Regulation of Potassium Transport in Leaves: from Molecular to Tissue Level

Abstract

Regulation of Potassium Transport in Leaves: from Molecular to Tissue Level

SERGEY SHABALA^*,1

¹ School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, Tasmania 7001, Australia

Annals of Botany 92: 627-634, 2003.

 

Abstract

Over millions of years, plants have evolved a sophisticatednetwork of K⁺ transport systems. This Botanical Briefing providesan overview of K⁺ transporters in various leaf tissues (epidermis,mesophyll, guard cells and vascular system) at both the cellularand organelle levels. Despite the tremendous progress in ourknowledge of genes encoding K⁺ transport systems in plants,understanding has not developed of coordinated functioning andoperation of these genes or proteins in the context of wholeplant physiology and plant–environment interaction. ThisBotanical Briefing is aimed at filling that gap by analysingelectrophysiological and molecular evidence for mechanisms coordinatingK⁺ transport between various leaf cells and tissues in changingenvironments.

Introduction

Potassium is the most abundant inorganic cation in non-halophytes,contributing up to 6 % of plant dry weight. As a majorinorganic osmolyte, K⁺ is crucial for cell osmoregulation andturgor maintenance and, hence, for cell expansion, stomatalfunction, tropisms and leaf movement. Being an activator ofa large number of enzymes, K⁺ plays a key role in photosynthesis,protein synthesis, and oxidative metabolism. It also affectsphloem transport and provides charge balance during vectorialion transfer across cellular membranes (Marschner, 1995).

Being complex and heterogeneous structures, plants have evolveda sophisticated and highly specialized K⁺ transport system tomeet the different requirements for K⁺ in various cells andtissues and many reviews addressing various molecular and electrophysiologicalaspects of K⁺ transport in plants have been published recently(Maathuis and Amtmann, 1999; Schachtman, 2000; Mäser etal., 2001, 2002; Very and Sentenac, 2002; Pilot et al., 2003).Most of these deal with regulation of K⁺ uptake and transportin roots. As almost all K⁺ found in plant tissues is taken upby roots, it is not surprising that K⁺ transport systems inroots are the most studied, although the majority of K⁺ in aplant is found in the stems and leaves. In contrast to rootcells, our knowledge of the properties and regulation of membraneK⁺ transport in leaves is much more limited (Pineros and Kochian, 2003).

Recent advances in molecular biology have allowed tremendousprogress in our knowledge of genes encoding K⁺ transport systemsin plants (Mäser et al., 2001). However, with a possibleexception of guard cells (Assmann and Shimazaki, 1999; Blatt, 2000;Schroeder et al., 2001), understanding of the coordinatedfunctions and operation of these genes/proteins in the contextof whole plant physiology and plant–environment interactionhas not developed at the same rate. In this Briefing, an attemptis made to fill this gap and address electrophysiological andmolecular aspects of coordination of K⁺ transport between variousleaf cells and tissues under natural conditions.

Heterogeneity of leaves

The heterogeneous nature of leaves is obvious: there are strikingdifferences between leaves in venation, stomatal alignment andstomatal density. It is not surprising, therefore, to find thatthe nutritional demands and, consequently, regulation of ionicuptake are remarkably different in various cells, even of oneleaf.

Heterogeneity of leaf ionic environment
The cells of the mesophyll and of the epidermis are very differentanatomically, with their vacuoles occupying around 61–75 %and up to 99 % of the cell volume, respectively (Karleyet al., 2000a). In addition, epidermal cells are virtually unableto produce organic solutes and rely heavily on inorganic ions(mainly K⁺) for osmotic adjustment (Fricke et al., 1994). Inmesophyll cells, the total contribution of organic osmolytesis much higher (20–30 %; Gonzales et al., 2002) thanin epidermal cells, although K⁺ remains the dominant osmoticum(Shabala et al., 2000; Gonzales et al., 2002). A differencein K⁺ composition between epidermal and mesophyll cells is anobvious consequence of their different solute compositions (Leigh, 2001).Cytosolic K⁺ homeostasis is a characteristic featureof mesophyll cells and is maintained at the expense of the supplyto the epidermis, where cytosolic K⁺ may decline to very lowlevels (Cuin et al., 2003). This homeostasis enables protectionand maintenance of optimal photosynthetic activity of the mesophyllcells. The process underlying differential accumulation of ionsby leaf cells can include both the regulation of supply of certainions and the capacity for ion uptake (Karley et al., 2000b).

Apoplastic free K⁺ concentrations are usually much lower thansymplastic and have been reported between 2 and 26 mM (Karleyet al., 2000a; Roelfsema and Hedrich, 2002). These values maychange dramatically as a result of altered light (Roelfsema and Hedrich, 2002)or salinity (Leigh, 2001).

Heterogeneity of leaf physiological responses
The heterogeneity of leaf physiology is illustrated by two examples:leaf extension growth and stomatal ‘patchiness’.

There are striking differences between growth patterns of differentregions of the leaf lamina and yet expansion of various leaftissues is highly coordinated. It is believed that the epidermisnormally restricts leaf expansion (Van Volkenburgh, 1999), presumablydue to the fact that epidermal cells usually have lower turgorthan mesophyll cells. Even within the epidermis, a significantdifference was measured between K⁺ uptake at the growing (leafbase) and at the fully extended (leaf tip) regions of corn leaves(S. Shabala and B. Zivanovic, unpub. res.). Although the molecularand ionic mechanisms underlying these differences are yet tobe studied, it is reasonable to suggest that either K⁺ concentrationin the apoplast or functional expression of K⁺ transporterswill vary significantly between the apical and basal regionsof the leaf, depending on growth patterns. It is also possiblethat post-transcriptional modulation of transport activity maydetermine these differences.

On a smaller scale, stomatal ‘patchiness’ is anotherexample of heterogeneity of leaf physiological characteristics.It has been demonstrated that in many species and under variousexperimental conditions, stomatal conductance and behaviourdiffer between regions of the leaf, forming patches up to severalmillimetres across (Mott and Buckley, 1998). Based on membranepotential measurements, Roelfsema et al. (2001) showed that,in planta, stomatal guard cells exhibit at least three differentphysiological states under apparently identical environmentalconditions. It was speculated that local production and distributionof chemical ‘signals’ facilitates these differences(Mott and Buckley, 1998). Keeping in mind the apoplastic couplingbetween epidermal and guard cells and the fact that leaf apoplasticK⁺ concentration is strongly correlated with the leaf venation(Shabala et al., 2002), it is possible to suggest that K⁺ distributionin the leaf apoplast is one of the factors determining stomatal‘patchiness’ and heterogeneity in leaf photosynthesis.

Functional expression of plasma membrane potassium transporters in leaf tissues

General features of K⁺ transporters in plants
A large number of genes encode proteins involved in K⁺ transportin plants. In arabidopsis, these transport mechanisms fall intoseveral distinct categories (Mäser et al., 2001, 2002;Very and Sentenac, 2002): (a) two families of K⁺ channels (Shaker-typeand KCO channels; 15 genes in total); (b) Trk/HKT transporters[Na⁺/K⁺ symporter (Schachtman, 2000); one gene]; (c) KUP/HAK/KTtransporters [H⁺/K⁺ symporter (Kim et al., 1998); 13 genes];(d) K⁺/H⁺ antiporter homologs (six genes); (e) cyclic-nucleotide-gatedchannels (CNGC; 20 genes in arabidopsis; Very and Sentenac, 2002);and (e) glutamate receptors (GLRs; 20 genes; Very and Sentenac, 2002).The Shaker-type channels are further subdividedinto SKOR and GORK channels (both depolarization-activated),KAT channels and AKT channels. AKT channels contain an ankyrin-bindingmotif, which is lacking in KAT type channels (Mäser etal., 2001). An important feature of plant Shaker-like K⁺ channelsis that they can form heterotetrameric structures (Pilot etal., 2003), allowing plants to tune the K⁺ transport activityin various cells, independently in each organ/tissue, in relationto environmental conditions. A brief summary of the functionalexpression of plasma membrane K⁺ channels in various leaf tissuesin Arabidopsis thaliana is given in Table 1, and some detailsof the functions and intracellular location of K⁺ transportersin leaves of various plant species are shown in Table 2. Thediversity of K⁺ transport mechanisms at the plasma membraneof a ‘generalized’ leaf cell is summarized in Fig.1.

Stomatal guard cells
Two major types of K⁺ channels are present at the plasma membraneof guard cells: voltage-dependent K⁺-selective inward (KIR)and outward (KOR) rectifying channels (Pilot et al., 2001; Schroederet al., 2001; Szyroki et al., 2001; Zimmermann et al., 2001).KIR channels are activated by membrane hyperpolarization andmediate stomatal opening, whereas KOR channels are opened byvoltages more positive than E_k and mediate stomatal closure.

Recent transcription–PCR experiments with isolated guardcell protoplasts showed that in addition to KAT1, the K⁺ channelsAKT1, AKT2/3, AtKC1 and KAT2 (all KIRs) were expressed (Szyrokiet al., 2001), suggesting that KAT1 inward-rectifying K⁺ channelsmay not play as dominant a role in K⁺ uptake in guard cellsas previously believed (Assmann and Wang, 2001). It was alsoshown that KAT1 and KAT2 can form heteromultimeric channels(Pilot et al., 2001; Zimmermann et al., 2001), leading to moreflexibility when adapting to altered developmental and/or environmentalconditions. Several other channels of unknown voltage-dependence(AtKC1, AKT5 and AKT6) were also shown to co-localize in arabidopsisguard cells (Dietrich et al., 2001). It is thought that sucha multiple ensemble of K⁺ channels provides greater versatilityand much more efficient regulation of K⁺ homeostasis in guardcells compared with only one type of KIR channel. The only certaincandidate in the arabidopsis genome to mediate stomatal closureis GORK, a voltage-gated outwardly rectifying K⁺ channel ofthe guard cell membrane (Hosy et al., 2003).

In addition to specific K⁺-selective channels, guard cells alsopossess a wide range of non-selective cation channels (NSCC),either depolarization- or hyperpolarization-activated (Demidchiket al., 2002). These channels are likely to be involved in releaseof solutes during turgor adjustment and, to some extent, functionallycomplement GORK channels. Finally, there is strong evidencethat guard cells possess mechanosensitive (or stretch-activated,SAS) channels at the plasma membrane (Cosgrove and Hedrich, 1991).These channels are K⁺ permeable and change their openprobabilities as a result of volume or turgor changes.

As well as their voltage-dependence, K⁺ channels in guard cellsare regulated by several other factors, the most obvious ofwhich is pH. Both apoplastic (Pilot et al., 2001; Roelfsema and Hedrich, 2002)and cytosolic (Dietrich et al., 2001) acidificationlead to the activation of inward K⁺ currents in guard cells.The effect is voltage-dependent (Roelfsema and Hedrich, 2002).Most other second messengers such as cytosolic Ca²⁺, IP³, GTP,G-proteins, polyamines, reactive oxygen species (ROS) and phosphorylationevents also exert direct control over K⁺ channel activity (Assmann and Shimazaki, 1999;Blatt, 2000; Schroeder et al., 2001; Kohleret al., 2003). It has also been shown that guard cells may utilizevoltage-dependent K⁺ channels as targets of the osmosensingpathway by regulating channel opening probability by the osmogradientacross the plasma membrane of guard cells (Liu and Luan, 1998).

Mesophyll
Molecular studies suggested that both AKT1 and AKT2/3 genesare expressed in arabidopsis leaf mesophyll (Dennison et al.,2001; Cherel et al., 2002). A specific feature of AKT2/3 channelsis their weak dependence on the membrane potential, sensitivityto ATP and an inverted pH regulation (Marten et al., 1999).Being only weakly controlled by the membrane potential, AKT2/3channels are able to conduct both inward and outward currents.

Electrophysiologically, K⁺-permeable channels at the plasmamembrane of mesophyll cells were characterized as KIRs (Kourie and Goldsmith, 1992;Karley et al., 2000a), and KORs (Spaldinget al., 1992; Blom-Zandstra et al., 1997; Pineros and Kochian, 2003).These KOR channels are regulated by Ca²⁺ and G-proteins(Krol and Trebacz, 2000) and may play a role in stabilizingcell membrane potential (Pineros and Kochian, 2003). Also, Ca²⁺-sensitive,depolarization-activated NSCCs were found at the plasma membranein arabidopsis mesophyll cells (Spalding et al., 1992).

In addition to KIR channels there is a need for active K⁺ transportersin mesophyll cells, since environmental fluctuations, such aslight/dark transitions, may leave E_m positive to E_k (Shabala and Newman, 1999),making the function of KIR channels impossible.Both HAK/KT/KUP and HKT type transporters are present at theplasma membrane of leaf mesophyll cells (Table 2; Leigh, 2001;Golldack et al., 2002; Su et al., 2002).

Epidermis
Only a few electrophysiological studies have specifically targetedK⁺ transporters from epidermal cells other than stomata. Twotime-dependent Ca²⁺-regulated K⁺-selective channels (resemblingguard cell KIR and KOR, respectively) were found in subsidiarycells of maize (Majore et al., 2002). Time-dependent inwardK⁺ currents were also reported for barley epidermis (Karleyet al., 2000a) and there is evidence that NSCCs may also bepresent in leaf epidermal cells (Elzenga and Van Volkenburgh, 1994;Majore et al., 2002). At the molecular level, expressionof AKT2 genes (Cherel et al., 2002) and the HAK/KT/KUP K⁺ transporters(Su et al., 2002) have been attributed to epidermal cells.

Vascular tissues
Phloem loading with assimilates is accompanied by a significantincrease in symplastic K⁺ concentration, required to maintainelectrical neutrality during vectorial H⁺ transfer. The mostabundant K⁺ channels in the phloem tissue are AKT3 (Marten etal., 1999; Cherel et al., 2002) and their homologues (Golldacket al., 2003). The unique characteristics of this channel (itsweak voltage dependence, inhibition by physiological concentrationsof external Ca²⁺ and by extracellular acidification, and theability to be open in the entire physiological voltage range;Marten et al., 1999), allow AKT3 to mediate both K⁺ influx andefflux, determining the diverse roles of AKT3 in the phloem.Another major type of K⁺ channel detected in minor veins isKAT2 (Pilot et al., 2001), involved in K⁺ loading into the phloemsap.

There is both electrophysiological and molecular evidence forthe presence of high affinity K⁺ transporters, in addition tochannels, in the leaf vascular system. McHAKs transcripts showedsignals in the leaf vascular bundles (Su et al., 2002) and theHKT1 transporter was localized in cell layers bordering thevascular tissue in leaves (Schachtman, 2000).

 

Potassium transport within leaf cells

Vacuole
Several types of K⁺ permeable channels are known to be presentin the tonoplast (Fig. 2). The most abundant are slow-activating(SV) and fast-activating (FV) vacuolar channels. The SV channelis permeable to both mono- and divalent cations and is activatedby cytosolic Ca²⁺ and positive vacuolar voltage. The FV channelis selective for monovalent cations only, activated by positivevoltages, and may be blocked by divalent cations (for a review,see Allen and Sanders, 1997). Both SV and FV channels are ubiquitousin plant tissues, including mesophyll and guard cell vacuoles(Allen and Sanders, 1997; Pottosin et al., 1997; Tikhonova etal., 1997). In addition, guard cells possess another channelspecifically selective for K⁺ (Allen and Sanders, 1997). Thisso-called VK channel is voltage-independent and activated byCa²⁺ (Schönknecht et al., 2002) as well as by cytosolicalkalization (Allen and Sanders, 1997). Recently, a two-poreA. thaliana KOR channel, named AtKCO1, was cloned and localizedto the tonoplast of both mesophyll cells and guard cells (Czempinskiet al., 2002; Schönknecht et al., 2002). Finally, thereis evidence for mechanosensory SAS channels to be present atthe tonoplast (Alexandre and Lassalles, 1991).

At least two other types of transporters may also contributeto K⁺ transport across the tonoplast. Firstly, Banuelos et al.(2002) targeted the rice OsHAK10 (a member of KUP/HAK/KT family)gene to the tonoplast and suggested that such a transportermay be needed to release K⁺ from the vacuole to the cytoplasmwhen the vacuolar concentration is low. Secondly, there is evidencethat NHX1 (a vacuolar Na⁺/H⁺ exchanger) has some affinity forK⁺ and may operate in K⁺ transport during low Na⁺ conditions[Blumwald et al. (2000) and references within].

Chloroplast
The transport barrier in the chloroplast is the inner membrane,which contains transporters for a selected numbers of low molecularweight substrates. The outer membrane contains specific pore-formingproteins and is permeable to substances with molecular weightof several kDa (Pottosin, 1992). Most of these ‘pores’are also able to conduct ions (for a review, see Neuhaus and Wagner, 2000).

Massive light-driven transport of H⁺ into the thylakoid lumenis electrically balanced by the counter flow of other ions (Hinnah and Wagner, 1998).This process is mediated by weakly voltage-dependentcation-selective channels, equally permeable to K⁺ and Mg²⁺(Pottosin and Schönknecht, 1996). Several types of cation-permeablechannels have been found at thylakoid membranes of differentspecies (Pottosin, 1992; Pottosin and Schonknecht, 1996; Hinnah and Wagner, 1998).All of them belong to the NSCC class. Channelconductance varied greatly from 60 pS (Pottosin and Schonknecht 1996)to very high values (non-selective porin-like maxi channelwith 1016 pS conductance; Pottosin, 1992). Most of thesechannels show bimodal gating (Pottosin, 1992). However, somechannels showed only moderate voltage dependence (Pottosin and Schönknecht, 1996),suggesting that additional mechanismsto regulate the thylakoid cation channel activity might be involved.

Mitochondria
ATP-sensitive K⁺ channels (mitoKATP) have been reported forvarious animal tissues (Ferranti et al., 2003). For plant mitochondria,studies on K⁺ transporters are still at an early stage. It appearsthat the only reported evidence comes from Petrussa et al. (2001),who provided evidence that plant mitochondria possess a K⁺ selective,voltage-dependent channel, which is opened by cyclosporin, regulatedby the redox state and inhibited by nucleotides. The hypotheticalrole of this new ATP-sensitive K⁺ channel was attributed tomitochondrial volume regulation, thermogenesis, apoptosis and/orprevention of ROS formation in plants. As studies on ROS haverecently received a great deal of attention from plant scientists(Demidchik et al., 2003; Kohler et al., 2003), there is obviouslya strong need for more studies on the role and mechanisms ofK⁺ transport across the mitochondrial membranes.

 

Regulation of k+ transporter activity by environmental stimuli

Light
Light stimulates expansion growth of leaves via cell wall acidificationand increased cell turgor, both resulting from light activationof H⁺-ATPase (Van Volkenburgh, 1999). The most rapid responseof growing cells to light is electrical depolarization (Elzengaet al., 1995; Van Volkenburgh, 1999) caused by an influx ofCa²⁺ (Shabala and Newman, 1999). This response could be eithera part of a signal transduction pathway leading to growth regulationor, more likely, reflect a part of the growth mechanism itself.

Guard cells.
There are both red light (RL) and blue light (BL) componentswithin the action spectrum of stomatal opening. RL stimulatesphotosynthetic activity within the chloroplast, thereby providingan energy source for H⁺ extrusion (Dietrich et al., 2001). Thisprocess is mediated by chlorophyll located in guard cell protoplasts.BL also drives guard cell photosynthesis acting via a cryptochromeor zeaxanthin (Assmann and Wang, 2001; Dietrich et al., 2001;Zeiger et al., 2002) receptor.

Several mechanisms mediate RL and BL control over activity ofK⁺ transporters in guard cells. First, most guard cell K⁺ channelsare voltage-dependent (see above) and thus are coupled withlight-induced stimulation of the H⁺-ATPase (Dietrich et al.,2001). Secondly, light-induced apoplastic acidification providesan additional mechanism for K⁺ gating (Blatt, 2000). Finally,many guard cell K⁺ channels are Ca²⁺ sensitive (Blatt, 2000;Dietrich et al., 2001), and light-induced elevation in cytosolicfree Ca²⁺ is a widely reported phenomenon (Schroeder et al.,2001). In addition to chlorophyll-mediated light signallingin guard cells, there is growing evidence that phytochrome isanother photoreceptor involved in control of stomatal aperture(Zeiger et al., 2002).

Epidermis.
Both BL and RL cause significant changes in net K⁺ flux acrossthe plasma membrane of epidermal cells (S. Shabala and B. Zivanovic,unpub. res.). It has been proposed that BL stimulates the H⁺pump by direct interaction between the BL photoreceptor andthe pump, while RL may influence pump activity indirectly bymodulating passive ion conductances of Ca²⁺ and K⁺ channels[Van Volkenburgh (1999) and references within]. Phytochromeis also likely to be involved as shown in experiments with pcd2phytochrome-deficient pea mutants (Elzenga et al., 2000).

The precise roles of light-stimulated ion fluxes in leaf epidermis,apart from H⁺ efflux, are obscure. As membranes cannot stretch,it was suggested that phytochrome-mediated Ca²⁺ influx acrossthe plasma membrane enhances vesicle fusion, wall synthesisand growth in leaf cells (Van Volkenburgh, 1999). The role ofphytochrome-mediated K⁺ fluxes from epidermal cells remainsunresolved.

Mesophyll.
Transient increase in K⁺ uptake across the plasma membrane ofleaf mesophyll cells has been reported (Spalding et al., 1992;Blom-Zandstra et al., 1997; Shabala and Newman, 1999), mostlikely due to an increase in the open probability of KIR channels(Spalding et al., 1992). As light-induced up-regulation of K⁺channel activity was not observed in chlorophyll deficient cells,it was concluded that this process is mediated by photosynthesis(Blom-Zandstra et al., 1997). This view is further supportedby the presence of a significant lag period (tens of seconds;Spalding et al., 1992; Shabala and Newman, 1999) between theonset of illumination and K⁺ flux changes (presumably due todiffusion of the unknown activator produced by illuminated chloroplasts).The physiological role of light-induced K⁺ influx in leaf mesophyllremains obscure. The transient character of this process (Shabala and Newman, 1999)suggests that K⁺ influx is involved in thecharge balance, rather than directly contributing to an increasein cell turgor.

Temperature
Information about direct effects of temperature on activityof ion channels (and, specifically, K⁺ channels) remains rudimentary.Ilan et al. (1995) reported that inward- and outward-rectifyingchannels in Vicia guard cell protoplasts had different temperature-dependentactivation kinetics. Temperature effects on the voltage-dependenceof the open probability of KIR channels were explained by temperature-inducedshifts in the electric field in the vicinity of the channel,whereas the bell-shaped response in the number of active KORchannels was explained by temperature effects on membrane fluidityand, thus, on the activity of the channels. Indeed, criticaltemperature of recovery of K⁺ transporters in corn epidermalcells showed strong correlation with the phase transition ofmembrane lipids (Shabala and Shabala, 2002). Therefore, decreasedavailability of KOR channels was interpreted as a reflectionof immobilization (‘locking’) of the channel gatesat some ‘superclosed’ conformation (Ilan et al.,1995). This issue remains highly speculative and requires furtherinvestigation.

Salinity
Plant adaptation to salt stress involves significant ‘reprogramming’of K⁺-channel gene expression, especially in leaves (Golldacket al., 2003; Pilot et al., 2003) and includes strong and progressiveincrease in the level of AtKC1 transcript and a decrease inAKT2 mRNA accumulation. This effect is especially pronouncedin the leaf epidermis (Dennison et al., 2001) and was specificto Na⁺ toxicity, but not to changes in osmotic potential. Expressionof McHAK2 and McHAK3 was stimulated in leaves of the ice plantin response to high salinity (Su et al., 2002). The most exceptionalstimulation was in phloem cells, while expression of HKT1 homologuesin rice and barley were both strongly inhibited by salinity(Su et al., 2002). At the intracellular level, salinity blocksa substantial portion of FV channels, presumably by increasingthe level of endogenous polyamines in the cell cytosol (Brüggemannet al., 1998).

Drought
K⁺ channel activity in guard cells is known to be regulatedby ABA (Assmann and Shimazaki, 2001; Luan, 2002). Biosynthesisof ABA in both root and shoot tissues is significantly enhancedby soil drying and/or salinity (reviewed in Wilkinson and Davies, 2002).Soil drying also increases the pH of the xylem stream,reducing the ability of the leaf symplastic compartments tosequester ABA, thus amplifying the ABA effects. In addition,in dry soil, there is a significant reduction in xylem sap K⁺,reducing K⁺ availability as a guard cell osmoticum (Wilkinson and Davies, 2002).Taken together, these factors are believedto be responsible for stomatal closure under drought conditions(Luan, 2002; Wilkinson and Davies, 2002).

Regulation of K⁺ channels may be mediated by either ABA-inducedincrease in cytosolic free Ca²⁺, which activates KIR (Assmann and Wang, 2001;Schroeder et al., 2001; Kohler et al., 2003),or via an ABA-induced Ca²⁺-independent pathway (Blatt, 2000).The latter is probably mediated by the pH_i effect on activityof FV and SV vacuolar channels (Assmann and Wang, 2001); notinvolvement of ROS is also possible (Kohler et al., 2003). Also,not only gating properties and/or open probability of K⁺ channels,but their expression per se might be affected by ABA (Pilotet al., 2003). Finally, mechanosensory (SAS) channel involvementcannot be ruled out (Cosgrove and Hedrich, 1991).

Conclusions

As stated by Assmann and Wang (2001), the ‘emergent collectivebehaviour’ of stomata adds one more layer of complexityto stomatal function under real-world conditions. In the caseof regulation of K⁺ transport in plant leaves, there is obviouslymore than one such layer of complexity. Precise intracellularand tissue locations of many K⁺ transporters remain to be elucidated,as well as their specific physiological functions and gatingmodes (see gaps in Fig. 1 insert). Our knowledge of mechanismscoordinating and ‘tuning’ a complicated networkof K⁺ transport systems in various leaf tissues in responseto environmental fluctuations remains in its infancy. Expressionstudies alone may not reliably map out distribution of genefunctions (Dennison et al., 2001). Also, results from the expressionof K⁺ transporters in various heterologous systems are artificialand sometimes confusing. There is a strong need for in plantafunctional studies of specific mutants to quantify the relativecontributions of particular members of a gene family to plantadaptive responses to the environment. An amalgamation of thepower of the tools of molecular biology with an understandingof the plant’s functioning as an ‘entirety’has always been and remains the highest priority for futureresearch.

Acknowledgments

My sincere apologies to many colleagues whose research was notcited due to space constraints. Many special thanks to ProfessorI. Pottosin (University of Colima) for valuable references onlocalization of K⁺ transporters within cell organelles. I wouldlike also to acknowledge Drs I. Newman, O. Babourina and L.Shabala for the critical reading of this manuscript and helpfulsuggestions, and my son Stas for technical assistance. The financialsupport from ARC and AusIndustry is greatly acknowledged.

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Table 1

Functional expression of plasma membrane K⁺ channels in various leaf tissues in arabidopsis

Class	Genes	Expression in leaves	Voltage dependence	Physiological function	Reference
Shaker-typeK+ channels	AKT1	Guard cells	Inward rectifier	Stomatal opening	Szyroki et al. (2001)
		Mesophyll		Charge balance (?)	Dennison et al. (2001)
	AKT2/3	Phloem	Weakly voltagedependent	Phloem loading;charge balance;osmoregulation;long-distance transport	Marten et al. (1999)
		Guard cells			Szyroki et al. (2001)
		Mesophyll			Dennison et al. (2001)
		Epidermis			Cherel et al. (2002)
	AKT5	Guard cells	Unknown	Unknown	Dietrich et al. (2001)
	AKT6	Guard cells	Unknown	Unknown	Dietrich et al. (2001)
	KAT1	Guard cells	Inward rectifier	Stomatal opening	Assmann and Wang (2001)
	KAT2	Guard cells	Inward rectifier	Stomatal opening	Szyroki et al. (2001)
		Vascular system		K+ loading into phloem	Pilot et al. (2001)
	AtKC1	Guard cells	Inward rectifier	Stomatal opening	Pilot et al. (2001)
		Epidermis		Extension growth (?)	Dietrich et al. (2001)
		Mesophyll		Charge balance (?)	Dennison et al. (2001)
	GORK	Guard cells	Outward rectifier	Stomatal closure	Mäser et al. (2001)

Table 2

Examples of K⁺ transporters, their functions and intracellular location in leaves of various plant species

Class	Genes	Species	Expression inleaves	Intracellularlocation	Physiological function	Reference
KUP/HAK/KTtransporters	McHAK1	Ice plant	Mesophyll	Plasmalemma	High (?) affinity K+ uptake	Su et al. (2002)
			Epidermis	Plasmalemma
	McHAK4	Ice plant	Mesophyll	Plasmalemma	High (?) affinity K+ uptake	Su et al. (2002)
			Epidermis	Plasmalemma
	OsHAK10	Rice	Epidermis	Tonoplast	K+ efflux from vacuole underK+ deficiency	Banuelos et al. (2002)
	HvHAK1	Barley	Phloem	Plasmalemma	High (?) affinity K+ uptake	Su et al. (2002)
HKT transporters	OsHKT1	Rice	Mesophyll	Plasmalemma	High-affinity K+ loading	Golldack et al. (2002)
	HKT1	Wheat	Vascular tissue	Plasmalemma	Xylem unloading	Schachtman (2000)
H+/Na+(K+)antiporter	AtNHX1	Arabidopsis	Epidermis	Tonoplast	K+ efflux from vacuole underK+ deficiency	Blumwald et al. (2000)
			Mesophyll

Figures

Fig. 1. Diversity of K+ transport mechanisms at the plasma membrane of a ‘generalized’ leaf cell. Four different types of K+-selective channels [inward- (KIR) and outward- (KOR) rectifying, stretch-activated (SAS) and weakly voltage-dependent (WDKC) channels], one non-selective cation channel (NSCC) and two K+ transporters (HKT and HUK) are shown. Their gating properties are shown in the figure inset (a tick denotes activation/deactivation by an external or internal factor; a question mark means the gating properties are not known). MP, membrane potential; CN, cyclic nucleotides; Pol, polyamines; ROS, reactive oxygen species. (Click image to enlarge)

Fig. 2. Potassium transport mechanisms at plant tonoplast. Five types of K+-permeable channels [slow (SV)- and fast (FV)-activated non-selective channels; K+-selective (VK) channel; K+ outward rectifiers (KCO); and stretch-activated (SAS) K+ channels] are shown. In addition, at least two transporters (HUK and NHX) may also contribute to K+ transfer across the tonoplast. (Click image to enlarge)

 

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