Download Regulation of Potassium Transport in Leaves: from Molecular to

Annals of Botany 92: 627±634, 2003
doi:10.1093/aob/mcg191, available online at www.aob.oupjournals.org
BOTANICAL BRIEFING
Regulation of Potassium Transport in Leaves: from Molecular to Tissue Level
SERGEY SHABALA*
School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, Tasmania 7001, Australia
Received: 8 May 2003 Returned for revision: 29 May 2003 Accepted: 11 August 2003 Published electronically: 19 September 2003
Over millions of years, plants have evolved a sophisticated network of K+ transport systems. This Botanical
Brie®ng provides an overview of K+ transporters in various leaf tissues (epidermis, mesophyll, guard cells and
vascular system) at both the cellular and organelle levels. Despite the tremendous progress in our knowledge of
genes encoding K+ transport systems in plants, understanding has not developed of coordinated functioning and
operation of these genes or proteins in the context of whole plant physiology and plant±environment interaction.
This Botanical Brie®ng is aimed at ®lling that gap by analysing electrophysiological and molecular evidence for
mechanisms coordinating K+ transport between various leaf cells and tissues in changing environments.
ã 2003 Annals of Botany Company
INTRODUCTION
Potassium is the most abundant inorganic cation in nonhalophytes, contributing up to 6 % of plant dry weight. As a
major inorganic osmolyte, K+ is crucial for cell osmoregulation and turgor maintenance and, hence, for cell
expansion, stomatal function, tropisms and leaf movement.
Being an activator of a large number of enzymes, K+ plays a
key role in photosynthesis, protein synthesis, and oxidative
metabolism. It also affects phloem transport and provides
charge balance during vectorial ion transfer across cellular
membranes (Marschner, 1995).
Being complex and heterogeneous structures, plants have
evolved a sophisticated and highly specialized K+ transport
system to meet the different requirements for K+ in various
cells and tissues and many reviews addressing various
molecular and electrophysiological aspects of K+ transport
in plants have been published recently (Maathuis and
Amtmann, 1999; Schachtman, 2000; MaÈser et al., 2001,
2002; Very and Sentenac, 2002; Pilot et al., 2003). Most of
these deal with regulation of K+ uptake and transport in
roots. As almost all K+ found in plant tissues is taken up by
roots, it is not surprising that K+ transport systems in roots
are the most studied, although the majority of K+ in a plant is
found in the stems and leaves. In contrast to root cells, our
knowledge of the properties and regulation of membrane K+
transport in leaves is much more limited (Pineros and
Kochian, 2003).
Recent advances in molecular biology have allowed
tremendous progress in our knowledge of genes encoding
K+ transport systems in plants (MaÈser et al., 2001).
However, with a possible exception of guard cells
(Assmann and Shimazaki, 1999; Blatt, 2000; Schroeder
et al., 2001), understanding of the coordinated functions and
operation of these genes/proteins in the context of whole
plant physiology and plant±environment interaction has not
developed at the same rate. In this Brie®ng, an attempt is
* For correspondence. E-mail [email protected]
made to ®ll this gap and address electrophysiological and
molecular aspects of coordination of K+ transport between
various leaf cells and tissues under natural conditions.
H E T E RO G EN E I T Y O F L EA V E S
The heterogeneous nature of leaves is obvious: there are
striking differences between leaves in venation, stomatal
alignment and stomatal density. It is not surprising,
therefore, to ®nd that the nutritional demands and, consequently, regulation of ionic uptake are remarkably different
in various cells, even of one leaf.
Heterogeneity of leaf ionic environment
The cells of the mesophyll and of the epidermis are very
different anatomically, with their vacuoles occupying
around 61±75 % and up to 99 % of the cell volume,
respectively (Karley et al., 2000a). In addition, epidermal
cells are virtually unable to produce organic solutes and rely
heavily on inorganic ions (mainly K+) for osmotic adjustment (Fricke et al., 1994). In mesophyll cells, the total
contribution of organic osmolytes is much higher (20±30 %;
Gonzales et al., 2002) than in epidermal cells, although K+
remains the dominant osmoticum (Shabala et al., 2000;
Gonzales et al., 2002). A difference in K+ composition
between epidermal and mesophyll cells is an obvious
consequence of their different solute compositions (Leigh,
2001). Cytosolic K+ homeostasis is a characteristic feature
of mesophyll cells and is maintained at the expense of the
supply to the epidermis, where cytosolic K+ may decline to
very low levels (Cuin et al., 2003). This homeostasis
enables protection and maintenance of optimal photosynthetic activity of the mesophyll cells. The process underlying differential accumulation of ions by leaf cells can
include both the regulation of supply of certain ions and the
capacity for ion uptake (Karley et al., 2000b).
Apoplastic free K+ concentrations are usually much lower
than symplastic and have been reported between 2 and
Annals of Botany 92/5, ã Annals of Botany Company 2003; all rights reserved
628
Shabala Ð Regulation of Potassium Transport in Leaves
TA B L E 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-type
K+ channels
AKT1
Guard cells
Inward recti®er
Stomatal opening
Szyroki et al. (2001)
AKT2/3
Mesophyll
Phloem
Charge balance (?)
Phloem loading;
charge balance;
osmoregulation;
long-distance transport
Dennison et al. (2001)
Marten et al. (1999)
AKT5
AKT6
KAT1
KAT2
AtKC1
GORK
Weakly voltage
dependent
Guard cells
Mesophyll
Epidermis
Guard cells
Guard cells
Guard cells
Guard cells
Vascular system
Guard cells
Epidermis
Mesophyll
Guard cells
Unknown
Unknown
Inward recti®er
Inward recti®er
Inward recti®er
Outward recti®er
Unknown
Unknown
Stomatal opening
Stomatal opening
K+ loading into phloem
Stomatal opening
Extension growth (?)
Charge balance (?)
Stomatal closure
Szyroki et al. (2001)
Dennison et al. (2001)
Cherel et al. (2002)
Dietrich et al. (2001)
Dietrich et al. (2001)
Assmann and Wang (2001)
Szyroki et al. (2001)
Pilot et al. (2001)
Pilot et al. (2001)
Dietrich et al. (2001)
Dennison et al. (2001)
MaÈser et al. (2001)
TA B L E 2. Examples of K+ transporters, their functions and intracellular location in leaves of various plant species
Class
KUP/HAK/KT
transporters
HKT transporters
H+/Na+(K+)
antiporter
Genes
Species
Expression in
leaves
Intracellular
location
Physiological function
Reference
McHAK1
Ice plant
Mesophyll
Plasmalemma
High (?) af®nity K+ uptake
Su et al. (2002)
McHAK4
Ice plant
Su et al. (2002)
Rice
Plasmalemma
Plasmalemma
Plasmalemma
Tonoplast
High (?) af®nity K+ uptake
OsHAK10
Epidermis
Mesophyll
Epidermis
Epidermis
Banuelos et al. (2002)
HvHAK1
OsHKT1
HKT1
AtNHX1
Barley
Rice
Wheat
Arabidopsis
Phloem
Mesophyll
Vascular tissue
Epidermis
Plasmalemma
Plasmalemma
Plasmalemma
Tonoplast
K+ ef¯ux from vacuole under
K+ de®ciency
High (?) af®nity K+ uptake
High-af®nity K+ loading
Xylem unloading
K+ ef¯ux from vacuole under
K+ de®ciency
Mesophyll
26 mM (Karley et al., 2000a; Roelfsema and Hedrich, 2002).
These values may change 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
different regions of the leaf lamina and yet expansion of
various leaf tissues is highly coordinated. It is believed that
the epidermis normally restricts leaf expansion (Van
Volkenburgh, 1999), presumably due to the fact that
epidermal cells usually have lower turgor than mesophyll
cells. Even within the epidermis, a signi®cant difference
was measured between K+ uptake at the growing (leaf base)
Su et al. (2002)
Golldack et al. (2002)
Schachtman (2000)
Blumwald et al. (2000)
and at the fully extended (leaf tip) regions of corn leaves
(S. Shabala and B. Zivanovic, unpub. res.). Although the
molecular and ionic mechanisms underlying these differences are yet to be studied, it is reasonable to suggest that
either K+ concentration in the apoplast or functional
expression of K+ transporters will vary signi®cantly
between the apical and basal regions of the leaf, depending
on growth patterns. It is also possible that post-transcriptional modulation of transport activity may determine these
differences.
On a smaller scale, stomatal `patchiness' is another
example of heterogeneity of leaf physiological characteristics. It has been demonstrated that in many species and
under various experimental conditions, stomatal conductance and behaviour differ between regions of the leaf,
forming patches up to several millimetres across (Mott and
Buckley, 1998). Based on membrane potential measure-
Shabala Ð Regulation of Potassium Transport in Leaves
629
ments, Roelfsema et al. (2001) showed that, in planta,
stomatal guard cells exhibit at least three different physiological states under apparently identical environmental
conditions. It was speculated that local production and
distribution of chemical `signals' facilitates these differences (Mott and Buckley, 1998). Keeping in mind the
apoplastic coupling between epidermal and guard cells and
the fact that leaf apoplastic K+ concentration is strongly
correlated with the leaf venation (Shabala et al., 2002), it is
possible to suggest that K+ distribution in the leaf apoplast is
one of the factors determining stomatal `patchiness' and
heterogeneity in leaf photosynthesis.
FUNCTIONAL EXPRESSION OF PLASMA
MEMBRANE POTASSIUM TRANSPORTERS
IN L EAF T I S S UE S
General features of K+ transporters in plants
A large number of genes encode proteins involved in K+
transport in plants. In arabidopsis, these transport mechanisms fall into several distinct categories (MaÈser et al., 2001,
2002; Very and Sentenac, 2002): (a) two families of K+
channels (Shaker-type and KCO channels; 15 genes in
total); (b) Trk/HKT transporters [Na+/K+ symporter
(Schachtman, 2000); one gene]; (c) KUP/HAK/KT transporters [H+/K+ symporter (Kim et al., 1998); 13 genes]; (d)
K+/H+ antiporter homologs (six genes); (e) cyclic-nucleotide-gated channels (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 subdivided into SKOR and GORK channels
(both depolarization-activated), KAT channels and AKT
channels. AKT channels contain an ankyrin-binding motif,
which is lacking in KAT type channels (MaÈser et al., 2001).
An important feature of plant Shaker-like K+ channels is
that they can form heterotetrameric structures (Pilot et al.,
2003), allowing plants to tune the K+ transport activity in
various cells, independently in each organ/tissue, in relation
to environmental conditions. A brief summary of the
functional expression of plasma membrane K+ channels in
various leaf tissues in Arabidopsis thaliana is given in
Table 1, and some details of the functions and intracellular
location of K+ transporters in leaves of various plant species
are shown in Table 2. The diversity of K+ transport
mechanisms at the plasma membrane of a `generalized'
leaf cell is summarized in Fig. 1.
Stomatal guard cells
Two major types of K+ channels are present at the plasma
membrane of guard cells: voltage-dependent K+-selective
inward (KIR) and outward (KOR) rectifying channels (Pilot
et al., 2001; Schroeder et al., 2001; Szyroki et al., 2001;
Zimmermann et al., 2001). KIR channels are activated by
membrane hyperpolarization and mediate stomatal opening,
whereas KOR channels are opened by voltages more
positive than Ek and mediate stomatal closure.
Recent transcription±PCR experiments with isolated
guard cell protoplasts showed that in addition to KAT1,
F I G . 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 ®gure 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.
the K+ channels AKT1, AKT2/3, AtKC1 and KAT2 (all
KIRs) were expressed (Szyroki et al., 2001), suggesting that
KAT1 inward-rectifying K+ channels may not play as
dominant a role in K+ uptake in guard cells as previously
believed (Assmann and Wang, 2001). It was also shown that
KAT1 and KAT2 can form heteromultimeric channels
(Pilot et al., 2001; Zimmermann et al., 2001), leading to
more ¯exibility when adapting to altered developmental
and/or environmental conditions. Several other channels of
unknown voltage-dependence (AtKC1, AKT5 and AKT6)
were also shown to co-localize in arabidopsis guard cells
(Dietrich et al., 2001). It is thought that such a multiple
ensemble of K+ channels provides greater versatility and
much more ef®cient regulation of K+ homeostasis in guard
cells compared with only one type of KIR channel. The only
certain candidate in the arabidopsis genome to mediate
stomatal closure is GORK, a voltage-gated outwardly
rectifying K+ channel of the guard cell membrane (Hosy
et al., 2003).
630
Shabala Ð Regulation of Potassium Transport in Leaves
In addition to speci®c K+-selective channels, guard cells
also possess a wide range of non-selective cation channels
(NSCC), either depolarization- or hyperpolarization-activated (Demidchik et al., 2002). These channels are likely to
be involved in release of solutes during turgor adjustment
and, to some extent, functionally complement GORK
channels. Finally, there is strong evidence that 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 open
probabilities as a result of volume or turgor changes.
As well as their voltage-dependence, K+ channels in guard
cells are regulated by several other factors, the most obvious
of which is pH. Both apoplastic (Pilot et al., 2001;
Roelfsema and Hedrich, 2002) and cytosolic (Dietrich
et al., 2001) acidi®cation lead 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 phosphorylation events also exert direct control over K+ channel activity
(Assmann and Shimazaki, 1999; Blatt, 2000; Schroeder et al.,
2001; Kohler et al., 2003). It has also been shown that guard
cells may utilize voltage-dependent K+ channels as targets of
the osmosensing pathway by regulating channel opening
probability by the osmogradient across the plasma membrane
of guard cells (Liu and Luan, 1998).
Mesophyll
Molecular studies suggested that both AKT1 and
AKT2/3 genes are expressed in arabidopsis leaf mesophyll
(Dennison et al., 2001; Cherel et al., 2002). A speci®c
feature of AKT2/3 channels is their weak dependence on the
membrane potential, sensitivity to ATP and an inverted pH
regulation (Marten et al., 1999). Being only weakly
controlled by the membrane potential, AKT2/3 channels
are able to conduct both inward and outward currents.
Electrophysiologically, K+-permeable channels at the
plasma membrane of mesophyll cells were characterized as
KIRs (Kourie and Goldsmith, 1992; Karley et al., 2000a),
and KORs (Spalding et 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 stabilizing cell membrane potential
(Pineros and Kochian, 2003). Also, Ca2+-sensitive, depolarization-activated NSCCs were found at the plasma membrane in arabidopsis mesophyll cells (Spalding et al., 1992).
In addition to KIR channels there is a need for active K+
transporters in mesophyll cells, since environmental ¯uctuations, such as light/dark transitions, may leave Em positive
to Ek (Shabala and Newman, 1999), making the function of
KIR channels impossible. Both HAK/KT/KUP and HKT
type transporters are present at the plasma membrane of leaf
mesophyll cells (Table 2; Leigh, 2001; Golldack et al.,
2002; Su et al., 2002).
Epidermis
Only a few electrophysiological studies have speci®cally
targeted K+ transporters from epidermal cells other than
stomata. Two time-dependent Ca2+-regulated K+-selective
channels (resembling guard cell KIR and KOR, respectively) were found in subsidiary cells of maize (Majore et al.,
2002). Time-dependent inward K+ currents were also
reported for barley epidermis (Karley et al., 2000a) and
there is evidence that NSCCs may also be present in leaf
epidermal cells (Elzenga and Van Volkenburgh, 1994;
Majore et al., 2002). At the molecular level, expression of
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
signi®cant increase in symplastic K+ concentration, required
to maintain electrical neutrality during vectorial H+ transfer.
The most abundant K+ channels in the phloem tissue are
AKT3 (Marten et al., 1999; Cherel et al., 2002) and their
homologues (Golldack et al., 2003). The unique characteristics of this channel (its weak voltage dependence,
inhibition by physiological concentrations of external Ca2+
and by extracellular acidi®cation, and the ability to be open
in the entire physiological voltage range; Marten et al.,
1999), allow AKT3 to mediate both K+ in¯ux and ef¯ux,
determining the diverse roles of AKT3 in the phloem.
Another major type of K+ channel detected in minor veins is
KAT2 (Pilot et al., 2001), involved in K+ loading into the
phloem sap.
There is both electrophysiological and molecular evidence for the presence of high af®nity K+ transporters, in
addition to channels, in the leaf vascular system. McHAKs
transcripts showed signals in the leaf vascular bundles (Su
et al., 2002) and the HKT1 transporter was localized in cell
layers bordering the vascular tissue in leaves (Schachtman,
2000).
PO T A S S I U M T RA N SP O R T W I TH I N L E A F
C EL L S
Vacuole
Several types of K+ permeable channels are known to be
present in the tonoplast (Fig. 2). The most abundant are
slow-activating (SV) and fast-activating (FV) vacuolar
channels. The SV channel is permeable to both mono- and
divalent cations and is activated by cytosolic Ca2+ and
positive vacuolar voltage. The FV channel is selective for
monovalent cations only, activated by positive voltages, and
may be blocked by divalent cations (for a review, see Allen
and Sanders, 1997). Both SV and FV channels are
ubiquitous in plant tissues, including mesophyll and guard
cell vacuoles (Allen and Sanders, 1997; Pottosin et al.,
1997; Tikhonova et al., 1997). In addition, guard cells
possess another channel speci®cally selective for K+ (Allen
and Sanders, 1997). This so-called VK channel is voltageindependent and activated by Ca2+ (SchoÈnknecht et al.,
2002) as well as by cytosolic alkalization (Allen and
Sanders, 1997). Recently, a two-pore A. thaliana KOR
channel, named AtKCO1, was cloned and localized to the
Shabala Ð Regulation of Potassium Transport in Leaves
631
ions (Hinnah and Wagner, 1998). This process is mediated
by weakly voltage-dependent cation-selective channels,
equally permeable to K+ and Mg2+ (Pottosin and
SchoÈnknecht, 1996). Several types of cation-permeable
channels have been found at thylakoid membranes of
different species (Pottosin, 1992; Pottosin and Schonknecht,
1996; Hinnah and Wagner, 1998). All of them belong to the
NSCC class. Channel conductance varied greatly from 60 pS
(Pottosin and Schonknecht 1996) to very high values (nonselective porin-like maxi channel with 1016 pS conductance; Pottosin, 1992). Most of these channels show
bimodal gating (Pottosin, 1992). However, some channels
showed only moderate voltage dependence (Pottosin and
SchoÈnknecht, 1996), suggesting that additional mechanisms
to regulate the thylakoid cation channel activity might be
involved.
Mitochondria
F I G . 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 recti®ers (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.
tonoplast of both mesophyll cells and guard cells
(Czempinski et al., 2002; SchoÈnknecht et al., 2002).
Finally, there is evidence for mechanosensory SAS channels
to be present at the tonoplast (Alexandre and Lassalles,
1991).
At least two other types of transporters may also
contribute to 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 transporter may be needed to release
K+ from the vacuole to the cytoplasm when the vacuolar
concentration is low. Secondly, there is evidence that NHX1
(a vacuolar Na+/H+ exchanger) has some af®nity for K+ 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 molecular weight substrates. The outer
membrane contains speci®c pore-forming proteins and is
permeable to substances with molecular weight of 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
lumen is electrically balanced by the counter ¯ow of other
ATP-sensitive K+ channels (mitoKATP) have been
reported for various animal tissues (Ferranti et al., 2003).
For plant mitochondria, studies on K+ transporters are still at
an early stage. It appears that the only reported evidence
comes from Petrussa et al. (2001), who provided evidence
that plant mitochondria possess a K+ selective, voltagedependent channel, which is opened by cyclosporin, regulated by the redox state and inhibited by nucleotides. The
hypothetical role of this new ATP-sensitive K+ channel was
attributed to mitochondrial volume regulation, thermogenesis, apoptosis and/or prevention of ROS formation in
plants. As studies on ROS have recently received a great
deal of attention from plant scientists (Demidchik et al.,
2003; Kohler et al., 2003), there is obviously a strong need
for more studies on the role and mechanisms of K+ transport
across the mitochondrial membranes.
REGULATION OF K+ TRANSPORTER
A C T I V I T Y B Y E N V I R O N M E N T A L ST I M U L I
Light
Light stimulates expansion growth of leaves via cell wall
acidi®cation and increased cell turgor, both resulting from
light activation of H+-ATPase (Van Volkenburgh, 1999).
The most rapid response of growing cells to light is
electrical depolarization (Elzenga et al., 1995; Van
Volkenburgh, 1999) caused by an in¯ux of Ca2+ (Shabala
and Newman, 1999). This response could be either a part of
a signal transduction pathway leading to growth regulation
or, more likely, re¯ect a part of the growth mechanism itself.
Guard cells. There are both red light (RL) and blue light
(BL) components within the action spectrum of stomatal
opening. RL stimulates photosynthetic activity within the
chloroplast, thereby providing an energy source for H+
extrusion (Dietrich et al., 2001). This process is mediated
by chlorophyll located in guard cell protoplasts. BL also
drives guard cell photosynthesis acting via a cryptochrome
or zeaxanthin (Assmann and Wang, 2001; Dietrich et al.,
2001; Zeiger et al., 2002) receptor.
632
Shabala Ð Regulation of Potassium Transport in Leaves
Several mechanisms mediate RL and BL control over
activity of K+ transporters in guard cells. First, most guard
cell K+ channels are voltage-dependent (see above) and thus
are coupled with light-induced stimulation of the H+ATPase (Dietrich et al., 2001). Secondly, light-induced
apoplastic acidi®cation provides an 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 cytosolic free Ca2+ is a
widely reported phenomenon (Schroeder et al., 2001). In
addition to chlorophyll-mediated light signalling in guard
cells, there is growing evidence that phytochrome is another
photoreceptor involved in control of stomatal aperture
(Zeiger et al., 2002).
Epidermis. Both BL and RL cause signi®cant changes in
net K+ ¯ux across the 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 and the pump,
while RL may in¯uence pump activity indirectly by
modulating passive ion conductances of Ca2+ and K+
channels [Van Volkenburgh (1999) and references within].
Phytochrome is also likely to be involved as shown in
experiments with pcd2 phytochrome-de®cient pea mutants
(Elzenga et al., 2000).
The precise roles of light-stimulated ion ¯uxes in leaf
epidermis, apart from H+ ef¯ux, are obscure. As membranes
cannot stretch, it was suggested that phytochrome-mediated
Ca2+ in¯ux across the plasma membrane enhances vesicle
fusion, wall synthesis and growth in leaf cells (Van
Volkenburgh, 1999). The role of phytochrome-mediated
K+ ¯uxes from epidermal cells remains unresolved.
Mesophyll. Transient increase in K+ uptake across the
plasma membrane of leaf mesophyll cells has been reported
(Spalding et al., 1992; Blom-Zandstra et al., 1997; Shabala
and Newman, 1999), most likely 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 de®cient cells, it was concluded that
this process is mediated by photosynthesis (Blom-Zandstra
et al., 1997). This view is further supported by the presence
of a signi®cant lag period (tens of seconds; Spalding et al.,
1992; Shabala and Newman, 1999) between the onset of
illumination and K+ ¯ux changes (presumably due to
diffusion of the unknown activator produced by illuminated
chloroplasts). The physiological role of light-induced K+
in¯ux in leaf mesophyll remains obscure. The transient
character of this process (Shabala and Newman, 1999)
suggests that K+ in¯ux is involved in the charge balance,
rather than directly contributing to an increase in cell turgor.
Temperature
Information about direct effects of temperature on
activity of ion channels (and, speci®cally, K+ channels)
remains rudimentary. Ilan et al. (1995) reported that inwardand outward-rectifying channels in Vicia guard cell
protoplasts had different temperature-dependent activation
kinetics. Temperature effects on the voltage-dependence of
the open probability of KIR channels were explained by
temperature-induced shifts in the electric ®eld in the vicinity
of the channel, whereas the bell-shaped response in the
number of active KOR channels was explained by temperature effects on membrane ¯uidity and, thus, on the activity
of the channels. Indeed, critical temperature of recovery of
K+ transporters in corn epidermal cells showed strong
correlation with the phase transition of membrane lipids
(Shabala and Shabala, 2002). Therefore, decreased availability of KOR channels was interpreted as a re¯ection of
immobilization (`locking') of the channel gates at some
`superclosed' conformation (Ilan et al., 1995). This issue
remains highly speculative and requires further investigation.
Salinity
Plant adaptation to salt stress involves signi®cant `reprogramming' of K+-channel gene expression, especially in
leaves (Golldack et al., 2003; Pilot et al., 2003) and includes
strong and progressive increase in the level of AtKC1
transcript and a decrease in AKT2 mRNA accumulation.
This effect is especially pronounced in the leaf epidermis
(Dennison et al., 2001) and was speci®c to Na+ toxicity, but
not to changes in osmotic potential. Expression of McHAK2
and McHAK3 was stimulated in leaves of the ice plant in
response to high salinity (Su et al., 2002). The most
exceptional stimulation was in phloem cells, while expression of HKT1 homologues in rice and barley were both
strongly inhibited by salinity (Su et al., 2002). At the
intracellular level, salinity blocks a substantial portion of
FV channels, presumably by increasing the level of
endogenous polyamines in the cell cytosol (BruÈggemann
et al., 1998).
Drought
K+ channel activity in guard cells is known to be
regulated by ABA (Assmann and Shimazaki, 2001; Luan,
2002). Biosynthesis of ABA in both root and shoot tissues is
signi®cantly enhanced by 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 to sequester
ABA, thus amplifying the ABA effects. In addition, in dry
soil, there is a signi®cant reduction in xylem sap K+,
reducing K+ availability as a guard cell osmoticum
(Wilkinson and Davies, 2002). Taken together, these factors
are believed to be responsible for stomatal closure under
drought conditions (Luan, 2002; Wilkinson and Davies,
2002).
Regulation of K+ channels may be mediated by either
ABA-induced increase 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 pHi effect on activity of FV and SV
vacuolar channels (Assmann and Wang, 2001); not
Shabala Ð Regulation of Potassium Transport in Leaves
involvement 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 (Pilot et al., 2003). Finally, mechanosensory (SAS)
channel involvement cannot be ruled out (Cosgrove and
Hedrich, 1991).
CONCLUSIONS
As stated by Assmann and Wang (2001), the `emergent
collective behaviour' of stomata adds one more layer of
complexity to stomatal function under real-world conditions. In the case of regulation of K+ transport in plant
leaves, there is obviously more than one such layer of
complexity. Precise intracellular and tissue locations of
many K+ transporters remain to be elucidated, as well as
their speci®c physiological functions and gating modes (see
gaps in Fig. 1 insert). Our knowledge of mechanisms
coordinating and `tuning' a complicated network of K+
transport systems in various leaf tissues in response to
environmental ¯uctuations remains in its infancy.
Expression studies alone may not reliably map out distribution of gene functions (Dennison et al., 2001). Also,
results from the expression of K+ transporters in various
heterologous systems are arti®cial and sometimes confusing. There is a strong need for in planta functional studies of
speci®c mutants to quantify the relative contributions of
particular members of a gene family to plant adaptive
responses to the environment. An amalgamation of the
power of the tools of molecular biology with an understanding of the plant's functioning as an `entirety' has
always been and remains the highest priority for future
research.
ACKNOWLEDGEMENTS
My sincere apologies to many colleagues whose research
was not cited due to space constraints. Many special thanks
to Professor I. Pottosin (University of Colima) for valuable
references on localization of K+ transporters within cell
organelles. I would like also to acknowledge Drs I.
Newman, O. Babourina and L. Shabala for the critical
reading of this manuscript and helpful suggestions, and my
son Stas for technical assistance. The ®nancial support from
ARC and AusIndustry is greatly acknowledged.
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