ELECTROCHEMICAL EVIDENCE OF ACTION OF IAA IN
MNIUM
1239
Electrochemical Evidence of Specific Action of Indole Acetic Acid
on Membranes in Mnium Leaves
U . LÜTTGE * , N . HIGINBOTHAM * * , a n d C . K . PALLAGHY * * *
Fachbereich Biologie — Botanik — Techn. Hochschule, Darmstadt, Germany
(Z. Naturforsch. 27 b , 1239—1242 [1972] ; received June 16, 1972)
Indole acetic acid (IAA), membranes, electrochemical potential, ion uptake, membrane potential
The effect of 5 - 1 0 ~ 7 and 5 - 1 0 - « M IAA on K + , Cl" and S 0 4 " " uptake by young and old
Mnium
gametophytes from 0.2 MM or 10 MM salt solutions has been investigated. These IAA concentrations selectively enhance K + uptake from 0.2 MM KCl or K 2 S 0 4 in old gametophytes. IAA
up to 1 0 - 5 M does not affect the membrane potential (P.D.). At 0.2 MM KCl the observed P.D.
of —200 to — 220 mV is more negative than the calculated Nernst potential of + 8 4 to + 1 0 7 mV
for Cl~ and —136 to —157 mV for K1". These results are discussed in relation to a recent model
of IAA regulation of cell elongation involving a proton extrusion pump.
In an earlier investigation
1
it was shown that the
ion uptake isotherm of young Mnium
Materials and Methods
leaves in the
The experiments described here extended over
several years. Tracer uptake experiments where perwhile old Mnium leaves showed a typical saturating
formed in 1967/68 by the first author (U. L.) in Darmstadt. Membrane potential measurements were made
system 2 2 isotherm. This finding was related to diflate in 1968 in Canberra (U. L. and C. K. P. at the
ferences in ultrastructure between young and old
Research School of Biological Sciences, Austral. Natl.
Mnium leaves 3 . I A A (indole acetic acid) is effective
Univ.) and in September 1971 in Darmstadt (N. H.).
4
6
in morphogenetical regulation in mosses
and we
The material for all these experiments was obtained
from forests surrounding Darmstadt. The material for
therefore endeavoured to unravel further aspects of
the Canberra experiments was airmailed in polythene
ion uptake by young and old Mnium leaves as inbags. Old gametophytes of Mnium cuspidatum were
fluenced by this growth regulator.
obtained in autumn through winter to spring. Young
gametophytes were collected in the early summer.
There has been a continuous argument as to
For tracer influx experiments the material was
whether growth regulators act at the transcriptional
treated as reported earlier 1 . Brandies were cut and
or the translational level in the cell or whether they
washed for 4 8 - 7 2 hrs in 1 0 - 4 M Ca ++ solutions. Calact at membranes, changing ion transport characcium was supplied in the form of CaCl 2 in experiments
teristics. Effects of I A A on ion transport have been
with 3 5 S 0 4 ~ a n d as CaS0 4 in experiments with 36C1~
and 8 6 Rb + .
repeatedly r e p o r t e d 7 - 1 7 . Recently HAGER and coAt the end of this pretreatment samples of about
workers 18 have put forward a detailed model of
150 mg fresh weight were blotted dry and transferred
I A A action in cell elongation based on specific I A A
to 25 ml of the uptake solution, in which they were
effects on metabolically controlled proton fluxes at
kept for 3 hrs at 25 °C. The uptake solutions contained the appropriate labelled ions at concentrations of
cell membranes. This focuses interest on specific
either 0.2 mM or 10 mM. The molarities of CaS0 4 or
interactions of I A A with ion fluxes at membranes.
CaCl2 were the same as those in the pretreatment
Therefore, although they do not explain the different
solutions. The labelled ions investigated were Cl~
nature of the ion uptake isotherm in young and old
(applied as KCl, labelled with 36 C1), S 0 4 " (applied
Mnium leaves 1 , results showing a specific enhanceas K 2 S 0 4 , labelled with 35 S, and K + (applied as KCl
or K 2 S 0 4 , labelled with 8 6 Rb a widely used tracer in
ment of K + uptake f r o m 0.2 MM solution by
+ transport studies in higher plants 1 9 ).
K
5 • 1 0 ~ 7 — 5 • 1 0 - 6 M I A A , while membrane potential
IAA (indole acetic acid) was added to the uptake
is unchanged, are reported here.
solution as an alcoholic solution. Similar amounts of
concentration range of 1 — 10 MM salt was linear,
Requests for reprints should be sent to Prof. Dr. U.
LÜTTGE, Fachbereich Biologie —Botanik, T.H. Darmstadt,
D-6100 Darmstadt,
Schnittspahnstr. 3 — 5.
* ULRICH LÜTTGE, Fachbereich Biologie —Botanik, Technische Hochschule, D-6100 Darmstadt, Schnittspahnstr. 3 — 5,
Germany.
** NOE HIGINBOTHAM, Department of Botany, Washington
State University, Pullman, Wash. 99163, U.S.A.
*** CHARLES K. PALLAGHY, Department of Botany, La Trobe
University, Bundoora Vic. 3083, Australia.
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U. LÜTTGE, N. IilGINBOTHAM, AND C. K. PALLAGHY
1240
ethanol without added IAA were pipetted to the controls, so that the final ethanol concentration was 0.1%
in all flasks. Solutions were unbuffered to avoid uptake
solutions of unduly complex ionic composition. Table I
gives the pH in the solutions used in relation to increasing IAA concentration. Up to 10~ 5 M there is
little discrepancy in pH of IAA solutions as compared
with controls. Above 10~ 5 M there is a significant acidiTable I. pH measured in the solutions used for tracer uptake
studies.
Experimental
solution
0.2 mM KCl
+
0.1 mM CaS0 4
10 mM KCl
+
0.1 mM CaS0 4
0.2 mM K,S0 4
+
0.1 mM CaCl2
10 mM K,S0 4
+
0.1 mM CaCl2
IAA
conc.
[M]
0
10-«
10-5
10-4
10-3
0
10-6
10-5
10-4
10-3
0
10-«
10-5
10-4
10~ 3
0
10-«
10-5
10-4
10-3
pH of IAA
solution
+ tissue
immediately
after adding
the tissue
pH of IAA
solution +
tissue after
3 hrs at 25 °C
6.1
6.1
5.8
4.7
3.9
5.8
5.8
5.7
4.6
3.9
6.0
6.1
6.1
4.5
3.9
5.9
6.0
6.0
4.7
4.1
5.6
5.4
5.3
4.8
4.1
5.2
5.2
5.2
4.6
4.1
5.1
5.3
5.1
4.8
4.1
5.1
5.1
5.0
4.7
4.2
the tissue to the planchets. 36C1" and 35 S0 4 ~~ labelled
tissue was extracted as reported earlier
and aliquots
were dried on the planchets. Radioactivity was measured
with a methane gas flow counter or a Geiger-Müller
tube.
Intracellular electrical potentials were measured
with glass microelectrodes filled with 0.3 or 3.0 M KCl
using calomel half cells (Canberra) or Ag/AgCl wire
electrodes (Darmstadt). Experiments were carried out
under microscope lighting conditions.
Results
1. Tracer uptake
experiments
Table II shows the effcet of I A A on tracer cation
fication. Interpretations presented here are therefore
based only on experiments with IAA at less than
10~ 5 M. Under all conditions described in Table I, there
is an increase in H + concentration in the external solutions after a 3 hrs uptake period. This would indicate
an apparent H + net-flux. However the experiment has
been designed solely to test possible pH discrepancies
between controls and IAA solutions, and it is not adequate to permit comparative conclusions on H + or OH"
fluxes.
In experiments with 35 S0 4 ~ ~ and 36C1" the moss
material was washed 2 x 1 5 min in ice cold solutions
of 10~ 4 M CaCl 2 or C a S 0 4 , respectively, at the end of
the uptake period in order to exchange the free space.
In experiments with 8 6 Rb + the washing solutions contained the same amount of unlabelled salt as the uptake solutions. No particular efforts for illumination or
darkening of the tissue during tracer uptake have been
made (room light!).
After washing the plant material was blotted dry
and the fresh weight determined. In experiments with
86 Rb the material was dried on planchets with the addition of a drop of \% aqueous gelatine solution to glue
and anion uptake by young and old gametophytes of
Mnium at two very different external salt concentrations. It can be seen that potassium uptake by old
gametophytes f r o m 0.2 MM KCl and K 2 S 0 4 solutions
is significantly enhanced by 5 1 0 ~ 7 and 5 - 1 0 - 6 M
Table II. The effect of IAA on cation and anion uptake by
Mnium gamephytes in % of the appropriate controls.
Potassium
Exter- IAA conc.
nal
K + uptake from:
salt
conc.
KCl
K,SO,
[mM] [M]
Cl"
uptake
from
KCl
Anions
S04"
uptake
from
K„S0 4
old Mnium gametophytes:
0.2
5-10-7
162 + 11 149 + 1 0
76 + 13
5-10-«
170 + 9 145 + 1 0
96 + 1 1
10
5 • 10~ 7
112 + 5
89 + 4 1 0 6 ± 1 1
5-10-«
115 + 7
94 + 3 103 + 7
117±
111+
111+
100±
5
7
7
4
young Mnium gametophytes:
0.2
5 • 10~ 7
97+ 8
5-10-«
102± 8
10
5-10-7
97+4
5-10-6
104+ 3
95 ±
95 ±
100+
100 ±
6
5
4
4
-
I A A . N o enhancement of tracer ion uptake is observed f o r any of the other conditions listed, i. e.
for anion uptake, f o r potassium uptake by young
gametophytes
and
for
potassium
uptake b y
old
gametophytes f r o m 10 MM KCl and K 2 S 0 4 solutions.
High
IAA-concentrations
( > 10~4
M)
caused
severe inhibitions of ion uptake under all conditions
tested. However, f o r the reasons stressed in the
methods section this shall not be further discussed in
this paper.
Absolute rates f o r tracer K + , Cl~, and S0 4 ~ ~
influx at various external concentrations are given
in ref. 1.
ELECTROCHEMICAL EVIDENCE OF ACTION OF IAA IN MNIUM
2. Membrane
Discussion
potentials
Since at 0.2 mM KCl specific effects of I A A on
cation uptake by old Mnium gametophytes have been
observed, potential measurements were performed
under these conditions. Resting potential between
the Mnium leaf cells and the 0.2 mM K C l + 0.1 mM
C a S 0 4 solution was approximately — 2 2 0 m V in the
Canberra experiments ( 1 9 6 8 ) and — 2 0 0 . 7 + 4.3
m V (n = 1 8 ) in the Darmstadt experiments (August
— September 1 9 7 1 ) . The ion content of the material
used in the latter series of experiments was as follows :
K+
90.1 ± 1.7//moles x g fresh w e i g h t - 1
(5 determinations)
Cl -
5.3 ± 0.4 //moles x g fresh weight - 1
(8 determinations)
Na +
12.8 ± 2.5 //moles x g fresh weight - 1
(5 determination).
Assuming
that these amounts
are
approximately
equivalent to activities ( a ; ) , the Nernst potential
The results clearly show a selective effect of IAA
on cation uptake by old Mnium
gametophytes.
Cation uptake is enhanced while anion uptake is not
affected. The different reactions of young and old
Mnium gametophytes are somewhat difficult to explain. They may, however, be due to different endogenous contents of I A A and of other interacting
growth substances. The salt concentration dependence of the enhancement of K + uptake by I A A
— observed at 0.2 mM KCl and K 2 S 0 4 but not at
10 mM salt concentration — may be due to nonspecific effects of high external salt concentration on
membrane permeability which have not been further
elucidated here.
The IAA-induced enhancement of K uptake in the
absence of any change in the electropotential gradient, might be explained as either an increase in
permeability or by a stimulation of a K influx pump
(where K influx is electrically balanced by anion
influx or cation efflux). Passive aspects of the
system may be described by the G o l d m a n voltage
equation 2 0 :
PK * j + P y a Naj+Pci Cl„
RT
calculated f o r 0.2 mM KCl in the external solution
(a 0 ) and 25 ° C amounts to
K + — 157mV,
1241
Cl~ + 84 mV.
In an older series of measurements gametophytes
treated f o r 4 8 — 7 2 hrs in 0.1 mM C a S 0 4 followed
by a 30 hr treatment with 0.2 mM KCl + 0.1 mM
C a S 0 4 gave K + and C l - contents of 4 0 and 13
//moles X g fresh w e i g h t - 1 respectively giving calculated Nernst potentials of — 136 m V and + 107 m V
respectively. The significance of these values will be
considered in the discussion.
In all experiments no elfect of I A A concentrations
up to 1 0 - 5 M on the membrane potential was observed. At 1 0 - 4 and 1 0 - 3 M I A A a reversible depolarisation of approximately 100 m V was found
both in the Canberra experiments and in some of
the Darmstadt experiments. This may b e an interesting phenomenon. However, although solutions
have been buffered and buffer controls have been
run in the Darmstadt experiments there remain some
uncertainties regarding its significance and reproducibility, and no further emphasis will be given to
it in the discussion.
zF
n
PK ^o+^-Va N a 0 + P c i Cli
1
where E is the membrane potential, K0,
K\, etc.
are the ion activities outside and inside the cell
respectively, and Pk , Ps,a a n d Pc\ are the permeability constants f o r potassium, sodium and chloride
respectively.
As noted under Results at K0 = 0.2 mM the
N e r n s t potential f o r K , EK,
is approximately
— 157 m V whereas the membrane potential is — 2 0 0
to — 2 2 0 mV. Thus the membrane potential is much
more negative than can be explained b y passive diffusion and an electrogenic pump may be inferred 2 1 ;
that is, E is comprised of the sum of the diffusion
potential, as expressed in Eq. ( 2 ) , plus a potential
derived f r o m a pump contributing a current. In any
event the driving force on K,
, should b e :
=E-E
K
(3)
thus giving E% equal to — 43 to — 63 m V . As a
result K influx is downhill and an IAA-induced increase in membrane permeability ( f o r K only)
might explain the enhanced uptake of K . The markedly different effect of I A A at low concentrations
on anion and cation influx by old Mnium leaves
might thus suggest, that in the absence of a signi-
ELECTROCHEMICAL EVIDENCE OF ACTION OF IAA IN MNIUM 1242
1242
ficant
change in the membrane potential, I A A in-
significant, and might be explained also as an effect
creases selectively the permeability constant of the
of
membrane for potassium.
ETHERTON
It might be argued that the data suggests a pas-
H.
Both
22
anions
are
actively
accumulated.
has previously suggested that active
K efflux may occur in pea and oat seedling tissue.
sive influx of K and an active efflux. If the cells are
W e thus conclude, that the data reported here are
at flux equilibrium, this would explain wdiy K\ is
in agreement with a theory like that of HAGER et
much less than predicted by Eqns. ( 1 ) or ( 2 ) . In
al.18,
this case the effect of I A A could be interpreted as
serve as a model f o r electrochemical evaluations of
and that these investigations with Mnium mav
decreasing the K-efflux pump, perhaps by inducing
membrane theories explaining I A A action on cell
localised increases in H concentrations at or in the
elongation in plants. However, more electrochemical
membrane and assuming that H interferes or com-
evidence is needed, and this should be
petes with active K transport but not with leakage
using materials more widely used in I A A studies
inward. This appears to be in accord with the theory
than moss gametophytes.
of
HAGER
et al.18
according to which I A A induces
a H-efflux dependent upon high energy phosphate.
The effect of I A A on the anion fluxes is less, or not
1
U . LÜTTGE a n d K . BAUER, P l a n t a 7 8 , 3 1 0
2
E. EPSTEIN, Nature [ L o n d o n ] 2 1 2 , 1 3 2 4
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4
7
The technical assistance of Frau K. V O N W I L L E R T
and Frl. E . B A L L and a grant from the Deutsche Forschungsgemeinschaft are gratefully acknowledged.
[1968],
5
6
collected