1 Department of Plant Sciences, University of Cambridge, Downing Street,
Cambridge CB2 3EA, UK
2 School of Agricultural Sciences, University of Tasmania, Hobart, Tasmania
7001, Australia
* Author for correspondence (e-mail: vd211{at}cam.ac.uk)
Accepted 1 October 2002
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Summary |
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Key words: Arabidopsis, Calcium, Channel, Free oxygen radical, Plasma membrane, Potassium
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Introduction |
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This study tests the hypothesis that, in plant cells as in animals, FORs
regulate channel activity. The study has focused on root epidermal cells as
sites of pathogen interaction, stress perception and gravitropism and on
hydroxyl radicals (OH) as the most potent, biologically
relevant FOR that can be produced in various plant systems, including
Arabidopsis roots (Becana and
Klucas, 1992; Moran et al.,
1994
; Shen et al.,
1997
; Van Doorslaeder et al.,
1999
; Smirnoff and Wheeler,
2000
; Joo et al.,
2001
). Moreover, weakly reactive oxygen species (such as hydrogen
peroxide and superoxide anion radicals) do not interact directly with most
target biomolecules, and thus need to be converted to OH,
which are capable of modifying targets
(Halliwell and Gutteridge,
1999
). Here, OH have been generated at the
external face of the epidermal plasma membrane using a
Cu2+/ascorbate mixture
(Halliwell and Gutteridge,
1999
). In planta, apoplastic FOR production could occur
by several mechanisms including the interaction of transition metals (such as
Cu2+) with ascorbate or H2O2
(Fry, 1998
). It is shown here
that FORs activate plasma membrane Ca2+- and
K+-permeable channels in the root epidermis. Activation by FORs
forms a novel pathway for the regulation of K+ channel activity in
plant cells and delineates a new family of plant ion channels
FOR-activated Ca2+-permeable nonselective cation channels. This
activation of cation conductances is involved in two crucial phenomena in root
cells: response to stress and elongation growth.
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Materials and Methods |
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For moving vibrating ion-selective microelectrode (MIFETM)
experiments, Columbia seedlings were grown for 3 to 6 days on standard medium
or paper rolls (Shabala et al.,
1997; Shabala,
2000
). All other plants used in MIFETM experiments
(Triticum aestivum L., Zea mays L., Vicia faba L.,
Spinacia oleracea L., Trifolium pratense L.) were from
commercial sources and were grown for 3 to 6 days on paper rolls.
Isolation of protoplasts from mature root epidermis and
pericycle
The method was adapted from Demidchik and Tester
(Demidchik and Tester, 2002).
Roots from 50 seedlings were cut into approximately 1 mm-long pieces in 4 ml
of enzyme solution. This comprised 1.5% Cellulase Onozuka RS (Yakult Honsha,
Tokyo, Japan), 1% cellulysin (CalBiochem, Nottingham, UK), 0.1% pectolyase
Y-23 (Yakult Honsha, Tokyo, Japan), 0.1% bovine serum albumin (Sigma), 10 mM
KCl, 10 mM CaCl2, 2 mM MgCl2, 2 mM MES, pH 5.6 with
Tris; 290 to 300 mOsM adjusted with 330 mM sorbitol. After shaking (60 rpm) in
the enzyme solution for 30 to 50 minutes at 28°C, protoplasts were
filtered (50 µm pore) and rinsed with holding solution (HS: 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 10 mM sucrose, 10 mM glucose, 2 mM
MES, pH 5.7 with Tris; 290 to 300 mOsM). Protoplasts were collected by 5
minutes centrifugation at 200 g and diluted with HS. Most
viable protoplasts isolated by this procedure were derived from mature
epidermis as judged by direct observation and comparison with protoplast
release from roots expressing GFP in the epidermis or pericycle. In patch
clamp experiments, pericycle protoplasts were identified by GFP expression,
while mature epidermal protoplasts were identified by direct observation
(light microscopy), larger diameter (20±1.5 µm diameter) and uniform
grey colour.
Isolation of protoplasts from root elongation zone epidermis
Root tips (3-4 mm) were isolated directly in the patch-clamp chamber and
exposed to the enzyme solution (no shaking) for 1-2 hours (22°C, dark).
Direct observation confirmed protoplast release solely from elongation zone
epidermis. Protoplasts (20±1.5 µm diameter) were washed with HS for
5 minutes prior to use.
Electrophysiology
Electrophysiological protocols were adapted from Demidchik and Tester
(Demidchik and Tester, 2002).
Osmolarity of all solutions was adjusted to 300 mOsM with D-sorbitol. The
pipette solution contained (mM): 50 K+, 10 Cl-, 40
gluconate-, 4 EGTA, 5 HEPES, pH 7.2 (KOH). For selectivity
estimates, 50 mM TEA+ or 25 mM Ba2+ were added as
chlorides. In preliminary assays, OH-induced currents (whole
cell configuration) were found to be independent of pipette solution
Ca2+ [1-200 nM: activities calculated using GEOCHEM
(Parker et al., 1995
)]. To
avoid uncertainties arising from ATP-dependent Cl- currents, ATP
was omitted from the pipette solution
(Demidchik and Tester, 2002
).
Control assay (bathing) solution contained (mM): 20 CaCl2, 2 MES,
pH 5.7 (Tris). To generate OH, 1 mM CuCl2 and 1 mM
ascorbic acid (Cu/a) (Halliwell and
Gutteridge, 1999
) were incorporated (pH 5.7) unless stated
otherwise. Selectivity estimates were made as described in Demidchik and
Tester (Demidchik and Tester,
2002
). In pharmacological assays, conductances before
(Gcontrol) and after addition of inhibitors (mM), 20
TEA+, Ca2+ or Ba2+, 0.05 La3+ or
Gd3+, 0.02 verapamil (Gblock), were calculated from
currents between -80 mV and -160 mV (for the inward Ca2+ current)
or between 0 mV and +80 mV (for the outward K+ current). A standard
patchclamp amplifier (IM/PCA, List, Darmstadt, Germany), Digidata 1200
digitiser and pClamp software, version 6.0.2 (Axon Instruments, Foster City,
CA) and 8-pole Bessel filter (Frequency Devices, Haverhill, MA) were used.
Current was sampled at the end of the 2 second voltage pulse. Liquid junction
potentials were measured and corrected as described elsewhere
(Ward and Schroeder, 1994
).
Curve fitting was performed using Statistica (StatSoft, Tulsa, OK) or Sigma
Plot (SPSS Science, Chicago).
K+ photometry
Excised Arabidopsis roots (0.1 g) were rinsed for 60 minutes in
deionised water and placed in 5 ml of assay solution (10 mM CaCl2,
5 mM MES to pH 5.7 with Tris). Hydroxyl radicals were generated by the
addition of 1 mM Cu/a. Samples (0.5 ml) were assayed for K+ by
flame photometry.
Luminometry
Standard luminometry procedures (Price
et al., 1994) were used for recording luminescence from about 50
excised roots. Lucigenin-based luminescence assays were adapted from Papadakis
and Roubelakis-Angelakis (Papadakis and
Roubelakis-Angelakis, 1999
). Six to 7 cm-long (from the apex)
pieces were placed in the recording cuvette with 2 ml of control assay
solution (mM: 0.5 CaCl2, 1 EDTA, 10 Tris, pH 8.7). Stresses were
applied as 150 mM NaCl or 1% cellulase (Onozuka) or cooling (2°C). After
30 minutes, the cuvette was placed in the luminometer and 2 mM lucigenin
(Sigma) was incorporated. Counting at 1 Hz was performed for 100 seconds. The
assay for aequorin luminescence was adapted from Kiegle et al.
(Kiegle et al., 2000b
) Excised
roots were immersed in recording solution (10 mM CaCl2, 2 mM MES,
pH 5.7 (Tris), 4 µg ml-1 coelenterazine [free base, NanoLight
Technology, Prolume, Pittsburgh, PA]) for 20-30 hours at 28°C (dark, 20
rpm). Excised root samples were exposed to inhibitors (incorporated into
recording solution) for 30 minutes prior to experimentation. Reactions were
initiated by the addition of Cu/a or CuCl2 and luminescence changes
were recorded at 1 Hz.
Moving vibrating ion-selective microelectrode technique
(MIFETM)
This technique is described in detail elsewhere
(Shabala et al., 1997;
Shabala, 2000
). Each plant was
mounted in a Perspex holder by an agar drop and roots were immersed in assay
solution comprising (in mM) 0.1 KCl, 0.1 NaCl, 0.1 MgCl2 and 0.05
CaCl2, pH 5.6 with 1 Tris/MES. The microelectrode was placed 20
µm above the root surface and net K+ and Ca2+ fluxes
were measured concurrently. Hydroxyl radicals were generated by the addition
of 1 mM Cu/a.
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Results |
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In intact biological systems, excess Cu2+ or ascorbate alone are
also capable of generating some OH, but are much less potent
than Cu/a (Halliwell and Gutteridge,
1999). Nevertheless, prolonged (15 minutes) exposure to external 1
mM Cu2+ or ascorbate alone did not activate currents in
Arabidopsis root protoplasts; moreover, Cu2+ alone almost
completely blocked control Cain and halved
Kout (corresponding to initial current decrease during the
first 2-4 minutes after Cu/a addition; Fig.
2A). Only long-term exposure to 1-5 mM Cu2+ (30 to 90
minutes) activated currents resembling Cu/a-induced currents (in 19% of
protoplasts tested; n=62). Activation of Cain and
Kout was observed even at 3 µM Cu/a (15 minutes
exposure; Cain measured at - 160 mV increased from
-41±4 pA to -63±9 pA; Kout measured at +100
mV increased from +107±18 to +177±34; n=11); maximal
activation was achieved at 0.3 to 1 mM Cu/a. The potent response of
Cain and Kout to OH
contrasts markedly with that to the non-radical H2O2. In
mature epidermal protoplasts, prolonged (15 minutes) exposure to 1 to 20 mM
H2O2 had no significant effect on
Cain, while Kout even decreased (at
+100 mV) by 34±3% (n=17).
Permeability of FOR-activated conductances
Selectivity estimates were made for OH-activated
conductances observed in protoplasts derived from mature epidermis of
Columbia. Since, in multi-cationic conditions, using ramps or reversal
potentials for measuring plant cation channel selectivity can be unreliable
(Demidchik and Tester, 2002),
selectivity estimates were obtained from values of conductance calculated from
currents at voltages between -80 mV and -160 mV (for Cain)
or between 0 mV and +80 mV (for Kout). For the
inward-rectifying conductance, this yielded a permeability sequence of
K+ (1.00)
NH4+ (0.91±0.13;
n=4)
Na+ (0.71±0.09; n=4)
Cs+ (0.67±0.09; n=4) > Ba2+
(0.32±0.01; n=10)
Ca2+ (0.24±0.01;
n=10) > TEA+ (0.09±0.01; n=7). These
data show that OH-induced Cain was
mediated by nonselective cation channels [NSCC
(Demidchik and Tester, 2002
;
Demidchik et al., 2002
)] rather
than the root hyperpolarisation-activated Ca2+ channel
(Véry and Davies, 2000
;
Miedema et al., 2001
). The
TEA+ permeability of the OH-activated
Cain was lower than that of the Arabidopsis root
plasma membrane NSCC described previously
(Demidchik and Tester, 2002
),
suggesting that FORs may selectively activate a particular type of NSCC. To
confirm that OH-activated Cain was
mediated by NSCC rather than an inwardly rectifying K+ channel,
protoplasts derived from mature epidermis of the Columbia akt1 mutant
[that lacks the root plasma membrane K+ inward-rectifying channel
(Hirsch et al., 1998
)] were
tested. No significant differences in the response to 1 mM Cu/a were found
between wild-type and akt1 (in akt1 after 15 minute exposure
to 1 mM Cu/a, Cain measured at -160 mV was -121±9
pA and Kout measured at +100 mV was +278±28 pA;
n=14). The permeability sequence of the outward-rectifying
conductance agreed with that previously determined for the
Arabidopsis root K+ outward rectifier
(Maathuis and Sanders, 1995
):
K+ (1.00) > Na+ (0.31±0.03; n=10)
> Ba2+ (0.06±0.01; n=4)
TEA+
(0.05±0.01; n=4).
Pharmacological analysis of FOR-activated conductances
Pharmacological analysis of OH-activated conductances
(Columbia mature epidermis; Fig.
2C) further confirmed that Cain and
Kout were mediated by different populations of cation
channels. The OH-activated Cain was
insensitive to the K+ channel blocker TEA+ (20 mM) but
was blocked significantly by the cation channel blockers La3+,
Gd3+ (both 50 µM) and verapamil (20 µM). This response to
TEA+ and lanthanides resembles that of the plasma membrane NSCC
conductance shown previously to mediate Na+ influx to
Arabidopsis and wheat roots
(Demidchik et al., 2002;
Demidchik and Tester, 2002
).
However, the verapamil sensitivity of the OH-activated
Cain is in marked contrast to the verapamil-insensitive
Na+ influx pathway and supports the premise that FORs activate a
distinct population of root NSCC. In contrast to Cain,
OH-activated Kout was sensitive to
TEA+, less sensitive to lanthanides and insensitive to verapamil.
The block of Kout by TEA+, Ca2+ and
Ba2+ agrees with previous observations on the constitutive
Kout observed in Arabidopsis root
epidermis/cortex protoplasts (Maathuis and
Sanders, 1995
) and tends to confirm that the latter is activated
by FORs.
Cell-specific FOR responses
The response of root epidermal cells to FORs has great physiological
significance since these cells are situated at the `root/environment
boundary'. These cells are exposed to the environment and are the first
challenged by pathogens and abiotic stresses. Cells situated beneath the
epidermis may have a less important role in FOR production and signalling as
they do not interact directly with the rhizosphere. Accordingly,
OH-induced effects in an inner tissue (the pericycle) were
examined. In pericycle protoplasts identified by cell-specific GFP expression
(Kiegle et al., 2000a), only
slight OH-induced increases in currents were found, occurring
after a prolonged lag-phase (23±6 minutes; n=10) between
application of 1 mM Cu/a and activation
(Fig. 3). These data show that
the response to OH is tissue-specific and stronger in cells
that directly interact with the environment.
|
A novel procedure of protoplast isolation specifically from the root
epidermal elongation zone permitted comparison of epidermal responses from
young and mature regions (Columbia). In elongation zone protoplasts under
control conditions, the background currents were small until approximately 1
hour after establishing the whole cell configuration. At this time,
constitutive hyperpolarisation-activated Ca2+ channel (HACC)
activity appeared that resembled HACC activity previously described from the
root elongation zone cortex, apical epidermis and root hair
(Kiegle et al., 2000a;
Véry and Davies, 2000
;
Demidchik et al., 2003
).
Properties of elongation zone epidermal HACC were dissimilar to the
OH-induced conductance (data not shown).
OH-induced currents were two- to threefold larger than in mature epidermis (n=10; Fig. 4). These results were consistent with OH-induced K+ efflux from intact roots (peak magnitude 262±54 and 95±25 nmol m-2 second-1 for elongation and mature zone respectively; n=5) measured non-invasively using the MIFETM technique. Thus, FOR channel activation varies with the developmental state of a given cell type.
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Biotic and abiotic stress stimulate FOR production by roots
The apical position of epidermal elongation zone cells and lack of
protection from the root cap mean that such cells are amongst the first to
respond to changing soil environment as the root descends. Their increased
sensitivity to FORs as a regulatory/stress signal could make them `antennae'
for receiving and amplifying external stimuli. To establish that
Arabidopsis root cells generate FORs in response to stress, FOR
production in intact Columbia roots was measured using lucigenin-based
chemiluminometry (Papadakis and
Roubelakis-Angelakis, 1999). Exposure (30 minutes) to high salt
(150 mM NaCl), cold shock (2°C) and elicitors (1% cellulase) increased
lucigenin chemiluminescence (which directly reflects FOR formation) by
224±14%, 253±17% and 632±55%, respectively, in comparison
with the resting level (84±9.3 counts second-1 for 100 mg
fresh weight; n=7). In in vitro tests, addition of 30-100 µM
Cu2+/asc to the lucigenin assay caused an increase in luminescence
comparable with that caused by cellulase. These results (in conjunction with
the patch clamp data) clearly demonstrate that Arabidopsis root cells
generate an FOR signal in response to (a)biotic stress and FORs activate
plasma membrane cation channels.
FORs and root growth
The enhanced sensitivity of elongation zone epidermis to FORs raises the
possibility that the latter are involved in growth regulation. According to
Cramer and Jones (Cramer and Jones,
1996), root growth (rate of cell elongation) approximately
linearly depends on free cytosolic Ca2+
([Ca2+]cyt), significantly accelerating with even slight
elevation in [Ca2+]cyt. Therefore the FOR activation of
Ca2+ influx channels in the elongation zone should stimulate root
growth. Comparative 12 day assays of Columbia root growth in medium containing
additional Cu2+ [ascorbate is present in the cell wall in at least
the sub-millimolar range (Smirnoff and
Wheeler, 2000
)] showed (mean±s.e.m.) 21.5±1.5,
39.4±1.9 and 29.2±2.1% higher growth rates in 0.3, 1 and 3 µM
Cu2+, respectively, than in control medium containing a
non-growth-limiting Cu2+ concentration of 0.1 µM (6.2±0.2
mm day-1; n=25). The cation channel blocker
Gd3+ (5 µM added to growth medium) suppressed this
Cu2+-induced stimulation of root growth. As Gd3+ blocked
the OH-activated Cain, these results
suggest that Cu2+-induced increased growth rates may have been due
to FOR-activated cation channels. Moreover, root growth was affected by the
OH scavenging agent, dimethyl sulfoxide (DMSO; 0.1% induced a
26±2.2% decrease of root elongation rate; n=15). Although it
was not possible to corroborate this result in patch clamp experiments (DMSO
promoted loss of the whole cell configuration), the effect of the
OH scavenging agent coupled with the particular
OH-sensitivity of root elongation zone protoplasts strongly
suggest a role for FORs in root growth.
Verification of FOR-activated K+ efflux and
Ca2+ influx at the whole root level
In whole root studies to verify cellular responses at the whole organ
level, Cu/a was still used to generate FORs as washing procedures and
prolonged exposure to solutions was likely to deplete the cell wall of
ascorbate (Smirnoff and Wheeler,
2000). Thus adding Cu2+ alone to generate FORs by
reaction with wall ascorbate would not have been a fair test. Whole root
K+ efflux from Columbia induced by 1 mM Cu/a was measured
photometrically over a 30 minute period. Mean (±s.e.m.) efflux was
significantly enhanced by the presence of Cu/a at all time points, thus
confirming the patch clamp data (n=5;
Fig. 5A).
|
The OH effect on Ca2+ influx in whole roots was
examined by applying chemiluminometry
(Price et al., 1994;
Kiegle et al., 2000b
) to
Columbia plants constitutively expressing aequorin targeted to the root cell
cytosol. In agreement with the OH-activated
Cain, OH raised
[Ca2+]cyt (Fig.
5B). With 10 mM external Ca2+, 1 mM Cu/a increased
[Ca2+]cyt from 0.114±0.01 µM to a peak of
19.15±0.89 µM (n=12). With 1 mM Ca2+
(Fig. 5B), the maximal
OH-induced [Ca2+]cyt increase was four
to five times smaller, demonstrating that the [Ca2+]cyt
increase was mainly due to Ca2+ influx from the external medium.
The whole root [Ca2+]cyt increase in response to Cu/a
was more rapid than the current response of protoplasts. The slower activation
time in protoplasts may result from the absence of cell wall that in vivo
would provide an additional pool of reactants that would effectively increase
the local concentration of hydroxyl radicals above that generated by Cu/asc.
It is also feasible that protoplast isolation and recording conditions render
channels less sensitive or responsive.
Significantly, the OH-scavenger DMSO reduced peak [Ca2+]cyt increase in response to Cu/a (Fig. 5C), thus confirming OH as the activator FOR species (n=12). To confirm that the increase in [Ca2+]cyt was the result of Ca2+ influx, roots were pretreated with cation channel blockers (Fig. 5C). TEA+ (10 mM) did not inhibit the peak OH-induced [Ca2+]cyt increase but verapamil (20 µM) and Gd3+ (100 µM) were strongly inhibitory (n=12). Qualitatively these results match the effects of these agents on the OH-induced Cain of mature epidermis and strongly suggest that the conductance observed in protoplasts was involved in the OH response of the whole root.
FOR epidermal flux activation across species
Having confirmed that channel activation can manifest at a multicellular
level, the in planta time-courses of the OH-activated net
K+ and Ca2+ fluxes of intact Arabidopsis
(Columbia) root mature epidermal cells were measured simultaneously using
MIFETM (Fig. 6). Again,
addition of 1 mM Cu/a caused significant K+ efflux
(Fig. 6A). Net Ca2+
flux shifted negative in the first 2 minutes after Cu/a addition suggesting a
transient influx decrease corresponding to the Cu2+ suppression of
Cain measured by patch clamp. This was followed by
long-term Ca2+ influx increase consistent with the previously
observed OH-activation of Cain
(Fig. 6B). The possible
ubiquity of FOR responses was then confirmed as similar effects were measured
in mature epidermal cells from a wide range of crop plants (including
dicotyledons, monocotyledons, C3 and C4 plants). In all species tested,
OH activated K+ efflux and Ca2+ influx
(Fig. 6). Moreover, flux
amplitude and time-course varied between species, suggesting species-specific
`flux signatures' in response to OH.
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Discussion |
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The application of Cu/a (as a OH-generating mixture) to
the extracellular face of root epidermal protoplasts activated two distinct
plasma membrane cation conductances Cain and
Kout. Two key lines of evidence demonstrate that channel
activation was regulatory in nature and not the result of loss of membrane
integrity: reversibility of activation and specificity of activation.
OH are not membrane-permeable and most probably exerted their
effect directly at the external membrane face, given that the pipette solution
could not readily have supported membrane-delimited phosphorylation-based
channel activation. Significantly, the FOR precursor
H2O2 failed to activate conductances of the mature
epidermis. This is in contrast to the sensitivity of guard cell
Ca2+-permeable NSCC to H2O2
(Pei et al., 2000) and
confirms the premise of Mittler and Berkowitz
(Mittler and Berkowitz, 2001
)
that H2O2 is not the sole oxygen-derived species capable
of signalling and regulation in plants. Moreover, the results demonstrate that
a given cell type can be responsive to specific oxygen-derived species.
It should be noted that, apoplastically applied, FOR species and
H2O2 are capable of differential gene activation
(Wisniewski et al., 1999;
Bowler and Fluhr, 2000
). It is
therefore feasible that OH-activated Cain
comprises part of a signalling mechanism for Ca2+-dependent gene
regulation.
Analysis of the OH-activated conductances revealed for the
first time that a plant outwardly rectifying K+ conductance
(Kout) can be activated by FORs.
Free-oxygen-radical-activated K+ channels have been previously
described in many animal preparations and play crucial roles in animal cell
physiology (Kourie, 1998).
Permeation and pharmacological profiles demonstrated that the
OH-activated Cain is mediated by a
specific group of NSCC that do not share identity with the
Arabidopsis NSCC involved in toxic Na+ influx
(Demidchik and Tester, 2002;
Demidchik et al., 2002
).
Whether this group of NSCC comprises more than one channel type is a question
that must now be addressed by extensive biophysical studies. The presence of
rapidly and slowly activated components of OH-activated
currents strongly suggests that several cation channel types are activated.
The finding of a novel group of channels points not only to differential
regulation and physiological role of root plasma membrane NSCC but also to
specific genetic identities.
The cell and tissue-specific responses to OH indicate
precise functions for FORs and the target channels. The epidermis marks the
boundary between the plant and its environment; the greater sensitivity of the
epidermis (relative to the pericycle) suggests that its
OH-activated Cain plays a signalling role
in the perception of and response to rhizosphere challenges. As whole root
studies showed that OH generates
[Ca2+]cyt transients, it is reasonable to suppose that
epidermal OH-activated NSCC function in this context (a view
supported by the similar blocker profiles of transients and
Cain). Abiotic and biotic stresses (such as salinity and
pathogen attack) are known to induce oxidative bursts which vary in time of
onset, cellular origin and duration [from minutes to several hours
(Bowler and Fluhr, 2000)]. That
Arabidopsis roots respond to salinity, cold shock and elicitors by
releasing FORs has been confirmed in the present study. Given the short half
life of FORs and their detection in the recording medium, it is likely that
the epidermis was involved in their production. The mechanism of
stress-induced apoplastic FOR production can only be speculated on but could
include the NADPH oxidase [involved in pathogen responses
(Torres et al., 1998
;
Bowler and Fluhr, 2000
)], cell
wall amine peroxidases and the conversion of H2O2 to
OH by cell wall Fe2+ or Cu2+. The root
is therefore capable of generating FORs in response to stress and the
epidermis could respond with a OH-activated
Cain- mediated [Ca2+]cyt elevation.
Additionally, K+ efflux is one of the earliest cellular stress
responses (Babourina et al.,
2000
; Bowler and Fluhr,
2000
). A mechanistic basis of that response has been demonstrated
here: FOR-activated K+ efflux through K+ outward
rectifying channels.
Calcium may lie upstream or downstream of stress-induced FOR production in
plant tissues (Bowler and Fluhr,
2000), illustrating the complexity of the signalling networks. It
is clear now that even if [Ca2+]cyt changes were
upstream, the OH-activated Cain is
competent to mediate any subsequent Ca2+ influx to amplify and
propagate the signal. In this respect, it is significant that the NADPH
oxidase thought to be responsible for pathogen-elicited oxidative bursts
contains an EF-hand indicative of Ca2+ stimulation
(Torres et al., 1998
).
Increased apoplastic superoxide anion production by a
Ca2+-stimulated NADPH oxidase would result in OH
at the external face of the plasma membrane through
superoxide/H2O2 interaction and
H2O2 breakdown catalysed by cell wall transition metals
(Fry, 1998
;
Halliwell and Gutteridge,
1999
). This would activate Cain. Moreover,
sustained [Ca2+]cyt elevation caused by
lanthanide-sensitive Ca2+ influx is required for defence-gene
transcription associated with radical production
(Blume et al., 2000
), and
Cain appears competent to mediate such a
[Ca2+]cyt elevation.
Plant roots grow in length by Ca2+-dependent cell elongation at
the root apex (Cramer and Jones,
1996). In the elongation zone, the OH-induced
Ca2+ currents were significantly larger than in mature cells,
therefore differential channel sensitivity to FORs can define the zone of root
Ca2+ uptake. That the OH-scavenger DMSO inhibited
root elongation supports the possibility of FOR involvement in regulating cell
extension. Moreover, as OH are implicated in xyloglucan
remodeling, which would increase wall extensibility in the elongation zone
(Fry, 1998
;
Vissenberg et al., 2000
), FORs
may be a co-ordinating factor for the Ca2+ influx and wall
extension necessary for growth. In this context, it is significant that
hydroxyl radical involvement in auxin-induced extension growth of coleoptiles
has been recently reported (Schopfer et
al., 2002
). Another possible FOR-regulated process (also centered
at the elongation zone) is the root gravitropic response. Gravistimulated FOR
production of the elongation zone greatly exceeds that of the mature region
(Joo et al., 2001
). Since
elevation of [Ca2+]cyt is implicated in the gravitropic
response (Joo et al., 2001
),
the finding here of potent OH activation of
Cain in the elongation zone is significant. Placing the
OH-activated Kout in these contexts is
problematic as its activity would depend on the equilibrium potential for
K+ (EK; which in turn depends on external K+)
relative to the resting membrane voltage. It has been proposed that
K+ efflux helps counteract the depolarizing effect of
Ca2+ influx on membrane voltage
(Miedema et al., 2001
), thus
allowing net Ca2+ influx to proceed. Harnessing both
Ca2+ and K+ conductances to FOR regulation in the
elongation zone may ensure secure membrane voltage regulation in this critical
region.
Overall, it is now clear from this study that root FORs have precise cellular targets and obvious functions. The effect of FORs is not restricted to the root cell of a model plant but is observed in those of monocots and dicots, C3 and C4 plants. The response of each species varies in time course and magnitude but the fundamental response of K+ efflux and Ca2+ influx is conserved. The observed effects of OH at the cell, tissue and species level have begun to reveal an intricate regulatory network involving apoplastic FORs, in which channel-mediated events will depend on the FOR species, its local concentration and duration of exposure.
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Acknowledgments |
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References |
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