Download this PDF file

Proceedings of the Indiana
(1991)
Volume
Academy
of Science
j
29
99, p. 129-136
THE EFFECT OF CHLORO-DERIVATIVES OF
INDOLEACETIC ACID
ON PLASMA MEMBRANE ELECTRON TRANSPORT
AND PROTON EXCRETION
Michael Bottger 2
and Frederick L. Crane
Rita Barr
1
,
1
Department of Biological Sciences
1
Purdue University
West Lafayette, Indiana 47907
and
Institut fur
Allgemeine Botanik 2
University of
Hamburg
Ohnhorststr. 18
D
2000 Hamburg 52
Federal Republic of
ABSTRACT: As shown
Germany
by Bottger and Hilgendorf [Plant Physiol. 86, 1038-1043 (1988)], the
natural auxin indoleacetic acid in high concentrations (10 |xM) inhibited both e
and proton efflux
by corn roots of undamaged plants measured with a pH-stat combined with a redoxstat.
we show
study
inhibited
that of various chloro-substituted indoleacetic acid derivatives
NADH
oxidation and several derivatives stimulated trans-membrane hexacyanoferrate
reduction in concentrations from 0.1
these cells
was
nM
to
10 jxM in cultured carrot cells. Proton excretion by
also stimulated by 5-, 6- and 5,7-chloro substituted indoleacetic acid, but the 4-
chloro derivative inhibited
in
In this
only 4-chloro-IAA
H+
excretion by the plasma
membrane H + -ATPase, while H +
excretion
presence of hexacyanoferrate was stimulated. These divergent effects of chloro-substituted
indoleacetic acid derivatives
proton
movement through
Abbreviations.
HCF
III
show
the
that the
membrane
trans-membrane redox system
in a
is
not directly related to
relationship.
1:1
potassium hexacyano ferrate or potassium ferricyanide.
INTRODUCTION
Indole-3-acetic acid (IAA)
sponses
in plant cells
is
a growth
(Brummell and Hall,
hormone which induces rapid cellular re1987). A hypothesis for the mechanism of
auxin action according to Brummell and Hall (1987)
to a receptor in the
plasma membrane, an
signal cascade (Boss and Morre, 1989)
in the stimulation
act
initiation
as follows: First, the auxin binds
which promotes the
by which cellular Ca 2+
of protein kinases, which prepare an
with auxin. The protein-auxin complex then induces
cell wall
is
is
unknown
m-RNA
whereas the addition of auxin
to
protein to
synthesis,
loosening and leads to growth. These responses require
and could, therefore, not be considered as the
inositol trisphosphate
released. This results
first
complex
which promotes
at least
10-20 min for
plant response to auxin,
measurement of plasma membrane electron transport
Indiana
130
Academy of Science
Vol. 99 (1991)
NADH
reactions gives an immediate response in terms of stimulation of
hexacyano
oxidation or
ferrate reduction with associated increase in proton release.
Chlorinated auxins were chosen for the study of their effects on plasma
electron transport because they
all
show high
position 7 on the benzene ring unlike
membrane
of plasma
biological activity, unless substituted in
1984). Since stimulation
itself (Pless et al.,
IAA and
electron transport by
chloro derivatives
its
mechanism for inducing
cascade proposed by Brummell and Hall, (1987).
response,
signal
IAA
it
could provide an
membrane
is
a fast
the inositol trisphosphate
initial
MATERIALS AND METHODS
Carrot cells were grown in suspension culture as previously described (Barr et
1985; and Barr et
a
DW-2A
1987).
al.,
NADH oxidation was assayed spectrophotometrically with
beam mode following a decrease
wavelength set at 430nm (Barr et al., 1985).
double beam spectrophotometer
of absorbance
340nm
at
Reactions were run
with reference
in the
dual
(lOmM KC1, lOmM NaCl
sucrose-salts solution
lOmM CaCl
and
(0.005gdrywt.). After a 3-min incubation period, 100|ulM
NADH
IAA
or
its
magnetic
24°C (Barr
cell at
et al.,
pH
electrode in a 5-
The assembly was gently
1987).
stirred
on
IAA
or
its
chloro derivatives added
+
in ethanol.
ferricyanide
The basal rate of H excretion was monitored for 5min, then 100|xM
was added to measure the contribution of protons from plasma membrane
electron transport with ferricyanide as the
HC1
to start the
during assays. The reaction mixture contained carrot cells
stirrer
(0.005gdrywt.), 25fxM phosphate buffer, pH7, and
cells.
cells
chloro derivatives were added in concentrations indicated in figures.
ml water-jacketed
of
and carrot
2)
was added
Proton excretion was measured with a Corning combination
a
Tris-Mes, pH7,
Reaction rates were calculated using a millimolar extinction coefficient of
reaction.
6.22.
25mM
24°C. The reaction mixture contained
at
al.,
impermeable electron acceptor on the outside
Proton excretion rates were calculated from the addition of
known amounts of
as described in (Barr et al., 1987).
IAA and
NAA
were purchased from the Sigma Chemical Co.
Louis,
(St.
MO).
The chloro derivatives of IAA were obtained from Dr. Bottger's laboratory, University
of Hamburg, F.R.G.
RESULTS AND DISCUSSION
(NAA)
Indole-3-acetic acid (IAA) and a-naphthalene acetic acid
promote growth
action
was
is
not clear and
to see if
IAA and
it
its
electron transport and
chloro derivatives, as well as
H
+
excretion.
A
Waldrum and Davies,
1981), with
NAA
and only twofold by 0.01 |xM
concentration had a greater effect on
IAA
H+
NAA,
NAA,
the
plasma membrane
H
membrane
which cannot be oxidized, shows
NADH
(Figs.
1
oxidation
-ATPase may lead
is
stimulated tenfold
IAA
A, 1C). However,
excretion than
NAA
be significant, considering that the acid growth hypothesis,
+
affected plasma
1984; Reinecke and Bandurski,
et al.,
differences on cultured carrot cells: the rate of
by
to
comparison of the effects of IAA, an auxin
which can be oxidized by plant tissue (Pless
1988; and
known
are
(Brummell and Hall, 1987), but the mechanism of auxin
may differ from species to species. The purpose of this study
in plant cells
in
which
to cell wall loosening
H
+
same
may
excreted by
and increased growth
(Hayashi, 1989; and Rayle and Cleland, 1982). The chloro derivatives of
IAA, 7-chloro IAA and 5,7-dichloro IAA) were
in the
(Figs. IB, ID). This
tested to find out
if
IAA
(5-chloro
they could act as
Botany:
Vol. 99(1991)
8
9
Barr, Crane
1.
The
;
nfifl
of
H+
C-rate of
cells.
A-rate of
NADH
oxidation
at
NADH
oxidation
IAA concentrations; Bvarious IAA concentrations;
various
excretion without HCFIII (O) and with HCFIII (O) in presence of
NADH
8
cmD" K
(IAA) and naphthalene acetic acid (NAA) on
effect of indole-3-acetic acid
and proton excretion by cultured carrot
rate
^9~
iO
_
mac no*
Figure
131
NAA concentrations; D-rate of H + excretion without HCFIII
various NAA concentrations. NADH oxidation was assayed
oxidation with various
and with HCFIII (#)
in
spectrophotometrically,
presence of
H+
excretion with a
pH
electrode, as described in Materials and Methods.
(O)
Indiana
132
Academy of Science
^CHlOftOIAflCmD
5-CHlOFtOlAH CfH]"
IAA) on
The
effect of 5-chloro-indole-3-acetic acid
NADH
(5-C1-IAA) and 7-chIoro-indole-3-acetic acid (7-C1-
oxidation and proton excretion by cultured carrot cells. A-rate of
various 5-C1-IAA concentrations; B-rate of
H+
7-CI-IAA concentrations.
H+
NADH
oxidation
at
excretion wihtout HCFIII (O) and with HCFIII
NADH
NADH
oxidation
at
excretion without HCFIII (O) and with HCFIII (•) in
presence of various 5-C1-IAA concentrations; C-rate of
concentrations; D-rate of
K
7-CHL0R0-mflCfTD"H
7-CHlORO-IAACrTD
Figure 2.
Vol. 99(1991)
various 7-C1-IAA
(•)
oxidation was assayed spectrophotometrically,
electrode as described in Materials and Methods.
in
H
+
presence of various
excretion with a
pH
Botany:
Vol. 99(1991)
Barr, Crane
133
5,7-DICHLORO-lflflCfTD
Figure
3.
The
carrot cells.
effect of 5,7-dichloro-indole-3-acetic acid
NADH
(5,7-CMAA) on
oxidation was assayed spectrophotometrically
as described in Materials
and Methods.
at
NADH
oxidation by cultured
various 5,7-Cl-IAA concentrations
—
V
Indiana
134
Academy of Science
Vol. 99 (1991)
O BflSfll
• AHCfUE
h
3
—
t
rH
5,7-DICHLORO-lflfl Cm:
Figure 4. The effect of 5,7-dichloro-indole-3-acetic acid (5,7-Cl-IAA) on proton excretion by cultured
carrot cells.
H+
excretion measured without HCFIII (O) and with HCFIII
concentrations, using a
pH
(•)
electrode as described in Materials and Methods.
at
various 5,7-Cl-IAA
Botany:
Vol. 99 (1991)
growth regulators as
noted
mode
in their
by carrot
cells, but
in
IAA was
A
IAA
H
+
cells in
H+
NADH
oxidation
less effective than 7-chloro or 5,7-dichloro
maximum
to achieve
IAA
stimulation of
NADH
oxidation (Fig.
excretion were most notable in case of 7-chloro (Fig. 2D) and
IAA (Fig. 3B),
5-chloro IAA showed a
2B). The
Here, again, differences were
itself.
thousand times higher concentration (0.1 (xM vs. 0.1 mM) was
both of which inhibited the basal rate of
5,7-dichloro
while
135
of action. All three chloro derivatives stimulated
5-chloro
necessary for 5-chloro
2A). Differences
compound, IAA
their parent
2A, 2C, 3A).
(Figs.
Barr, Crane
of ATPase-mediated
slight stimulation
membrane
excretion mediated through plasma
H
+
H+
excretion,
excretion (Fig.
electron transport by carrot
presence of ferricyanide was not remarkable, showing only slight stimulation
or inhibition.
Previous studies by our group on cultured carrot cells (Crane
transformed versus non-transformed tobacco cells (Barr
transplasma
membrane
HCF
electron transport with
et al.,
III
et al.,
1983) or
1985) have shown that
as the
impermeable electron
acceptor respond to plant growth regulators and that an increase in proton excretion,
H+
aside from
membrane H + -ATPase, is seen when low conused (< |jlM). Until the discovery of NADH oxidase
excreted by the plasma
centrations of plant
hormones are
from soybean hypocotyls (Barr
1
1985), the stimulation of
et al.,
HCF
III
reduction by
NADH oxidation
isolation of a hormone-sensitive NADH
1988), the stimulation of NADH oxidation
growth regulators was relatively small (about 25%), but stimulation of
can be 10X, as seen
1C. Since the
in Fig.
oxidase by Brightman etal. (Brightmanetal.,
by
IAA and
its
shown here can
chloro derivatives
in the future
be related to plant growth
in general.
The stimulation by IAA and
its
chloro derivatives of
excretion of cultured carrot cells seen here
and Hilgendorf, 1988),
who found
is
NADH
oxidation and
contrary to Bottger and Hilgendorf (Bottger
inhibition of
HCFIII reduction and
H+
excretion by
intact
maize seedling roots, when treated with IAA. HCFIII reduction by carrot
in this
study was not significantly affected by
These differences may be due
may be
H+
IAA
or
its
cells
derivatives (data not presented).
to using tissue culture cells versus intact seedlings, or
it
a species-related difference. In an earlier study, Bottger and Luthen (1986)
found a correspondence between
NADH oxidation and proton excretion by roots of Zea
mays.
In
NADH
summary,
it
is
concluded
NAA, IAA
and
its
chloro derivatives stimulate
oxidation by cultured carrot cells in low concentrations (0.1
whereas only the
H + -ATPase
Unlike oxidation products of
mM
to
1(jlM),
generated protons are affected by these growth regulators.
IAA metabolism,
such as oxindole-3-acetic acid (Ernstsen
1987; Henderson and Patel, 1972; and Nonhebel and Bandurski, 1984), which
et al.,
were found
to
be inactive as growth promoters, the chloro derivatives of
no inhibition of
NADH oxidase activity.
by 7-chloro and 5,7-dichloro
is
that
IAA may
The
inhibition of
also be significant,
H
if
+
IAA showed
-ATPase-excreted protons
the acid
growth hypothesis
correct.
LITERATURE CITED
Barr, R.,
T.A. Craig and F.L. Crane. 1985. Transmembrane ferricyanide reduction
in carrot cells.
Biochim.
Biophys. Acta 812:49-54.
Barr, R., F.L.
J.
Plant
Crane and T.A. Craig. 1984. Transmembrane ferricyanide reduction
Growth Regul. 2:243-249.
in
tobacco callus
cells.
Academy of Science
Indiana
136
Barr, R., O. Martin,
Jr.
Vol. 99 (1991)
and F.L. Crane. 1987. Redox-induced proton excretion by cultured carrot
affected by protonophores and inhibitors of
ATPase. Indiana Acad.
cells
is
Sci. 96:139-144.
Barr, R., A.S. Sandelius, F.L. Crane and D.J. Morre. 1985. Oxidation of reduced pyridine nucleotides by
plasma membranes of soybean hypocotyl. Biochem. Biophys. Res.
Boss, W.F. and D.J. Morre, eds.
Liss,
Bottger,
New
York, 348
M. and
1989. Second Messengers in Plant
Commun.
131, 943-948.
Growth and Development. Alan R.
p.
F. Hilgendorf.
Hormone
1988.
H+
action on transmembrane electron and
transport. Plant
Physiol. 86:1038-1043.
Bottger,
M. and H. Luthen.
mays
L. roots.
J.
1986. Possible linkage between
NADH-oxidation and proton
secretion in
Zea
Exp. Bot. 37:666-675.
Brightman, A.O., R. Barr, F.L. Crane and D.J. Morre. 1988. Auxin-stimulated
NADH
oxidase purified
from plasma membrane of soybean. Plant Physiol. 86:1264-1269.
Brummell, D.A. and J.L. Hall. 1987. Rapid
article. Plant, Cell
cellular responses to auxin
and the regulation of growth: review
and Environment 10:523-543.
Crane, F.L., T.A. Craig, P.C. Misra and R. Barr. 1983. Hormonal control of transplasma
transport and proton flux. Proceed. 10th
membrane
Annu. Meeting Plant Growth Regul. Soc. of America,
electron
vol. 10
(H. R. Cooke, ed.), p.70-75.
Ernstsen, A., G. Sandberg and K. Lundstrom. 1987. Identification of oxindole-3-acetic acid, and metabolic
conversion of indole-acid to oxindole-3-acetic acid
Hayashi, T. 1989. Xyloglucans
primary
in the
in
cell wall.
Pinus sylvestris seeds. Planta 172:47-52.
Ann. Rev. Plant Physiol. Plant Molecular
Biol.
40:139-168.
Henderson, J.H.M. and C.S. Patel. 1972. Oxindole-3-acetic acid: physical properties and lack of influence
on growth. Physiol. Plant. 27:441-442.
Nonhebel, H.M. and R.S. Bandurski. 1984. Oxidation of indole-3-acetic acid and oxindole-3-acetic acid
2,3-dihydro-7-hydroxy-2-oxo-l
H
indole-3-acetic acid-7'-0-3-D-glucopyranoside in
Zea mays
to
seedlings.
Plant Physiol. 76, 979-983.
Pless, T.,
M.
Bottger, P.
and correlation of
its
Hedden and
J.
Graebe. 1984. Occurrence of 4-Cl-indole acetic acid
level with seed
in
broad beans
development. Plant Physiol. 74:320-323.
Rayle, D.L. and R. Cleland. 1972. The in vitro acid-growth response: relation to in vivo growth responses
and auxin action. Planta 104:282-296.
D.M. and R. S. Bandurski. 1988. Oxidation of
an enzyme preparation from Zea mays. Plant Physiol.
Reinecke,
Waldrum, J.D. and
1307.
indole-3-acetic acid to oxindole-3-acetic acid by
86, 868-872.
E. Davies. 1981. Subcellular localization of
IAA
oxidase
in peas. Plant Physiol.
68:1303-