Annals of Botany 82 : 331–336, 1998
Article No. bo980686
Auxin Stimulates Cl− Uptake by Oat Coleoptiles
O L G A B A B O U R I N A*, S E R G E Y S H A B A LA and I A N N E W M AN
Department of Physics, UniŠersity of Tasmania, GPO Box 252-21, Hobart, Tas 7001, Australia
Received : 10 February 1998
Returned for revision : 20 April 1998
Accepted : 15 May 1998
The effects of auxin on net ion fluxes near parenchyma of oat coleoptiles were studied using the non-invasive MIFE
system to measure specific ion fluxes using ion selective microelectrodes. Application of 10 µ 1-naphthaleneacetic
acid (NAA) for 3 h caused doubling of coleoptile segment growth, without changing the pH of the unbuffered bathing
solution from pH 5±4 during that time. Short term experiments revealed that auxin led to an immediate three-fold
increase of chloride influx to 1200 nmol m−# s−", maintained for at least 1 h. In the first minutes after auxin
application, proton fluxes were small (®25 nmol m−# s−", an efflux) and tended to decrease, whereas potassium and
calcium fluxes changed little, fluctuating from ®100 to 0 nmol m−# s−" and from ®15 to 0 nmol m−# s−", respectively.
It is suggested that one target of auxin action in plant cells is the plasma membrane chloride transport system
mediating increased chloride uptake.
# 1998 Annals of Botany Company
Key words : Auxin, chloride transport, ion flux, AŠena satiŠa L., oat.
I N T R O D U C T I ON
Auxin plays an important role in plant life, regulating cell
division and elongation, tropic responses and tissue differentiation (Wareing and Phillips, 1981). However, mechanisms of its action in plants as well as the signal
transduction pathways still remain obscure. In spite of the
lack of detailed knowledge, there is little doubt that auxin
perception in plants is mediated by various ion transporters
located in the cell membrane.
In the first 30 min, reactions of plant cells to additional
auxin include increased proton and free calcium concentrations in the cytosol (Felle, 1988 ; Gehring, Irving and
Parish, 1990), stimulation of the K+}H+ exchange rate
(Haschke and Lu$ ttge, 1975), and increased efflux of protons
and calcium (Arif and Newman, 1993). However, the
primary physiological action of auxin in plants is still
unknown.
Recent molecular analysis of the mutant tobacco line axi
4}1, which was regenerated from auxin-independent callus,
showed that the plant gene axi4 encodes a protein with
significant homology to a family of animal cation-chloride
co-transporters (Harling et al., 1997). This transporter is not
yet described electrophysiologically, but the direct link
between auxin action and a chloride transport system is
obvious. Thomine et al. (1997) indirectly confirmed the
involvement of anion channels in cell growth processes that
were regulated by auxin, when anion-channel blockers were
able to counteract the growth inhibition induced by auxins
at their high concentrations. Therefore the chloride transport system could be a primary target for auxin action in
plants.
To date our knowledge of chloride transport systems at
the plasma membrane of higher plant cells includes the
following : (1) high- and low-affinity uptake transporters
(Elzam, Rains and Epstein, 1964) ; and (2) chloride outwardand inward-rectifying channels through the plasma membrane (Schroeder and Keller, 1992 ; Skerett and Tyerman,
1994). Under normal conditions, with no external excess of
chloride salts, the Nernst potential ECl is positive. Therefore
chloride uptake into the plant cell must be performed by
active transport that drives chloride against its electrochemical barrier. In addition, tonoplast active pumps and
channels regulate cytoplasmic and vacuolar chloride concentrations. Recent findings for plant cells suggest involvement of chloride ions in regulation of turgor, stomatal
movement, nutrient transport, metal tolerance, signal
perception and transduction and possibly also signal
propagation as they are also responsible for the generation
of action potentials (Tyerman, 1992 ; Hedrich and Becker,
1994 ; Schroeder, 1995). However, the mechanism of chloride
uptake into plants is still poorly described and controversial.
Haschke and Lu$ ttge (1975) did not find a correlation
between the rate of Cl− uptake and indolacetic acid (IAA)
exposure, whereas Rubinstein and Light (1973) reported the
enhancement by IAA of $'Cl− uptake into AŠena coleoptiles.
Patch-clamp studies on isolated protoplasts revealed auxin
effects on plasma membrane chloride channels (Marten,
Lohse and Hedrich, 1991 ; Lohse and Hedrich, 1995). This
indicates that auxin directly affects plasmalemma chloride
transport systems in plants, and this effect should be
investigated using different approaches. One of them is to
measure net ion fluxes using a non-invasive microelectrode
technique such as the MIFE system used by Shabala,
Newman and Morris (1997). This system allows measurement of ion concentrations and fluxes close to intact cells,
thus avoiding abnormalities of plant protoplasts, such as
the loss of the cell wall, the high concentration of osmoticum
outside and the quite artificial medium inside.
* For correspondence. E-mail Olga.Babourina!utas.edu.au
0305-7364}98}090331­06 $30.00}0
# 1998 Annals of Botany Company
Babourina et al.—Auxin Stimulated Cl − Uptake
332
The aim of this work was to investigate the effect of auxin
application on chloride transport.
MATERIALS AND METHODS
Plant material
Etiolated 4-d old coleoptile segments of oats (AŠena satiŠa
L. ‘ Victory ’) were used in experiments. Seeds were obtained
from Svalof A.B. (Svalof, Sweden). After 48 h germination
in Petri dishes on wet filter paper in darkness at 26³2 °C,
seedlings were transferred onto cotton gauze covering 400 ml
vials filled with distilled water in the same growth cabinet.
Whilst being transferred, seedlings were illuminated by dim
red light for 1 h to slow down the growth of the mesocotyl.
Seedlings were used for measurements when coleoptiles
reached 2±5 to 3±5 cm in length.
A 15 mm long segment was taken, starting 5 mm below
the apex. The segment was deleafed using a pin. A cut
around half of the perimeter, about 10 µm deep, was made
5 mm from the apical end of the segment using a razor
blade, and the segment was partly peeled by removing
epidermal strips using fine tweezers (No 5, ‘ Sigma ’).
Segments were pre-incubated in room light for 4 to 5 h in
unbuffered 100 µ CaCl (pH 5±4) to allow recovery from
#
wounding. A segment was then placed in a perspex
measuring chamber, of 3 ml volume, and fixed horizontally
5 mm above the chamber floor on two fine partitions using
a cotton thread to prevent floating. The chamber was filled
by fresh bath solution (1 m KCl­100 µ CaCl ), put onto
#
the microscope stage in the Faraday cage, and allowed to
adapt to experimental conditions including the microscope
light, for about 30 min.
Microelectrode fabrication
The microelectrodes were made from borosilicate glass
tubing (GC 150-10, Clark Electromedical Instruments,
Pangbourne, Berks, UK). Electrodes were pulled, oven dried
at 200 °C for 5 h, and silanized with one-two drops (C 15 µg)
of tributylchlorosilane (90796, Fluka Chemical, Buchs,
Switzerland) for 10 min in the same oven under a steel
cover. The cover was then removed, and the electrodes left
to dry for another 30 min. To fill the microelectrodes,
silanized electrodes were broken back to give an external tip
diameter of 3 to 5 µm. The electrodes were back filled with
solutions (0±15 m NaCl­0±4 m KH PO adjusted to
# %
pH 6±0 using NaOH for proton electrode ; 0±5  CaCl for
#
calcium ; and 0±5  KCl for potassium and chloride). The
electrode tips were then filled with commercially available
ionophore cocktails (hydrogen 95297 ; calcium 21048 ;
potassium 60031 ; chloride 24902 ; all from ‘ Fluka ’). The
electrodes were calibrated in a known set of standards (pH
from 4±6 to 7±8 ; Ca#+ from 0±1 to 10 m ; K+ and Cl− from
1 to 100 m). Electrodes with responses less than 50 mV per
decade for monovalent ions and 25 mV per decade for
calcium were discarded.
Ion flux measurements
Fluxes of H+, Cl−, K+ and Ca#+ were measured close to the
parenchyma tissue of a peeled region of the coleoptile
segment using the non-invasive MIFETM system (Unitas
Consulting, Hobart, Australia) generally as described by
Shabala et al. (1997). Additional information about the
MIFE system and technique is available on the internet at
http:}}www.phys.utas.edu.au}physics}biophys. This system allowed measurement of three ions simultaneously. We
used proton, chloride, calcium in one group of experiments,
and proton, chloride, potassium in the other group. Briefly,
the microelectrodes filled with specific ionophores were
moved up and down above the tissue in a square-wave
manner with a 10 s cycle. The differences in the electrochemical potential were measured for each of the specific
ions between two positions, 50 and 85 µm above the tissue,
using the calibrated Nernst slope of the electrode. From the
electrochemical potential difference between the two
positions, the net flux was calculated for each ion assuming
cylindrical geometry of flux diffusion. The average concentration of each ion between the two positions was also
calculated from the recorded data. We maintain our
convention that net influx into tissue is positive.
Experimental procedure
Transient responses to 1-naphthaleneacetic acid (NAA).
Fluxes of specific ions were measured for about 15 min,
three different ions at the same time. 30 µl of 1 m NAA
stock solution was then added into the measuring chamber
6 mm from the measured site. The bath solution was
thoroughly mixed by sucking and expelling it from a 1 ml
pipette six–ten times. The final concentration of NAA in the
bath was 10 µ. The time required for NAA addition,
mixing, and establishing the diffusion gradients was discarded from the analysis and appears as a gap in the figures ;
it was about 3 min for H+, K+ and Ca#+ microelectrodes,
and 5 min for Cl− due to its ‘ slow ’ ionophore adjustment to
large concentration changes. In most cases, fluxes were
measured for another 30–40 min after NAA addition. The
NAA stock solution was made freshly every day before
experiments by dissolving NAA in concentrated KOH and
adjusting the pH with HCl.
As the NAA addition was expected to produce specific
responses of the plant tissues, no repeated experiments were
possible on the same plant. In studying the flux dependence
on NAA for different external chloride concentrations each
point in Fig. 4 (­NAA line) represents data from a separate
plant. For this particular case, each Cl− flux shown is the
average over 10 min at the steady part of the transient curve
(starting 10–15 min after NAA addition). In controls
(®NAA) it was possible to use several concentrations of
external chloride on the same plant. After fluxes were
measured at one concentration for 10–15 min, a drop of
KCl stock was added into the bath, mixed thoroughly as
described above, and fluxes measured for another
10–15 min. The concentrations were increased randomly.
Three to four concentrations were applied to each plant,
and each point shown in the ‘ ®NAA ’ line in Fig. 4
represents average Cl− flux over a 10 min interval from
three–four plants. Eight plants were used in total.
Elongation and pH recording experiments. In elongation
experiments up to ten peeled, deleafed 15 mm coleoptile
Babourina et al.—Auxin Stimulated Cl − Uptake
Long term effect of auxin on elongation, pH changes and
H + fluxes
The stimulating effect of auxin on plant elongation is
strongly dependent on the ion composition and pH of
solution, its metabolic supplement, the type of auxin, and
the way the auxin was dissolved (Cohen and Nadler, 1976 ;
Claussen et al., 1997). These factors normally vary from
experimenter to experimenter. Besides, there is a certain
genotypic and organ specificity in plant sensitivity to auxin :
roots, coleoptiles and hypocotyls require different concentrations for growth stimulation and inhibition. Elongation
tests were necessary to link the auxin effect to chloride fluxes
under our conditions.
There was no significant variation in the stimulating effect
of NAA on coleoptile elongation in 1 m KCl­100 µ
CaCl solution over the concentration range of NAA from
#
0±1 to 50 µ (Fig. 1). As the exposure time rose from 3 to
20 h, the stimulating effect of NAA became more evident in
the concentration range from 10 to 50 µ, reaching as high
as 300 %. These observations are in good agreement with
reports from other investigators who applied this particular
auxin to oat coleoptile tissue (Keller and van Volkenburgh,
1996). In all subsequent experiments we used 10 µ NAA.
According to the classic hypothesis of ‘ acid growth ’,
auxin activates the plasma membrane proton pump causing
a pH drop in the apoplast, which results in cell wall
loosening and growth (Hager, Menzle and Krauss, 1971).
To compare growth response with changes in pH, we made
long term measurements, up to 8 h, on both medium pH
and H+ flux near the coleoptile parenchyma. The results
showed that after 3 h, at which time growth response to
10 µ NAA was doubled, there was no difference in medium
pH between control and NAA-exposed conditions (Fig.
2 A). Differences between control and treated H+ fluxes were
not significant until 4 h after auxin application (Fig. 2 B).
Therefore, in our long term experiments we have also
encountered the critical point in the acid growth theory : the
apparent disagreement ‘ between the magnitude of acidification induced by auxin and the growth responses ’ (Rayle
and Cleland, 1992).
Short term auxin effect on ion fluxes
Disagreement between the change in H+ flux (starting at
3 h, Fig. 2 B) and the growth response in long term
Growth, % of control
300
250
200
150
100
50
0
0.01
0.1
1
10
NAA, µ M
100
1000
10 000
F. 1. Growth as a function of concentration of NAA at 3, 6 and 20 h.
Segments were 15 mm long (n ¯ 30) for each concentration. The final
length of control segments, taken as 100 % for each concentration, was
(³s.e.) : 15±9³0±5, 16±2³0±4 and 17±1³0±5 mm, respectively.
5.7
A
5.6
5.5
5.4
5.3
pH
R E S U L T S A N D D I S C U S S I ON
350
5.2
5.1
5
4.9
4.8
4.7
0
0
1
2
3
5
4
Hours
6
7
8
9
1
2
3
5
4
Hours
6
7
8
9
B
–20
H+ flux (nmol m–2 s–1)
segments were threaded on a nylon line, measured using a
ruler and placed in 30 ml unbuffered solutions (1 m
KCl­100 µ CaCl ) in 100 ml beakers. In long term
#
experiments on the effect of auxin, the partly peeled
coleoptile segments were freely floated in beakers and gently
shaken on a rotary shaker. After being exposed to 10 µ
NAA in a beaker, a coleoptile was transferred to a chamber,
filled by solution from the beaker. Each point in Fig. 2
represents the average, for three independent coleoptiles, of
10 min pH and H+ flux measurements made 15 min after
transferring the chamber into the MIFE system.
333
–40
–60
–80
–100
–120
–140
–160
–180
0
F. 2. Time dependence of pH changes of bathing solution (A) and H+
fluxes (influx positive) near parenchyma of oat coleoptiles (B) : without
NAA (+) and with 10 µ NAA (U).
experiments (already doubled at 3 h, Fig. 1) suggested that
the most interesting events involved in growth processes
occurred in the first 3 h after auxin application, and
obviously required an involvement of other ions contained
in the bathing solution. Figure 3 shows transient responses
in pH and the fluxes of H+, Cl−, K+ and Ca#+ measured noninvasively near the parenchyma tissue in the first 50 min
following NAA application. The bath solution was unbuffered 1 m KCl­100 µ CaCl at pH 5±4. Because we
#
334
Babourina et al.—Auxin Stimulated Cl − Uptake
applied NAA in KCl as an added solution into the chamber
we thereby increased the bath concentrations of K+ and Cl−
to about 6 m KCl. This was the reason for using KCl as a
control in these experiments.
The most pronounced effect was on Cl− transport (Fig.
3 C). Ten min after NAA application (10 µ), we observed
a significant increase in chloride uptake by the plant tissue
from about zero to 1100–1300 nmol m−# s−". Addition of
KCl alone led to a much smaller increase of chloride influx
(300–500 nmol m−# s−").
The stimulating effect of NAA on Cl− uptake was very
much greater than the increased influx of chloride caused
merely by increasing the external concentration of chloride
from 0±1 to 10 m KCl (Fig. 4). In the range of external
chloride concentrations used, the dependence of chloride
fluxes on external chloride concentrations was nearly linear.
In the presence of NAA the slope of the line was about three
times higher than in control conditions. This increase could
not be a result of pH changes, as pH was monitored in each
experiment and it fluctuated near 5±4 (Fig. 3 A). Other
experiments have shown that the maximum activity for
chloride uptake was found at pH 5±3 (data not shown).
There was a slight decrease in the size of the H+ efflux
from ®20 to about ®10 nmol m−# s−" as a result of NAA
application (Fig. 3 B). The smallest efflux (negative) values
were observed in 15–30 min, after which proton fluxes
returned to higher efflux, close to pre-treatment values. In
the control case of addition of KCl (Fig. 3 B), proton fluxes
followed their own rhythms and fluctuated near the pretreatment value. Our results for H+ flux changes are slightly
different from those reported previously in this laboratory
(Arif and Newman, 1993) ; however, the conditions of the
experiments (pH, composition of the bathing solution,
auxin and the way it was dissolved) were different. The
different results emphasize the dependence of auxin effects
on pH and ion composition of the bathing solution.
Addition of auxin to the external medium did not affect
K+ and Ca#+ fluxes (Fig. 3 D and E). Their values fluctuated
from ®100 to 0 nmol m−# s−" for potassium and from ®15
to 0 nmol m−# s−" for calcium. A minor trend to influx for
Ca#+ after application of NAA solution was similar to the
change in Ca#+ flux after control addition of KCl and so was
probably due to solution mixing and consequent small
changes in some membrane behaviour.
Auxin effect on H + and Cl − fluxes
There are numerous reports that auxin causes noticeable
acidification of the bathing medium starting within 1 h of
exposure. In our experiments using 1 m KCl­100 µ
CaCl we always observed an immediate small reduction of
#
the normal net H+ efflux (Fig. 3 B), with a corresponding
slight increase in the medium pH in the first 30 min followed
by acidification (Fig. 3 A). This time course is in good
agreement with reports on cytoplasmic acidification of plant
cells by auxin (Gehring et al., 1990). Close to the end of the
first hour of NAA exposure, the pH of the medium was
similar to the starting value (Figs 2 A, 3 A).
At the same time, there was a three-fold increase in Cl−
uptake, compared with controls, caused by NAA treatment
(Fig. 3 C). As our technique measured the net Cl− flux, the
increased chloride influx could occur either through increased active chloride inward transport or through decreased conductance of channels that release chloride into
the medium. Although both mechanisms are possible, it is
more likely that it was increased active uptake of chloride,
since Marten et al. (1991) and Lohse and Hedrich (1995) did
not observe changes in chloride channel conductivity after
auxin application. Observed increased influx of chloride
under auxin application is in good agreement with the work
of Rubinstein and Light (1973) and Stevenson and Cleland
(1981) performed on oat coleoptiles.
According to Mistrik and Ulrich (1996) there are at least
six different hypotheses of mechanisms of anion uptake by
plant cells. One of them is nH+}A− symport, with n from 1
to 4 stoichiometry. The symport is well characterized in
Characean cells (Sanders, 1980 ; Beilby and Walker, 1981)
and there are a few reports that provide evidence for a
similar mechanism in cells of higher plants (Ulrich and
Guern, 1990 ; Felle, 1994 ; Mistrik and Ulrich, 1996).
The shift of proton fluxes towards influx (Fig. 3 B) is
consistent with an H+}Cl− symport theory. However,
supposing that only this one type of chloride transporter is
present, we should observe a shift in proton flux equal to the
shift in Cl− flux. That such changes in net proton flux were
not observed could be due to (1) increased H+ extrusion
pumping masking the big shift in H+}Cl− symport flux or (2)
the induction of an additional H+ antiport transport system
(this possibility is being investigated). The requirement for
overall charge balance for a cell is not met by the fluxes we
have measured (Fig. 3). The small size of the K+ flux is
perhaps surprising as it is quite likely to be able to enter cells
passively at these external concentrations. The balancing
ion has yet to be identified and must be the subject of future
experiments. Efflux of malate or bicarbonate should be
considered.
What is the physiological function of NAA-induced Cl −
uptake ?
Assman (1993) described general processes that occur
within guard cells, the classical system for physiological
studies of swelling}shrinking processes. She noticed that
swelling of these cells always coincided with increased H+pumping from the cells hyperpolarising the plasma membrane. Both of these processes correspond with passive
potassium uptake and active chloride uptake. The entering
ions are stored primarily in the vacuole. As intracellular
osmotica increase, guard cells take up water, balancing their
water potential, and they begin to swell.
In oat coleoptiles, a classical system for plant physiologists
studying auxin action and growth by cell expansion, we
could be dealing with a similar chain of events, assuming
that chloride ions are a key inorganic osmoticum. The auxin
leads to initial depolarisation of the plasma membrane that
may reflect H+-IAA symport. Keller and Van Volkenburgh
(1996) found this first stage of auxin action on plasma
membrane potential (2–3 min) is highly dependent on the
external chloride concentration, lower chloride in the
medium caused greater depolarisation. Their explanations
Babourina et al.—Auxin Stimulated Cl − Uptake
4500
A
Cl– flux (nmol m–2 s–1)
5.54
5.52
pH
5.5
5.48
5.46
5.44
5.42
H+ flux (nmol m–2 s–1)
5.4
0
–5
Cl– flux (nmol m–2 s–1)
3500
3000
2500
y = 33·434x2 – 59·791x + 579·95
R2 = 0.9435
2000
y = 70·823x + 12·97
2
R = 0·8853
1500
1000
500
2
4
6
8
Cl– in media (mM)
10
12
F. 4. Effect of 10 µ NAA (U) on chloride net influx for parenchyma
of oat coleoptiles depending on external concentration of chloride. KCl
solution was used as control (®NAA ; +).
–10
–15
about the activation of chloride channels releasing chloride
from the cells are plausible. This proposition is in good
agreement with Hedrich et al. (1994) who report on the
Michaelis-Menten dependence of activation of chloride
channels upon external chloride concentrations. However,
we can add that the higher Cl− influx (Fig. 4) at higher
external chloride concentrations could also cause less
depolarisation of the plasma membrane of cells exposed to
auxin. Unfortunately in our experiments we could not
observe the immediate transient response of chloride fluxes
because of the electrode settling time required after solution
mixing.
Observations of the hyperpolarisation stage of membrane
potential reaction, 10–15 min after auxin application (Bates
and Goldsmith, 1983 ; Felle, Peters and Palme, 1991 ; Keller
and Van Volkenburgh, 1996), suggest increased H+-extrusion
or inward Cl−-pumping. This transport leads to cation and
osmotic water uptake, as was found for potassium ions
(Haschke and Lu$ ttge, 1975), and simultaneous increased
chloride pumping into the vacuole, that consequently leads
to coleoptile cell elongation. Hence, in a medium containing
only chloride as a mineral anion, Cl− could act as the main
anionic osmoticum in maintaining cell turgor in coleoptile
growth.
Auxin has more wide-ranging effects than just increasing
the volume of cells, as we observe for coleoptiles. In a recent
report, Harling et al. (1997) used a cell line which could
grow (divide and elongate) without external auxin supplement. They found that these cells overproduce a protein
which has high homology with an animal chloride transporter. Moreover, auxin independence for the wild line was
achieved by transforming cells by genes expressing the
animal K+-Na+-2Cl− transporter. Volume regulation of cell
division seems to have taken place in these experiments.
Thus, from our work and reports of other experimenters
we can conclude that one target of auxin action in plant cells
is the plasma membrane chloride transport system mediating
increased chloride uptake.
–20
–25
1400
C
1200
1000
800
600
400
200
0
–200
10
Ca+ flux (nmol m–2 s–1)
4000
0
B
–30
1600
D
5
0
–5
–10
–15
–20
E
–10
K+ flux (nmol m–2 s–1)
335
–30
–50
–70
–90
–110
–130
–150
0
10
20
30
Min
40
50
60
F. 3. Effect of 10 µ NAA (U) on pH changes (A) and on ion fluxes
near parenchyma of oat coleoptiles : H+ (B), Cl− (C), Ca#+ (D) and K+
(E). Addition of KCl was used as a control (+). Points are means of
three plants with 12 measurements each during 1 min intervals. Error
bars indicate ³s.e.
336
Babourina et al.—Auxin Stimulated Cl − Uptake
A C K N O W L E D G E M E N TS
This work was supported by an Australian Research Council
grant to I. A. Newman.
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