+ uptake (Glass and Siddiqi, 1982; Siddiqi and Glass,

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1984). However, recent studies with Neurospora (Rodriguez-Navarro and Ramos, 1986; Rodriguez-Navarro et al., 1986; Blatt and Slayman, 1987; Slayman et al., 1988) and Zea mays roots (Kochian and Lucas, 1987; Kochian et al., 1987, 1988; Newman et al., 1987) have provided evidence consistent with the hypothesis that a K+-Hf co-transport system may operate in the plasmalemma of fungal and higher plant cells. The evidence supporting the various models based on different modes of coupling between K+ and H+ fluxes will now be considered in more detail. 1. Unequal CationlAnion Uptake Charge Balanced by H' It has long been recognized that both intact and excised roots can maintain disparate rates of cation and anion absorption, depending on the composition of the salt solution bathing the roots. Even in early studies on root ion absorption, it was observed that, for example, excess cation absorption could occur from a solution containing K2S04,while excess anion uptake was often seen in solutions containing CaCl, or Ca(NO& (Brooks, 1929; Hoagland and Broyer, 1940). Uptake from KCl solutions usually results in a balance between cation and anion fluxes. During these early studies, it was recognized that the processes which transport net charge across the plasmalemma could not continue indefinitely, due to the axiom that electroneutrality must be maintained within living cells. (Additionally, we now realize that such processes could not continue in an uncompensated fashion due to the dielectric properties and limited capacitance of biological membranes.) Therefore, compensatory reactions must occur, in order to maintain charge balance both across membranes and within cells. Ulrich (1941) was the first to propose an interrelationship between unequal catiodanion uptake, cytoplasmic pH, and organic acid metabolism. He found that under conditions of excess cation uptake, organic acid content increased in proportion to the cation excess; the converse was found for uncompensated anion absorption. Ulrich hypothesized that under conditions of excess cation (usually K f ) absorption, H+ were exchanged for K+ ions. This could give rise to an alkalinization of the cell sap, stimulating the synthesis of organic acids which would help to buffer internal pH. Subsequent work from a number of laboratories extended these initial speculations. Several researchers dealt with the mechanism of K+ (and other cation) uptake in terms of a K+-H+ exchange and anion absorption as an anion-OH- antiport. Both processes would result in a trend towards electroneutrality (Jacobsen et al., 1950; Jackson and Adams, 1963). It was also amply established that during excess Kf uptake, the pH and buffering capacity of expressed root sap increased, as did dark COP fixation and organic acid (usually malate) synthesis (Jacobson and Ordin, 1954; Jacobson, 1955; Torii and Laties, 1966; Hiatt, 1967a; Hiatt and Hendricks, 1967; Jacoby and Laties, 1971). It is still not clearly understood how excess K+ absorption stimulates organic acid synthesis in the root. It has been proposed

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that the K+-H+ exchange involved in charge balance should increase cytoplasmic pH, and such a pH shift has been suggested from pH measurements of total expressed root sap. This pH shift would “turn on” carboxylating enzymes in the cytoplasm (PEP carboxylase + malate dehydrogenase) and result in the synthesis of malic acid. Subsequent dissociation of the malic acid carboxyl groups would yield H+ that could both help maintain cytoplasmic pH and permit continued K+-H+ exchange (Hiatt, 1967a,b). This model has been promoted as a regulatory mechanism involved in the maintenance of cytoplasmic pH (Davies, 1973; Raven and Smith, 1974; Smith and Raven, 1976). Alternatively, other models have been proposed which involve alterations in the levels of substrate (C02 versus HCO;) available for organic acid synthesis (Jacoby and Laties, 1971), feedback regulation of PEP carboxylase by cytosolic malate levels (Osmond, 1976), and ionic effects on carbon metabolism (Osmond, 1976; Osmond and Greenway, 1972; Schnabl, 1987). The above discussion has dealt more directly with regulatory aspects of K+ (and general cation) uptake, and not specifically with mechanisms of K+ absorption. It was important to introduce this topic in this section because this area of research was instrumental in developing the concept that K+ and H+ fluxes were, in some fashion, correlated. For a more detailed consideration of the interactions of ion absorption, carbon metabolism, and cytoplasmic pH regulation, the reader is referred to reviews by Osmond (1976), Smith and Raven (1976,1979), and Davies (1979).

2. Direct Coupling: K + - H + Antiport It has been well documented that plant cells accumulate K+ in the cytoplasm and generally maintain electrochemical potential gradients for K+ across the plasmalemma (Lauchli and Pfluger, 1979;Leonard, 1984,1985). Numerous studies have demonstrated that K+ uptake into root tissue is extremely sensitive to metabolic inhibitors. Uptake is energy-dependent, and a close correlation with cellular levels of ATP (modified through the application of metabolic inhibitors) has been demonstrated (Petraglia and Poole, 1980). However, the problems of elucidating underlying mechanisms on the basis of such studies is all too obvious. Nevertheless, by combining electrophysiological, kinetic, and inhibitor studies, it has been possible to demonstrate that at least at low external K+ levels, uptake is a thermodynamically active process (see Pitman, 1976; Cheeseman and Hanson, 1980). Additionally, compelling evidence has been accumulated over the past 20 years that electrogenic transport processes operate across both the plasmalemma and tonoplast of lower and higher plant cells. Further, it is now well accepted that these transport systems are membrane-bound ATPases that are involved (at the very least) in the active transport of H+ (for reviews see Poole, 1978; Spanswick, 1981; Leonard, 1982; Serrano, 1984; Marrk and Ballarin-Denti, 1985;Sze, 1985). During this same period, the chemiosmotic

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Pathway 1

K+

ADP

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Fig. 4. Possible mechanisms for K+ transport into plant cells. The plasma membrane H+-ATPase produces a pH gradient and a membrane potential during ATP hydrolysis. K+ transport can be either driven by the electrical component of the membrane potential with movement occurring through a membrane protein (pathway 1) or by direct transport via a K+/H+-ATPase (pathway 2). It is also possible that both pathways can occur. Modified from Briskin (1986), with permission.

hypothesis, developed by Mitchell (1970) to explain energy transduction in mitochondria and chloroplasts, had alerted plant physiologists to the possibility that H+ gradients could be coupled to other ion fluxes. Because plant transport scientists already had observed associations between K+ uptake and H+ efflux, it was only natural that all of these factors would influence thinking about active K+ influx. Hence, a number of mechanistic models were developed that coupled Hf to Kf fluxes. As mentioned previously, two types of coupling were hypothesized, and these are detailed in Fig. 4. For a number of different root tissues it has been demonstrated that as external pH is increased, a concomitant increase in K+ influx occurs (e.g. Poole, 1966, 1974; Lin, 1979; Glass and Siddiqi, 1982). This apparent pH dependency for washed corn root segments and intact barley roots is shown in Fig. 5 . Poole (1974), working with slices from red beet roots, observed that as external pH was increased from 5.5 to 8.0, there were parallel stimulations of Hf efflux and K+ uptake, and a hyperpolarization of the Em. He was probably the first to suggest that the plasmalemma electrogenic proton pump of plant cells was actually a Kf-Hf exchange ATPase, facilitating K+ uptake in response to active Hf efflux.

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Fig. 5 . Effect of external pH on K+(‘‘Rb’) influx into 4-h-washedcorn root segments (0) and intact barley roots (m). Where present, error bars represent +SE. Note the apparent pH insensitivity from pH 6-9, (Scale on right-hand side for intact barley roots.) 0: Data replotted Data replotted from Glass and Siddiqi (1982). from Lin (1979). .:

Hanson and co-workers, working with intact and excised corn root segments, have been among the more vocal proponents of a direct K+-H+ exchange mechanism (Hanson, 1977).Lin and Hanson (1976) demonstrated that the sulphhydryl-containing compound dithioerythritol (DTE) could “activate” a passive and stoichiometric (1: 1) K+-H+ exchange system. However, as they note, in their experiments this antiporter was involved in the release of K+ from corn root cells. They speculated that this passive antiport mechanism could be involved in K+ uptake under conditions where the thermodynamic gradients favoured K+ influx (K+ directed inwardly andor H+ out). However, this seems to be unlikely for the thermodynamic gradient for H+ would rarely be directed out of the cell. Lin and Hanson presented a model in which an active K+/H+-ATPase(for which no evidence was presented) was coupled to the observed DTE-induced passive K+-H+ exchange system as well as to an active anion uptake system (anion-OHantiport). An alternative function could exist for the passive K+-H+ antiport system, if it is physiologically significant. As the authors mention, this system could be involved in osmotic regulation and/or control of internal K+ under conditions of high K+ status. A precedence exists for such a situation. Active K+ uptake into the photosynthetic bacterium, Chromatiurn vinosum, is mediated via a K+-ATPase that exhibits a high affinity for K+, while a low-affinity K+-H+ antiport system (which mediates K+ efflux) is involved

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in osmotic balance and cytoplasmic pH regulation under conditions of high internal K+ (Davidson and Knaff, 1981,1982). In a subsequent paper investigating ion transport (K+ and Pi) in corn root segments, the differential effects of pH and several chemical modifiers on K+ and Pi transport were shown and used to support the existence of separate (and active) K+-H+ and Pi-OH- exchange systems (Lin, 1979). The apparent stimulation of K+ uptake at high pH values (Fig. 5 ) was correlated with increased values of H+efflux. These results were considered to be consistent with an active exchange system whose H+ efflux activity was increased in response to a reduction in the A& opposing H+ extrusion as the pH was increased. Lin found that the fungal toxin, fusicoccin, which has been shown to stimulate both H+ efflux and cation uptake, and to hyperpolarize the Em of plant cells (Marrk, 1979), rapidly stimulated K+ uptake while having little influence on phosphate transport. Additionally, the ATPase inhibitor diethylstilbestrol (DES) and the H+ ionophore carbonyl cyanide 4-(trifluoromethoxy)hydrazone (FCCP) both immediately inhibited Kf uptake while having an apparently less direct effect on Pi uptake. These results were taken by Lin asfurther evidence that K+ uptake occurs by an active, electrogenic K+-H+ exchange (even though no evidence for such a mechanism was presented in earlier papers). It should be noted that these results are quite circumstantial, and can be used to support other models for K+ transport, as will be discussed later. Cheeseman and Hanson (1979a,b) conducted a detailed study investigating the influence of anoxia and uncouplers on K+ influx and Emover a range of K+ concentrations. They analysed their data using the Goldman equations [used to describe passive ionic fluxes (Goldman, 1943; Hodgkin and Katz, 1949; Hodgkin and Huxley, 1952)] in order to determine the contributions of passive flux to the total K+ influx. They proposed the model illustrated in Fig. 6, which indicates that at low external K+ concentrations, active K+ influx is coupled to the H+-translocatingATPase. As K+ concentrations are increased in the mechanism I range, saturation of this carrier occurs and the increasing inward K+ current would have a depolarizing effect. As external K+ increases into the mechanism I1 range, K+ influx occurs via a passive electrophoretic uniport. However, in this concentration range, external K+ is thought to have a stimulatory effect on the ATPase. Cheeseman and Hanson originally suggested that both K+ transport functions were carried out by the same system, utilizing a single type of H+-ATPase. However, in a later publication (Cheeseman et al., 1980) they revised their model. After analysing the influence of ATPase inhibitors on Em and K+ influx, they hypothesized that at low K+ levels an electrogenic system dominates that is sensitive to ATPase inhibitors, and is associated with active K+ influx and H+ efflux. A second electrogenic system was proposed which operates at all external K+ concentrations and becomes dominant at higher K+ concentrations. This system drives passive K+ influx,

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EAT: Mechanirm I

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and is insensitive to ATPase inhibitors. Essentially, what Cheeseman and co-workers were proposing was a model that incorporates both types of coupling illustrated in Fig. 4. At low external K+ levels, active Kf uptake would occur via a K+-H+ exchange ATPase (pathway 2 of Fig. 4). At higher external Kf concentrations, passive K+ uptake would be mediated by a K+ channel, with the driving force arising primarily from the electrical potential difference developed across the plasmalemma by a H+-ATPase. Recently, evidence in support of this model was presented following a re-evaluation of the electrogenic nature of K+-H+ fluxes in corn roots (Thibaud et al., 1986). It should be noted that if Kf uptake is active at low K+ levels, then the K+ transport system must be coupled either directly to an ATPase (K+-ATPase or K+-H+ exchange ATPase), or to the transmembrane proton electrochemical gradient (PMF) via a Kf-H+ co-transport system. Indirect coupling (pathway 1, Fig. 4)can only be invoked for the passive K+ uniport. Early evidence for the existence of a Kf-transporting ATPase came from work which correlated Kf influx in corn, wheat, oat, and barley roots with K+-stimulated ATPase activity located in a microsomal membrane preparation isolated from these roots (Fisher and Hodges, 1969; Fisher et al., 1970). The putative plasmalemma ATPase was further characterized in subsequent studies (Hodges et al., 1972; Leonard and Hodges, 1973; Leonard and Hotchkiss, 1976). The enzyme showed an acidic pH optimum for activity, and a requirement for Mg2+,and was further stimulated by

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"0

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KCI concentration ( mM Fig. 7. Correlation between the kinetics of 42K+influx into excised oat roots (a) and the stimulation of a microsomal ATPase activity as a function of K+ present in the reaction medium influx and (b). ATPase activity was determined by the amount of Pi released. Insets show 42K+ Pi released over the concentration range of 0.01-0.35 mM KCI. Data from Leonard and Hodges (1973).

monovalent cations, particularly K+ and Rb'. Leonard and Hodges (1973) showed that the complex kinetics for K+ absorption, in oat roots, were quite similar to the kinetics observed for K+ stimulation of ATPase activity (Fig. 7). It seemed appropriate, therefore, to correlate K+ transport in plants with the H+/K+-ATPase of the gastric mucosa and the Na+/K+ATPase of animal cells, since these systems all exhibited cation-induced stimulation and transported K+ directly (Hodges, 1976; Cantley, 1981; Faller et al., 1982; Leonard, 1984; Briskin, 1986a). The correlation between plasma membrane-associated, K+-stimulated ATPase activity and K+ influx, and the similarity between the sequence for monovalent cation stimulation of ATPase activity (K' > NH: > Rb+ > Cs+ > Li') and the specificity of monovalent cation uptake into roots (Sze and Hodges, 1977), has been used as further evidence that this ATPase is involved in K+ uptake, putatively as a K+-H+ exchange system. It has also been demonstrated that microsomal membrane vesicles isolated from

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tobacco callus, and believed to be plasmalemma in origin, exhibited a stimulation of K+-ATPase activity in the presence of nigericin (which facilitates electroneutral K+-H+ exchange), or valinomycin plus a protonophore (Sze, 1980). These results again suggest that the plasmalemma ATPase may be involved in K+-H+ exchange. Recent work with isolated plasma membranes from corn roots (Briskin and Leonard, 1982a,b), oat roots (Vara and Serrano, 1983), and red beet storage tissue (Briskin and Poole, 1983a,b), has demonstrated that the plasmalemma ATPase forms a covalently bound, phosphorylated intermediate during the course of ATP hydrolysis. Briskin and Leonard (1982a,b) showed that K+ mildly stimulated the breakdown of this phosphorylated intermediate, using a crude preparation from corn roots, and concluded that this stimulation is further evidence for a K+ transport function by the ATPase. An analogy was again drawn with the reaction mechanism for the Na+/K+-ATPaseof animal cells, which also exhibits a K+ stimulation of dephosphorylation, and transports K+ directly (Cantley, 1981). However, it should be noted that the Na+/K+-ATPase phosphoenzyme intermediate has a much higher turnover rate, and the influence of K+ on the rate of dephosphorylation is considerable. Similar K+ effects on the dephosphorylation step were obtained with the plasmalemma ATPase isolated from red beet (Briskin and Poole, 1983a,b; Briskin and Thornley, 1985;Briskin, 1986b). However, in these studies the authors appear to place less emphasis on ascribing a K+ transport function for the ATPase. In contrast to the above studies, Vara and Serrano (1983) could find no effect of K+ on the phosphorylation and dephosphorylation of the plasmalemma ATPase from oat roots. This result led Serrano (1984) to propose that the ATPase is not a K+-dependent enzyme involved in K+ transport. As he points out, this interpretation is supported by the observation that, in the studies of the corn root microsomal membranes, the turnover of the phosphoenzyme was quite slow, and the K+ stimulation of the phosphoenzyme breakdown was much too small to support the concept of a direct role for K+ in the reaction kinetic scheme for this enzyme. Recently, Spanswick and Anton (1988) presented a model describing the kinetic characteristics of a H+-translocatingATPase purified from isolated plasmalemma vesicles of tomato roots (Fig. 8). They observed that the kinetics for the ATPase were similar to those for other cation-transporting ATPases that form phosphorylated intermediates. Their model does not ascribe a K+ transport role to the ATPase. Instead, stimulation of the breakdown of the phosphorylated intermediate by cytoplasmic K+ appears to be sufficient to explain the K+-stimulation of ATPase activity, without invoking a K+ transport role for the ATPase. It was proposed that K+ transport occurs by a separate mechanism operating in conjunction with the H+-ATPase. There are additional criticisms that can be directed at the work published in support of a K+ transport function for the plant plasmalemma ATPase.

L. V. KOCHIAN AND W. J. LUCAS

108

t

K:Vi

-“-f E2-p

Fig. 8. A reaction scheme for the plasma membrane ATPase proposed by Spanswick and Anton (1988). The binding of H+ at the cytoplasmic face of the plasma membrane occurs via reaction 4 to give HE1, subsequent hydrolysis of ATP via reaction 1 produces HE, P and from this state the H+ is released to the outer surface of the plasma membrane, via reaction 2. Potassium is proposed to act from the cytoplasmic face of the membrane to accelerate the dephosphorylation of E2-P, to return the ATPase to the state in which it can again bind H+. (Vi stands for inorganic vanadate which acts as a competitive inhibitor on the E2complex.)

-

First, the degree of K+ stimulation of ATPase activity is quite low when compared with total ATPase activity, and the K+ concentrations often used to achieve this stimulation are excessively high (50 mM) when considering Kf concentrations likely to be present in the soil solution or root apoplasm. These results again suggest that the K+effect may be a general salt effect, or activation by cytoplasmic K+, and not evidence for Kf requirement by a K+ transporting ATPase. Additionally, K+ stimulation is observed only at pH values below 7 (Leonard and Hodges, 1973; Leonard and Hotchkiss, 1976). These considerations led Serrano (1984), in his quite comprehensive review of fungal and higher plant plasmalemma ATPases, to state, “Thus, the plasma membrane ATPase is not a potassium-dependent enzyme although it is specifically stimulated by this ion”. An alternative view can be taken which would fit both mechanistic views. The dominant transport protein in the plant plasmalemma could be the H+-translocatingATPase, which might not be expected to exhibit Kf-stimulated activity. However, a Kf-ATPase could also be present, at much lower levels. This could explain the small K+ stimulation of ATPase activity and phosphoenzyme turnover, which would be measured against a large background of activity due to the H+-ATPase. It is not unrealistic, in the light of work from animal and bacterial systems, to suggest that a number of

POTASSIUM TRANSPORT IN ROOTS

109

different ATPases might be contained in the various plant membranes, operating in response to different signals and participating in various cell functions. The resolution of this controversy may ultimately come from demonstrations of ATP-dependent K+ and H+ transport in purified and reconstituted systems (Briskin, 1986a). Much of the early work on plasma membrane ATPases shared the rather common problem of contamination by other membranes. For example, it was only recently that ATP-dependent transport was demonstrated with membrane fractions isolated from plant roots. Using microsomal membranes isolated from corn, several different groups have now been able to demonstrate the existence of an ATP-dependent proton pump (Sze and Churchill, 1981; Churchill and Sze, 1983; DuPont et al., 1982; Mettler et al., 1982; Stout and Cleland, 1982; Bennett and Spanswick, 1983). However, it was unclear at the time whether the membrane vesicles exhibiting H+ transport originated from the plasmalemma. It appears that much of the early examples of ATP-dependent H+ pumping were being conducted with tonoplast vesicles (Mettler et al., 1982; Serrano, 1984). It has now been amply demonstrated that at least two distinct ATPases were contained in these microsomal membrane preparations: a K+-stimulated, vanadate-sensitive, NO;-insensitive ATPase that is plasma membrane in origin, and a C1--stimulated, vanadate-insensitive, NO;inhibited ATPase that is located in the tonoplast (Marrb and Ballarin-Denti, 1985). ATP-dependent Hf pumping has been demonstrated with both types of membrane vesicles. Recent improvements in plant membrane fractionation techniques have allowed for the isolation of fairly pure membrane fractions. Hence, it is now possible to reconstitute partially purified preparations of plasmalemma ATPases into proteoliposomes (Vara and Serrano, 1982; O'Neill and Spanswick, 1984). However, an unequivocal demonstration of ATP-dependent K+transport is still lacking. When partially purified oat root (Vara and Serrano, 1982) and red beet storage tissue plasmalemma ATPases (O'Neill and Spanswick, 1984) were reconstituted into liposomes, only H+ transport was observed. Vara and Serrano demonstrated ATP-dependent Hf pumping with inverted liposomes containing K+-free solutions (Fig. 9), which suggested to them that the ATPase did not mediate K+-H+ exchange. Additionally, it can also be seen in Fig. 9 that introducing K+ produced an instantaneous stimulation of H+ transport, which would be expected if K+ was acting on the active site of the ATPase facing the external medium (cytoplasmic side of inverted vesicles). They interrupted this result as further evidence against a K+-H+ transport function, since a lag in the stimulation by Kf would be expected as K+ diffused into the vesicle and became available for transport. Because of the demonstrated H+-pumping capacity of these membranes, and a lack of K+ transport ability, the present consensus of most researchers

110

L. V. KOCHIAN AND W. J. LUCAS

ATP

KCI\

ATP K W \

,_

I

Fig. 9. Changes in 9-amino-6-chloro-2-methoxyacridine fluorescence upon energization of Kf-free proteoliposomes prepared from oat root plasma membrane ATPase. Assay medium contained either 25 mM MgS04 (A, B and C), or 25 mM Mg(N03)2(D). Tris-ATP (1.25 mM), K2S04(25 mM), KCI (50 mM), KN03 (50 mM), imidazole-HC1 (pH 6.5,20 mM), and gramicidin D (5 pg ml-') were added as indicated. The initial rate of quenching expressed as per cenrof total fluorescence min-' is indicated. Data from Vara and Serrano (1982).

in this area appears to be that the plant plasmalemma ATPase acts as an electrogenic Hf transport system rather than a K+/H+-ATPase. However, this is an area of research in which rapid progress is being made, and it may well be that this consensus will be modified in the near future. Very recently, evidence suggesting ATP-dependent K+ transport in plasmalemma vesicles isolated from red beet storage tissue has been presented (Giannini et af., 1987). In this study, inverted plasmalemma vesicles were loaded with 86Rb+-labelled K+ solutions by a freeze-thaw technique. Although these vesicles were leaky for K+, the addition of ATP stimulated Kf efflux over and above that observed in the absence of ATP. The ATP-dependent efflux was completely inhibited by vanadate, but only partially inhibited by carbonyl cyanide N-chlorophenylhydrazone (CCCP), which should have abolished the H+ electrochemical gradient. Giannini et al. suggest that K+ transport may be mediated by two systems; one system would be indirectly coupled to the H+ gradient, while the other would be directly coupled to the ATPase, as a K+-translocating ATPase. It should be noted that the results of Giannini et af. do not support the K+-H+ exchange hypothesis. As noted by Serrano (1984), H+ ionophores

POTASSIUM TRANSPORT IN ROOTS

111

should not influence the transport function of ATPases that directly transport K+, either as a Kf-ATPase or as a K+/H+-ATPase.However, since H+ ionophores dissipate both the chemical and electrical components of the protonmotive force generated by a Hf-ATPase, any indirect coupling of K+ transport to a H+-ATPase would be significantly inhibited. This would include K+ uniports driven by the electrical component of the PMF and H+-K+ co-transport. Evidence for the co-transport of K+ with H+ will be discussed in Section II.B.4. 3. Indirect Coupling: Electrophoretic K + Uniport A number of researchers have argued that K+ uptake is not directly coupled to the H+-translocatingATPase, but is associated indirectly with the activity of the H+ pump through a K+ transport system driven by the electrical component of the PMF (Pitman et al., 1975; Marrb, 1979 and references therein). Pitman and co-workers were the first to propose this type of electrophoretic coupling, following studies on the effects of fusicoccin (FC) on K+ and H+ fluxes in low-salt and salt-saturated barley roots (Pitman et al., 1975). Their studies were generally conducted at high external K+ levels (5 mM). In low-salt roots, the FC-stimulated H+ efflux was similar, whether the roots were exposed to 5 mM KCl or 5 mM NaC1. However, in saltsaturated roots, which have a greater passive permeability to K+ than Na+ (in relation to low-salt roots), H+ efflux was stimulated to a greater degree in KC1 solutions. Hence, they suggested that FC acted to stimulate passive K+ influx, through the increased electrical gradient created when H+ extrusion was enhanced by FC. Subsequent studies, which further support the hypothesis of an electrical coupling between the H+ efflux and K+ influx, are based on the application of FC to various plant tissues at K+ concentrations (>1 mM) where uptake is considered to be passive (Marr5, 1977, 1979). It has been consistently observed that under these conditions FC stimulates both H+ efflux and K+ uptake, and elicits a hyperpolarization of Em.A large body of circumstantial evidence has been accumulated, indicating that FC acts directly on the plasmalemma H+-ATPase to stimulate H+ translocation (MarrP, 1979). Since, in either the direct or indirect mode of coupling, a stimulation of H+ efflux should enhance K+ uptake, it is not possible to differentiate between the two models simply through the application of FC. Obviously, other approaches must be used in conjunction with FC experiments. Marrb (1979), in his review on FC, has summarized the observations in support of indirect (electrophoretic) coupling. They are as follows: 1. The stoichiometry of the FC-stimulated K+-H+ exchange is often quite close to 1: 1 (at least for corn roots and pea stem segments), particularly when corrections are made to account for H+ consumption during anion uptake. Although this observation can be used in support of either mode of

112

L. V. KOCHIAN AND W. J. LUCAS

coupling, it appears to support indirect coupling when considered in conjunction with the following observations. 2. Under the conditions in which FC-stimulated fluxes are generally studied, Kf is in passive equilibrium across the plasmalemma (Pitman etal., 1975; Cocucci et al., 1976). Hence, any stimulation of net Kf influx would involve an increase in passive uptake. 3. It has been demonstrated that lipophilic cations, such as tributylbenzylammonium and tetraphenylphosphonium, can substitute for K+ in its role in promoting FC-stimulated H+ efflux (Bellando et al., 1979; Bellando and Trotta, 1980). Presumably, these cations cross the plasmalemma nonspecifically, by permeating the lipid bilayer. When this occurs, it has been shown that Em is depolarized and H+ efflux is stimulated. Therefore, it has been argued that the apparent dependency of H+ efflux on K+ is due to a K+ influx-induced depolarization of Em,which would activate the electrogenic H+-translocating ATPase. Conversely, chemical modifiers that stimulate the proton pump (such as FC) would hyperpolarize the membrane potential and increase the driving force for passive, electrophoretic K+ influx. Interpretation of FC experiments is based on the premise that FC acts specifically on the plasma membrane ATPase to increase its activity. The rapidity of the FC effects on plasmalemma ion transport, and the similar inhibitions of FC-stimulated Kf and H+ fluxes, and ATPase activity, by known plasmalemma ATPase inhibitors, are often used in support of this premise (Marrb et al., 1974a,b). Additionally, [3H]-FC has been shown to bind specifically to a protein component of the plasmalemma-enriched fraction isolated from corn coleoptiles (Dohrmann et al., 1977), and FC is known to stimulate ATPase activity associated with plasmalemma-enriched membrane fractions (Beffagna et al., 1977). However, caution must be exercised when interpreting these data. Several groups have solubilized the putative FC-binding protein from plasmalemma-enriched membrane fractions of corn coleoptile (Pesci et al., 1979; Tognoli et al., 1979) and oat roots (Stout and Cleland, 1980). In each case, it has been shown that the FC-binding protein can be separated from the Kf-stimulated plasmalemma Hf-translocating ATPase. Although it has been suggested in each of the above studies that the FC-binding protein could be a subunit of a multisubunit ATPase, the possibility exists that FC may act at sites totally separate from the Hf-ATPase. In a recent electrophysiological study on Viciafaba guard cells, Blatt (1987) obtained evidence that FC may not act on the pump, but via an effect on the related co-transport processes. Additionally, his current-voltage data indicated that FC may act to block a Kf channel involved in K+ efflux from guard cells. Such a mode of action could also explain the FC effects on other plant cell membranes, and it may be necessary to conduct a complete re-evaluation of these FC data. The use of the electrophoretic coupling model to explain Kf uptake over all K+ concentrations is subject to a number of criticisms, some of which

113

POTASSIUM TRANSPORT IN ROOTS

have been rather eloquently summarized (Glass and Siddiqi, 1982; Siddiqi and Glass, 1984). They point out that K+ influx often greatly exceeds H+ efflux, an observation that we have also stressed (Kochian et al., 1987,1988; Newman et al., 1987). This is a feature which has been presented in work dealing with K+ and H+ fluxes, although it has often gone unmentioned in the texts of these papers (e.g. Poole, 1974; Pitman et al., 1975; Lado et al., 1976; Lin and Hanson, 1976). It is difficult to reconcile a model where K+ influx is energetically dependent on the membrane potential (which, in turn, is dependent primarily on H+ efflux), when the H+ fluxes are usually much smaller than the associated K+ fluxes. Sometimes this disparity has been explained on the basis that the measured, “apparent” net H+ efflux is considerably smaller than the true unidirectional efflux, due to processes that also consume H+. These processes might include anion-H+ co-transport, passive “leaks” that would tend to return H+ into the cell, and H+ captured by the cell wall. However, Glass and Siddiqi (1982) conducted a careful assessment of the contribution of these processes to the underestimation of “true” H+ efflux, and concluded that for their system (low-salt barley roots) the measured net H+ efflux is a good approximation of the unidirectional flux. Furthermore, the data presented in Fig. 10 indicate that H+ efflux may be dependent upon external K+, or K+ influx, per se. Clearly, as stressed by Glass and Siddiqi (1982), this is the converse of what would be expected if K+ uptake were dependent upon H+ extrusion. They also noted that at

-0

-0

E, X

2 1

5 -

r

iii

f

+

I

+

Y

0

0 0.01

0.1

10

1

Potassium Concentration

(

rnol rn-3

)

Fig. 10. Potassium influx (0, A) and H+ efflux (0,A ) as a function of K’ (K,SO,) in the 0) bathing medium. Experiments were conducted on two varieties of barley, var. Fergus (0, andvar. Conquest ( A , A). Data from Glass and Siddiqi (1982).

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L. V. KOCHIAN AND W. J. LUCAS

higher K+ levels (>1 mM), H+ efflux often levels off or declines slightly (see Fig. 10). This saturation is somewhat surprising, considering earlier published reports which specifically linked K+ and H+ fluxes at higher K+ concentrations (Pitman, 1970; Marr2, 1977). Because it was found that anion influx [SO:- in the case of Glass and Siddiqi (1982)l was greatly increased at higher substrate levels (>0.5 mM K2S04), these potentially anomalous results could be explained by a reduction in net H+ efflux due to the consumption of H+ during SO:- influx, if uptake were mediated by an anion-H+ co-transport. However, these data, and most of the results used in support of models coupling K+ and H+ fluxes, could be explained equally well by a H+ extrusion mechanism involved in the maintenance of charge balance (Glass and Siddiqi, 1982), as originally suggested by Ulrich (1941). The constraints of charge balance are most evident when low-salt roots are transferred from a CaS04medium to one containing various inorganic ions, whence they begin to increase their internal salt levels. The roots would then be in a transition between two regulatory states. Pitman (1970) proposed that the stimulated K+ uptake, H+ efflux, and excess cation absorption exhibited as salt status is increased, would be a reflection of this transitional stage. Siddiqi and Glass (1984) have extended this hypothesis, by suggesting that these transitional changes are the result of a concerted effort, by the root, to maintain electrical neutrality during the alteration of salt status. This attempt to maintain electroneutrality does not appear to be specific for H+ efflux. Under certain conditions, plants can use other ion transport processes in order to effect charge balance (Siddiqi and Glass, 1984). For example, both K+-stimulated Na+ efflux and NHi-stimulated K+ efflux have been observed in plant roots. Thus, the apparent coupling of K+ and H+ fluxes may reflect a more general feature of plants, i.e. that cation exchanges (H+, K+, Na', Ca2+etc.) may be needed to maintain charge balance during times when high rates of cation uptake occur (i.e. during the transition from low-salt to high-salt status). Finally, it seems unlikely that a single type of mechanism, or coupling mode, will suffice to account for K+ uptake in higher plants. This is particularly apparent when one considers the potentially wide range of soil K+ concentrations that a root may experience. There are many examples in the literature for plant, bacterial, and animal transport systems, where the uptake of a particular solute is mediated by two or more types of transport mechanisms. Since it appears that K+ uptake in higher plants is active at low K+ levels, and thermodynamically passive at higher concentrations, it seems plausible to speculate that two different K+ transport systems might exist. The existence of K+ channels has been known for many years in animal and bacterial membranes, and increasingly strong evidence for K+ channels in higher plants has been accumulating (Schroeder et al., 1984,1987; Bentrup et al., 1985; Kochian et al., 1985; Kolb et al., 1987). Hence, it is quite possible that K+ channels in the plasmalemma facilitate passive K+ uptake (or release) at higher (>0.5 mM) concentrations. The direction and magnitude

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POTASSIUM TRANSPORT IN ROOTS

of these fluxes would depend upon the value of the membrane potential and on any voltage-dependent characteristics that these channels may possess. However, at low K+ concentrations, where active K+ uptake most probably occurs, a different type of transport system must operate. Uptake must either be directly coupled to an ATPase, as a K+-ATPase (or the less likely case of a K+/H+-ATPase),or active K+ uptake may be coupled to the H+ gradient, via a K+-H+ co-transport system. The K+-H+ co-transport hypothesis will be considered in the next section. Other Modes of Coupling: K + - H + Co-transport? Another approach that has been taken in order to simultaneously study K+ and H+ transport in roots involves the use of ion-selective microelectrodes for K+ and H+ (Kochian and Lucas, 1987; Kochian et al., 1988; Newman et al., 1987). An ion-selective microelectrode system was developed that could quantify and map the extracellular electrochemical potential gradients for K+, H+, and C1- along the roots of 4-day-old corn seedlings. From an analysis of the extracellular ion gradients, it is possible to simultaneously determine and monitor the net H+ and K+ fluxes associated with a few cells at the root surface (due to the small electrode tip diameter of 0.5-1.0 pm). Because this system provides a high degree of spatial (and temporal) resolution, it has proven to be a useful method for studying the coupling of K+ and H+ fluxes at the cellular level. A diagrammatic representation of the system used in these studies is shown in Fig. 11. Data collected with this system indicated that at any point along the root, large fluctuations in the fluxes (particularly H+ fluxes) occurred with time. On many occasions, H+ efflux was near zero while K+ influx was “normal”; the converse was also occasionally observed. Additionally, when repeated measurements were made at the same location on the root, both H+ efflux and K+ influx could vary significantly, and usually the variation of the two fluxes did not show any correlation. The data presented in Table I illustrate 4.

TABLE I Measurement of net H + and K+ influx in low-salt-grown corn roots K+ Influx

Time (min)

H+ Efflux (pmol (g fresh wt)-’ h-’)

(pmol (g fresh wt)-’ h-’)

0 3 8 12 20

1.87 0.76 0.00 0.00 1.12

2.92 3.18 2.52 2.89 2.27

Measurements were made with pH and K+ microelectrodes at a position 3.0 cm from the root apex at distances of 50 and 125 pm from the root surface. Bathing solution consisted of 0.1 mM K2S04 + 0.2 mM CaS04. Data taken from Kochian and Lucas (1987).

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L. V. KOCHIAN AND W. J. LUCAS

(- ,

f

6 ( a )

€'=c E,v-€; 3

+ c v-vo I,-I

Rootradius = 500pm

v, C O

__

!I/

E;

Ell+EKt

I

' Centre of corn roo1

I -

Portion of an intact

root or root segment

1 5 p m j r M i c r o e l e c t r o d e tipslocated within15-20pm

(b 1

_ -/ Cno,r////

root

'///////'Surface

of corn root

Fig. 11. Schematic representation of the ion-specific microelectrode system used by Newman et al. (1987) to measure the Kt and H+ electrochemical potential gradients which develop at the surface of intact corn roots. Individual voltage components are shown in (a): EL and E r represent the EMF of the half-cells for the reference and ion-selective electrode, respectively; E: is the EMF of the half-cell for the extracelluar electric potential electrode; E, is the EMF developed across the ion-exchange resin; V and V , are the extracellular potentials near the root surface and in the background bathing solutions, respectively. A scaled representation of the relative positions of Kt,Htand extracellular potential (EI) microelectrodes is shown in (b). Here the microelectrodes (0.5 pm tip diameter) ae shown at their closest position to the corn root surface.

this point. Here, five separate measurements of K+ influx and H+ efflux were made over a 20-min period, and it can be seen that H + efflux varied rather dramatically, while K+ uptake was relatively stable. From these studies no fixed stoichiometry was found for the two fluxes. Generally, net K+ uptake was significantly larger than H+ efflux; the K+ :H + flux ratio ranged from 3.6: 1 to 1:2.75. A similar lack of correlation between K+ and H+ fluxes was observed in barley roots (Glass and Siddiqi, 1982). Increasing the bathing medium pH resulted in a gradual, but small, increase in K+ influx (see Fig. 5 ) . However, H+ efflux did not exhibit a similar pH dependency, but rather appeared to vary independently of the pH value. In this situation, the K+:H+ flux stoichiometries varied from 1:1 to 12: 1, again with no apparent pH dependency. The response of the cortical cell membrane potential to changes in

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POTASSIUM TRANSPORT IN ROOTS

7 -100 E

Y

-0 -120.-

E -1400

c

0

a -160-

0.2rnM CaS04

I-7Z-l 0.I rnM K2S04

0)

n

-180-

E -200-

5

I

Time (min)

Fig. 12. Response of the cortical membrane potential of intact low-salt-grown corn roots to changes in the concentration of external K+ (as K2S04). (CaSO,, was present at 0.2 mM in all solutions.) Data from Newman et al. (1987).

external K+ was also investigated in these studies. Unlike the case in earlier studies (Cheeseman and Hanson, 1979a,b), the low-salt corn root membrane potential was extremely sensitive to external K+. Increasing the Kf concentration from 0.2 to 20 p~ caused a rapid 70 mV depolarization, from - 190 mV to - 120 mV (see Fig. 12). A further increase in K+ to 200 p~ only elicited a further 7 mV depolarization. Newman et al. (1987) made simultaneous measurements of Em and K+ fluxes in response to changes in external K+,using a three-microelectrode system (one to measure Em,and two K+ microelectrodes for K+ flux measurements). Both the kinetics (dependence on external K') for K+ influx and the depolarization of the membrane potential yielded similar (and quite low) apparent K m values in the 6-9 p~ range. These results indicated that low-salt corn roots possess a very high affinity K+ uptake system that is extremely electrogenic (Newman et al., 1987). In view of the highly electrogenic nature of this system, it is possible that other cations may be transported into the cell with Kf. Since these measurements were made in simple salt solutions (10 p~ K2S04 0.2 mM CaS04), H+ appears to be the most likely candidate for co-transport with K ' . In such a situation, active K+ influx would be achieved using the energy gained from the movement of H+ down their A&. Strong evidence for the existence of a K+-H+ co-transport system in fungi has been recently provided by work on Neurospora (Rodriguez-Navarro and Ramos, 1986; Rodriguez-Navarro et al., 1986;Blatt and Slayman, 1987; Slayman et al., 1988). Slayman and co-workers have described a high-affinity (K, = 1-10 p ~K+ ) uptake system, in K+-starved Neurosporu cells, that is highly electrogenic, as demonstrated by the response presented in Fig. 13. Measurements of internal and external K+, and Em,suggested that this uptake system may mediate active K+ uptake, and the system appears to be

+

118

L. V. KOCHIAN AND W. J. LUCAS ( 0

1

(b)

=-o

-I

100 urn Sdutlan

flow

~

IK'1,-

I

50~M

[K+l,

2

++

3 -304

5aM

,

4n - 2 1 8 +

-305

2014

1 2 0 9 + +

-307

5 0 ~ M

++

200g

Fig. 13. Effect of added extracellularKCon the membrane potential measured in low-K+ spherical cells of Neurospora crassa. (a) Diagram of the arrangement of the cell, the impaling electrode and a K+-floodingpipette for rapid introduction and removal of K+. (b) Depolarization of the Emwith 50 p~ K'. (c) Condensed record from a single cell, showing the Emresponse to four different K+ concentrations. Symbols I and I1 indicate cell impalement and electrode removal, respectively. Data from Rodriguez-Navarroet al. (1986).

coupled to the very active plasmalemma H+-ATPase of Neurospora. A net stoichiometry of one H+ out for one K+ in was demonstrated, which could be taken as evidence for K+-H+ exchange. However, current-voltage analysis conducted by Slayman's group indicated that the K+-associated inward current was twice that of the net K+ influx (see Fig. 14). Thus, one additional positive charge enters with every K+. In addition, the following points must be considered: (1) the H+-ATPaseoperates in parallel with the K+ uptake system; (2) almost every charge absorbed must be balanced by an extruded H+; and (3) only a single H+ is measured (released to the external solution) for every Kf taken up. Hence, the second charge coming in with the K+ must be a H+,which indicates that the high-affinity K+ uptake system operates as a K+-H+ symport (Rodriguez-Navarro et al., 1986; Slayman et al., 1988). Slayman and co-workers speculate that the data presented for higher plants could also be explained by this type of co-transport system coupled to the plasmalemma H+-ATPase. As discussed previously, it has been proposed that a similar co-transport system exists in the plasmalemma of corn root cells (Newman et al., 1987). Further support for this hypothesis comes from the observed similarities between the K+ uptake system seen in K+-starved Neurospora cells and that described above for low-salt corn roots (Kochian et al., 1987,1988; Newman et al., 1987). Both are very high affinity K+ uptake systems with almost identical K , values for K'. Furthermore, K+ uptake through both systems is

119

POTASSIUM TRANSPORT IN ROOTS

? C

2

A

30

0

40

80

120

External K + Concentration

200

160 (

p~

Fig. 14. Stoichiometry of the K+-Hf co-transport system of Neurosporu crassa. The smooth curves are Michaelis-Menten functions fitted to the two sets of data, using a common value of 14.9 ~ L MKt for the apparent K,. Separate values of V,,, are 15.3 k 1.0pmol cm-'s-' for the flux, and 30.1 f 1.6 pmol cm-2 s-' for the measured current. The stoichiometric ratio of the current to net K+ flux is very close to two. Data from Rodriguez-Navarroet af. (1986).

highly depolarizing (compare Figs 12 and 13). The similarities between the two systems, taken in conjunction with the lack of evidence, in low-salt corn roots, for a coupling between K+ influx and H+ efflux, strongly suggest that such a co-transport system could be operating in higher plants. On a kinetic basis, it can be reasoned that a K+-H+ co-transport system should reflect a sensitivity to changes in both extracellular and cytoplasmic pH values. Previous reports of increasing K+ influx, in response to decreasing H+ concentrations in the bathing medium, appear to be somewhat at variance with this prediction. However, in these reports, K+ influx was insensitive to external pH values from 6 to 9 (see Fig. 5 ) . To further investigate this hypothesis for K+ influx into corn roots, Kochian et al. (1987) used K+-selective microelectrodes and 86Rb+to measure K+ influx as a function of pH. Varying the external pH from 4 to 8 had no effect on either net K+ influx (measured with K+ microelectrodes) or unidirectional K+(86Rb+)influx. Additionally, Kochian et al. found that the K+-induced depolarizations of Em (with solution of 50 PM K+) also exhibited absolutely no pH dependence from pH 4 to 8. These results suggest that the K+-H+ co-transport system may have an extremely high affinity for H+. However, an alternative interpretation is that K+ influx is mediated by a different molecular mechanism, e.g. a K+-ATPase,which would not, apriori,show a sensitivity to external pH values.

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L. V. KOCHIAN AND W. J. LUCAS

C. UPTAKE AT HIGH K+ CONCENTRATIONS: THE LINEAR COMPONENT

As discussed in Section II.A, studies on the kinetics of K+ uptake, for various plant tissues, have generally focused on the generation of complex and often discontinuous kinetic curves, via either the operation of multiple Michaelis-Menten transport systems (such as Epstein's mechanisms I and 11), or by complex, multisite carriers. However, there are numerous examples, particularly in association with studies involving animal and bacterial systems, of complex transport kinetics that appear to be due to the parallel operation of one or more saturable transport mechanisms plus a system that exhibits nonsaturating, or first-order uptake kinetics (linear component). For example, a linear transport component has been demonstrated for the uptake of amino acids and sugars in animal cells (Akedo and Christensen, 1962; Christensen and Liang, 1966; Munck and Schultz, 1969; Cohen, 1975, 1980; Debnam and Levin, 1975), and for the uptake of lactose (Maloney and Wilson, 1973), K+ (Rhoads et al., 1976; Epstein and Laimins, 1980) and amino acids (Wood, 1975; Iaccarino et al., 1978) in E. coli. In many of these studies, nonsaturating solute uptake was dismissed as a physiologically insignificant process, because it was considered to reflect passive diffusion across the lipid portion of the plasma membrane. However, in work on K+ uptake in E. coli, linear Kf uptake was hypothesized to be mediated by a transport protein, which was presumably a Kf channel (Rhoads etal., 1976; Epstein and Laimins, 1980). Furthermore, Christensen and Liang (1966) demonstrated that nonsaturating amino acid uptake in Ehrlich tumour cells was substrate-specific, and exhibited considerable sensitivity to pH and temperature. Thus, passive diffusion was discounted in favour of a more complex system involving a transport protein. In recent years, there has been an increasing interest in linear, nonsaturating solute uptake kinetics from studies involving plant tissues. First-order kinetics have been observed for the uptake of Fe2+in rice roots (Kannan, 1971), sucrose and 3-0-methylglucose in Ricinus cotyledons (Komor, 1977; Komor et al., 1977), sucrose in sugar beet leaf and petiole sections (Maynard and Lucas, 1982a,b), soybean cotyledons (Lichtner and Spanswick, 198l), Vicia leaves (Delrot and Bonnemain, 1981) and red beet vacuoles (Willenbrink and Doll, 1979), and sucrose, fructose, and glucose in Allium leaf discs (Wilson et al., 1985), and for amino acid transport in Lemna (Fischer and Luttge, 1980), and suspension-cultured tobacco cells (Blackman and McDaniel, 1978). Separation of the contribution made by the linear transport component from the saturable mechanisms has been achieved through the use of various inhibitors (Debnam and Levin, 1975; Polley and Hopkins, 1979; Maynard and Lucas, 1982b; Van Be1 et al., 1982). For K+ uptake into corn roots, Kochian and Lucas (1982a,b, 1983) have shown that the complex kinetics

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could be resolved into saturable and first-order kinetic components, through the use of sulphhydryl modifiers. When corn root segments were subjected to a series of increasing N-ethyl maleimide (NEM) exposures (e.g. 0,10, and 30 s NEM exposures for the data illustrated in Fig. 3b), saturable K+ uptake was specifically inhibited and subsequently abolished, while linear uptake was relatively unaffected. Hence, it was suggested that the saturable and linear kinetic components represented separate K+ transport systems. Saturable K+ uptake would be analogous to Epstein's mechanism I; however, nonsaturating K+ uptake, which dominates uptake in the concentration range associated with Epstein's mechanism 11, appears to be distinctly different from the transport system described by Epstein and co-workers. Therefore, subsequent work was carried out in order to characterize this linear component for Kf uptake (Kochian and Lucas, 1984; Kochian et al., 1985); certain features of this transport system will now be discussed.

I . Anion Involvement in Nonsaturating K + Uptake Early work by Epstein et al. (1963) demonstrated that in barley roots K+ uptake by mechanism I1 was dramatically inhibited when C1- was replaced by SO:- in the uptake solution. Similarly, it was shown that linear K+uptake in corn roots was partially dependent on the presence of CI- in the uptake solution (Kochian et al., 1985). As shown in Fig. 15, replacing C1- in the

0

2

4

6

8

10

K+concentration (mM

Fig. 15. Influence of the accompanyinganion on K+(86Rb+)influx intocom root segments grown in 0.2 mM CaSO, (low-salt status). The first-order rate coefficients, k , in pmol (g fresh wt)-' h-' m K ' , for the linear component of K+ influx were as follows: 0.5 (Cl-), 0.21 (SO$-), and 0.24 (H2P0,). Data from Kochian et al. (1985).

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uptake solution with SO$-, H2PO; or NO; resulted in a 60% reduction in K+ uptake by the linear component in low-salt roots, while causing only a 15% reduction in the V,,, for the saturable system. In high-salt roots (grown on 5 mM KCl), a similar reduction in linear K+ uptake was seen, while saturable uptake was unaffected. This association between the linear component for K+ uptake and the presence of C1- was shown to be related to a coupling via the saturable C1-

Control

.

I , L

6 DIDS -a

=

OCO”

o

0

.

2

4

6

8

10

8

10

CI-concentration (mM 1

2

4

6

K+concentration (mM)

Fig. 16. Effect of the anion transport inhibitor DIDS on the influx of wl- (a) and K+ (=Rb’) (b) into low-salt corn root segments. For K+ influx, k had values of 0.40 and 0.19 pmol (g fresh wt)-’ h-’ mM-’for the control and DIDS treatment, respectively. Data from Kochian etal. (1985).

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acid influx process. Application of 1mM 4,4-diisothiocyano-2,2’-disulphonic stilbene (DIDS), which has been shown to be an anion transport inhibitor in red blood cells (Cabantchik et al., 1978), corn roots (Lin, 1981) and Chum (Keifer et al., 1982), abolished the saturable component for C1- influx into low-salt corn roots (Fig. 16a). In the presence of this inhibitor, the linear component of K+ uptake was suppressed to an identical degree to that seen when C1- was replaced in the uptake solution by other anions (compare Figs 15 and 16b). These results strongly suggest that the linear component of K+ influx in corn roots is, in some way, linked, at least partially, to saturable C1- uptake.

2. Involvement of K’ Channels? The effect of the quaternary ammonium salt, tetraethylammonium chloride (TEA), which has been shown to block K+ channels in excitable membranes, such as the plasma membrane of nerve fibres (Tasaki and Hagiwara, 1957; Armstrong, 1969), and in Chara (Keifer and Lucas, 1982), was studied on K+ uptake in low- and high-salt corn roots. In high-salt roots, TEA caused a dramatic (75%) and specific inhibition of the linear component of K+ influx (Fig. 17), which suggests that K+ channels may be involved. However, although low-salt roots possess a similar linear component for K+ influx, it was found to be insensitive to TEA, which seems at variance with the above interpretation. Uptake studies with [I4C]-TEAindicated that high-salt corn roots exhibited much higher rates of TEA accumulation than did low-salt roots, presumably via a transport system for quaternary ammonium salts

K+concentration (mM Fig. 17. Influence of 10 mM TEA-CI on K+ (86Rb+) influx into high-salt-growncorn roots. Roots were pretreated with a solution containing 10 mM TEA-C1,5 mM KCI, and 0.2 mM CaS04 for 30 min prior to K+ (“Rb’) uptake (10 mM TEA-CI was included in the uptake solutions). The first-order rate coefficients, k, for the linear component were 0.31 and 0.09 pmol (g fresh wt)-’ h-’ mM-’ for the control and TEA-C1, respectively. Data from Kochian et al. (1985).

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that has been demonstrated in higher plants (Michaelis et al., 1976). Hence, it was proposed that corn root tissue behaves in a similar manner to nerve axons, where it is necessary to inject the TEA into the axoplasm in order to block Kf channels (Tasaki and Hagiwara, 1957; Armstrong, 1969). Highsalt roots may be able to accumulate more TEA in the cytoplasm relative to low-salt roots. If this were the case, then K+ channels could be involved in nonsaturating K+ influx in both low- and high-salt roots; but only in high-salt roots would the cytoplasmic TEA concentration rise to a level high enough to block the putative Kf channels at the cytoplasmic face of the channel. In recent years, the application of the patch-clamp technique to plant cell membranes has provided direct evidence for the existence of K+ channels in higher plant cell membranes. For example, K+ channels have been demonstrated in the plasmalemma of guard cells (Schroeder et al., 1984, 1987), Samanea pulvinar cells (Moran et al., 1987) and corn root cells (Ketchum et al., 1987), and in the tonoplast of Chenopodium suspension cells (Bentrup et al., 1985) and barley mesophyll cells (Kolb et al., 1987). Therefore, it seems reasonable to speculate that one or more classes of K+ channels could operate in the plasmalemma of root cells, in order to facilitate passive K+ uptake at high external levels of K+. Although it is axiomatic that any transport system should have a finite transport capacity, it is possible that a system like an ion channel would not exhibit saturation kinetics under physiological conditions. As Cohen (1975) has pointed out, such a channel-mediated process should eventually saturate at high substrate concentrations. However, in his system (amino acid uptake into mouse brain slices), at high substrate levels, the medium changes from isotonic buffered saline to hypertonic buffered amino acid saline. Thus, any changes in transport kinetics may be due to changes in media composition. The same applies for K+ influx in corn roots. Potassium uptake has been studied from a range of concentrations up to 50 mM. At these high K+ levels, there was no significant change in nonsaturating K+ uptake (Kochian and Lucas, 1982a). Uptake was not studied from solutions of higher concentration, because it was felt that ionic and osmotic effects could alter membrane lipid/protein structure and make data interpretation tenuous.

3. Root Salt Status and Nonsaturating K + Uptake Uptake into low-salt barley roots, at low external K+ levels, is highly specific for K', while at higher K+ concentrations (>0.5 mM), Na+ can competitively inhibit K+ influx (Epstein et al., 1963). A similar response was also observed with low-salt corn roots. Inclusion of 3 mM NaCl in the uptake solution (K+ concentration was from 0.1 to 10 mM) caused a 50% inhibition of the linear component for K+ uptake, while saturable uptake was unchanged (Kochian et al., 1985). The interesting feature is that within the mechanism I1 concentration range, high-salt corn roots exhibit a much higher selectivity for K+ influx over other cations (Pitman, 1967, 1970; Pitman et al., 1968; Kochian et al., 1985). If we accept that the linear component represents the

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operation of a Kf channel, these data indicate that some aspect of tissue salt status modifies the channel characteristics from relatively nonspecific to highly K+-specificin high-salt roots. It will be interesting to see whether this prediction can be confirmed using the patch-clamp technique.

4. Alternative Explanation f o r the Linear Component Sanders (1986) has developed a generalized model to explain biphasic or otherwise complex transport kinetics, based on the co-transport of a solute with a driver ion. For Kf influx into roots, a K+-H+ co-transport system would be driven by the inwardly directed A& generated by the H+-ATPase. In Sander’s reaction kinetic model, the assumption is made that both solute (K’)and driver ion (H’) can bind randomly to the carrier, and the limitation is made that the carrier can cross the membrane only as the fully loaded complex (influx) and can return to the outside free of ligand. The reaction kinetic scheme for this model is shown in Fig. 18. Numerical analysis

Fig. 18. Membrane transport modelled on the basis of a reaction kinetic scheme. (a) Reaction kinetic scheme for random binding of solute (S) and H+ to a membrane-bound carrier (X)which catalyses the transport of S across the membrane. Carrier is represented as transporting positive charge in the loaded form. (b). As in (a), but with loaded carrier being neutral and charge transfer occurring on the unloaded form of the carrier. (c) Generalized reaction kinetic scheme for the charged and uncharged models. Concentration (density) of carrier state “j” is designated as Ni, with rate constants (not shown) from carrier state i tostate j designated kii.Redrawn from Sanders (1986), with permission.

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simulation of this model demonstrated that it generates biphasic kinetics under conditions resembling those present in many uptake experiments. The utility of this model is that it makes a number of predictions that are experimentally testable. These predictions are (in terms of a K+-H+ cotransport system): 1. As [H'], is increased to saturating levels, the kinetics for K+ uptake should change from a complex biphasic to a monophasic profile. 2. At saturating [H'],, increasing levels of internal K+ should result in an uncompetitive inhibition of K+ influx.

Fig. 19. Comparison between the experimental data (W) obtained for 6-deoxyglucose influx into Chorella, as a function of bathing medium pH (Komor and Tanner, 1975), and the theoretical simulation of Sanders' (1986) reaction kinetic model. The solid lines represent a simulation of the charged carrier model, simplified for very negative Em and internal 6-deoxyglucose concentration set at zero. Data from Sanders (1986).

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3. As [H’l0 is decreased, the relative contribution to the uptake isotherm made by the “apparent” low-affinity K+ transport should increase.

As Sanders (1986) noted, there are few published examples of data concerning pH effects on complex isotherms for solute uptake in plants that permit his predictions to be properly tested. However, such kinetic isotherms do exist for the H+-sugar co-transport system of Chlorella (Komor and Tanner, 1975). As shown in Fig. 19, these data give a reasonable fit to the first prediction of the Sanders model. However, if one then examines the pH dependency of the detailed kinetics for sucrose uptake into sugar beet leaves obtained by Maynard and Lucas (1982a), it is apparent that as the external pH is decreased from 9.0 to 4.0, both the saturable and nonsaturable components of sucrose uptake are stimulated. Clearly, these data do not fit the first prediction of the Sanders (1986) model. Furthermore, it is difficult to reconcile K+ uptake into high-salt corn roots with this Sanders model, since, in this system, it was possible to speciJically abolish saturable K+ uptake with NEM, while leaving nonsaturable uptake relatively unaffected (Fig. 3b). Conversely, it was possible to specifically inhibit the linear transport component either by replacing C1- in the uptake solution with other anions (Fig. 15), or by treating the roots with the K+ channel blocking agent, TEA (Fig. 17). At present it is not obvious how these perturbations, which appear to specifically resolve the complex kinetics for K+ uptake into separate kinetic components, can be explained by the Sanders reaction kinetic model. 5. Physiological Role for Nonsaturating K + Uptake? It has been noted that nonsaturating solute uptake often occurs over a concentration range which exceeds the levels normally experienced by roots in the soil, and so it is difficult to assign a physiological role for such a transport system. Reisenauer (1966) has pointed out that the majority of soil Kf is below 2 mM; yet, in corn roots, nonsaturating K+ uptake becomes significant above 1 mM external K’. What then would be the physiological relevance of a transport system that operates at substrate levels rarely experienced by the plant? The answer to this question may come from transport studies conducted on E. coli and Neurospora. In both organisms, it has been well documented that multiple carrier systems are often involved in the transport of a single solute. These organisms tend to combine constitutive, low-affinity,high-capacity transport systems with derepressible high-affinity systems; glucose and phosphate uptake into N . crassa are excellent examples of this strategy (Lowendorf et al., 1974; Scarborough, 1970). These systems give the organism the adaptive advantage of most effectively obtaining nutrients whose concentrations may vary considerably over a period of time. A root growing through the soil may often experience extremely low K+ levels. Therefore, a high-affinity system may be necessary in order for the

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plant to satisfy its requirements for this essential nutrient. However, if the growing root encounters a localized region of high soil K', the high-affinity system may not have the capacity to utilize excess K'. In such a situation, a low-affinity, high-velocity system would allow the plant to effectively utilize these pockets of high soil K+. Furthermore, as soil water content (potential) declines, the root system must respond in terms of osmotic adjustment. If only a high-affinity K+ transport system were present, the root would not be able to take advantage of the increase in the K+ concentration of the soil solution that would occur along with the decline in soil moisture. The presence of a high-velocity system may provide the plant with an advantage in terms of its response to water stress. In this way, the plant may maintain relatively efficient methods of dealing with its varying environment. D. SUMMARY

A summary of the mechanisms by which K+ may enter the symplasm of the root is given in Fig. 20. Uptake at low external K+ levels is facilitated by a

H+ f

H't-

K'Channel

-

-

- -

~

F e e d b a c k on c a t i o n specificity

Cell Wall

Fig. 20. Schematic representation of possible K+ transport systems operating at the plasmalemma to facilitate K+ uptake at both low and high external K+ concentrations. At low K+ levels, a high-affinity system is hypothesized to be either a K+-ATPase, or a H+-K+ co-transport system, coupled to the H+-ATPase. Both systems would be subject to feedback control by internal K+. Under high K+ levels, nonsaturating K+ uptake involves a K+channel transport. The degree of K+ specificity exhibited which is, in some way, coupled to anion (CI-) by this channel may be influenced by cytoplasmic K+ levels.

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very high affinity transport system that is strongly depolarizing, and can transport K+ against its electrochemical potential gradient. Several possible models appear to fit the published data equally well. In our evaluation, the most likely candidates would either be a K+-translocating ATPase or, as in Neurospora, a Kf-Hf symport coupled to the H+-translocating ATPase. The possibility exists that both types of active transport could be functioning in parallel. This transport system would appear to be subject to kinetic and thermodynamic control, and would also be subject to feedback (allosteric) regulation from Kf contained in an internal compartment (see Section V. A). Potassium uptake at higher external levels would be due to a lower-affinity transport system that would transport K+ passively, down its electrochemical potential gradient. This system could be a K+ channel, or a less specific cation channel, which is coupled, in some way, to a saturable C1uptake system which may function as a H+-Cl- co-transport system (Sanders, 1980b; Jacoby and Rudich, 1980). This system does not appear to be subject to feedback regulation by internal K+ levels, but does exhibit changes in substrate specificity as internal salt levels are altered.

111. REDOX-COUPLED PLASMALEMMA TRANSPORT OF K+ The influence of exogenous electron donors and acceptors has been studied in a variety of plant cell types (Bienfait, 1985; Lin, 1985; Liittge and Clarkson, 1985; Moller and Lin, 1986). Although much of this work has concerned the role of plasmalemma electron transport (redox) systems in Fe” reduction at the root surface (Luttge and Clarkson, 1985; Mdler and Lin, 1986), several studies have addressed the more general topic of the involvement of plasma membrane redox systems in the energetics of solute transport into plant cells. Crane and co-workers (Craig and Crane, 1980, 1981; Misra et al., 1984) investigated the effects of exogenous NADH and ferricyanide on membrane transport and cell growth in suspension cells of carrot. They reported that carrot suspension cells can oxidize exogenous NADH with a concomitant increase in O2consumption (30% stimulation), and that this oxidation results in a stimulation (-60%) of K+ influx. Misra et al. (1984) postulated that a plasma membrane redox system operates in close association with the H+-translocating ATPase of this membrane to exert an influence on K+ transport. In experiments conducted on corn root cortical protoplasts, Lin (1982a,b, 1984) reported that addition of NADH tripled O2consumption, and caused a two- to three-fold increase in K+ influx, a marked stimulation of H+efflux and a moderate (20 mV) hyperpolarization of the E m . Although the extent of the NADH influence was less, Lin reported that similar results were obtained on excised corn root segments. Consistent with the results of earlier workers, Lin (1984) proposed a model in which the oxidation of

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exogenous NADH would result in the establishment of a transplasma membrane H+ gradient. The relationship between this H+ gradient and K+ influx remains to be established (but see Rubinstein and Stern, 1986). A. INFLUENCE OF EXOGENOUS NADH ON K+ INFLUX

Almost all of the above-mentioned NADH studies were conducted at one particular K+ concentration, usually 0.2 or 1.0 mM. However, Lin (1984) showed that the addition of 1.5 mM NADH to corn root protoplasts had its main effect on K+ influx within the mechanism I range. In these protoplast experiments the apparent K,,, for K+ influx (0.3 mM) was not affected by NADH, but the V,,, underwent a four-fold stimulation. Uptake of K+ over the higher concentration range did not appear to be affected by exogenous NADH. Although in some cases a stimulatory effect of exogenous NADH has been observed on K+ uptake, a clear discrepancy exists between these reports and the findings of Kochian and Lucas (1985) and Thom and Maretzki (1985). Figure 21 illustrates the inhibitory effect that 1.5 mM NADH had on K+ influx into corn root segments (Kochian and Lucas, 1985). At low external K+ concentrations (C0.5 mM)) influx was inhibited by 80%, and a similar degree of inhibition of K+ influx into protoplasts, prepared from sugar cane suspension cells, was reported by Thom and Maretzki (1985). (Leucine, arginine and 3-0-methyl glucose influx into

, I" r

ol

Kt concentration ( mM 1

Fig. 21. Influence of 1.5 mM NADH on @Rb+ influx into high-salt-grown corn root segments. (A similar response was observed in NADH experiments conducted on low-salt corn roots.) Data from Kochian and Lucas (1985).

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these protoplasts were similarly inhibited by NADH.) Interestingly, K+ influx into corn roots was also inhibited by the electron acceptor ferricyanide (Kochian and Lucas, 1985; Rubinstein and Stern, 1986). It has been suggested that these two chemicals elicit this inhibition by separate mechanisms, based on the different K+influx kinetics elicited after exposure to NADH or ferricyanide, and the different time courses for recovery from inhibition. B. MEMBRANE TRANSPORT AND THE WOUND RESPONSE

Kochian and Lucas (1985) proposed that exogenous NADH reacts with the outer surface of the plasmalemma to signal a tissue wound response. Hanson and co-workers have shown that corn roots are a particularly sensitive tissue, and that physical handling of the roots, imposition of cold shock etc., can elicit a wound response (Leonard and Hanson, 1972; Gronewald and Hanson, 1980; Chastain and Hanson, 1982; Zocchi and Hanson, 1982). This response is generally characterized by a reduction in K+ influx and a stimulation of efflux, a reduction in H+ efflux, and a depolarization of the Em.A recovery period of about 4 h is usually required before the “wounded” tissue returns to control levels of physiological functioning. It was shown that NADH does not affect K+ influx if the corn root segments have not yet recovered from excision-associated wounding (Kochian and Lucas, 1985). Additionally, if cycloheximideis included in the recovery medium, K+ influx does not return to the control level, and again NADH has no effect on K+ influx. These results indicate that activation of a wound response blocks the NADH effect. Kochian and Lucas (1985) demonstrated that in this state no NADH-stimulated O2consumption can be detected. Only in the recovered state could they measure a 30% stimulation of O2 uptake upon addition of 1.5 mM NADH. Interestingly, although addition of NADH perturbed K+ influx (and efflux), it continued to be oxidized by these perturbed root segments. As mentioned earlier, when the redox system in corn roots (or protoplasts) is stimulated by NADH, net apparent H+ efflux was reported to increase (Lin, 1984). However, Kochian and Lucas (1985) found that NADH elicited a significant decrease in H+ efflux in both washed corn root segments and intact roots (see also Lucas and Kochian, 1987). In some cases an alkalinization of the external medium was observed after addition of NADH, which implies net apparent H+ influx. A similar situation has also been reported for sugar cane protoplasts (Thorn and Maretzki, 1985). Transferring NADH-treated roots to fresh control solution (minus NADH) resulted in an almost immediate recovery of net apparent H+ efflux to pretreatment values (Kochian and Lucas, 1985). This imbalance between recovery of H+ efflux and K+ influx has also been observed with roots that

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are recovering from excision wounding (Gronewald and Hanson, 1982; Kochian and Lucas, 1985). Wounding the root elicits a depolarization of the Em,a response which is in direct contrast to the NADH-induced hyperpolarization of the potential reported by Lin (1982a). In recent studies conducted by Lucas and Kochian (1987), NADH was found to cause a significant depolarization of the corn root Em.In some cases the potential remained in this depolarized state, while in others it repolarized very slowly towards the pre-NADH resting potential. However, consistent with the response of H+ efflux discussed above, removal of NADH always resulted in a repolarization of the potential. Certainly, then, the NADH influence on K+ influx, H+ efflux, and the Em is consistent with the hypothesis that exogenous application of this redox reagent elicits some form of wound response in corn root tissue, while in protoplasts and suspension-cultured cells its application appears to stimulate these physiological processes. C. DEVELOPMENT OF AN INTEGRATED NADH MODEL

Although there is a clear conflict concerning the reported effects of exogenous NADH on plasmalemma transport of K+, there is little doubt that bona fide redox systems are located within this membrane (De Luca et al., 1984; Rubinstein et al., 1984; Buckhout and Hrubec, 1986; Bottger and Liithen, 1986; Macri and Vianello, 1986;Pupillo et al., 1986; Luster et al., 1987). The challenge is to develop a working model of the way in which these putative redox systems interact with the various transport systems functioning within the plasmalemma of plant cells. Such a model would have to account for these reported extremes in tissue response to NADH. In the present context, we will confine our attention to the effects of NADH on H+ and K+ fluxes and the membrane potential. Firstly, the inability of freshly cut, or wounded, corn roots to oxidize exogenous NADH must be explained in terms of the inability of NADH to further inhibit K+ influx, once the K+ transport system has received the wound response “signal(s)”. In analysing the NADH response we have, perforce, assumed that Kf transport occurs via a K+-H+ co-transport system. Zocchi el al. (1983) have shown that cold shock treatment of corn roots increased the phosphorylation of microsomal membrane proteins. These changes were correlated with a decrease in ATPase activity and, since the major changes in phosphorylated proteins occurred within the 92 to 100 kDa range, they concluded that one effect of injury is the blockage of H+ efflux via phosphorylation of the plasma membrane H+-ATPase. This hypothesis is consistent with the observed changes in adenine nucleotide content of corn roots during the injury recovery period (Zocchi et al., 1983). The critical step is the mechanism by which tissue injury is “sensed” by the cells and transduced into protein phosphorylation.

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Several possible control systems appear to be tenable. Calcium influx has been shown to increase upon wounding (Zocchi and Hanson, 1982; De Quintero and Hanson, 1982), and the resultant change in the cytosolic Ca2+ level may activate a Ca2+-calmodulin system. One consequence of this activation could be the stimulation of a specific protein kinase. Alternatively, the turnover of phosphatidylinositol may function as a biochemical signal transduction system in roots in a manner analogous to its function in animal tissues (Berridge, 1983;Berridge and Irvine, 1984;Nishizuka, 1984). In animal cells, various external stimuli activate a phospholipase C on the inner plasma membrane surface, which then hydrolyses phosphatidylinositol 4,5-bisphosphate to release myoinositol 1,4,5-triphosphate (IP,) into the cytosol and diacylglyceride (DG) which remains within the membrane. IP3 can stimulate numerous biochemical and biophysical processes, including the release of internally sequestered Ca2+ and activation of certain kinase systems. Recent studies on plant tissues have demonstrated the presence and metabolic turnover of IP, (Boss and Massel, 1985; Morse et al., 1986; Sandelius and Sommarin, 1986; Rincon and Boss, 1987). These reports, along with the central role played by IP, in regulatory phenomena in animal tissues, provided the impetus to incorporate the IP3 cycle as a central component in our NADH “wound” model outlined in Fig. 22. When the alternative agonist membrane receptor (MR) has been stimulated by a wound “signal”, the membrane transmitter (MT) activates the signal transduction system (STS; phospholipase C), and this produces an increase in the level of IP3. The consequences of this increase in IP3are:

1. Activation of a protein kinase which results in phosphorylation of a protein subunit of the H+-ATPase near the Pi release site. This causes a reduction in H+ efflux; the effect will depend on the extent of phosphorylation and the dephosphorylation capability of the cell. 2. Ca2+ release, which may or may not exert its effects on cellular metabolism via Ca*+-calmodulin(CaCM). 3. Direct or indirect effects of IP3 on the gating (G) of ionic channels in both the plasma membrane and tonoplast. An important consequence of this wound activation of MT is that in this state the transmission system is incapable of accepting alternative stimuli. As indicated in Fig. 22, we suggest that an external NADH-oxidizing site can function to stimulate the MT system. Evidence for the presence of this NADH oxidation system comes from a reinterpretation of Lin’s trypsin data (Lin, 1982b; see also Buckhout and Hrubec, 1986). Although the SDSpolyacrylamide gel electrophoretogram of the TCA-precipitated protein obtained from the supernatant of trypsin-treated corn root protoplasts indicates a high level of endogenous protease activity, Lin (1982b) was able to show that the supernatant was capable of NADH oxidation. If endomem-

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EXOGENOUS

NADH

+2H+

I

I

+

H'

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brane contamination (mitochondria and endoplasmic reticulum) was absent (but see Komor et al., 1987), this NADH oxidation may be attributed to the presence of a 42 kDa polypeptide released by mild trypsin action (Lin, 1982b, 1984). This protein appears to be able to oxidize NADH and reduce O2 (provided that O2is present); these properties are inconsistent with the NADH redox model proposed by Lin (1984). Note that in the model presented in Fig. 22, exogenous NADH will result in the following: 1. A stimulation of the MT and STS, with the consequences outlined above. 2. A continued stimulation of O2consumption, even though the cell has received a wound signal. 3. Proton consumption at the outer surface of the plasmalemma; depending on the degree to which the total H+-ATPase system is inhibited, this may give rise to alkalinization of the root surface. 4. Continual exogenous NADH oxidation is required to maintain the enhanced IP3 level; removal of the exogenous NADH results in a decline in IP3with the rate being dependent on the levels and/or activation states of the enzymes involved in its breakdown and resynthesis to phosphatidylinositol 4,5-biphosphate. These properties would account for all of our experimental observations on the perturbative influence of exogenous NADH on K+ fluxes in corn roots and corn root segments (Kochian and Lucas, 1985;Lucas and Kochian, 1987). How, then, is it possible to explain the NADH-mediated stimulation of K+ influx observed in corn root segments, corn root protoplasts (Lin, 1982a, 1984) and carrot suspension cells (Misra el al., 1984)? Based on recent reports of plasmalemma-bound electron transport systems, we have included two forms of NADH redox systems in our model (Fig. 22). We propose that these systems would function only when the protoplast, cell or tissue is not in a “wound” state. The protoplast is the easiest system to

Fig. 22. Model summarizing the various effects of NADH on K+ influx into corn roots (or protoplasts). Two transplasmalemma NADH redox systems are shown. The Lin (1984) model (top) utilizes endogenous or exogenous NADH and transports both electrons and H+. The Rubinstein and Stern (1986) model transfers only electrons across the plasmalemma. In this model, generation of the H+ in the cytoplasm, by the oxidation of NAD(P)H, is compensated for by transport out of the cell by the H+-ATPase. In both models, stimulation of K+ uptake would occur by H+-K+ co-transport. We propose that the NADH-induced “wound response” exhibited by corn, is mediated by the coupling of a membrane-bound signal transduction system (STS) to the IPS cycle. Two agonist membrane receptors (MR) are postulated, one being an NADH oxidase [the 44 kDa protein from Lin (1984)], and the other being an alternative receptor mediating other types of wound response. Binding to the MR activates the STS (phospholipase C) via a membrane transmitter (MT). The resultant release of IP3 acts via the activation of protein kinases andor the release of Ca2+, to modify transport proteins (H+ATPase, cation channels) at the plasmalemma and tonoplast. Redrawn from Lucas and Kochian (1988).

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rationalize, because lysis during preparation could release proteases that actually remove the NADH MR protein. Buckhout and Hrubec (1986) also reported that washing their isolated plasmalemma preparation (with or without salt) resulted in a partial loss of NADH reductase as well as oxidase activity. In this state, the protoplasts may oxidize NADH (either exogenously supplied or via the cytosol) to cause an increase in the A& across the plasmalemma by direct transport of H+ (Lin, 1984; Bottger and Luthen, 1986) or by coupling to the H+-ATPase (Rubinstein and Stern, 1986). The IP, recycling system may also vary from cell to cell, or from one tissue to another, as a consequence of the physiological prehistory of the plant system. Since lithium acts as an inhibitor of IP, conversion to inositol and hence its resynthesis to phosphatidylinositol 4,5-bisphosphate, growing cultures in the presence of lithium may reduce the sensitivity of the IP, regulatory system, thereby allowing the NADH redox system to function. Introduction of myoinositol should re-establish the wound response. A further test of the IP,-regulation/wounding hypothesis may be achieved using FC. Chastain and Hanson (1982) have shown that in wounded corn roots FC can override the endogenous control mechanism that inhibits the plasmalemma H+-ATPase. It will be of interest to see whether FC can negate the perturbative influence that exogenous NADH has on K+ influx into roots. Purification of these putative redox systems of the plasmalemma should greatly assist the elucidation of their function. Questions of sidedness, direction of electron transport, and natural substrates (donors and acceptors) are all important issues that need to be resolved. Reconstitution of these proteins into liposomes may provide an unambiguous answer to the question of whether or not they are important in energizing K+ transport into roots.

IV. RADIAL K+ TRANSPORT TO THE XYLEM A symplasmic pathway for K+ movement, from the epidermis to the xylem parenchyma, was first proposed by Crafts and Broyer (1938). In their hypothesis, the cortex was the site where nutrients, like K’, were taken up into the symplasm by active, 02-dependent processes. Movement to the stele was via plasmodesmata, and release into the xylem vessels was by diffusion, or leakage, since the compact tissue of the stele was thought to be low in oxygen. Some 38 years later, Anderson (1976), in his review on solute transport across the root, concluded that, “In the stele, the most common proposal is that the parenchyma either leaks or secretes solutes which then diffuse or are swept along into the vessels.” Figure 23 is a diagrammatic representation of the pathway discussed by Anderson (1976). Based on Anderson’s reviews, it

POTASSIUM TRANSPORT IN ROOTS Epidermis

Cortex

~

Endodermis

near- unity 0 value

w

137

e

low d value

7

External

-

APOPl and water fluxes

probably turgor drlven

Arrows show @ fluxes

Apo$asmic barrier

Fig. 23. Diagrammatic sketch of the root in cross-sectional view showing what Anderson (1976) considered to be the “usually accepted mechanisms of ion and water through-putto the xylem vessels.” Stippled areas represent the cytosol which, in this Anderson model, also extends in the xylem vessel. From Anderson (1976).

would appear that over the period from 1938 to 1976 the only improvement to the Crafts and Broyer hypothesis was the concept that solutes might be secreted into the xylem vessels. However, the mechanistic basis for this “secretion” remained poorly defined. Note also that Fig. 23 suggests that the xylem vessels may be symplasmically connected to the surrounding parenchyma and contain parietal cytoplasm. For those who are quick to be critical of this concept, we should point out that the controversy of whether living xylem vessels are engaged in the release of K+ to the translocation stream is still unresolved (Lauchli et al., 1978; McCully et al., 1987). Clearly, the complexity of the root has made it difficult to obtain a rigorous test of the Crafts and Broyer hypothesis. However, experiments of the past decade have provided some new insights. A. SITE OF K+ ENTRY INTO THE SYMPLASM

1. Epidermis or Cortex? Most current textbooks on ion transport and plant physiology discuss K+ movement into the cortex in terms of two parallel pathways, namely the apoplasmic and symplasmic routes. However, there is still some question as to the relative importance of each component (Anderson, 1976; Lauchli, 1976; Van Iren and Boers-van der Sluijs, 1980; Kochian and Lucas, 1983). Using a theoretical model for Rb’ uptake into barley roots, Bange (1973) found that the model only generated reasonable profiles when transport was

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restricted to the epidermis. The studies of Van Iren and Boers-van der Sluijs (1980) offered experimental support for this hypothesis. These workers investigated K+ uptake by applying the assumption that plasmolysis would sever plasmodesmata, thereby symplasmically isolating each cortical and epidermal cell of a barley root. Autoradiographic localization of 86Rbf, accumulated by the root following plasmolysis, was generally limited to the root periphery. These results suggested that when roots are exposed to low K+ concentrations (<1 mM), transport into the symplasm occurs at the epidermis; i.e. the apoplasmic pathway and subsequent uptake by the cortical cells is of little significance. The criticism which must be levelled at this work is that it may be erroneous to base a hypothesis concerning “normal” transport function on data obtained from a system where transport physiology may have been dramatically changed by plasmolytic treatment. Furthermore, the work of Anderson and Reilly (1968) concerning fluid exudation from the xylem of corn roots indicated that the cortical cells do have the capacity to absorb ions. They found that surgical removal of the epidermis and outer cortex of excised corn roots did not prevent significant fluxes of ions and H 2 0 to the xylem. Kochian and Lucas (1983) examined this issue by using radioactively labelled sulphhydryl reagents, [ 3H]N-ethylmaleimide (NEM) and [*03Hglpchloromercuribenzenesulphonic acid (PCMBS). A brief (30-60 s) exposure of corn roots to either NEM or PCMBS dramatically reduced K+ influx into the root, without affecting root respiration. Autoradiographic localization studies revealed that sulphhydryl binding occurred almost exclusively in the cells of the root periphery (see Fig. 24). However, protoplasts isolated from corn root cortical tissue exhibited significant “Rb+ influx, the kinetics of which were identical in shape to those obtained on corn roots (Kochian and Lucas, 1982a, 1983). These results suggest that although cortical cells possess the capacity to absorb ions at the low concentrations likely to be present in normal soils, Kf influx into the root symplasm most probably occurs at the root periphery.

2. Aerenchyma Induction The role of the cortex in K+ uptake and radial movement into the stele has been investigated by the induction of aerenchymatous tissue (Drew et al., 1980; Drew and Saker, 1986;Deacon etal., 1986). Drew et al. (1980) showed that an 02-stresstreatment of young corn roots induced aerenchyma in the developing, adventitious roots (Fig. 25). Microscopic examination of this root tissue revealed extensive breakdown of cells within the mid-cortex, while the epidermis, endodermis and stele remain unaffected. A comparison of this aerenchymatous tissue with control roots revealed that the volume of the cortex was reduced by 36% and that there was an 80% reduction in the symplasmic pathway available for K+ movement from the epidermis to the stele. Thus, if the cortex were involved in Kf uptake, the flux into these

POTASSIUM TRANSPORT IN ROOTS

139

60

5 8

so

30 a NEM Trootmont

40

a a

3

10 0

Fig. 24. Microautoradiographic localization of [3H]N-ethyl maleimide (NEM) following a

3fJ-9exposure of corn root segments to 0.3 mM NEM. Numerous cross-sections were analysed

for grain distribution with an Imanco Quantimet 720 Image Analysing Computer. These data have been averaged and presented in the histogram below the micrograph. Data from Kochian and Lucas (1983).

Fig. 25. Scanning electronmicrograph illustrating the aerenchyma that develops in nodal, adventitious roots of corn grown in non-aerated culture solution. Root sections were cut from the region 8-10 cm from the root tip and lyophilized before being viewed in the electron microscope. Symbols are as follows: C, cortical air spaces; W, wall residues of collapsed cells; I, intact cells linking inner and outer cortex. (Bar represents 500 pm.) From Drew etal. (1980), with permission.

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aerenchymatous roots should be reduced relative to control roots. Drew and Saker (1986) investigated the ion-transporting capability of such corn roots and found that, with respect to K', PO$- and C1-, aerenchymatous roots were at least as effective as control roots. These results obtained using aerenchymatous roots are consistent with the hypothesis that ion uptake, at low concentrations, occurs primarily at the epidermis. In view of this lack of effect of a reduction in the symplasmic pathway, it would also appear that the radial flux to the stele is rate-limited by transport into the symplasm (see Anderson, 1976). However, a note of caution is needed in the interpretation of these flux data. The experimental sys;tem used by Drew et al. (1980) and Drew and Saker (1986) imposed a relatively thick unstirred layer at the surface of both control and aerenchymatous roots. It is possible that the equivalent performance of the two root types was due to a limitation in the supply of nutrients to the actual root surface. This possibility should be investigated using a well-mixed bathing medium. Alternatively, flux analysis using the technique of Newman et al. (1987) might be informative. B. RADIAL PATHWAY

1 . Symplasmic Route Although the symplasmic pathway is the currently accepted route for K+ movement from the epidermis to the stele, there have been only a few studies on plasmodesmatal structure, frequency and function in root tissues. Spanswick (1972) was the first to demonstrate that corn root cortical cells are electrically connected via plasmodesmata. Clarkson and Robards (1975) showed that plasmodesmatal connections exist between cortical, endoderma1 and stelar parenchyma cells in the mature, transporting region of the root. The elegant work by Overall et al. (1982) and Overall and Gunning (1982) provided important information on the substructure of the plasmodesma in Azolla roots but, unfortunately, this work focused on the root apex. Consequently, their findings with respect to symplasmic coupling cannot be extended to the mature region of terrestrial root systems. Plasmodesmatal frequency distribution studies have been conducted on the root apex of corn (Juniper and Barlow, 1969), and this work indicated that as the cells matured, the number of plasmodesmata per unit area decreased. However, this study also focused on developing tissue. Perhaps the most interesting study, from the viewpoint of radial transport, is the electrical systems analysis of Lipidium sativum roots reported by Behrens and Gradmann (1985). By employing a combination of microelectrodes located at various positions along the root, it was possible to detect electrical anisotropy within the root, there being a preference for longitudinal over transverse (or radial) coupling. These results suggest that further studies are

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needed on the spatial distribution of plasmodesmata within the cortical tissue of mature roots. It seems almost inconceivable that the concept of symplasmic K+ transport is so well entrenched, in view of the almost complete absence of supporting cytological data obtained on mature root tissue.

2. Compartmentation and the Endoplasmic Reticulum? In the Robards (1971,1975) model of the plasmodesma, there is continuity between the endoplasmic reticulum (ER) of neighbouring cells, via the desmotubule. Lauchli and others have extended this concept of an ER continuum in terms of providing a specialized K+ transport compartment in roots (see Fig. 26). Much of the justification for this special compartment comes from X-ray microprobe analysis and cytohistochemical precipitation data. Lauchli et al. (1971) analysed the radial distribution of K+ within the roots of 8-day-old corn seedlings. Roots were pretreated for 4 h in 0.2 mM KCI plus 0.5 mM CaS04,before the terminal 1cm was removed and subsequent 1 mm sections cut and immediately frozen in liquid N2. Following freezesubstitution and embedding in Spurr's resin, anhydrous sections were cut (1-2 pm) and analysed for K+ distribution using X-ray microprobe. The reported line scan indicated a high level of K+ in the epidermis and within the cells of the stele; the xylem parenchyma appeared to contain the highest levels of K+. Clearly, there are problems in evaluating these X-ray microprobe data, the question of artefacts generated during tissue preparation being the most serious. Of course, there is also the problem of spatial resolution, with the possibility of contamination from the vacuole and, to a lesser extent, the cell walls. To circumvent these problems, Lauchli et al. (1977) used the bulktissue frozen-hydrated specimen protocol developed by Lauchli (1975). Barley root segments, obtained from a position 1-2 cm from the apex of soil-grown tissues, were frozen in liquid N2 and then fractured under liquid Xylem

Vessc1

Fig. 26. Pathways of ion (K') transport in roots, involving the endoplasmic reticulum (ER) as a special compartmentof the symplasmicroute. Symbols: C, cytoplasm; C,, Casparian strip; V, vacuole. From Lauchli (1976), with permission.

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N2. This tissue was then quickly transferred to an SEM cryostage (held at - 15OOC). A comparison of the X-ray spectra collected from epidermal and xylem parenchyma cells again revealed that K+ was higher in the xylem parenchyma. However, the K+ level in the metaxylem vessels was suspiciously high, perhaps indicating the presence of developing vessel elements (McCully et al., 1987). Although one might have more confidence in the K+ distribution determined on frozen-hydrated tissue, one must not lose sight of the fact that barley roots are still quite thick and so ice crystal artefacts cannot be discounted, especially deep within the root where the rate of cooling would be the slowest. Spatial resolution on bulk frozen-hydrated tissue is also a serious limitation, especially when the SEM is operated at 10 keV. Since the cytosolic compartment in the barley xylem parenchyma cells was less than 0.5 pm [see Fig. 5 of Lauchli et al. (1977)], it is highly unlikely that the system could resolve K+within the cytosol. Thus, provided that ice crystal artefacts are not seriously affecting the K+ concentrations, the above data establish that the K+ level in the vacuole of the xylem parenchyma is higher than elsewhere in the root. The problem of spatial resolution on frozen-hydrated root tissue was further investigated by Echlin et al. (1981) and Pitman et al. (1981). In these studies, and especially in the latter, it is clear that the SEM system was operating at the limit of resolution. Additionally, the images showed clear evidence of ice crystal artefacts and so X-ray microprobe data on such tissue must be interpreted with caution. Finally, Pitman et al. realized that their data on relative distributions of K+ and Na’ were of a preliminary nature with respect to assigning valid concentrations. The problem of chemical activities versus “bulk” concentrations remains a formidable problem. Even with the above-mentioned limitations of the X-ray microprobe technique, data collected on root tissues by several groups show that variations in K+, Na+ and C1- distributions, among individual cells of the root, do not correlate with their spatial position in the root (Lauchli et al., 1977; Echlin et al., 1981; Pitman et al., 1981; Chino, 1981; Harvey, 1985). As stressed by Pitman et al. (1981), these spatial inhomogeneities in ionic distribution draw into question one of the most basic assumptions underlying radioisotope flux analysis, namely, a homogeneous distribution of the label. Finally, at present, it is our assessment that statements made about K+ concentration gradients across the root must be interpreted in terms of vacuolar changes. It is anticipated that X-ray microprobe studies, conducted with more advanced systems capable of higher spatial resolution and lower accelerating voltages, will provide the much needed information on cytosolic versus vacuolar concentrations at all sites across the root. Without such data, it will be difficult to make progress in elucidating the role played by the xylem parenchyma in K+ transport into the xylem. A final comment on the putative role of the ER in K+ transport is

POTASSIUM TRANSPORT IN ROOTS

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necessary. Although no unique model of the plasmodesma is accepted at present, the model proposed by Overall et al. (1982) (see also Lopez-Saez et a f . , 1966) appears to be consistent with most studies on symplasmic permeability (Erwee and Goodwin, 1983, 1984; Erwee et a f . , 1985; Madore et a f . , 1986; Terry and Robards, 1987). In this model, the “desmotubule” is presented as a solid phospholipid rod, not an open tubule as in the Robards model. Movement through the plasmodesma is considered to occur via the “cytoplasmic annulus” and not via the ER-associated desmotubule. Hence, the role of the E R in intercellular K+ transport should be viewed as being entirely speculative and perhaps at variance with the current view of the structure of the plasmodesma. C . LAG PHASE IN XYLEM LOADING

An extensive literature exists on the application of compartmental flux analysis to K+ movement across the root. Theoretical aspects of this work have been reviewed by Walker and Pitman (1976). For a broad description of compartmental flux analysis, as it pertains to ion transport across the root, the reader is referred to Jeschke (1980) and Pitman (1982). For the present, we will focus on the phenomenon of the lag phase in K+ transport into the xylem, to illustrate the complex nature of the processes controlling K+ movement into the various compartments of the root (see also Section V). When low-salt-grown plants are transferred from their CaS04medium to a solution containing K+ (or “Rb’), there is immediate uptake into the symplasm of the root.,However, as shown by the data presented in Fig. 27, transport into the xylem translocation stream is delayed, or lags behind root uptake by a period of 1-4 h (Hooymans, 1976; Bange, 1977; Glass, 1978; Van Iren e t a f . ,1981). Van Iren etal. (1981) showed that this lag was not due to a time-dependent penetration of K+ into the stele. Also, the extent of the lag phase was independent of both the amount of K+ in the bathing medium and the extent of Kf uptake by the root (Fig. 27). However, it would appear that the abrupt transition from root accumulation to transport to the xylem is influenced by the rate at which the root is taking K+ into the symplasm (see Fig. 27c). In subsequent studies, Bange (1977,1979) has shown that during this lag phase, %Rb+accumulation into the vacuole starts slowly, even though the cytoplasmic “compartment” seems to fill quite rapidly. Consequently, the difference between the lack of K+ accumulation in the barley roots bathed in 5 and 10 ,UM K+ (Fig. 27a,b) and the accumulation found in 1.0 mM K+ (Fig. 27c), must reflect the operation of a regulatory mechanism that in some way senses the K+ requirement for xylem translocation. When this requirement is exceeded, vacuolar transport of K+ appears to be activated [but see Glass (1978) for an alternative explanation]. At present no informa-

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L. V. KOCHIAN AND W. J. LUCAS

(a 1 5pM Kt

c

I

I I

1 2 3 4 5 6 7 8

0

root

I

1 2 3 4 5 6 7 8

12 34 5 6 7 8

Time (hours) Fig. 27. Influence of extracellular K+ levels on Kf uptake into intact barley roots (0)and as a function of time after roots were introduced to subsequent translocation to the shoot (0) K2S04.Experimental solutions contained 5 mM CaCI2,0.1 mM Ca(HC03)2and 5, 10 or 1000 ~ L K+ M in (a), (b) and (c), respectively. Note the pronounced lag in the supplyof K+ to the shoot. Data from Hooymans (1976).

tion exists as to the nature of this putative sensing mechanism (see Section V for a further discussion on regulation of K+ transport within the root). Hooymans (1976) argued that the insensitivity of the lag phase to both external and bulk root K+ concentration supported the hypothesis that a specialized compartment or “organelle” was involved in K+ transport to the xylem. Glass (1978) offered a seemingly more plausible hypothesis that the lag phase reflected an inductive period associated with establishing the conditions necessary for K+ transport into the xylem. Induction by high symplasmic Kf concentration (Glass, 1978) would be inconsistent with Hooymans’ (1976) data; consequently, a threshold K+concentration may be involved in the induction process. Van Iren et al. (1981) attempted to test this induction hypothesis by giving low-salt-grown barley plants a brief exposure to K+. They argued that this brief treatment would allow sufficient K+ to enter the symplast to activate the induction process. If this were correct, the lag would be either absent or considerably shortened when these pretreated plants were resupplied with K’. In experiments where a 5-min K+pretreatment was employed there was no observable reduction in the lag period. It is unfortunate that Van Iren et al. did not investigate the effect of longer pretreatment periods.

POTASSIUM TRANSPORT IN ROOTS

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Another approach to studying the nature of this lag phase would be to employ p-fluorophenylalanine (FPA), an analogue of the amino acid phenylalanine, which produces abnormal or defective proteins. A review of the effect of this compound on transport across the root is given by Pitman (1977). When high-salt barley roots are treated with FPA, transport into the root remains unaffected, whereas transport into the xylem becomes inhibited (Schaefer et al., 1975). The response to FPA is quite rapid, which indicates that the essential “transport” protein(s) involved in the release of K+ or C1- to the xylem turns over quite rapidly. Low-salt-grown roots could be pretreated with FPA, for various times, prior to introducing K+ to commence the induction phenomenon. Roots pretreated in FPA should not commence transport into the xylem, but upon FPA removal transport should commence almost immediately if a threshold level of cytoplasmic K+ is involved. Studies by Jeschke (1984) also implicate a role for the phloem in the duration of the lag period. On a speculative note, it may be that the induction of transport to the xylem may be affected by the level of K+ recirculating to the root via the phloem. In any event, the abrupt transition to xylem loading (Fig. 27) is at variance with loading via K+ leakage (see Fig. 23), and at first sight seems inconsistent with K+ release into the translocation stream by late-maturing xylem elements (McCully et a l . , 1987). D. K+ TRANSPORT INTO THE XYLEM

Roots grown in a full nutrient medium (high-salt status) take up K+ and transport a major part of this influx to the shoot. By contrast, in low-saltgrown roots a definite lag period exists between K+ entering the root and its transfer to the xylem (see Fig. 27). Numerous studies have contributed to this type of phenomenological characterization of the transport of mineral nutrients from the soil solution to the shoot (Pitman, 1977,1982). Although it was necessary to utilize a flux compartmental analysis approach in many of these studies, the data could be evaluated without the assignment (or knowledge) of specific transport mechanisms. The active or passive nature of these specificfluxes (to the symplasm and the xylem vessels) was deduced from studies involving various metabolic inhibitors and chemical agents, as well as from frustratingly difficult attempts at determining the electrochemical potential gradients for the various ions (Lauchli, 1976; Bowling, 1981; Pitman, 1982). Although it may come as a surprise to the reader, it was not until 1978 that Mitchell’s chemiosmotic hypothesis was applied to solute transport at both boundaries of the symplasm; i.e. at the plasmalemma of the epidermal and xylem parenchyma cells. Hanson (1978) developed a “speculative” model in which he placed two “opposing” H+-translocating ATPases in the plasma

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membranes of the outer cortex and the stelar parenchyma. The impermeable nature of the Casparian strip of the endodermis separated the apoplasmic solutions of the cortex and stele, with the symplasm operating as a “bridging” osmotic unit. Transport of cations and anions was proposed to occur by various uniport and antiport systems. It is worth stressing that K+ transport into the xylem was hypothesized to take place by a K+-H+ antiporter with the flux being dependent on the A&. An important prediction of the Hanson (1978) hypothesis was that the transroot potential, measured between the apoplasm of the xylem and the bathing medium, should have two opposing (or “antagonistic”) electrogtnic components. Although many electrophysiological studies had been conducted on the nature of the transroot potential, no study indicated the presence of an electrogenic component at the xylem parenchyma plasma membrane (Shone, 1968,1969; Davis and Higinbotham, 1969; Ansari and Bowling, 1972; Bowling and Ansari, 1972). In general, these studies suggested that the transroot potential (measured from the xylem with respect to the bathing medium) could be interpreted based on the transmembrane potential difference equivalent to that of a single cell. Although their experiments were conducted on an excised hypocotyl-root system from Vigna sesquipedalis, Okamoto et al. (1978, 1979) were able to demonstrate the existence of opposing electrogenic pumps between the surface of the hypocotyl and the xylem vessels. De Boer et al. (1983) constructed a similar experimental system to investigate the transroot potential of two Plantago species. The elegant system developed by De Boer et al. (1983) is illustrated in Fig. 28 (the reader is directed to the original paper for important technical details). An investigation of the response of excised Planfago roots to changes in O2partial pressure revealed three classes of transroot potentials. One class, having transroot potentials of approximately -100 mV, was comparable to the earlier reports of Davis and Higinbotham (1969) and Shone (1968, 1969). The second and third class of roots had transroot potentials from 0 to -20 mV and -50 to -70 mV, respectively, with the latter class being the less frequently observed of the three classes. The response of the second class of Planfago roots to changes in the O2 partial pressure is illustrated in Fig. 29. Anoxia, and recovery from anoxia, elicited a transient change in the transroot potential (Fig. 29a), while exposure of the root to 10% oxygen resulted in a shift in the transroot potential from -20 mV to -60 mV (Fig. 29b). An electrical analogue of Hanson’s (1978) model is presented in Fig. 29c. As discussed by De Boer et al. (1983), this model can readily account for the observed responses in the transroot potential when the excised Plantago root undergoes changes in oxygen status. If the two opposing electrogenic components are functioning and almost equal, their net effect on the transroot potential will be small, and the value of the potential will depend

Fig. 28. Experimental system used by De Boer et uf.(1983) to simultaneously measure the transroot potential(TRP), ionic activitiesfor H', K+and Na', and the xylem sap flow rate (Jv) on an excised Pfuntugo root. Symbols: A, amplifier;A', precision intrumentationamplifier; SOS, slotted optical switch. From De Boer et al. (1983), with permission.

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-80

I 40

-60

'

-, E

30 20

-40

10

-

oe 40

4

30 -40

20

-20

10

0

50 ( C )

Time

100 150 minutes 1

1

200

(

Potential Profile

I

Caaparian strip

Fig. 29. Response of the transroot potential of Plantago to changes in the O2tension of the medium bathing the roots. (a) Roots brought under anoxia and then returned to 30% 02. (b) Oxygen tension reduced from 30% to 10% and then subsequently returned to 30%. (c) Schematic representation of the electrophysiological organization of the Planfago root. The Em of the epidermaUcortical cells ( E l ) and the xylem parenchyma cells (Ez) result from the combined EMF of the passive diffusion potential (EDland E D 2 , respectively) and the electrogenic pump (Epl and Ee2, respectively). The hyperpolarization produced by the electrogenic pumps (EHypIand EHm) will depend upon the EMF and conductances ( g ) of the pump and passive diffusion systems. The transroot potential is given by: TRP = El - E2 = (EHYPI + EDI)- (EHY~L + ED^). Data from De Boer et al. (1983).

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upon the difference between the diffusion potentials generated at the boundary of the cortex and the xylem parenchyma. When the O2level in the bathing solution is lowered from 30 to 10% (Fig. 29b), the O2supply to the inner regions of the root will be reduced, causing a decline in the available ATP level, and so the activity of the electrogenic pump on the xylem parenchyma plasmalemma will be decreased. The outer region of the root will still have an adequate O2supply, and so the continued operation of the outwardly directed H+-ATPase will cause the transroot potential to shift to a more negative value (Fig. 29b). Anoxia eventually reduces the activity of both H+-ATPases, but the differing time courses of the decline in ATP give rise to the observed transient change in the transroot potential (Fig. 29a). The electrophysiological data obtained on Pluntugo roots (De Boer et al., 1983; De Boer and Prins, 1984, 1985) and the related measurements conducted on Vigna hypocotyls (Okamoto et ul., 1978, 1979) provide support for the most important aspect of the Hanson model for ion transport into the xylem. Further support is also offered by the xylem perfusion studies of Clarkson et al. (1984) and Clarkson and Hanson (1986). Using a special xylem perfusion system, these workers introduced unbuffered solutions of different pH values into excised roots of AZZium cepa. Irrespective of whether the initial pH value was acidic (pH 3.9) or basic (pH 9.3), the pH value of the perfusate leaving the root was adjusted to within a range of pH 5.5-6.5. Furthermore, the extent of H+ efflux into the xylem perfusate could be increased greatly by buffering the medium at alkaline pH values. It is also interesting to note that malate has been reported to be a normal constituent of the xylem sap in some roots (Butz and Long, 1979); the pK,(2) of malate is -5.3. These pH shift data were interpreted in terms of pH regulation of the xylem sap, in situ; a process which would be essential if K+ transport into the xylem were to occur via a K+-Hf antiporter as suggested by Hanson (1978). Figure 30 represents a synthesis of current data on K+ transport into the symplasm of the root and its efflux across the xylem parenchyma to the xylem vessel. Unfortunately, the complexity of the root, and especially that of the stele, has made it extremely difficult to conduct the necessary biophysical experiments to gain further data in support of the Hanson model. Although various attempts have been made to determine electrochemical gradients across the cells of the root (Dunlop and Bowling, 1971; Bowling, 1972; Dunlop, 1973,1982;Davis and Higinbotham, 1976), these studies generally involved making successive electrode impalements through cells of the epidermis, cortex and stele. As stressed by Anderson and Higinbotham (1975), great caution should be used when evaluating these data. Thus, with the spatial resolution of the X-ray microprobe system(s) used to examine K+ distribution across the root being such that only vacuolar estimates can be deemed reliable, and the microelectrode studies on the stelar tissue being

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Fig. 30. Schematic representation of the transport processes thought to be involved in the acquisition of K+ from the soil solution and in its subsequent transport from the symplasmic compartmentof the root into the xylem vessels. This model has been developed on the basis of the chemiosmotic scheme originally proposed by Hanson (1978) and later refined by Clarkson and Hanson (1986). (Open “arrowed” circles on tonoplast represent regulation of K+ movement between the cytoplasm, vacuole and radial transport “pools”, and rectangles represent channels.)

somewhat dubious, precious little information is currently available concerning the Kf gradients across the root. The only reliabledata concerns the K+ concentrations within the xylem vessels, and even here one must be concerned with extrapolation from in vitro measurements to the intact root. As a consequence of these limitations, we can only make general comments on the thermodynamic nature of the Kf fluxes at the two boundaries of the root symplasm. Provided that the Donnan charges within the epidermal cell wall do not result in a significant increase in the K+ concentration at the outer surface of this plasma membrane, the typical gradients illustrated in Fig. 30 indicate that net Kf influx into the symplasm is clearly an active process. Our comments concerning the situation at the xylem parenchyma plasmalemma cannot be made with the same degree of certainty. If the K+ concentration in the cytoplasm is within the range expected for normal physiological functioning (250mM), and the steady-state level in the xylem vessels is approximately 5 mM, the thermodynamic status of K+ efflux will depend very much on the magnitude of the potential across this membrane. If the value is similar to that deduced for the Plantugo root system, then K+ efflux would necessarily occur by an active process. Although the K+-H+ antiport hypothesis is still purely speculative, if K+ were to be transported by such a mechanism, with a 1: 1stoichiometry, the Em would not influence the thermodynamics of the situation, but it could have a significant influence on Kf efflux through its effect on the kinetic parameters of the putative transport system.

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It is perhaps indicative of the complexity of the root that hypotheses like the Crafts and Broyer (1938) and Hylmo (1953) models have been so difficult to disprove. It is interesting to note that the Hylmo model was recently resurrected by McCully et al. (1987). The Hylmo model proposes that ion accumulation in the root involves uptake into the vacuoles of living xylem vessels, which upon maturation release their contents into the translocation stream. Although this model received some degree of support in the mid-1970s (Davis and Higinbotham, 1976), Lauchli et al. (1978) provided irrefutable evidence that ion transport occurs across the stele into mature xylem vessels. However, in the recent report by McCully et al. (1987), X-ray microprobe was used on bulk, frozen-hydrated tissue, to examine the K+ concentrations within the late-maturing xylem vessels of field-grown corn plants. Surprisingly, K+ values in the range of 150-400 mM were detected; the microprobe system was “calibrated” against known KC1 standards that were vacuum-infiltrated into the mature xylem vessels. Clearly, these levels of K+ could only be established by transport across the tonoplast of these maturing vessels. In view of these findings, McCully et al. claim that since the living, late-maturing xylem elements contain the highest K+ concentrations of any cells in the root, they “represent the end point of potassium accumulation from the soil before it is released as a slug of concentrated solution to the transpiration stream in the mature vessel”. In view of all the available data, it seems to us that a combination of the two routes may not be completely unrealistic.

V. REGULATION OF K+ FLUXES WITHIN THE PLANT In contrast to the situation in animal cells where homeostasis is mediated primarily at the tissue or organ level, plants must be able to regulate their metabolism at the cellular level. This is particularly true for the cells of a root growing in the soil, which can be exposed to a wide range of environmental conditions. Glass and Siddiqi (1984) suggest that two primary priorities for the regulation of inorganic nutrients in the plant are the maintenance of ionic composition of the cytoplasm and nutrient allocation to the shoot. These priorities are particularly applicable to K+.Although the levels of K+ do not appear to be precisely regulated, the cytoplasmic values seem to be constrained within certain limits. In glycophytic plants, cytoplasmic K+ concentrations range from 100 to 200 mM (Leigh and Wyn Jones, 1984). It has been suggested that maintenance of K+ within these limits is important because of its roles in activating a number of cytoplasmic enzymes and in protein synthesis (Lauchli and Pfluger, 1979; Wyn Jones et al.,1979). Unfortunately, direct measurements of cytoplasmicK+ have been difficult to accurately obtain. However, it has been well documented that “bulk” K+ concentrations are generally maintained at fairly constant levels, even in the

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face of widely varying Kf supply to the root. For example, Asher and Ozanne (1967) studied the growth and K+ content of 14 plant species grown at external K+ concentrations held constant at levels varying from 1 to . found that at 24 PM K+, eight of the species reached 1000 p ~ They maximum yield, while at 95 p~ K', the remaining species reached maximum yield, and increases in external K+ above 95 p~ had little further effect on K+ content or root :shoot ratios. Although our knowledge of ion transport regulation is far from complete, most of the work that has been conducted has been directed at the cellular level. A large portion of this work has focused on the regulation of K+ uptake across the plasmalemma of higher plant roots, undoubtedly because of the important metabolic and osmotic roles played by this ion. An examination of the pathway of K+ movement from the root surface to the shoot indicates a number of potentially important regulatory sites. These include the plasmalemma of epidermal cells, the tonoplast of epidermal and cortical cells, the plasmodesmata hypothesized to be involved in maintaining symplasmic continuity along this pathway, and the xylem parenchyma, which may form the site of K+ transfer into the xylem translocation stream. The location where ions are transported into the xylem vessels appears to be an important focal point for root-shoot interaction, in terms of regulatory signals moving between the shoot and root. We will advance the concept that there is a hierarchy of regulatory mechanisms operating to regulate K+ within the plant which extend from the subcellular to the whole plant level. These various levels of regulation are thought to establish a feedback system which influences K+ uptake across the plasmalemma of root epidermal cells. We will deal with regulation of K+ transport at the level of the plasmalemma. Regulation of transport across the root will then be examined in terms of the importance of the shoot in the regulation of root Kf fluxes. A. ALLOSTERIC REGULATION OF K+ TRANSPORT

A number of studies that have involved either bulk tissue K+ measurements, or indirect measurements of cytoplasmic K+ (i.e. compartmental analysis), have suggested that cytoplasmic K+ is maintained at fairly constant levels, while vacuolar K+ concentrations can vary more dramatically (e.g. Glass and Siddiqi, 1984; Leigh and Wyn Jones, 1984; Memon et al., 1985). Hence, a number of researchers have sought to uncover the regulatory mechanisms involved in the maintenance of cytoplasmic K+ levels. Certainly the most widely discussed and studied model involves allosteric regulation of the plasmalemma K+ transport system by cytoplasmic Kf concentration. I . Feedback by Internal K + A negative correlation between K+ uptake and internal K+ concentration ([KIi) has been reported for a number of plant tissues (Johansen et al., 1970;

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Young and Sims, 1972; Glass, 1975, 1976, 1977; Jensen and Pettersson, 1978; Cram, 1980; Kochian and Lucas, 1982a). In many of these studies, K+ influx was correlated with bulk tissue K+ levels. However, in the work of Young and Sims (1972) and Glass (1977), the adjustment of K+ influx and [K], following either an increase or decrease in the external Kf concentration was quite rapid and correlated with known half-times for the turnover of cytoplasmic K+. These results provide circumstantial evidence in support of the hypothesis that a regulatory feedback loop exists between cytoplasmic K+ levels and K+ influx across the plasmalemma. The K+ transport protein(s) may possess both catalytic (transport) and regulatory (allosteric) sites (Cram, 1976; Glass, 1975, 1976, 1977, 1978; Jensen and Pettersson, 1978; Pettersson and Jensen, 1978, 1979). Binding of cytoplasmic K+ to internal allosteric sites on the transporter may induce a conformational change in the protein which would result in a reduction of K+ influx. Further evidence in support of the allosteric regulation hypothesis for K+ influx into roots has been provided by the work of Glass and co-workers (for reviews see Glass, 1983; Glass and Siddiqi, 1984). Glass (1975) observed that when low-salt barley roots were being exposed to high exogenous K+ levels, in order to increase [K], , Kf influx was inversely related to the square of [K],, a result previously reported for Lemna (Young and Sims, 1972). Subsequently, the kinetics for Kf influx at low external K+ concentrations ( ~ 0 . mM) 5 were obtained on barley root tissue in which different [K], levels had been established (Glass, 1976). As shown in Fig. 31a, as [K], increased from 26.3 pmol g-' to 130.5 pmol g-', the V,,, for the saturable component of K+ influx decreased, and the related K,,, increased. Replotting these data

( b ) \O-

r

- 3 c

+-

E

Y

1 0 40

Extemal K+ConcentraUon (mM)

80

120

Root K+ Content ( pnd g-' 1

Fig. 31. Evidence for allosteric control of K + influx into barley roots. (a) Influence of pretreatment in 50 mM KCI upon the influx kinetics obtained on low-salt-grown barley roots. 0 h; 0 ,3 h; W, 6 h; A, 12 h duration in 50 mM KCI. (b) Relationship between Symbols: 0, influx values from solutions containing various KCI concentrations as a function of [Kli. Symbols(mMKCI):0,0.32;0,0.16; 0,0.08; W,0.04; A,0.02; A,O.Ol. DatatakenfromGlass (1976).

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as a function of [K], indicated that a sigmoidal relationship exists between K' influx and [K], (Fig. 31b). Glass (1976) noted that these observations are characteristic of an enzyme exhibiting allosteric regulation. He applied a transformation of the Hill equation to these kinetic data to determine the number of allosteric binding sites [a description of the use of the Hill equation can be found in Segel (19791. Linear Hill plots were obtained from which Glass deduced that four allosteric sites were operational on each transport protein. Glass has quite cautiously suggested (due to the dangers inherent in extrapolating from simple enzyme kinetics to a complex tissue such as a root) that in low-salt barley roots the K+ carrier possesses one binding site for external K' and four allosteric binding sites for [K],. Similar results have been subsequently obtained for K+ influx into the roots of a number of different plants (Jensen and Pettersson, 1978; Pettersson and Jensen, 1979). In low-salt-grown roots, the reduction in net K' influx, by internal K + , was not due to a change in K+ efflux (Glass and Siddiqi, 1984). This is not surprising, since it is well established that in low-salt roots net K+ uptake is equivalent to unidirectional K + influx, indicating that K' efflux is quite small (Johansen et al., 1970; Glass and Siddiqi, 1982; Newman et al., 1987). It had been previously reported that as excised barley roots were loaded with KCl, K+ influx remained constant but efflux increased (Jackson and Edwards, 1966). However, the fluxes in this study were measured in 10 mM KCI, which is in the range of passive K+ uptake that we suggest is mediated by K+ channels. In this concentration range, K+ influx does not respond significantly to [K], (Glass and Dunlop, 1978). It would appear that only the high-affinity K' uptake system (K'-H+ symport or K+-ATPase?) is subject to feedback control by internal K + (see Fig. 20). 2. Involvement of Protein Synthesis andlor Degradation? The response of the K+ transport system to changes in [KIi can be quite rapid, which indicates a direct control on the transport system, as opposed to regulation via synthesis or degradation of transport carriers, or regulatory proteins (Glass, 1977; Glass and Siddiqi, 1984). Glass (1977) found that the reduction in K+ influx, in response to increasing [K],, showed no lag and appeared to be independent of RNA, DNA and protein synthesis. Pettersson and Jensen (1979), on the other hand, have hypothesized that a combination of both allosteric regulation and carrier synthesis occurs either simultaneously or sequentially during the period of adjustment following a perturbative treatment. Unfortunately, for higher plants, techniques have not been available that allow for the demonstration of transport regulation through carrier synthesiddegradation. As Glass and Siddiqi (1984) point out, even though direct evidence is lacking, a realistic model for transport regulation would involve short-term adjustments involving rapid responses of transport systems to

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changes in internal ion levels, in conjunction with long-term adaptations (hours or days) involving control of carrier synthesis and degradation. In this context, recent work by Fernando etal. (1987) indicated that K+ deprivation in barley roots resulted in an increase of both a 34 and a 44 kDa plasmalemma protein, as well as K+ uptake. However, the increase in K+ uptake occurred before any changes in the plasmalemma protein complement could be detected. Fernando et al. (1987) speculated that allosteric regulation occurs prior to a stimulation in the synthesis of specific plasmalemma proteins (in response to Kf deprivation). Clearly, further studies in this area are needed to confirm this hypothesis.

3. Regulation of Tonoplast K + Transport? The complex structure of the root makes it difficult to measure tonoplast fluxes and vacuolar content. In general, only approximate values can be obtained through radioisotope compartmental analysis studies. Glass and Siddiqi (1984) have analysed this literature and conclude that as the root is loaded with K+, both plasmalemma and tonoplast K+ fluxes are reduced, with the greater reduction being observed at the tonoplast. Leigh and Wyn Jones (1984) have also proposed that a coupling exists between the cytoplasmic and vacuolar K+ pools. They developed a model which attempts to explain the relationship between growth and tissue Kf levels. The model is based on the obsecvations that: (1) K+ is not replaceable in the cytoplasm; (2) K+ salts in the vacuole that are involved in turgor generation can be replaced by other solutes like Na’, Mg2+,Ca2+and organic molecules, during periods of K+ deficiency; and (3) K+ is often unequally distributed between the vacuole and cytoplasm. They propose that as the Kf concentration in a plant tissue declines (during periods of K+ deficiency), cytoplasmic Kf concentrations are initially maintained while vacuolar Kf is depleted and replaced by other solutes. A minimum vacuolar K+ level (usually around 20 mM) is envisaged; when the vacuolar K+ reaches this minimum, cytoplasmic K+ begins to decline and metabolism and growth become negatively affected. Support for the Leigh and Wyn Jones (1984) model was provided by the K+-use efficiency study of Memon et al. (1985). In this work three barley varieties were examined and one was found to exhibit “typical” K+ deficiency symptoms when grown at 10 PM K’. The important point is that this same variety was capable of accumulating K+ to higher levels, in the root and shoot, than the other varieties used in this study. Using a compartmental analysis approach, it was shown that the variety which exhibited K+ deficiency symptoms retained more of its K+ in the vacuole. This failure to transport vacuolar K+ to the cytoplasm resulted in a depletion of cytoplasmic K+,which then caused the onset of K+ deficiency symptoms. These studies suggest that the relative levels of cytoplasmic and vacuolar K+ must, in some fashion, be integrated. Furthermore, these concepts can be extended to indicate that the transport systems at both membranes must

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share some common controls. Observations have been made of membrane perturbations acting specifically at the outer face of the plasmalemma (applications of PCMBS, NADH, ferricyanide) that have resulted in significant alterations in tonoplast K+ fluxes (Kochian and Lucas, 1982a, 1985). In these studies, it was suggested that signalling mechanisms operate between the two membranes. The mechanism(s) of K+ transport at the tonoplast is poorly developed, and information concerning regulation of transport across the tonoplast is essentially nonexistent. Recent studies with tonoplast vesicles purified from roots and storage tissue have shown that the tonoplast contains two H+translocating enzymes, an ATPase and a pyrophosphatase (Blumwald, 1987). Both function to generate an electrochemical gradient for H+ across the tonoplast. This gradient is dominated by the activity component, which is considered to be approximately two pH units (the vacuole being acidic), while a small electrical component is also thought to exist. A number of H+-associated antiport mechanisms have recently been described for the tonoplast. Examples include Ca2+ uptake into the vacuole of oat roots (Schumaker and Sze, 1985) and Na+ uptake into the vacuole of red beet storage tissue (Blumwald and Poole, 1985). Evidence consistent with a K+-Hf antiport for tonoplast vesicles from red beet was recently presented (Sarafian and Poole, 1987). In terms of mechanisms for the transport of K+ into and out of the vacuole, one could speculate that K+ uptake may be mediated by a K+-Hf antiport, while K+ efflux from the vacuole could be facilitated by gated K+ channels, where the driving force for K+ efflux would be derived from the small (interior-positive) membrane potential. Of course these speculations do not describe potential regulatory processes for the tonoplast K+ transport systems, or offer explanations for how the plasmalemma and tonoplast might “communicate”. Answers to these questions must be sought in future studies. 4.

Criticisms of the Allosteric Model The major criticism of the allosteric regulation model is based on our inability, due to technical limitations, to make accurate measurements of cytoplasmic K+ concentrations. As Sanders (1980a) has pointed out, it is impossible to demonstrate a direct interaction between cytoplasmic K+ and the plasmalemma, since changes in root tissue content reflect primarily those occurring within the vacuole. Furthermore, according to the model of Leigh and Wyn Jones (1984), changes in vacuolar K+ content may not necessarily reflect what is occurring in the cytoplasm. Conversely, alterations in the cytoplasmic concentration of an ion can occur without corresponding changes in vacuolar concentration, as has been observed for C1- in perfused Chum cells (Sanders, 1980a). Consequently, caution must be exercised when attempting to generate models dealing with ionic levels of cytoplasmic compartments from data based on average (or bulk) tissue content.

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This cautionary note is supported by the occasional observation that at times Kf fluxes can be altered even though no change in [KIiwas recorded. An excellent example of this type can be found in the article by Pettersson and Jensen (1978). These workers attempted to alter the internal K+ content of barley roots (grown in full nutrient solution that contained either 0.1 or 10 mM K+) by placing the low-Kf seedlings in 10 mM KCl solution and the high-K+ seedlings in 0.1 mM KN03 solution; these pretreatments were applied for up to 80 min. Pettersson and Jensen found that K+ fluxes either decreased or increased significantly, while root Kf content did not change (Fig. 32). The authors argued that this was evidence for allosteric regulation,

Time of pretreatment in 0.1mM K t min Fig. 32. Investigation of the apparent relationship between [KIi and Rb+(8hRb+)influx into barley roots. (a) Roots grown in the presence of 0.1 mM K+ in the cultivation medium and then transferred to a 10 mM K+ pretreatment solution for the times indicated. (b) As in (a), except 10 mM K+ in cultivation medium and 0.1 mM K" in pretreatment solution. Rb+(86Rbf) uptake (0)was measured from a complete nutrient medium containing 1.0 mM Rb'. At each pretreatment time internal Kf ([KIi) was also determined (A).Data from Pettersson and Jensen (1978).

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because it indicated (to them) that the cytoplasm was filling rapidly and thus influencing allosteric sites, while bulk K+ concentration, which represented the vacuole, remained constant. Unfortunately, although these events could be occurring, there is no possible way to extrapolate from the data to the conclusions drawn by Pettersson and Jensen. Obviously, precise measurements of cytoplasmic K+ concentrations must be made before such extrapolations can be accepted. Certainly those who have championed the allosteric model are aware of this criticism, and some have attempted to deal with it via indirect measurements of cytoplasmic K+. However, direct measurements are required. Hopefully, recent technological advances in ion-selective microelectrodes may enable routine (?) measurements of cytoplasmic K+ (Felle and Bertl, 1986). Furthermore, the ability to measure K+ influx while making instantaneous changes in the K+ concentration at the cytoplasmic face of the plasmalemma, as has been done for perfused Chum cells, would be a powerful technique in the study of allosteric regulation. The use of the patch-clamp technique to study plasmalemma (and tonoplast) K+ currents, while controlling the ionic environment on each side of a membrane, in either the “patch” or “whole cell” (“whole vacuole”) mode, may be the answer to this technical problem. However, because the patch-clamp technique, of necessity, involves the use of protoplasts and isolated vacuoles, the regulatory aspects of transport involved in the integration of cells and subcellular compartments at the tissue level will be lost. Many of the studies concerning both regulatory and mechanistic aspects of K+ uptake have utilized low-salt-grown roots. As we previously indicated, low-salt roots are in a transitional stage between two regulatory states. Glass and Siddiqi (1984) have defined uptake experiments in which plants are grown under one set of conditions and then exposed to uptake media that differ from the growth solutions, as a perturbation technique. The use of low-salt roots for the generation of kinetic curves for K+ influx is a prime example of a perturbation technique. As they have noted, this technique has been useful for the elucidation of transport mechanisms, but its utility as an approach for studying transport regulation is limited, primarily because this technique is incapable of documenting long-term adaptations. Glass and Siddiqi (1984) illustrate their point with a comparison of two kinetic curves for K+ influx (over identical external K+ concentrations) for barley plants grown under different growth regimes. The fluxes measured via the perturbation technique were obtained by using intact plants that had been grown in a balanced inorganic nutrient solution containing 40 PM K’. A kinetic profile for K+ influx was determined by transferring these plants to new solutions containing from 1 to 100 PM K+ (10 min uptake). Steady-state K+ influx, on the other hand, was determined by growing barley plants under conditions of continuous K+ supply at the same concentrations used to obtain the K+ fluxes. For example, plants grown in 10 PM K+ solution were

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/

Steady- state flux

~oco-o-o-o-o-o-o-o-o.

20

40

60

80

100

Potassium concentration ( p m )

Fig. 33. Comparison between “steady-state’’and “perturbation”kinetic profiles obtained for Kt influx. Steady-state experiments were conducted on barley roots that were grown at the Perturbation experiments were conducted concentrationused in the K+ influx experiment (0). on barley plants that were grown in 40 /AM K+ and then transferred to the indicated K+ concentrationsto determine the “perturbationflux” (A).Data from Glass and Siddiqi (1984).

used only for flux determinations from an uptake solution containing 10 p~ K’. As shown in Fig. 33, the two kinetic profiles are dramatically different. The curve obtained using the perturbation technique yielded a V,,, and K , of 16.8 pmol g-’ h-’ and 26 p ~respectively, , while the steady-state fluxes were considerably smaller and were independent of external concentrations above about 10 p~ K+. The differences in the two kinetic curves graphically illustrate the influence that growth conditions can have on K+ fluxes. Although their point is well taken, there are some inconsistencies in the data presented by Glass and Siddiqi (1984). At an external K+ concentration of 40 p ~one , would expect that the fluxes obtained from both techniques would be quite similar, since the seedlings used for this particular concentration were grown at that concentration. However, the data in Fig. 33 indicate that the perturbation flux at 40 p~ K+ is approximately 10 times the steady-state flux. If one examines the original paper from which these data originate, the apparent discrepancy is explained. Siddiqi and Glass (1983) obtained the data for the perturbation fluxes from 8-day-old barley seedlings, while the steady-state fluxes were obtained with seedlings grown for 18 days. Therefore, a significant portion of the reduction seen in the steadystate fluxes was due to an increase in [KIi. For example, the root K+ content of the 8-day-old seedlings grown in 40 p~ K+ solution and used for the perturbation flux determinltions was 13.7 pmol g-’, while the roots used for steady-state fluxes that were grown in 10-50 )(LMKf solutions for 18 days contained between 35 to 68 pmol K+ g-’. Although we still agree with the ideas that were proposed, a better choice of data could have been made in order to illustrate the concept.

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Another caveat concerning the use of low-salt roots for studies on ion transport regulation was made by Cheeseman (1985) in his study on the influence of Na' on K+ uptake in both low-salt corn seedlings and corn seedlings grown in full nutrient solution. In an earlier study on excised roots from 3-day-old low-salt corn roots, Cheeseman (1982) found that inclusion of Na+, over a concentration range from 0.02 to 20 mM, had no effect on K+ uptake from solutions containing 2 PM to 20 mM K'. However, when Cheeseman (1985) conducted his experiments using the roots of 8-day-old corn seedlings that had been grown on a full nutrient solution (containing 1 mM K') he found that the inclusion of Na+ in the uptake solution caused a marked stimulation of K+ influx. When the kinetics for K' uptake were determined from solutions containing from 0.05 to 2 mM KCl and Na' at 45 times the K' concentration, influx values quite similar to those for low-salt roots were obtained. On the basis of these results, Cheeseman proposed that transport regulation in full-nutrient-grown plants may be qualitatively different from that operating in low-salt roots. This idea is similar to those expressed earlier by Pitman (1970) and Siddiqi and Glass (1984) concerning transitions between different regulatory modes as a low-salt root alters its salt status. It should be noted that with respect to this Na' response, there are some differences in terms of K' uptake between the low-salt seedlings used by Cheeseman (1982) and those used by other laboratories. The lack of inhibition of K+ influx, at high external K' concentrations (>1 mM), by Na' in Cheeseman's investigation is rather surprising, considering the measurements made by Epstein et al. (1963). In this work conducted on low-salt barley roots, mechanism I1 K' uptake was inhibited by Na'. Additionally, Kochian et al. (1985) have demonstrated, for low-salt corn roots, that the inclusion of 3 mM Na+ in the uptake solution inhibits the linear component for K+ uptake by approximately 50%. The possibility exists that, in addition to the existence of qualitative differences between Cheeseman's low-salt and full-nutrient-grown roots, there may be some qualitative differences between his low-salt roots and those grown by other laboratories. It is clear that in studies involving regulation of root ion transport, the various growth conditions used to produce experimental material may have a significant influence on the results obtained and the models generated from these data. Therefore, particularly when work is conducted with the relatively non-physiological low-salt roots, special care must be taken when evaluating the data. However, this does not mean that earlier work with low-salt roots is invalid, or that they are not still a useful research material. In fact, the use of nutrient-starved roots may be necessary in order to unmask transport mechanisms that would otherwise not be detected in nutrient-rich roots. For example, the high-affinity K' uptake system recently observed in low-salt corn roots (Newman et a f . ,1987) could not be detected in high-salt-grown roots. The same is true for the high-affinity K' transport

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system operating in Neurospora. Earlier K+ uptake studies by Slayman and Tatum (1965) were carried out with K+-replete cells, and were conducted in the absence of extracellular Ca2+.In these cells, K+ uptake occurred via a low-affinity,high-velocity transport system. However, if K+-starved Neurospora cells were grown (analogous to low-salt roots), and K+ uptake studied in the presence of Ca2+, the high-affinity K+ transport system (K+-H+ symporter) was revealed (Rodriguez-Navarro and Ramos, 1986;RodriguezNavarro et al., 1986). 5. Alternatives to the Allosteric Model Although most of the root work concerned with feedback control of K+ uptake by [KIi has centred on allosteric models, other models can also account for regulation of ion transport in plants. At the cellular level, control of K+ uptake by cytoplasmic K+ could also be explained on the basis of a reaction kinetic model (see Fig. 18). Here, feedback inhibition by high cytoplasmic K+ levels can be accounted for in terms of a reduction in K+ dissociation from the cytoplasmic face of the carrier; i.e. in an analogous way to that proposed for the H+-Cl- co-transport system of Chara (Sanders, 1980a,b; Sanders and Hansen, 1981). Unfortunately, due to technical limitations, it has not yet been possible to conduct the appropriate experiments, in roots, that would enable a differentiation between these two models. Recently, a regulatory model was presented which suggested that under conditions of increased shoot demand, allosteric regulation of K+ transport at the root epidermal and cortical cell level is superseded by a more complex regulatory mechanism that focuses on ion transport to the xylem as a key point for root-shoot interactions. Drew et al. (1984) and Drew and Saker (1984) investigated the effects of K+ deficiency (along with PO!- and Cl-) on K+ uptake and translocation. In their initial study, they determined the kinetic parameters for K+ uptake into barley roots by monitoring rates of depletion of labelled ions from dilute K+ solutions (Claassen and Barber, 1974). By this means, they were able to measure K+ fluxes over a full range of concentrations (as the roots depleted external K+ from the initial value of 100 PM) on the same sets of plants. With 14-day-oldplants, they found that 1 day of K+ starvation caused the K , to decline dramatically (from 53 to 11 p ~ )while , the V,,, remained unaffected. Further K+ starvation (4 or 7 days) had no influence on the K,, while the V,,, increased two-fold. Bulk K+ content of the roots and shoots declined significantly after each period of starvation (1,4 and 7 days). Drew et al. (1984) proposed that the initial rapid decrease in K , reflected allosteric regulation of the carriers by decreasing [KIi. For the starvation periods longer than 1day, they speculated that the increase in V,,, was the result of protein synthesis which increased the number of available transport proteins with a high affinity for K'.

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In the accompanying work, Drew and Saker (1984) conducted dividedroot experiments with young barley plants. These plants were grown such that one seminal root was segregated from the remaining four or five seminal roots. This single root was continuously supplied with full nutrient solution (including K'), while the remaining roots were supplied with the same solution (controls), or with the same solution minus K+ for either 2 or 4 days. Using 42K+in the compartment containing the single root, net uptake of 42K+ into the root was monitored, as well as 42K+translocation to both the shoot and the K+-starved roots. Additionally, bulk Kf concentration of the single absorbing root, the other roots, and the shoot were determined at the end of the experimental period. The results of this study are presented in Table 11. Following K+ starvation of the other roots, for either 2 or 4 days, there was a marked stimulation in K+ uptake into the single fed root, as well as dramatic stimulations of K+ translocation to the shoot and other roots (compared with controls). However, the bulk K+ concentration of the fed root remained unchanged following either 2 or 4 days of K+ starvation of the other roots. The bulk K+ concentrations of the other roots declined significantly after both 2 and 4 days of starvation, while the shoots exhibited a significant decline in K+ content only after 4 days. Drew and Saker (1984) concluded that the marked stimulation of K+ uptake and translocation to the shoot that occurred during the period of K+ TABLE I1 Potassium uptake and internal ion concentration in barley plants with roots divided between K+-free (- K ) and K+-containing ( + K ) nutrient solution

Control (single root +K, other roots + K) Net uptake of 42K-labelledK+ by single root (pmol (g dry wt root)-' day-') Single root Shoots Other roots Total translocated Tissue K+ concentration ( w o l (g dry w9-l) Single root Shoots Other roots

Potassium-deficient (single root +K, other roots -K) 2 day

'

4 day

452' 481" 53" 533"

634 1280' 188' 1470'

826' 1830' 252' 2090"

1730" 1550" 1590"

1700" 1430" 970'

1600' 1280' 500'

Values across the table followed by a different letter are significantly different from each other (P < 0.05, N = 8). Data taken from Drew and Saker (1984).

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deprivation, without any detectable change in Kf concentration within the absorbing root, was incompatible with the operation of an allosterically regulated transport mechanism. They suggested that allosteric regulation had been overriden by an alternative mechanism involving transport across the root; i.e. K+ uptake into the root was regulated by transport to the xylem. The implications of such a model are that allosteric regulation will be the primary regulatory mechanism when K+ flux to the xylem is not significant. An example of such a situation would be during the lag in K+ transport to the shoot which is characteristic of low-salt roots undergoing the transition to salt saturation. Furthermore, such a model suggests the integration of regulatory mechanisms (and signals) over the entire plant, because it indicates that increased nutrient demand on a root would influence the uptake characteristics of that root. This type of transport regulation would involve a hierarchy of regulatory mechanisms ranging from control at the cellular or membrane level, which might include allosteric and/or kinetic control, to transport across the root, which might involve a feedback loop from the xylem parenchyma to the epidermis. At yet a higher level, signals from the shoot might influence root ion transport processes via the Kf transport systemsinvolved in releasing K+ into the xylem (presumably at the plasmalemma of the xylem parenchyma). Siddiqi and Glass (1987) have challenged the concept that shoot demand directly controls root K+ levels. Various combinations of experimental protocols were used in order to manipulate barley root and shoot Kf levels. Usually, the trends in changing root and shoot Kf levels were similar; i.e. root and shoot K+ increased or decreased in concert. However, some of their experimental conditions resulted in situations where alterations in the K+ content of shoots and roots did not correlate. In these cases, the kinetic parameters for Kf influx were always correlated with root K'. Based on such analyses, Siddiqi and Glass proposed that although other types of transport regulation may operate, in addition to allosteric control, negative feedback from root K+ content is the primary effector in terms of regulating Kf uptake into the root. B. Kt CYCLING WITHIN THE PLANT: AN INTEGRATION OF REGULATORY MECHANISMS

When one considers the complexities involved in nutrient cycling within the plant, particularly for a highly mobile and important nutrient such as K', it seems reasonable to hypothesize a multicomponent regulatory system for the uptake and translocation of K'. This would particularly apply to the fully autotrophic plant growing in the field, which would encounter a wide range of different environmental variables for the root versus the shoot, and would also impose the constraints of shoot and root growth upon the regulation of

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K+ uptake and partitioning. Hence, a regulatory model which simply involves the control of K+ influx across the plasmalemma of the epidermis/ cortex appears to lack the level of integration necessary to achieve a balance of K+ between the shoot and root (Drew and Saker, 1984; Jeschke et al., 1985; Cheeseman and Wickens, 1986a,b). A number of reports have shown that alterations in root K+ uptake are not negatively correlated with root K+ content (e.g. Jensen and Pettersson, 1978; Pettersson and Jensen, 1978;Wild etal., 1979; Drew and Saker, 1984; Jeschke, 1984;Pettersson, 1986;Cheeseman and Wickens, 1986a,b). Generally, non-allosteric regulation has been observed when environmental conditions have been manipulated in order to change factors such as relative growth rate (RGR) or shoot: root ratio. As Siddiqi and Glass (1987) have argued, it is possible that an alteration of factors such as environmental conditions or shoot parameters indirectly influences regulation, by changing the quantitative relationship between root [KIi and K+ influx. That is, the magnitude of the change in K+ influx in response to a particular incremental change in [KIi could be altered in response, for example, to a change in shoot growth. This explanation would still be based on the premise that only root K+ levels directly control K+ transport in the plant. Alternatively, a model that relates the needs of the shoot to the root, via signals directed to the site of K+ transfer into the xylem, with subsequent changes in transport activity then feeding back (ultimately) to K+ uptake at the root surface, would appear to be better suited to the needs of the entire plant. Such a model would still be ultimately based on feedback control of root K+ uptake by root [KIi, but in a more complex manner. Unfortunately, few studies have addressed the relationship between growth and K+ uptake specifically in terms of transport regulation. Pitman (1972) looked at the influence of shoot growth rate (modified through variations in the photoperiod) on root K+ uptake and translocation to the shoot. He found that under conditions of low shoot growth, K+ uptake and translocation were decreased in relation to the situation observed under conditions of rapid shoot growth. These results suggested that “shoot demand” for K+ could override regulatory processes operating in the root. Pitman suggested that phytohormones, such as ABA and cytokinins, could be participating in feedback control of K+ transport. Although hormones are often considered as likely candidates for the signal between the shoot and root, the available data appears contradictory. Thus, we can only conclude that hormones may play a role in the regulation of ion transport within the plant and that this area of research awaits further clarification (for review, see van Steveninck, 1976). Jeschke (1982) has examined the effect of shoot :root ratios on K+ and Na+ fluxes in intact barley seedlings. The shoot : root ratio was increased by excising all but one seminal root. This resulted in an increase in plasmalemma K+ influx, net K+ uptake, transport to the xylem, and cytoplasmic

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K+. The positive correlation that was observed between K+ influx and cytoplasmic K+ levels again suggests a situation where allosteric regulation has been overriden. Based on this work, Jeschke proposed that increasing the shoot :root ratio (which would be analogous to increasing shoot demand) altered the feedback control of root ion fluxes by the shoot, possibly through the participation of hormonal signals. In a subsequent study, Jeschke (1984) studied the influence of increased transpiration on K+ fluxes in barley seedlings in which the root system had been reduced. It was found that accelerated water flow stimulated K+ uptake and translocation to the shoot, while root K+ content decreased. These observations appear to be consistent with the allosteric model. However, compartmental analysis performed under these conditions indicated that vacuolar K+ declined, while cytoplasmic K+ remained constant, or increased slightly. These results further underscore the need for measurements of cytoplasmic K'. In terms of transport regulation, Jeschke has commented on the marked similarities between the effects of increased transpiration and increased shoot :root ratio on K+ fluxes and compartmentalization within the plant. He speculated that water flow and K+ recycling within the plant could play a role in regulatory interactions between the shoot and root. This would be particularly true for seedlings where the root system has been reduced to a single root. In this situation very high rates of phloem translocation are maintained from the shoot to the root (Passioura and Ashford, 1974). The participation of recycled K+ as a signal between the shoot and root is discussed below. Cheeseman and Wickens (1986a,b) conducted a detailed study on the relationships between Kf uptake, tissue K+ content, root : shoot ratios, and growth in intact, vegetative Spergularia marina plants grown under full nutrient conditions. Potassium uptake into S. marina was negatively correlated with root weight (or root :shoot ratio), but positively correlated with root K+ content. Again, these results are incompatible with the concept that allosteric regulation of uptake functions as the prime regulatory mechanism. In all of the studies relating ion transport to growth, the focus has been placed, quite logically, on the needs of the shoot. However, there are situations where root nutrient demands may change the way a regulatory network might operate. It is well documented that nutrient deficiency often results in an increase in root growth and, therefore, a decrease in shoot :root ratio (Clarkson and Hanson, 1980). Any regulatory scheme for K+ transport in the whole plant must take into account growth factors of both the shoot and root. In terms of an integrated regulatory scheme for K+ transport throughout the plant, one logical choice for the signal between the root and shoot would be K+ recirculated from the shoot to the root, via the phloem. At least three important sites need to be considered when developing a regulatory model

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for K+ transport. These include the site of entry for K+ (the root surface), the site of transfer to the shoot (the xylem parenchyma), and the site of K+ recirculation (the shoot). The critical “fulcrum” for such a model would be the xylem parenchyma, which would be the point of signal reception from the shoot, and also the site from which root K+ uptake would be modulated, presumably through the feedback control that exists between root [KIi and plasmalemma K+ influx. As Drew and Saker (1984) have proposed, K+ recirculated to the root via the phloem could be the signal that would affect xylem K+ transport, and, ultimately, root K+ uptake. Potassium recirculation within the plant, via the phloem, has been well documented (Greenway and Pitman, 1965; Pate, 1975; Armstrong and Kirkby, 1979; Jeschke et al., 1983). Although cytological studies within the root, detailing the relationship between various cells of the phloem and the xylem parenchyma, are lacking (and much needed), the close proximity of the xylem parenchyma to the phloem does make it possible that K+ released from the phloem could influence K+ transport by these cells. Recently, elegant studies on ion recirculation (K+, Mg2+,Ca2+,and Na’) throughout

Fig. 34. Model of K+ transport and accumulation in nodulated Lupinus albus plants. Rates of ion flows (figure at arrows) and of accumulation during growth (figures in boxes) are given in pmol (g fresh wt)-’ h-’. Arrow thicknesses relate to net uptake (1) into the root of 100%. Numbers represent: accumulation in the root (2) and shoot (6); (3) “direct” accumulation from the external medium; transport via xylem (4) and phloem ( 5 ) ; phloem to xylem transfer in the root (7). Data from Jeschke et al. (1985).

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the plant have been conducted by Jeschke and co-workers on both nodulated white lupin and barley (Jeschke et al., 1985,1987;Wolf and Jeschke, 1987). Initially, Jeschke and co-workers chose white lupin because of its ability to bleed from both the xylem and phloem. Xylem and phloem sap was collected, and rates of ion flow and cycling between the shoot and root were calculated from the measured carbon :ion weight ratios in the xylem and phloem streams. These determinations were based on an empirical model previously developed for carbon and nitrogen partitioning in lupin (Pate et al., 1979).The model for K+ circulation in lupin is shown in Fig. 34. The data indicate that 52% of the absorbed K+ is recirculated back to the root, and that 76% of the phloem-borne K+ returning to the root re-enters the xylem stream and is transported again to the shoot! Clearly, since a large portion of the K+ moving in the xylem transpiration stream is supplied by the phloem, the concept gains strength that K+ transfer, from the phloem, influences the ionic environment of the root xylem parenchyma. An even more detailed study of K+ and Na+ recirculation in barley has been developed to take into account the interactions between older and younger leaves (Wolf and Jeschke, 1987). The Wolf and Jeschke model is presented in Fig. 35, and from this the complexities involved in K+ partitioning within the plant are clearly evident. A number of points are brought out by this model:

1. The older leaves were the major sinks for xylem-borne K+. Subsequently, most of this imported K+ was re-exported out of the older leaves via the phloem. 2. Most of the K+ supplied to the young leaves came frnm the older leaves, via the phloem. However, the oldest leaves retranslocated their K+ primarily to the root, with a smaller proportion going to the younger leaves. 3. A large portion of the shoot K+ was recirculated to the root, and subsequently returned to the shoot via the xylem. It was estimated that 43% of the K+ absorbed by the root was recirculated back from the shoot to the root. From these models, it is apparent that the K+ flows are strongly influenced by photoassimilate partitioning, as evidenced by the large proportion of K+ that is conducted in the phloem. When the details of such complicated flow schemes are examined in the context of K+ transport regulation within the whole plant, it becomes apparent that regulatory models based on the control of a single K+ transport system (i.e. allosteric regulation of root K+ uptake) are inadequate to account for the control that would be necessary to direct these flows. Instead, it should be recognized that feedback control of K+ uptake, at the cellular level, is probably the most critical component of a multicomponent, hierarchical regulatory system that would integrate K+ fluxes at the cellular, organ, and whole plant level. It should be made clear that, at this time, such a speculative regulatory model is only a conceptual

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Fig. 35. Rates of K+ flow between single leaves and the root of barley plants. Figures represent ion movements and increments in individual organs [pmol plant-' (5 days)-'] for the period 14-19 days after germination (four-leaf stage). Arrow thicknesses are drawn proportional to transport rates, with striped arrow indicating flows in the xylem and solid arrows flows in the phloem. Squares in leaves or roots represent ion increments due to growth andor to increases in tissue ion concentrations; areas are drawn in proportion to ion increments. The indicated rate of phloem to xylem transfer within the root represents a minimum estimate. Data from Wolf and Jeschke (1987).

framework from which future work can be directed. Certainly almost nothing is known, for example, about how alterations in the activity of K+ transport systems in the plasmalemma of the xylem parenchyma might feed back upon K+ uptake systems in the root epidermal plasmalemma. Obviously, as Cheeseman and Wickens (1986a,b) noted earlier, future research must be directed at the elucidation of the physiological basis for the signals and transduction systems operating within the plant, and also the mechanisms by which the plant integrates these components into an interactive and coherent system.

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VI. FUTURE RESEARCH AND PROSPECTS We began this chapter by noting the lack of understanding and knowledge concerning the mechanisms and regulatory aspects of K+ transport in roots. It can certainly be said that progress in this area has not been rapid, primarily because of the complexities of the processes being studied. However, our examination of the literature indicates that over the past 10 years significant insight has been gained into both the mechanisms of K+ transport across the root and the control processes involved. Progress has come primarily through the application of a range of recent technological advances. In order to maintain this progress, we must not only continue to take advantage of new technologies as they become available, but we must also apply them in an integrated manner. In terms of mechanisms of plasmalemma and tonoplast K+ transport, a combination of biophysical, biochemical and molecular techniques should prove extremely fruitful. The application of patch-clamp studies (both whole cell and vacuole, and excised patch) will enable the investigation of the coupling and stoichiometry of K+ and Hf fluxes. In this way, it should be possible to study the putative Kf-Hf co-transport system in the most rigorous of ways. Additionally, we are pleased to note that the study of Kf channels with the patch-clamp technique has already begun and should yield important information on mechanisms and control of passive K+ transport systems. Recent improvements in cell fractionation and purification techniques should also enable researchers to obtain purified preparations of transport proteins (at least the more ubiquitous ones) which can be used for further reconstitution and transport studies. This approach should complement patch-clamp and traditional electrophysiological techniques in discriminating between Kf-ATPases, K+-H+ co-transport systems, and KfH+ exchange ATPases. Furthermore, the combination of membrane purification techniques with immunology and molecular genetics is enabling researchers to obtain the gene sequences for some transport proteins. Further progress in this exciting field will allow for the molecular characterization of various transport proteins. By integrating these new techniques into existing programmes, plant transport physiologists should be able to study the different Kf transport systems at the mechanistic/molecular level. The future prospects for research into Kf transport regulation, at the cellular, root, and whole plant level, are very exciting. A t the cellular level, the patch-clamp technique should again be very useful. This approach will enable researchers to study K+ transport while carefully controlling the ionic environment on both sides of the plasmalemma (or tonoplast). Hence studies into kinetic and allosteric control can be conducted. Additionally, this technique can be used to study the interactions between membraneassociated redox systems, K+ transport, and the control of growth. Recent

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advances in ion-selective microelectrode technology should enable researchers to use K+ and Hf-selective microelectrodes to obtain direct measurements of cytoplasmic and vacuolar pH and K+ activities, and to monitor changes in these activities as the root absorbs and translocates K+. Finally, the application of high-resolution SEM X-ray microprobe analysis should prove to be a powerful tool in the investigation of K+ compartmentalization within the various cells of the root, especially during alterations in the status of this ion. The integration of K+ transport across the root with root-shoot interactions is an important area of research that is gaining the attention of a number of plant physiologists. Studies must be focused on the roles of the xylem parenchyma in K+ transport to the shoot and Kf recirculation within the plant. Furthermore, possible modes of coupling between the xylem parenchyma and root epidermis, in terms of regulating K+ transport across the root, should be investigated. This will require the integration of cell cytology, biophysics, biochemistry and whole plant physiology. In view of the models indicating that Kf recirculation from the shoot to the root may play an important role in regulating K+ transport in the plant, it is obvious that an extensive cytological study is needed to establish whether symplasmic continuity exists between the phloem and xylem tissues of the stele. Additionally, the use of detopped roots for the study of both transroot electrical potentials and ion transport to the xylem, using combinations of various nutritional regimes and chemical probes directed at the xylem parenchyma, are needed to further characterize K+ transport across the root. Finally, further whole plant studies quantifying Kf flows within the plant, particularly under conditions where Kf concentrations and flow in the phloem are artificially modified, should prove of particular value. In conclusion, it is apparent that as we gain further understanding and insights concerning K+ transport within the plant, we also gain a growing appreciation of the complexities involved in this highly regulated physiological system. In the future, it will often be necessary to dissect the system into its cellular, subcellular and molecular components, even down to the gene level. However, we should take every opportunity to integrate these new findings to yield a highly cohesive approach to the study of K+ transport in the plant. We feel that only in this way will we be able to build upon the knowledge that has been yielded up so grudgingly by our beloved plant!

ACKNOWLEDGEMENTS Work on Kf transport conducted in our laboratories has been supported by grants from the United States National Science Foundation (W.J.L.) and the United States Department of Agricultural Research Service (L.V.K.). We also wish to thank Lyn Noah and Cheryl Redlich for their help in typing

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t h e manuscript a n d t h e many scientists who provided us with reprintdpreprints of their work. W e offer special thanks to Drs Glass, Drew and Jeschke for valuable discussion. Finally, we would like t o thank D i a n a Lucas for her patience and encouragement throughout t h e preparation of this manuscript.

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