Download REVIEW ARTICLE. Calcium Channels in the Plasma

Annals of Botany 81 : 173–183, 1998
REVIEW ARTICLE
Calcium Channels in the Plasma Membrane of Root Cells
P H I L I P J. W H I T E
Department of Cell Physiology, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK
Received : 11 July 1996
Returned for revision : 18 September 1997
Accepted : 8 October 1997
Calcium is an essential and major plant nutrient. It is required for structural, osmotic and signalling purposes. In this
review the pathway and molecular mechanisms are described by which Ca#+ enters the root and is delivered to the
xylem. The characteristics of inwardly-directed Ca#+ fluxes, Ca#+ currents and Ca#+-permeable channels in the plasma
membrane of root cells are compared. It is concluded that all root plasma membrane Ca#+ channels characterized to
date have a similar voltage-dependence (all are activated by membrane depolarization) but at least three distinct
classes can be defined based on their differential sensitivities to La$+, Gd$+ and verapamil. It is anticipated that further
classes of Ca#+ channels will be identified in the future, including stretch-activated (mechanosensitive) and second
messenger-activated Ca#+ channels. The roles of Ca#+ channels in mineral nutrition, intracellular signalling and
polarized growth are discussed.
# 1998 Annals of Botany Company
Key words : Calcium (Ca#+), channel, chilling, electrophysiology, endodermis, influx, root hair, plasma membrane.
INTRODUCTION
Calcium is an essential and major plant nutrient. It is
required in its ionic (Ca#+) form (1) extracellularly, for a
variety of structural roles, (2) as a cytoplasmic secondary
messenger, linking a range of external stimuli to their
physiological responses, and (3) in the vacuole, as a countercation for inorganic and organic anions (Marschner, 1995).
A shoot calcium content in excess of 0±1 to 1 % dry weight
is required for maximal growth. This requirement is larger
in legumes and herbs than in cereals and grasses, the
difference being attributed to the contrasting cation
exchange capacities of their cell walls. Sufficient calcium
may be obtained from flowing nutrient solutions with
calcium concentrations as low as 2±5 to 100 µ. However, if
the concentration of other cations is increased, or the
solution pH is lowered, a higher calcium concentration is
necessary. This is a consequence of competition between
cations for extracellular binding sites. Although mature
tissues may accumulate considerable calcium, due to the
immobility of calcium in the phloem, the growth of the
developing parts of a plant (such as fruits, young leaves and
the immature regions of the root) is dependent upon the
concurrent uptake of calcium. Furthermore, root apical
cells, which are not supplied via the xylem, must take up the
Ca#+ they require from the soil solution directly.
There is a considerable electrochemical gradient for Ca#+
movement into all root cells. Not only is there a cytoplasmnegative voltage difference across the plasma membrane,
but there is also a steep Ca#+ concentration gradient
between the rhizosphere (millimolar Ca#+) and cytoplasm
(submicromolar Ca#+ ; Clarkson, Brownlee and Ayling,
1988 ; Felle, 1988 ; Gehring, Irving and Parish, 1990 ; Tretyn,
Wagner and Felle, 1991 ; Felle, Tretyn and Wagner, 1992 ;
0305-7364}98}020173­11 $25.00}0
Ayling, Brownlee and Clarkson, 1994 ; Cramer and Jones,
1996 ; Herrmann and Felle, 1995 ; Felle and Hepler, 1997 ;
Legue! et al., 1997). Thus, Ca#+ influx into root cells is likely
to be mediated by ion channels which facilitate the rapid
movement of Ca#+ down its electrochemical gradient. Here
the term ‘ Ca#+ channel ’ is used to describe all ion channels
permeable to Ca#+.
The root is composed of many different cell types, each
specialized to a specific task (Fig. 1). The Ca#+ channel
complement of each cell type probably differs. It may be
possible to predict the characteristics of Ca#+ channels in
certain cell types on the basis of their physiology. If current
ideas about the pathway of Ca#+ movement through the
root to the xylem are correct, Ca#+ channels involved in
supplying the shoot with calcium are expected to be located
primarily in the plasma membrane of root endodermal cells
(but see ‘ Transport of calcium within the root ’ below). To
transfer Ca#+ across the Casparian strip, catalytically active
Ca#+ channels would be located on the cortical side of the
endodermal cell. How the activity of these Ca#+ channels
might be controlled is unknown. Expanding cells, such as
root hair cells and cells in the elongation zone, require
elevated cytoplasmic Ca#+ concentrations ([Ca#+]cyt) to
maintain their growth (Herrmann and Felle, 1995 ; Cramer
and Jones, 1996), and mechanosensitive Ca#+ channels by
which to regulate vesicle fusion and osmotically significant
solute fluxes are credible components of their plasma
membranes (see ‘ Plasma membrane calcium channels of
root cells ’ below). Finally, all root cells must respond to
environmental signals and many cells must respond to
specific developmental cues. It is expected that voltagedependent Ca#+ channels will provide a generalized signal
for plasma-membrane integrity, and that ligand-modulated
Ca#+ channels in competent cells will initiate specific
bo970554
# 1998 Annals of Botany Company
174
White—Calcium Channels in the Plasma Membrane of Root Cells
F. 1. A transection illustrating the cell-types within a wheat root (adapted from Esau, 1965, by permission of John Wiley & Sons Inc.).
developmental responses. These might be randomly distributed or clustered into discrete functional domains.
TRANSPORT OF CALCIUM WITHIN THE
ROOT
Calcium must be acquired by the root from the soil solution
and delivered to the xylem at rates sufficient for shoot
growth. In a cereal seedling calcium must be translocated at
rates approximating 40 nmol h−" g−" f.wt root (calculated
assuming a shoot f.wt}root f.wt ratio of 2 and a relative
growth rate of 0±2) to maintain a minimal shoot calcium
content of 0±1 % d. wt. It is thought that all root cortical
cells can transport Ca#+ in either direction across their
plasma membranes and are joined by plasmodesmata.
However, since calcium mobility through the symplasm
might be restricted by low [Ca#+]cyt, it is often assumed that
the bulk of radial Ca#+ movement within the root cortex
occurs by an apoplastic pathway. Net Ca#+ uptake into the
root is greatest in apical zones (less than 5 mm from the root
tip) and much smaller in mature regions (Ryan, Newman
and Shields, 1990 ; Huang, Grunes and Kochian, 1992). In
mature regions, Ca#+ uptake is largely determined by the
translocation of Ca#+ to the shoot (Clarkson, 1984, 1993 ;
White, Banfield and Diaz, 1992).
The delivery of Ca#+ to the xylem is restricted to regions
of the root at specific developmental stages (Clarkson, 1984,
1993 ; Peterson and Enstone, 1996). It is maximal in the
apical region. In this region, endodermal cells possess a
Casparian strip in their radial and transverse (end) walls,
which is firmly attached to the plasma membrane and which
contains lignin, but rarely suberin (State I endodermis). The
Casparian strip prevents Ca#+ movement through the
apoplast into the stele and Ca#+ must be taken up into the
root symplasm prior to entering the xylem. Ion channels
probably mediate Ca#+ influx from the apoplast into the
cytoplasm, but the loading of the xylem (which contains
millimolar Ca#+, White et al., 1981) against the Ca#+
electrochemical gradient must be effected by either a Ca#+transporting ATPase and}or countertransport with protons.
Upon the deposition of suberin lamellae in the endodermis,
Ca#+ delivery to the xylem is severely restricted, perhaps
because the plasma membrane of endodermal cells is no
longer accessible from the apoplast. Thereafter, some Ca#+
entry to the xylem may occur in basal zones if lateral roots
penetrate the endodermis.
It is astounding that root cells can attain the large
throughput of calcium to the xylem required for shoot
growth whilst maintaining low [Ca#+]cyt. It has been proposed
that low [Ca#+]cyt is achieved both by efficient Ca#+-buffering
in the cytoplasm (which may contain 10 to 15 m total
calcium, White et al., 1992) and perhaps by restricting Ca#+
influx to localized regions, such as the endodermal cell itself
(Clarkson, 1984, 1993). If the Ca#+ flux occurred exclusively
though endodermal cells in the cereal seedling described
above it would approach 480 pmol per cell h−", or 27 nA per
cell (assuming all endodermal cells contributed equally,
occupied 5 % of the root volume and each had a volume of
600¬10−' cm$). This might be achieved by a spatial
separation of Ca#+ channels mediating Ca#+ influx at the
cortical side and Ca#+ efflux transporters (eg. Ca#+-ATPase)
at the stelar side of the Casparian strip. This hypothetical
arrangement is favoured by many workers and can be tested
by the immunolocalization of plasma membrane Ca#+
channels and Ca#+-ATPases within the intact root. Antibodies have been raised to Ca#+-ATPases (LCA1 and
BCA1) and immunoreactive proteins colocalize with both
plasma membrane marker enzyme and Ca#+-ATPase
activities during membrane fractionation studies (Ferrol
and Bennett, 1996 ; Askerlund, 1997) but, to date, no in situ
localizations have been published. To support the Ca#+ flux
through an endodermal cell with a membrane potential of
®100 mV, assuming the current through a single channel
approximates 4 pA (Pin4 eros and Tester, 1995), would
require 7 000 open Ca#+ channels. This is equivalent to a
channel density of about 2 µm−# and is feasible. However,
assuming a transport activity of 10 µmol Ca#+ mg−" min−"
White—Calcium Channels in the Plasma Membrane of Root Cells
for the plasma membrane Ca#+-ATPase (based on an
abundance of 0±03 % total membrane protein and a
transport activity of 3 nmol Ca#+ mg−" min−" ; see White
et al., 1992), each endodermal cell would require 4¬10"!
Ca#+-ATPase molecules or 0±8 ng Ca#+-ATPase protein. In
these cells the abundance of the Ca#+-ATPase (about
1±3 mg g−" f.wt) would be greater than the average (total)
protein content of the root plasma membrane. Clearly this
is improbable and alternative routes for Ca#+ transport to
the xylem should be considered. While the anatomy of the
root requires Ca#+ transfer across the endodermis to be
symplastic, it does not require Ca#+ influx and Ca#+ efflux to
be restricted to endodermal cells. Epidermal and cortical
cells might contribute to Ca#+ influx and all living cells
within the stele could contribute to Ca#+ efflux. The argument
sometimes presented against this arrangement is that low
[Ca#+]cyt restricts Ca#+ mobility in the symplasm. However,
the [Ca#+]cyt is only a minute fraction of the total cytoplasmic
calcium pool (White et al., 1992), most of which may be
available not only to buffer [Ca#+]cyt but to sustain diffusive
fluxes by providing Ca#+ ions.
It is difficult to estimate unidirectional Ca#+ fluxes across
the plasma membrane of root cells using tracer techniques
and all estimates of Ca#+ influx based on these techniques
should be viewed with caution (see White et al., 1992, and
Reid and Tester, 1992, for an overview). This is a
consequence of (1) the high capacity and high affinity of cell
walls for binding divalent cations and (2) the rapid
equilibration of cytoplasmic Ca#+ due to the exceedingly low
[Ca#+]cyt. The first problem may be avoided by using
protoplasts, but the trauma associated with generating
protoplasts from root cells may create its own problems.
Unfortunately, no other techniques are available to measure
unidirectional Ca#+ fluxes. The vibrating Ca#+-selective
electrode technique (e.g. Schiefelbein, Shipley and Rowse,
1992 ; Huang et al., 1992 ; Hush, Newman and Overall,
1992 ; Herrmann and Felle, 1995 ; Jones, Shaff and Kochian,
1995 ; Felle and Hepler, 1997) yields data on net Ca#+ fluxes,
membrane-patch voltage-clamp electrophysiological techniques (e.g. Thion et al., 1996 b) measure net current (which
may be carried by other ions in addition to Ca#+) and
techniques which monitor [Ca#+]cyt merely illustrate the net
result of many transport activities confounded with Ca#+buffering within the cytoplasm.
Lanthanides inhibit Ca#+ influx into plant root cells, but
the classical organic inhibitors of animal L-type Ca#+
channels (diltiazem, verapamil and nifedipine) are relatively
ineffective. Lanthanum, but not verapamil, inhibited Ca#+
influx into maize roots (Rincon and Hanson, 1986) and La$+
and Gd$+ inhibited distinct Ca#+ influx pathways in maize
root protoplasts (Marshall et al., 1994). Furthermore, La$+,
but not diltiazem, verapamil or nifedipine, inhibited the
electrical response initiated by rapid cooling of cucumber
roots, which is thought to be due to Ca#+ influx (Minorsky
and Spanswick, 1989). The trivalent cation Al$+ inhibited
Ca#+ influx into barley roots (Nichol et al., 1993), wheat
roots (Huang, Grunes and Kochian, 1995) and fine roots of
spruce (Widell, Asp and Jense! n, 1994). Unfortunately,
inhibitors of animal L-type Ca#+ channels, La$+ and Al$+ are
notoriously non-specific in plants. They interact with a
175
variety of transport functions including K+ channels (Terry,
Findlay and Tyerman, 1992 ; Gassmann and Schroeder,
1994 ; Thomine et al., 1994 ; Wegner, De Boer and Raschke,
1994 ; White, 1996), which may affect Ca#+ influx indirectly
via effects on the cell membrane potential. Thus, an
unequivocal pharmacology of Ca#+ channels can only be
determined by assaying their activity directly. This can be
achieved under defined ionic conditions by measuring %&Ca#+
influx into voltage-clamped plasma membrane vesicles
(Huang, Grunes and Kochian, 1994 ; Marshall et al., 1994)
or cation currents through Ca#+ channels using voltageclamp electrophysiological techniques (White, 1997 ; Pin4 eros
and Tester, 1997). The next section describes the properties
of plasma membrane Ca#+ channels studied under appropriate conditions.
PLASMA MEMBRANE CALCIUM
CHANNELS OF ROOT CELLS
The previous definition of a Ca#+ channel, simply as a
channel permeable to Ca#+, tacitly assumed that its
physiological function was to mediate Ca#+ influx from the
apoplast into the cytoplasm. Although several channels
described as non-specific outward-rectified (K+) channels
(NORC) clearly fit the definition (see White, 1997, for a
review), providing a mechanism for Ca#+ influx is unlikely to
be their main physiological role. For this reason they have
not been considered here. The NORC differ from the Ca#+
channels discussed below by activating at positive voltages,
showing no inactivation and exhibiting low unitary conductances.
Plasma membrane Ca#+ channels from plant roots have
been characterized both from %&Ca flux measurements in
isolated vesicles and electrically, either after incorporating
vesicles into planar lipid bilayers (PLB) or by patchclamping root cell protoplasts. All studies indicate the
presence of depolarization-activated Ca#+ channels with
contrasting pharmacologies (Table 1). These may co-reside
in the plasma membrane of an individual cell type or reside
in different cell types. In general, the pharmacology of these
channels resembles that of Ca#+ influx into roots and root
cell protoplasts.
Two distinct Ca#+ channel activities have been observed
when plasma membrane vesicles derived from rye (White,
1993, 1994) or wheat roots (Pin4 eros and Tester, 1995) were
incorporated into PLB. One has a high unitary conductance
and is termed the maxi cation channel (White, 1993). The
inward Ca#+ flux through the maxi cation channel is
inhibited by ruthenium red, but diltiazem, verapamil and
quinine at micromolar concentrations and TEA+ at millimolar concentrations inhibited the outward K+ flux through
this channel only (White, 1996). The second Ca#+ channel
observed in PLBs has a lower unitary conductance and is
termed voltage-dependent cation channel two (VDCC2 ;
White, 1997). Based on commonalities in pharmacology
(Table 1), conductance, selectivity (Table 2) and the voltagedependence of both gating and inactivation kinetics, it can
be concluded that VDCC2 was studied by both White
(1994) and Pin4 eros and Tester (1995, 1997).
—
—
—
—
—
—
—
ND
Ineffective
Slow block* (100 µ)
Ineffective
Slow block* (100 µ)
Immediate & complete
Slow block* (100 µ)
No effect†
(100 µ)
Voltage-dependent
Fast & intermediate†‡
(K ¯ 700 µ, δ ¯ 0±75 ;
d
K "¯ 140 mM, δ ¯ 4±24)
d#
Voltage-independent
Fast & slow block†
(50 µ)
Voltage-dependent
Stabilized substates†
(100 µ)
ND
Mg#+
Sr#+
Mn#+
Cd#+
Cu#+
Ni#+
Zn#+
Al$+
La$+
Nd$+
Nifedipine
ND
ND
ND
Voltage-dependent
Intermediate block
(1 µ)
ND
ND
ND
Inhibited
(1 mM)
ND
—
—
—
—
—
—
—
—
Inhibited
(" 10 m)
ND
White (1997)
VDCC2
Rye roots
Voltage-dependent
Intermediate block*‡
(120 µ)
No effect*‡ (100 µ)
Inhibition*‡
(K ! 10 µ)
d
Voltage-dependent
Fast block*†
(K ¯ 17 µ ; δ ¯ 0±03)
d
Increased P and G
o
(" 6 n ; α [Ca] )
ext
Voltage-dependent
Intermediate block*†‡
(K ¯ 25 µ ; δ ¯ 0±59)
d
Voltage-dependent
Fast block*†
(K ¯ 10 µ ; δ ¯ 0±11)
d
Voltage-dependent
Fast block*†
(K ¯ 3 µ ; δ ¯ 0±05)
d
ND
Voltage-dependent*
(K ¯ 205 µ)
d
—
—
—
—
—
—
—
Pin4 eros (1995)*
Pin4 eros and Tester (1995)†
Pin4 eros and Tester (1997)‡
Inhibited*
(K ¯ 1±7 m ; δ ¯ 0)
d
ND
VDCC2
Wheat roots
No effect
(100 µ)
ND
Inhibited‡ (K ¯ 75 µ)
i
Inhibited
(K % 15 µ)
i
No effect
(100 µ)
No effect
(100 µ)
Inhibited
(K ¯ 20 µ)
i
No effect
(500 µ)
Inhibited
(K ¯ 40 µ)
i
Inhibited
(K ¯ 5 µ)
i
No effect
(100 µ)
No effect
(100 µ)
No effect
(100 µ)
No effect
(100 µ)
ND
No effect
(100 µ)
No effect
(100 µ)
ND
ND
ND
Marshall et al. (1994)
La$+-sensitive
Maize roots
ND
ND
No effect*
(500 µ)
Inhibited*
(K " 500 µ)
i
ND
ND
Inhibited*‡
(K ¯ 2 µ)
i
Inhibited†‡
(K ¯ 2 to 10 µ)
i
ND
ND
ND
ND
No effect*
(500 µ)
Inhibited*
(K " 500 µ)
i
Inhibited*
(K " 500 µ)
i
ND
Huang et al. (1994)*
Huang et al. (1996)†
Sasaki et al. (1994)‡
No effect*
(10 m)
ND
La$+-sensitive
Wheat roots
ND
No effect
(100 µ)
Inhibited
(K % 15 µ)
i
No effect
(100 µ)
No effect
(100 µ)
No effect
(500 µ)
Inhibited
(K ¯ 20 µ)
i
No effect
(500 µ)
No effect
(500 µ)
No effect
(100 µ)
No effect
(100 µ)
Inhibited
(100 µ)
Inhibited
(100 µ)
ND
Inhibited
(100 µ)
Inhibited
(100 µ)
ND
ND
ND
Marshall et al. (1994)
La$+-insensitive
Maize roots
Pharmaceuticals were applied to the extracellular face. The maximal concentration of pharmaceutical tested, or its Ki or Kd value, is given in parenthesis. The inhibitor constant (Ki) was defined
as the concentration which inhibited %&Ca influx by 50 %. The equilibrium binding constant (Kd) was defined at zero volts and δ is the apparent distance through the electrical field at which the
pharmaceutical binds. Blockade is classified as described by Hille (1992). Agonistic effects on channel open probability (Po) and unitary conductance (G) are also indicated. Data from Marshall
et al. (1994) assume that cations inhibit only one class of channel, which is inhibited completely.
Bepredil
Diltiazem
Ruthenium Red
Verapamil
Gd$+
Ba#+
Quinine
TEA+
White (1995)*
White (1996)†
White and Ridout (1998)‡
No effect†
(100 m)
Voltage-dependent
Fast & intermediate
block† (100 µ)
—
References
Maxi cation
Rye roots
T     1 . Inhibitor sensitiŠities of Ca#+ channels in the plasma membrane of root cells
176
White—Calcium Channels in the Plasma Membrane of Root Cells
White—Calcium Channels in the Plasma Membrane of Root Cells
T
2.
177
SelectiŠities of cation channels in the plasma membrane of root cells characterized following
incorporation into PLB
Conductance (pS) :
Relative permeability :
Maxi-cation
Rye roots
VDCC2
Rye roots
K & Rb " Cs " Na " Li
451 438 389 278 149
Ba " Sr " Ca " Mg " Co " Mn
213 162 135
88
78
57
Cs " K " Rb " Na
206 174 157
98
Ba & Ca
46
40
K & Rb " Cs " Na " Li
1±00 1±00 0±85 0±78 0±51
Ba " K
2±56 1±00
Ba " Sr " Mn D Mg & Co D Ca
1±00 0±92 0±84 0±82 0±79 0±77
K " Rb " Na
1±00 0±95 0±68
Ca " Ba " K
2±60 1±66 1±00
VDCC2
Wheat roots
K " Na
164 105
Ba & Sr "
32
29
Zn " Mn &
15
14
Ca " Co & Mg &
24
17
17
Ni & Cd & Cu
13
11
10
Ca " Rb " Li " K " Na D Cs
1±00 0±25 0±17 0±10 0±07 0±07
Ba & Sr " Ca " Zn & Ni D
1±64 1±25 1±00 0±60 0±47
Mg D Mn " Co " Cu " Cd
0±47 0±46 0±25 0±22 0±15
Unitary conductances for the maxi cation channel and VDCC2 from rye roots were determined in the presence of symmetrical 100 m cation
chloride. Unitary conductances for VDCC2 from wheat roots were determined between Erev and ®100 mV at (apparently saturating)
extracellular : cytoplasmic concentrations of 1 m cation chloride : 1 m CaCl (for divalent cations), 100 µ CaCl : 140 m KCl (for K+) and
#
#
150 m NaCl : 50 µ CaCl (for Na+). Relative permeabilities for the maxi cation channel and VDCC2 from rye roots were determined in
#
extracellular : cytoplasmic concentrations of 100 m cation chloride : 100 m KCl (for monovalents and for Ba#+ and Ca#+) or 100 m cation
chloride : 100 m BaCl (for divalents). Relative permeabilities for VDCC2 from wheat roots were determined in extracellular : cytoplasmic
#
concentrations of 1 m cation chloride : 1 m CaCl . Data for the maxi cation channel and VDCC2 from rye roots were taken from White (1993,
#
1994). Data for VDCC2 from wheat roots were taken from Pin4 eros (1995) and Pin4 eros and Tester (1995, 1997).
Pharmacological evidence suggests that Ca#+ influx to
plasma membrane vesicles and protoplasts from maize
roots is mediated by two separate classes of Ca#+ channels
(Marshall et al., 1994). But it must be recognized that
pharmaceuticals might influence Ca#+ fluxes indirectly via
effects on the membrane potential in these systems, and that
transport studies with isolated vesicles can suggest (but
cannot prove) that these fluxes are mediated by Ca#+
channels. One class of Ca#+ channels appears to be inhibited
by La$+, but not Gd$+, and mediates 70 % of the Ca#+ influx.
This may be the exclusive Ca#+ channel present in plasma
membrane vesicles from wheat roots (Huang et al., 1994,
1996 ; Sasaki, Yamamoto and Matsumoto, 1994). The other
class of Ca#+ channels, which mediates the remaining 30 %
of the Ca#+ influx into maize plasma membrane vesicles,
appears to be inhibited by Gd$+ but not by La$+ (Marshall
et al., 1994). Based on pharmacological criteria, it is possible
that the maxi cation channel of rye roots belongs to this
class of La$+-insensitive, Gd$+-sensitive Ca#+ channels (Table
1). Since Ca#+ influx through VDCC2 is inhibited by La$+,
Gd$+ and verapamil it is pharmacologically distinct from
the channels mediating Ca#+ influx to plasma membrane
vesicles from maize and wheat roots.
The pharmacological studies described above suggest that
plasma membrane fractions from plant roots contain at
least three distinct classes of Ca#+ channels, which can be
defined by their differential sensitivities to inhibition by
La$+, Gd$+ and verapamil. However, the use of pharmacological criteria to differentiate between classes of Ca#+channels is not unequivocal. Apparent differences in channel
pharmacology may arise artefactually : (1) through the
disruption of pharmaceutical binding sites during membrane
isolation, protein purification or protoplast preparation ; (2)
from differences in the experimental techniques employed,
for example whether electrophysiological or Ca#+-flux
measurements are made ; and (3) from differences in the
ionic conditions under which assays are performed. It will
be necessary, therefore, to confirm the pharmacologies of
contrasting Ca#+-channel activities with further electrophysiological studies.
Finally, a depolarization-activated Ca#+ current has been
reported in protoplasts from root cells of Arabidopsis
thaliana (Thion et al., 1996 b). This current is stable in
protoplasts from the Arabidopsis ton2 mutant, which has a
disorganized cytoskeleton, and in protoplasts from wildtype roots in the presence of colchicine, which induces a
disorganization of microtubules. Its properties resemble
those of the Ca#+ current described in protoplasts from
carrot (Daucus carota L.) suspension cells (Thuleau et al.,
1994 a, b ; Thion et al., 1996 a). The latter channels are
permeable to Ba#+, Sr#+, Ca#+ and Mg#+ (a permeability to
K+ was not excluded), show ‘ recruitment ’ (activation of
quiescent channels) upon depolarization to positive voltages,
and exhibit slow and reversible inactivation at extreme
negative voltages. These properties are shared by both the
maxi cation channel and VDCC2 when incorporated into
PLB (White, 1993, 1994 ; Pin4 eros and Tester, 1995, 1997).
They are distinct from the (non-selective) Ca#+-permeable
channels formed by the phenylalkylamine-binding protein
purified from carrot suspension cells (Thuleau et al., 1993),
which are blocked by bepredil and verapamil.
The Šoltage dependence of plasma membrane Ca#+
channels
Despite their contrasting pharmacologies, the voltagedependencies of %&Ca fluxes into plasma membrane vesicles,
Ca#+ channels observed in PLB and Ca#+ currents recorded
178
White—Calcium Channels in the Plasma Membrane of Root Cells
2
B
1
0
0
1
Current (pA)
Ca2+ influx [nmol (mg min)–1]
A
2
3
–1
–2
–3
4
–200
–150
–100
–50
Voltage (mV)
0
–4
–200
50
–150
–100
–50
Voltage (mV)
0
50
–150
–100
–50
Voltage (mV)
0
50
10
D
C
30
0
Current (pA)
Current (pA)
20
10
0
–10
–20
–10
–20
–200
–150
–100
–50
Voltage (mV)
0
50
–30
–200
F. 2. A, The relationship between voltage and %&Ca#+ influx into plasma membrane vesicles from wheat roots assayed over a 3 min period in
the presence of 50 m K SO plus contaminant Ca#+ on the cytoplasmic side and 5 µ to 175 m K SO plus 100 µ CaCl on the extracellular
# %
# %
#
side of the membrane. The voltage was calibrated from the distribution of TPP+ with linear interpolation between measurements. Data were taken
from Huang et al. (1994). B, The relationship between voltage and the net inward current through a single VDCC2 channel from wheat roots
in a PLB assayed over a 5 min period in the presence of symmetrical 1 m CaCl . The inward current was calculated as the product of the unitary
#
current (27 pS) and T (the proportion of time VDCC2 was open over the 5 min period), which was defined by the equation T ¯ 1}(1­exp
(®128®V)}6). Data and equations were taken from Pin4 eros and Tester (1995). C, The relationship between voltage and ionic current through
the maxi cation channel from rye roots incorporated into a PLB. The channel was assayed in the presence of 100 m KCl plus contaminant Ca#+
on the cytoplasmic side and 1 m KCl plus 4 m CaCl on the extracellular side of the channel. Data represent the mean current through a bilayer
#
containing two channels subjected to a 7±8 s voltage-ramp ranging from ®100 to ­100 mV and is the average of five experiments. D, The
relationship between voltage and ionic current in protoplasts from patch-clamped carrot suspension cells subjected to a 4 s voltage-ramp ranging
from ®161 to ­59 mV. The pipette solution equilibrated with the cytoplasm contained 2 m MgCl , 10 m MgATP, 10 m Tris EGTA, 200 µ
#
#
GTPγS, 10 m HEPES-Tris (pH 7±2) and sorbitol to 620 mosmol kg−". The extracellular solution contained 40 m CaCl , 1±6 m Ca(OH) , 10 m
#
#
−
"
HEPES (pH 6±7) and sorbitol to 620 mosmol kg . This figure is taken from Thuleau et al. (1994 b) by permission of Oxford University Press.
in root cell protoplasts are all remarkably similar (Fig. 2).
At extreme negative voltages the Ca#+ channels are closed.
This would be their state under physiological conditions in
the resting cell. As the voltage is driven more positive, which
equates with depolarization of the plasma membrane, the
probability of the channel opening (Po) is increased. This
can be attributed to both an increased Po of activated Ca#+
channels, as well as a lengthening of the period prior to their
inactivation (White, 1993, 1994 ; Pin4 eros and Tester, 1995,
1997). Maximal Ca#+ current and Ca#+ influx is generally
observed at about ®100 mV, but this value may depend on
the exact ionic composition of assay media since the
voltage-dependence of (for example) the maxi cation channel
and VDCC2 vary in parallel with the zero-current reversal
potential (Erev) of the channel (White, 1993, 1994). As the
voltage is driven to more positive values, towards ECa, both
the Ca#+ current and Ca#+ influx decline. This is a
consequence of reducing the electrophoretic driving force.
White—Calcium Channels in the Plasma Membrane of Root Cells
In physiological terms, Ca#+ influx through these channels
would be initiated by plasma membrane depolarization.
Depolarization could occur via a number of mechanisms,
such as stalling of the plasma membrane H+-ATPase or the
opening of channels mediating cation influx or anion efflux,
and in response to a variety of abiotic and hormonal stimuli
(Thuleau et al., 1994 b). Ca#+ influx would potentiate plasma
membrane depolarization. The duration of the depolarizing
phase would be related to the ability of the cell to reduce the
depolarizing currents and to repolarize the plasma membrane by compensatory ion fluxes (mediated by OR K+
channels and}or H+-ATPase activity). The total Ca#+ influx
would depend, in a complex manner, on both the magnitude
and duration of plasma membrane depolarization and the
magnitude of any rise in [Ca#+]cyt. When active, the Ca#+
channels discussed here could catalyse substantial Ca#+
influx and contribute to both plant nutrition and cellular
regulation by [Ca#+]cyt (see ‘ Plasma membrane calcium
channels and intracellular signalling ’ below).
The selectiŠity of plasma membrane Ca#+ channels
Ionic selectivity cannot be determined solely from the
effects of cations on Ca#+ influx into plasma membrane
vesicles (Huang et al., 1994, 1995, 1996 ; Marshall et al.,
1994 ; Sasaki et al., 1994). In such experiments no direct
estimate of cation influx is made and it cannot be assumed.
However, the ionic selectivity of Ca#+ channels incorporated
into PLB has been estimated both by comparison of ionic
conductances and from permeability ratios, calculated from
the Erev obtained when contrasting permeant cations were
present on either side of the channel (Table 2). Both the
maxi cation channel and VDCC2 were permeable to a wide
variety of monovalent and divalent cations. However,
contrasting selectivity sequences were obtained depending
upon the exact ionic composition of solutions and whether
conductances or relative permeabilities were compared. In
particular, the calculated PCa : PK varied markedly for both
cation channels depending upon solution ionic composition.
A larger PCa : PK was obtained at lower [Ca#+]ext for both the
maxi cation channel (White, 1993, 1997) and for VDCC2
(White, 1994 ; Pin4 eros, 1995 ; Pin4 eros and Tester, 1995,
1997). This may indicate that both cation channels have
complex pore structures and that several cations may
occupy the pore of the channel simultaneously and interact,
electrically or sterically, with each other. If this is correct,
then appropriate comparisons between channels of conductance and permeability ratios can only be made under
identical ionic conditions (Hille, 1992). The activation of
either the maxi cation channel or VDCC2 by depolarization
of the plasma membrane to voltages more positive than EK
will result in simultaneous Ca#+ influx and K+ efflux through
the pore. The K+ efflux may prevent excessive plasma
membrane depolarization, which might be important in
maintaining appropriate electrochemical gradients for the
transport of other ions across the plasma membrane. The
(lack of) selectivity of these channels suggests that, in
addition to facilitating Ca#+ influx, they might have a
physiological role in the uptake of other monovalent and
divalent cations ; essential, beneficial and toxic.
179
Stretch-actiŠated and second messenger-actiŠated Ca#+
channels
In addition to the (primarily) voltage-dependent Ca#+
channels described above, it is likely that both stretchactivated (mechanosensitive) and second messengeractivated Ca#+ channels are also present in the plasma
membrane of root cells.
Mechanosensitive Ca#+ channels in the plasma membrane
of root cells might resemble those characterized in protoplasts from onion-bulb epidermal cells (Ding and Pickard,
1993). Mechanosensitive ion channels may be involved in
the regulation of turgor and in determining the allometry of
cell expansion and morphogenesis, whereas depolarizationactivated Ca#+ channels are thought to provide a generalized
signalling mechanism indicating a breach in plasma membrane integrity and priming the cell for response. That
depolarization-activated Ca#+ channels are constituents of a
generalized signal follows because depolarization is not
only a non-specific response to many environmental, developmental and pathological stimuli, and may occur by one of
many contrasting mechanisms (Thuleau et al., 1994b), but
also because depolarization generates a global signal, which
is likely to increase [Ca#+]cyt throughout the periphery of the
cell and, therefore, is unlikely to encode directionality. A
specific role for mechanosensitive Ca#+ channels in root hair
elongation is discussed below.
Indirect activation via internal signalling cascades of Ca#+
channels in the plasma membrane of root cells might be
analogous to that proposed for the elicitor-activated, Ca#+permeable channels in the plasma membrane of protoplasts
from tomato (Gelli, Higgins and Blumwald, 1997) and
parsley (Zimmermann et al., 1977) suspension cells. The
activity of these channels is increased greatly when the
protoplast is challenged by an appropriate elicitor. This
activation is indirect. In tomato, the heterotrimeric Gprotein dependent activation of a protein kinase modulates
Ca#+ channel opening (Gelli et al., 1997). It is thought that
such second messenger-activated Ca#+ channels are required
to transduce specific stimuli into changes in [Ca#+]cyt.
PLASMA MEMBRANE CALCIUM
CHANNELS AND INTRACELLULAR
SIGNALLING
The [Ca#+]cyt is of critical importance for the physiological
poise of a plant cell and, since the activities of many proteins
and enzymes are modulated in response to altered Ca#+
concentration, changes in [Ca#+]cyt may exert effects on
metabolism, gene expression and integrated physiological
processes including cell division and cell elongation (Bush,
1995). In the resting cell, low [Ca#+]cyt is maintained by
energy-dependent processes which transport Ca#+ against its
electrochemical gradient into either the apoplast or internal
stores such as the endoplasmic reticulum or vacuole (Bush,
1995). Low [Ca#+]cyt confers sensitivity to signals mediated
by changes in [Ca#+]cyt. The opening of Ca#+ channels allows
[Ca#+]cyt to increase rapidly from the nanomolar to the
micromolar range in response to an appropriate signal. The
Ca#+ influx required for signalling is quite small. Thuleau et
180
White—Calcium Channels in the Plasma Membrane of Root Cells
al. (1994b) estimate that a current of 5 pA per cell could
increase [Ca#+]cyt by 0±3 µ s−". This contrasts starkly with
the projected nutritional Ca#+ flux through root endodermal
cells (27 nA per cell). The specificity of [Ca#+]cyt signals is
thought to be encoded by different amplitude, temporal or
spatial changes in [Ca#+]cyt (Bush, 1995 ; Berridge, 1997).
The influx of Ca#+, and subsequent rise in [Ca#+]cyt, has
been implicated in the responses of root cells to a variety of
environmental and hormonal stimuli (Bush, 1995). These
include thigmotropic and gravitropic responses (Trewavas
and Knight, 1994), responses to wounding (Hush et al.,
1992) and pathogens (Dixon, Harrison and Lamb, 1994),
the initiation of nodulation (Ehrhardt, Wais and Long,
1996) and acclimation to low temperatures (Minorsky,
1989). In addition, the opening of Ca#+ channels is important
in raising [Ca#+]cyt, which stimulates cell expansion
(Herrmann and Felle, 1995 ; Cramer and Jones, 1996) and,
if localized, maintains polarized elongation of root hairs
(see below).
The following examples were chosen to illustrate the role
of Ca#+ channels in signalling (1) the direction, and rate, of
root hair elongation and (2) a breach in plasma membrane
integrity to initiate low-temperature acclimation.
Approaches to study the Ca#+ channel activities involved in
these processes are suggested.
(Garrill et al., 1993 ; Levina, Lew and Heath, 1994) or
rhizoids of Fucus serratus (Taylor et al., 1996). It is
noteworthy that these channels are inhibited by La$+ but
not by nifedipine or verapamil, and that the peripheral Factin network establishes and}or maintains their clustering.
When root hairs are exposed to a fluorescently-labelled
dihydropyridine (BODIPY-DHP), which inhibits animal Ltype Ca#+ channels in a manner similar to nifedipine, label
accumulates at the apex of the hair cell and mirrors the
[Ca#+]cyt gradient (Bibikova and Gilroy, 1997). However,
since the affinity and specificity of dihydropyridines for
plant Ca#+ channels appears to be low (see ‘ Plasma
membrane calcium channels of root cells ’), it will be
necessary to extend these observations by direct measurements of Ca#+-channel activities. Results from the application of laser microsurgery to access the plasma
membrane at the root hair apex combined with patch-clamp
electrophysiological techniques (cf. Henriksen et al., 1996)
are eagerly awaited.
It is expected that the isolation of mutants in root hair
elongation, which phenocopy root hairs grown at low
[Ca#+]ext (Schiefelbein et al., 1992), will allow the genes
involved in this process to be identified (Schiefelbein and
Somerville, 1990 ; Schiefelbein et al., 1993). These will
(hopefully) include the genes encoding plasma membrane
Ca#+ channels.
Ca#+ channels and the growth of root hairs
In the root hair, a Ca#+ current (net Ca#+ flux) enters the
cell exclusively at the apex (Schiefelbein et al., 1992 ;
Herrmann and Felle, 1995 ; Jones et al., 1995 ; Felle and
Hepler, 1997). This Ca#+ current is confined to the apical 20
to 50 µm of the root hair and depends critically on external
pH and [Ca#+]. A parallel gradient in [Ca#+]cyt is observed in
this region (Clarkson et al., 1988 ; Felle et al., 1992 ;
Herrmann and Felle, 1995 ; Felle and Hepler, 1997 ; Wymer,
Bibikova and Gilroy, 1997). The [Ca#+]cyt at the apex
(378–831 n), where secretory vesicles fuse with the plasma
membrane in a Ca#+-dependent process (reviewed by Battey
et al., 1996), is several-fold greater than [Ca#+]cyt in the basal
region (98 to 253 n). These phenomena appear to be
specifically associated with root hair elongation. Neither the
apical Ca#+ current (Schiefelbein et al., 1992 ; Jones et al.,
1995) nor the gradient in [Ca#+]cyt (Wymer et al., 1997) are
observed in mature, non-growing root cells or in root hairs
of the rhd2 Arabidopsis mutant which is defective in root
hair elongation. In addition, factors which reduce [Ca#+]cyt
or remove the [Ca#+]cyt gradient inhibit root hair elongation
(Herrmann and Felle, 1995 ; Felle and Hepler, 1997 ; Wymer
et al., 1997). The Ca#+ current, internal [Ca#+]cyt gradient
and root hair elongation are all eliminated in the presence of
external La$+ (Felle and Hepler, 1997) or verapamil (Wymer
et al., 1997)
It is thought that the inward Ca#+ current, which generates
the [Ca#+]cyt gradient, is mediated by the clustering of
catalytically active (perhaps mechanosensitive) Ca#+
channels at the apex of the root hair. This arrangement
would be analogous to the apical clustering of mechanosensitive Ca#+ channels involved in osmoregulation and
extension of hyphae of the oomycete Saprolegnia ferax
The role of Ca#+ channels in low-temperature acclimation
The acclimation of chilling-resistant plants to growth at
low temperatures is thought to be initiated by Ca#+ influx
across the plasma membrane and mediated by the consequent increase in [Ca#+]cyt (Minorsky, 1989 ; Monroy and
Dhindsa, 1995 ; Knight, Trewavas and Knight, 1996). Rapid
cooling of plant roots to low non-freezing temperatures
evokes an initial increase in plant [Ca#+]cyt followed by its
restoration (Knight et al., 1991, 1993, 1996 ; Campbell,
Trewavas and Knight, 1996). The kinetics of these changes
are related to the rate, magnitude, duration and
repetitiveness of root cooling. Similar dependencies are
observed in the electrical responses of root cells to cooling
(Minorsky and Spanswick, 1989). The electrical response is
termed the slow action potential (SAP) and comprises
plasma membrane depolarization followed by repolarization
(Minorsky, 1989). It is believed, but not verified, that the
SAP results from the successive opening of depolarizationactivated Ca#+ channels, Cl− channels and OR K+ channels.
Ding and Pickard (1993) suggest that mechanosensitive
Ca#+ channels are involved in this response.
It should be possible to identify ionic currents underlying
the SAP using the action potential-clamp technique described by Thiel (1995). The changes in membrane potential
recorded during the SAP could be used as the clamp-voltage
for patch-clamp studies and the currents associated with
each class of channel identified from compensation currents
in the presence of appropriate pharmaceuticals. To identify
Ca#+ currents, La$+ would be used since both the SAP
(Minorsky and Spanswick, 1989) and the increase in [Ca#+]cyt
(Knight et al., 1996) upon cooling are inhibited by La$+ but
White—Calcium Channels in the Plasma Membrane of Root Cells
not by diltiazem, verapamil or nifedipine. Again it is also
hoped that the isolation of chilling-sensitive mutants
(Schneider, Hugly and Somerville, 1995) or mutants which
do not show an increase in [Ca#+]cyt upon chilling (M. R.
Knight, unpubl. res.) will help identify the genes for Ca#+
channels and their effectors.
181
colleagues at Horticulture Research International and Dr
D. E. Evans (Oxford Brookes University, UK), Dr S.
Gilroy (Pennsylvania State University, USA), Dr M. R.
Knight (University of Oxford, UK) and Dr M. Tester
(University of Cambridge, UK).
LITERATURE CITED
PERSPECTIVE
It is clear that further characterization of Ca#+ channels in
the plasma membrane of root cells is required at the
molecular level. This may be achieved by applying patchclamp electrophysiological techniques either in situ, using
laser microsurgery to access the plasma membrane
(Henriksen et al., 1996), or to protoplasts of root cells
(Thion et al., 1996 b). In this respect the ability to mark
protoplasts from specific cell-types using marker genes
under the control of tissue-specific promoters should provide
a useful experimental approach (White et al., 1996). In
tandem, the regulation of Ca#+ channels should be investigated. This might be addressed simply by manipulating
ionic or enzymatic activities within the cytoplasm during
patch-clamp experiments, or by identifying genes which
impact on Ca#+ channel activity. The relationship between
Ca#+ channel activities studied in patch-clamp experiments,
in membrane vesicles and in PLB should also be determined.
The identification of genes for plant Ca#+ channels is a
priority. None are known. Conventional approaches based
on (1) the purification of Ca#+ channel proteins (reviewed by
White and Tester, 1994), (2) genetic homologies with known
Ca#+ channel genes, (3) functional complementation of cells
lacking analogous Ca#+ channels (e.g. Winstanley, 1997)
and (4) the analysis of mutants exhibiting altered Ca#+
transport, or physiological responses mediated by changes
in [Ca#+]cyt, should be actively pursued.
Finally, the involvement of plasma membrane Ca#+
channels in physiological processes should be addressed
more thoroughly. In particular, when certifying their role in
intracellular signalling it should be demonstrated (cf. Jaffe,
1980) : (1) that physiological responses are preceded by the
opening of Ca#+ channels ; (2) that the specific blockade of
plasma membrane Ca#+ channels eliminates these responses ;
and (3) that changes in [Ca#+]cyt comparable to those
generated by active Ca#+ channels initiate comparable
physiological responses. With regard to nutritional studies,
it is now important to determine the location of Ca#+
channels within the root and, in particular, their spatial
distribution in endodermal cells. Although our knowledge
of plasma membrane Ca#+ channel proteins in root cells,
and their involvement in physiological processes, is rudimentary at present, the powerful combination of molecular genetic, Ca#+ imaging and electrophysiological techniques should allow us to address these questions in the near
future.
A C K N O W L E D G E M E N TS
This work was supported by the Biotechnology and
Biological Sciences Research Council (UK). For fruitful
discussions on the topic of Ca#+ dynamics I thank my
Askerlund P. 1997. Calmodulin-stimulated Ca#+-ATPases in the
vacuolar and plasma membranes in cauliflower. Plant Physiology
114 : 999–1007.
Ayling SM, Brownlee C, Clarkson DT. 1994. The cytoplasmic streaming
response of tomato root hairs to auxin ; observations of cytosolic
calcium levels. Journal of Plant Physiology 143 : 184–188.
Battey N, Carroll A, van Kesteren P, Taylor A, Brownlee C. 1996. The
measurement of exocytosis in plant cells. Journal of Experimental
Botany 47 : 717–728.
Berridge MJ. 1997. The AM and FM of calcium signalling. Nature 386 :
759–760.
Bibikova TN, Gilroy S. 1997. The role of cytoplasmic calcium in
directing root hair formation and growth in Arabidopsis thaliana.
Plant Physiology 114S : 32.
Bush DS. 1995. Calcium regulation in plant cells and its role in
signaling. Annual ReŠiew of Plant Physiology and Plant Molecular
Biology 46 : 95–122.
Campbell AK, Trewavas AJ, Knight MR. 1996. Calcium imaging shows
differential sensitivity to cooling and communication in luminous
transgenic plants. Cell Calcium 19 : 211–218.
Clarkson DT. 1984. Calcium transport between tissues and its
distribution in the plant. Plant, Cell and EnŠironment 7 : 449–456.
Clarkson DT. 1993. Roots and the delivery of solutes to the xylem.
Philosophical Transactions of the Royal Society (London) B 341 :
5–17.
Clarkson DT, Brownlee C, Ayling SM. 1988. Cytoplasmic calcium
measurements in intact higher plant cells : results from fluorescence
ratio imaging of fura-2. Journal of Cell Science 91 : 71–80.
Cramer GR, Jones RL. 1996. Osmotic stress and abscisic acid reduce
cytosolic calcium activities in roots of Arabidopsis thaliana. Plant,
Cell and EnŠironment 19 : 1291–1298.
Ding JP, Pickard BG. 1993. Modulation of mechanosensitive calciumselective cation channels by temperature. The Plant Journal 3 :
713–720.
Dixon RA, Harrison MJ, Lamb CJ. 1994. Early events in the activation
of plant defense responses. Annual ReŠiew of Phytopathology 32 :
479–501.
Ehrhardt DW, Wais R, Long SR. 1996. Calcium spiking in plant root
hairs responding to Rhizobium nodulation signals. Cell 85 :
673–681.
Esau K. 1965. Plant anatomy. 2nd edn. New York : Wiley International.
Felle H. 1988. Cytoplasmic free calcium in Riccia fluitans L. and Zea
mays L. : Interaction of Ca#+ and pH ? Planta 176 : 248–255.
Felle HH, Hepler PK. 1997. The cytosolic Ca#+ concentration gradient
of Sinapis alba root hairs as revealed by Ca#+-selective microelectrode tests and fura-dextran ratio imaging. Plant Physiology
114 : 39–45.
Felle HH, Tretyn A, Wagner G. 1992. The role of the plasmamembrane Ca#+-ATPase in Ca#+ homeostasis in Sinapis alba root
hairs. Planta 188 : 306–313.
Ferrol N, Bennett AB. 1996. A single gene may encode differentially
localized Ca#+-ATPases in tomato. The Plant Cell 8 : 1159–1169.
Garrill A, Jackson SL, Lew RR, Heath IB. 1993. Ion channel activity
and tip growth : tip localized stretch-activated channels generate
an essential Ca#+ gradient in the oomycete Saprolegnia ferax.
European Journal of Cell Biology 60 : 358–365.
Gassmann W, Schroeder JI. 1994. Inward-rectifying K+ channels in
root hairs of wheat. A mechanism for aluminium-sensitive lowaffinity K+ uptake and membrane potential control. Plant
Physiology 105 : 1399–1408.
Gehring CA, Irving HR, Parish RW. 1990. Effects of auxin and abscisic
acid on cytosolic calcium and pH in plant cells. Proceedings of the
National Academy of Sciences of the USA 87 : 9645–9649.
182
White—Calcium Channels in the Plasma Membrane of Root Cells
Gelli A, Higgins VJ, Blumwald E. 1997. Activation of plant plasma
membrane Ca#+-permeable channels by race-specific fungal
elicitors. Plant Physiology 113 : 269–279.
Henriksen GH, Taylor AR, Brownlee C, Assmann SM. 1996. Laser
microsurgery of higher plant cell walls permits patch-clamp access.
Plant Physiology 110 : 1063–1068.
Herrmann A, Felle HH. 1995. Tip growth in root hair cells of Sinapis
alba L. : significance of internal and external Ca#+ and pH. New
Phytologist 129 : 523–533.
Hille B. 1992. Ionic channels of excitable membranes. 2nd edn.
Sunderland, Massachusetts : Sinauer Associates.
Huang JW, Grunes DL, Kochian LV. 1992. Aluminium effects on the
kinetics of calcium uptake into cells of the wheat root apex.
Quantification of calcium fluxes using a calcium-selective vibrating
electrode. Planta 188 : 414–421.
Huang JW, Grunes DL, Kochian LV. 1994. Voltage-dependent Ca#+
influx into right-side-out plasma membrane vesicles isolated from
wheat roots : Characterization of a putative Ca#+ channel.
Proceedings of the National Academy of Sciences of the USA 91 :
3473–3477.
Huang JW, Grunes DL, Kochian LV. 1995. Aluminium and calcium
transport interactions in intact roots and root plasmalemma
vesicles from aluminium-sensitive and tolerant wheat cultivars.
Plant and Soil 171 : 131–135.
Huang JW, Pellet DM, Papernik LA, Kochian LV. 1996. Aluminium
interactions with voltage-dependent calcium transport in plasma
membrane vesicles isolated from roots of aluminium-sensitive and
-resistant wheat cultivars. Plant Physiology 110 : 561–569.
Hush JM, Newman IA, Overall RL. 1992. Utilization of the vibrating
probe and ion-selective microelectrode techniques to investigate
electrophysiological responses to wounding in pea roots. Journal
of Experimental Botany 43 : 1251–1257.
Jaffe LF. 1980. Calcium explosions as triggers of development. Annals
of the New York Academy of Science 339 : 86–101.
Jones DL, Shaff JE, Kochian LV. 1995. Role of calcium and other ions
in directing root hair tip growth in Limnobium stoloniferum. I.
Inhibition of tip growth by aluminium. Planta 197 : 672–680.
Knight MR, Campbell AK, Smith SM, Trewavas AJ. 1991. Transgenic
plant aequorin reports the effects of touch and cold-shock and
elicitors on cytoplasmic calcium. Nature 352 : 524–526.
Knight MR, Read ND, Campbell AK, Trewavas AJ. 1993. Imaging
calcium dynamics in living plants using semi-synthetic recombinant
aequorins. Journal of Cell Biology 121 : 83–90.
Knight H, Trewavas AJ, Knight MR. 1996. Cold calcium signaling in
Arabidopsis involves two cellular pools and a change in calcium
signature after acclimation. The Plant Cell 8 : 489–503.
Legue! V, Blancaflor E, Wymer C, Perbal G, Fantin D, Gilroy S. 1997.
Cytoplasmic free Ca#+ in Arabidopsis roots changes in response to
touch but not gravity. Plant Physiology 114 : 789–800.
Levina NN, Lew RR, Heath IB. 1994. Cytoskeletal regulation of ionchannel distribution in the tip-growing organism Saprolegnia
ferax. Journal of Cell Science 107 : 127–134.
Marschner H. 1995. Mineral nutrition of higher plants. 2nd edn.
London : Academic Press.
Marshall J, Corzo A, Leigh RA, Sanders D. 1994. Membrane potentialdependent calcium transport in right-side-out plasma membrane
vesicles from Zea mays L. roots. The Plant Journal 5 : 683–694.
Minorsky PV. 1989. Temperature sensing by plants : a review and
hypothesis. Plant, Cell and EnŠironment 12 : 119–135.
Minorsky PV, Spanswick RM. 1989. Electrophysiological evidence for
a role for calcium in temperature sensing by roots of cucumber
seedlings. Plant, Cell and EnŠironment 12 : 137–143.
Monroy AF, Dhindsa RS. 1995. Low-temperature signal transduction :
Induction of cold acclimation-specific genes of alfalfa by calcium
at 25 °C. The Plant Cell 7 : 321–331.
Nichol BE, Oliveira LA, Glass ADM, Siddiqi MY. 1993. The effects of
aluminium on the influx of calcium, potassium, ammonium,
nitrate, and phosphate in an aluminium-sensitive cultivar of barley
(Hordeum Šulgare L.). Plant Physiology 101 : 1263–1266.
Peterson CA, Enstone DE. 1996. Functions of passage cells in the
endodermis and exodermis of roots. Physiologia Plantarum 97 :
592–598.
Pickard BG, Ding JP. 1993. The mechanosensory calcium-selective ion
channel : Key component of a plasmalemmal control centre ?
Australian Journal of Plant Physiology 20 : 439–459.
Pin4 eros MA. 1995. Single channel characterisation of a calcium-selectiŠe
channel from wheat roots. PhD Thesis, University of Adelaide,
Australia.
Pin4 eros M, Tester M. 1995. Characterization of a voltage-dependent
Ca#+-selective channel from wheat roots. Planta 195 : 478–488.
Pin4 eros M, Tester M. 1997. Calcium channels in plant cells : selectivity,
regulation and pharmacology. Journal of Experimental Botany
Special Issue 48 : 551–557.
Reid RJ, Tester M. 1992. Measurements of Ca#+ fluxes in intact plant
cells. Philosophical Transactions of the Royal Society (London) B
338 : 73–82.
Rincon M, Hanson JB. 1986. Controls on calcium ion fluxes in injured
or shocked corn root cells : Importance of proton pumping and cell
membrane potential. Physiologia Plantarum 67 : 576–583.
Ryan PR, Newman IA, Shields B. 1990. Ion fluxes in corn roots
measured by microelectrodes with ion-specific liquid membranes.
Journal of Membrane Science 53 : 59–69.
Sasaki M, Yamamoto Y, Matsumoto H. 1994. Putative Ca#+ channels
of plasma membrane vesicles are not involved in the tolerance
mechanism of aluminium tolerant wheat (Triticum aestiŠum L.)
cultivar. Soil Science and Plant Nutrition 40 : 709–714.
Schiefelbein J, Galway M, Masucci J, Ford S. 1993. Pollen tube and
root-hair tip growth is disrupted in a mutant of Arabidopsis
thaliana. Plant Physiology 103 : 979–985.
Schiefelbein JW, Shipley A, Rowse P. 1992. Calcium influx at the tip of
growing root-hair cells of Arabidopsis thaliana. Planta 187 :
455–459.
Schiefelbein JW, Somerville C. 1990. Genetic control of root hair
development in Arabidopsis thaliana. The Plant Cell 2 : 235–243.
Schneider JC, Hugly S, Somerville CR. 1995. Chilling sensitive mutants
of Arabidopsis. Plant Molecular Biology Reporter 13 : 11–17.
Taylor AR, Manison NFH, Fernandez C, Wood J, Brownlee C. 1996.
Spatial organization of calcium signaling involved in cell volume
control in the Fucus rhizoid. The Plant Cell 8 : 2015–2031.
Terry BR, Findlay GP, Tyerman SD. 1992. Direct effects of Ca#+channel blockers on plasma membrane cation channels of
Amaranthus tricolor protoplasts. Journal of Experimental Botany
43 : 1457–1473.
Thiel G. 1995. Dynamics of chloride and potassium currents during the
action potential in Chara studied with action-potential clamp.
European Biophysics Journal 24 : 85–92.
Thion L, Mazars C, Thuleau P, Graziana A, Rossignol M, Moreau M,
Ranjeva R. 1996 a. Activation of plasma membrane voltagedependent calcium-permeable channels by disruption of microtubules in carrot cells. FEBS Letters 393 : 13–18.
Thion L, Thuleau P, Mazars C, Ranjeva R. 1996 b. Activation of plasma
membrane voltage-dependent calcium-permeable channels by
disruption of microtubules in plant cells. Plant Physiology and
Biochemistry Special Issue, 172.
Thomine S, Zimmermann S, Van Duijn B, Barbier-Brygoo H, Guern J.
1994. Calcium channel antagonists induce direct inhibition of the
outward rectifying potassium channel in tobacco protoplasts.
FEBS Letters 340 : 45–50.
Thuleau P, Graziana A, Ranjeva R, Schroeder JI. 1993. Solubilized
proteins from carrot (Daucus carota L.) membranes bind calcium
channel blockers and form calcium-permeable ion channels.
Proceedings of the National Academy of Sciences of the USA 90 :
765–769.
Thuleau P, Moreau M, Schroeder JI, Ranjeva R. 1994 a. Recruitment of
plasma membrane voltage-dependent calcium-permeable channels
in carrot cells. The EMBO Journal 13 : 5843–5847.
Thuleau P, Ward JM, Ranjeva R, Schroeder JI. 1994 b. Voltagedependent calcium-permeable channels in the plasma membrane
of a higher plant cell. The EMBO Journal 13 : 2970–2975.
Tretyn A, Wagner G, Felle HH. 1991. Signal transduction in Sinapis
alba root hairs : Auxins as external messengers. Journal of Plant
Physiology 139 : 187–193.
Trewavas A, Knight M. 1994. Mechanical signalling, calcium and plant
form. Plant Molecular Biology 26 : 1329–1341.
White—Calcium Channels in the Plasma Membrane of Root Cells
Wegner LH, De Boer AH, Raschke K. 1994. Properties of the K+
inward rectifier in the plasma membrane of xylem parenchyma
cells from barley roots : Effects of TEA+, Ca#+, Ba#+ and La$+.
Journal of Membrane Biology 142 : 363–379.
White PJ. 1993. Characterization of a high-conductance, voltagedependent cation channel from the plasma membrane of rye roots
in planar lipid bilayers. Planta 191 : 541–551.
White PJ. 1994. Characterization of a voltage-dependent cationchannel from the plasma membrane of rye (Secale cereale L.) roots
in planar lipid bilayers. Planta 193 : 186–193.
White PJ. 1995. Separation of K+ and Cl−-selective ion channels from
rye roots on a continuous sucrose density gradient. Journal of
Experimental Botany 46 : 361–376.
White PJ. 1996. Specificity of ion channel inhibitors for the maxi cation
channel in rye root plasma membranes. Journal of Experimental
Botany 47 : 713–716.
White PJ. 1997. Cation channels in the plasma membrane of rye roots.
Journal of Experimental Botany Special Issue 48 : 499–514.
White MC, Baker FD, Chaney RL, Decker AM. 1981. Metal
complexation in xylem fluid. II. Theoretical equilibrium model
and computational computer program. Plant Physiology 67 :
301–310.
183
White PJ, Banfield J, Diaz M. 1992. Unidirectional Ca#+ fluxes in roots
of rye (Secale cereale L.). A comparison of excised roots with roots
of intact plants. Journal of Experimental Botany 43 : 1061–1074.
White PJ, Moore HC, Marchant A, May ST, Bennett MJ. 1996.
Identifying the physiological roles of ion transport proteins in root
cells. Plant Physiology and Biochemistry Special Issue, 161.
White PJ, Ridout MS. 1998. The estimation of rapid rate-constants
from current-amplitude frequency distributions of single-channel
recordings. Journal of Membrane Biology (in press).
White PJ, Tester MA. 1994. Using planar lipid-bilayers to study plant
ion channels. Physiologia Plantarum 91 : 770–774.
Winstanley M. 1997. ISIS. BBSRC Business, January 1997, 17.
Widell S, Asp H, Jense! n P. 1994. Activities of plasma membrane-bound
enzymes isolated from roots of spruce (Picea abies) grown in the
presence of aluminium. Physiologia Plantarum 92 : 459–466.
Wymer CL, Bibikova TN, Gilroy S. 1997. Cytoplasmic free calcium
distributions during the development of root hairs of Arabidopsis
thaliana. Plant Journal 12 : 427–439.
Zimmermann S, Nu$ rnberger T, Frachisse J-M, Wirtz W, Guern J,
Hedrich R, Scheel D. 1997. Receptor-mediated activation of a
plant Ca#+-permeable ion channel involved in pathogen defense.
Proceedings of the National Academy of Sciences of the USA 94 :
2751–2755.