Protoplasma (1997) t97:182-187
PflOTOPL MA
Protein synthesis and Golgi-mediated vesicle traffic is not required to maintain
a polarised membrane voltage in the presence of auxin
G. ThieP'*, A. Briidern 1, and A. Moroni 2
Pflanzenphysiologisches Institut, Universitiit G~Sttingen, G~Sttingen and 2 Sezione di Fisiologia e Biochimica della Piante, Universita
di Milano, Milano
Received October 28, 1996
Accepted January 7, 1997
Summary. The contribution of protein synthesis and secretion to
indol acetic acid (IAA) induced polarisation of the plasma membrane
voltage (VM)was investigated. The VMof coleoptiles from Zea mays
was measured in the presence of known inhibitors of protein- and
RNA synthesis, as well as those of Golgi-mediated vesicle secretion.
Inhibitors were applied under conditions at which they are known to
abolish IAA stimulated H+ secretion and cell elongation effectively.
Cycloheximide (CHI), an inhibitor of protein synthesis, caused depo-
larisation of I.q,~iwith a half maximal concentration of approximately
20 vtM.At 100 viM CHI, k~ depolarised to a new stable voltage with
a half time of 9.8 + 0.6 min. The temporal similarity of CHI-induced
depolarisation and cessation of coleoptile elongation suggests that
the induced change in VM underlies inhibition of elongation. CHI
evoked membrane depolarisation to a final voltage of about
-100 mV irrespective of the presence or absence of auxin in the
external medium. Thus, CHI probably affected constitutive mem-
brane transport properties independently of IAA-induced modulation
of transport proteins. Cordycepin (COR), an inhibitor of RNA syn-
thesis, had no significant effect at 400 ~tM on VM of IAA-treated
cells, suggesting that gene transcription for transport- or regulatory
protein synthesis was not essential for IAA-generated polarisation of
VM. Brefeldin-A (BFA), an inhibitor of Golgi-mediated vesicle
secretion in maize coleoptiles, had no perceivable effect at 20 rag/1
on VMof IAA-treated coleoptile cells, demonstrating that constitu-
tive or IAA-stimulated protein secretion was not essential for the
mechanism underlying IAA-evoked V• polarisation. Hence, IAA-
stimulated and COR/BFA-depressed H+ extrusion in elongating
coleoptiles may not be entirely mediated by auxin-enhanced ATPase
activity.
Keywords: Auxin; Maize coleoptiles; Membrane potential; Cyclo-
heximide; Protein synthesis.
*Correspondence and reprints: Pflanzenphysiologisches Institut,
Universitfit G~Sttingen, Untere KarspiJle 2, D-37073 G6ttingen,
Federal Republic of Germany.
9 Springer-Verlag 1997
Printed in Austria
Abbrevations: COR cordycepin; CHI cycloheximide; BFA
Brefeldin-A; IAA indol acetic acid.
Introduction
Inhibitors o f protein synthesis have been found to
abolish auxin-induced H + efflux and cell elongation
rapidly in a variety o f g r o w i n g cell systems (Bates
and Cleland 1979, C h o and H o n g t995, H a g e r et al.
1991, E d e t m a n n et al. 1989). F r o m the rapid effect o f
these inhibitors it has been c o n c l u d e d that protein
synthesis is o f central importance to the m e c h a n i s m
o f h o r m o n e - s t i m u l a t e d cell elongation (Edelmann
et al. 1989, C h o and H o n g 1995). As a m o d e l for a
causal link between protein synthesis and elongation
it was speculated that the auxin indole acetic acid
( I A A ) controls synthesis and secretion o f a short-
lived protein to regulate the activity o f the proton
p u m p and thereby H + extrusion (Lado et al. 1977, Cho
and H o n g 1995). A n alternative hypothesis explains
the link b e t w e e n protein synthesis and coleoptile
elongation by the ability of auxin to stimulate a rapid
rise in the concentration o f ATPase molecules in the
plasma m e m b r a n e (Hager et al. 1991). Such a hor-
m o n e - g e n e r a t e d rise in ATPase concentration and
consequently H + p u m p i n g m a y be the m e c h a n i s m
underlying acidification o f the cell wall, a process
believed to be essential for cell elongation (Hager
et al. 1991). Since the rise in ATPase concentration
was also rapidly reversed by the protein synthesis
inhibitor c y c l o h e x i m i d e or the R N A synthesis
G. Thiel et at.: Maintenance of polarised membrane voltage
inhibitor cordycepin, it was assumed that the ATPase
protein is subject to rapid turnover, and therefore that
gene transcription (Frias et al. 1996), protein synthe-
sis and secretion from the ER to the plasma mem-
brane is essential for cell elongation (Hager et al.
t99t). The latter hypothesis is mainly based on the
finding that auxin enhances transcription of the plas-
ma membrane H+-ATPase in coleoptiles (Frias et at.
1996) and that the rate of cell elongation closely fol-
lows the time course of IAA-stimulated and inhibitor-
decreased ATPase concentration in the plasma mem-
brane. The proposed scenario would also explain why
inhibitors of intracellular vesicle traffic such as
Brefeldin-A and/or monensin rapidly inhibit IAA-
stimulated acidification of the cell wall and elonga-
tion of growing tissue (Cunninghame and Hail 1986,
Schindler et al. 1994, Cho and Hong 1995). The miss-
ing evidence in support of the hypothesis is that it has
not yet been shown that IAA and inhibitor-generated
changes in ATPase concentration correlate with
ATPase activity in the plasma membrane (Napier and
Venis 1995). Furthermore, the predicted dependency
of in vivo ATPase activity on continuous secretion
from the ER to the plasma membrane remains to be
tested. To address these questions we measured the
membrane voltage (rv~) of maize coteoptile cells as
an indirect measure of ATPase activity (Senn and
Goldsmith 1988). The effects of known inhibitors of
protein and RNA synthesis and of intracellular vesicle
traffic were compared with the predictions of the
hypotheses, namely that IAA stimulates de novo syn-
thesis and secretion of proteins which regulate
ATPase activity (Lado et ai. 1977, Cho and Hong
1995) or synthesis and incorporation of active ATPase
molecules into the plasma membrane (Hager et al.
1991).
Material and methods
Coleoptiles were obtained from 4-day-old, dark-grown corn (Zea
mays L. cv. Mutin; KWS Saatzucht, Einbeck, Federal Republic of
Germany) seedlings. Sub-apical coleoptile segments (10 mm long)
were separated from the primary leaf and from the co|eoptile apex
(5 ram). After gently abrading with diatomaceous earth coleoptiles
were preqncubated in standard experimental medium (in mM:
0.1 KCI, 0.1 NaC1, 0.1 CaCla, 2.5 Tris/2-(N-morpholino) ethanesul-
phonic acid (MES)) for ~_1 h prior to the experiment in order to
deplete any endogenous auxin. For voltage recordings, coleoptile
segments were fixed horizontally in an experimental chamber and
cells from the outer epidermis impaled with a conventional micro-
electrode containing 300 mM KCt. The voltage difference between
the intracellularly positioned electrode and a reference in the bath
was measured with a voltage amplifer (giP; Wye Science, Wye,
U.K.). Elongation assays wei~e carried out with abraded coleoptile
I83
segments (1 cm long) threaded on a steel rod and kept in aerated
solution (2 mM KC1, unbuffered) in the dark. Coleoptile length was
obtained from the projection (75 fold magnification) of a coleoptile
placed on an overhead projector.
Results
The membrane voltage (VM) across the plasma mem-
brane of maize coleoptile cells can be explained as the
voltage at which an ATPase-generated outward H § is
balanced by an inward K + current through a K +
inward rectifier (Rfick et al. 1993, Hedrich et al.
1995, Thiel et al. 1996). Assuming that the IAA-
induced polarisation of the membrane is predomi-
nantly due to an increase in H+-ATPase activity
(reviewed in Napier and Venis 1995), recording VM is
a convenient, albeit indirect, assay of H+-32FPase
activity. On this basis the hypothesis that IAA
increases the concentration of active H+-ATPases in
the plasma membrane and that the hormone-induced
rise in pump concentration is dependent on rapid pro-
tein synthesis and secretion via the Golgi apparatus
(Hager et al. 1991) can be tested.
Figure 1 A shows a typical recording of VM from a
A
> -100
E 15 min
-120
1 00 BM CHI
B
-100
E -11o
-120
t O0 gM CHI
Fig. 1. A, B. Indol acetic acid-evoked polarisation and cyclohex-
irnide evoked depolarisation of coleoptile cells. A An auxin-depleted
and abraded coleoptile was first bathed in standard buffer before
10 uM IAA was added. After reaching a stable polarised VM in IAA
the wotein synthesis inhibitor CHI (100 gM) was added. B CHI (100
btM) was added directly to the standard bathing medium (-IAA) of
an auxin-depleted coleoptile. Arrows indicate addition of IAA and
CIII, respectively
184
AVM
2O
10
.4;
/
0 CHt
0l 1
20
I
40
t
60
I
80
I
100
CHI / gM
Fig. 2. Mean inhibitor-generated membrane depolarisation (AVM) of
coleoptile cells as a function of CHI concentration. Four abraded
coleoptiles were kept in standard experimental medium containing
10 gM tAA and challenged with increasing concentrations of CHI.
Fitting of mean AV.~ data _+SE with the Michaelis-Menten equation
(dotted line) yields a concentration of 17 gM CHI for half maximal
depolarisation
maize coleoptile cell. In standard experimental medi-
um the resting voltage was stable at-108 mV. Adding
IAA (10 ~tM) to the bathing medium caused the well
known slow polarisation of VM (Fig. 1 A). Subsequent
administration of CHI (100 aM) evoked a slow posi-
tive shift of TV~,levelling off, in the present example,
at a new stable voltage o f - l l 0 m V . Comparable
results were obtained in all the cells tested and the
mean data are summarised in Table 1. Figure 2 illus-
trates that the effect of CHI on VM was concentration-
dependent, with a concentration of 17 ~tM CHI
required for half maximal AVM.
The mean half time (tl/2) of CHI action, i.e., the time
between addition of 100 ~tM CHI and half maximal
AVM, was 9.8 + 0.6 min (n = 8). This is very similar
to the CHI generated decay of ATPase concentration
(hn = 12 min; Hager et al. 1991) and somewhat faster
than CHI-induced decay of the coleoptile elongation
rate (q/2 = 21 min; Hager etal. 1991) obtained in
maize coleoptiles under the same conditions.
The depolarising effect of CHI was not dependent on
pre-treatment of coleoptiles with IAA. Figure 1 B
shows that addition of 100 ~M CHI to the bathing
medium of an auxin-depleted coleoptile also resulted
in a slow (tu2 = 8.2 + 1 min, n = 4) positive shift of
VM from -113 to -103. The average CHI-induced
AVM in auxin-depleted cells is summarised in Table 1.
For tentative characterisation of VM in the presence of
CHI, coleoptile cells were incubated in standard
experimental medium containing 1 mM NaCN plus
G. Thiel et al.: Maintenance of polarised membrane voltage
-80 -
> 1 0 min
I=: -120 -
_
-160 -
4 O0 aM
t
CCR 1 00 gM
Fig. 3. Insensitivity of Vt4 o f the IAA-treated coleoptile cell to RNA-
synthesis inhibitor COR. VMwas stable in the standard bathing medi-
um containing IAA and remained unaffected after adding 400 #M
COR. Subsequent addition of CHI (100 gM) evoked depolarisation
of VM. Arrows indicate addition of COR and CHI, respectively
> -80 j
E -12o
-1 60 f t
20 mg/I 100 ,uM
BFA CHI
Fig. 4. Insensitivity of VM of an IAA-treated coleoptile cell to inhibi-
tion of Golgi-mediated vesicle secretion. VM of the coleoptile cell
was stable in the standard bathing medium containing 10 vM IAA
and remained unaffected by adding 20 mg/t BFA. Subsequent addi-
tion of CHI (100 laM) evoked depolarisation of VM. Arrows indicate
addition of BFA and CHI, respectively
0.4 mM salicylhydroxamic acid to inactivate H +-
ATPase by a rapid metabolic block (Slayman et al.
1973, Blatt 1987). In coleoptile cells this procedure
caused rapid depolarisation to about -100 mV (data
not shown), comparable to the mean l/~,~ recorded in
saturating CHI (Table 1).
Cordycepin, an inhibitor of RNA synthesis (Delseny
et al. 1975), also reverses rapidly in maize coleoptiles
at 400 ~tM IAA-stimulated elongation (tl/2 = 24 min;
Edelmann et al. 1989: fig 5) and auxin enhanced
ATPase concentration (Hager et al. 1991). The effect
of COR on VM was tested using coleoptile cells with
stable polarised voltages in the presence of 10 ~M
IAA. Figure 3 shows that addition of 400 gM COR
produced no significant effect on VM over the 45 min
recording period. The same insensitivity of VM to
COR treatment was observed in all five coleoptiles
tested (Table 1). Figure 3 also shows that subsequent
addition of CHI (100 FM) in the presence of COR
resulted in the typical slow positive shift of VM pro-
duced by the former inhibitor (cf. Fig. 1). This shows
G. Thiel et al.: Maintenance of polarised membrane voltage I85

Table 1. Effect of IAA and inhibitors on membrane voltage of maize coleoptile cells

Mean Vu Mean AVMd

controP CHI u NaCN+SHAM ~ BFA COR
-IAA +IAA -IAA +IAA -IAA 30 rain 45 rain 30 min 45 rain

-116• -135_+2(22) -99_+4(4) -107_+6(8) -97• 0.25+2(8) 1.25+2(6) -3+2(5) -3_+I(3)

10 • 0.6 22 • 3.3

Mean control VM in standard experimental solution before (-IAA) and after maximal potarisation in 10 ~tM IAA (+IAA)
b Mean stable VM and (AVMbelow) achieved after addition of 100 gM CHI to standard solution with or without 10 ,aM IAA
Stable VM after adding 1 mM NaCN and 0.4 mM salicylhydroxamic acid (SHAM) to standard experimental solution (-IAA)
Mean AVM obtained 30 rain and 45 rain after adding 20 rag/1 BFA or 400 gM COR to standard experimental solution containing 10 uM IAA
VM values given in mV of n experiments (in brackets) as mean _+SE

mann 1996), but the inhibitor had no significant

Z
c
~9
impact on ~t~ over the 45 rain recording period. The
E same insensitivity of VM tO 20 rag/1 BFA was found in
0.1 seven comparable experiments (Table 1). Figure 4
E also shows that adding 100 #M CHI as well as BFA
E
resulted in the characteristic CHI-induced depolarisa-
0 tion. This again shows that extracellularly applied
03 inhibitors are accessible to the impaled cell.
o To relate the effect of individual inhibitors on VM to
i ! i their impact on coleoptile elongation, the latter was
0 45 90 measured in the presence of the various inhibitors.
Figure 5 shows auxin-enhanced coleoptile elongation
t / min
in the presence and absence of inhibitors. To allow
Fig 5. Elongation of abraded coleoptile segments in the presence of comparison between VM and elongation measure-
20 gM IAA without (9 control), with addition of BFA (Q, 20 rag/l), merits, coleoptile segments were also abraded and the
COR (~5,400 ~tM) and CHI (11,100 gM). Auxin and inhibitors were
inhibitors added at the same concentration as for VM
added at time zero. Data points are means of 3 batches of 5 coleop-
tile segments recordings. Figure 5 shows that all three drugs inhibit
coleoptile elongation; COR and CHI cause drastic
inhibition, BFA moderate inhibition. The effect of
that absence of a VM response to COR is not due to individual inhibitors for 45 rain periods on VM did not
limited accessibility of the impaled cell to extracellu- correlate with their impact on coleoptile elongation
larly applied inhibitors. (Fig. 4 and Table 1).
If IAA-stimulated activity of the H+-ATPase required
synthesis of short-lived ATPase proteins (Hager et al. Discussion
1991) or regulatory proteins (Cho and Hong 1995), It is generally accepted that the auxin-generated
the contribution of the H+-pump to VM must be hyperpolarisation of the membrane in maize coleop-
decreased by inhibitors of intracellular vesicle traffic. tiie cells is due to an increase in H+-ATPase activity
To test this postulated relationship, coleoptiles were (reviewed in Napier and Venis 1995). Against this
treated with BFA, an effective inhibitor of Golgi- background, l/~i recordings in intact coleoptite cells
mediated vesicle transport in coleoptiles (Schindler et can be seen as a simple indirect assay of changes in
al. 1994, Edelmann and Volkmann 1996). Figure 4 pump activity. The aim of the present investigation
shows an example in which 20 rag/1 BFA was added was to test in vivo whether the auxin-stimulated rise
to the bathing medium of an abraded coleoptile. At in ATPase activity is due to hormone-enhanced syn-
this concentration BFA rapidly and severely inhibits thesis and incorporation of ATPase molecules into the
coleoptile elongation (Schindler et at. t994: fig. 5) plasma membrane (Hager et al. 1991) or to hormone-
and the gravitropic response (Edelmann and Volk- enhanced synthesis and secretion of a short-lived reg-
186
ulatory protein (Lado et al. 1977, Cho and Hong
1995) which might control ATPase activity.
The present voltage recordings conflict with this
hypothesis in several key aspects: first, the protein
synthesis inhibitor CHI reverses the IAA-induced rise
in ATPase concentration (Hager etal. 1991) and
should therefore only reverse the IAA-generated
hyperpolarisation. However, the inhibitor caused
membrane depolarisations irrespective of the pres-
ence of auxin (see also Lado et al. 1977). Thus, CHI
must affect constitutive properties of membrane
transport unrelated to any IAA-generated rise in
ATPase concentration (Hager et al. 1991) or other
IAA-dependent regulatory proteins (Cho and Hong
1995). Secondly, the membrane voltage in auxin-
treated coleoptile cells is not affected by BFA, the lat-
ter being a potent inhibitor of vesicle transport in
grass coleoptiles (Schindler et al. 1994, Edelmann
and Votkmann 1996) under these conditions. There-
fore, it is unlikely that the auxin-stimulated hyperpo-
larisation is due to hormone-controlled synthesis of
ATPase molecules (Hager et al. 1991) or proteins
which regulate ATPase activity (Cho and Hong .1995).
Their contribution to the membrane voltage should be
inactivated when Golgi-mediated vesicle traffic, the
general pathway for proteins transport from the ER to
the plasma membrane in eukaryotic cells (Staehelin
and Moore 1995), is blocked by BFA. Thirdly, the
RNA synthesis inhibitor COR reverses the IAA-
induced rise in ATPase activity (Hager et al. 1991), an
effect attributed to the continuous synthesis of
ATPase molecules (Hager et al. 1991) or proteins
required for H+-ATPase regulation (Cho and Hong
1995). Since equivalent treatment of coleoptiles with
COR produced no short-term effect on l/~u, correla-
tion between constitutive or IAA-promoted transcrip-
tion with membrane transport processes can be dis-
counted.
In summary, the voltage recordings show that auxin-
dependent ATPase- and general protein synthesis
and/or delivery through the secretory pathway is not
significant for the maintenance of an auxin-dependent
hyperpolarisation in the short term. Thus, the hor-
mone-stimulated rise in pump activity, the process
underlying hyperpolarisation, must be due to kinetic
modulation of the ATPase. The latter must occur via a
mechanism that does not involve a short-lived regula-
tory protein.
Interpretation of the present data is in agreement with
the finding that IAA modified the transcription level
of P-type ATPases in tomato hypocotyl, but the
G, Thiel et al.: Maintenance of polarised membrane voltage
modulated transcription level was not correlated with
the time course of IAA-induced growth (Ewing and
Bennett 1994). The present data are also in accor-
dance with recent investigations reporting a lack of
auxin-stimulated rise in ATPase concentration in
plasma membrane fractions from maize coleoptiles
(Jahn et al. 1996) or sunflower hypocotyls (Cho and
Hong 1995). Jahn and co-workers (1996) speculated
that the lack of a detectable rise in ATPase concentra-
tion in fractionated membrane preparations might be
due to the fact that the latter is confined to outer epi-
dermal cells. In this case the effect may be masked by
the bulk fraction of cortical cell membranes. Howev-
er, in the present study data were obtained from the
outer epidermis of intact coleoptite cells which makes
it unlikely, that an auxin-generated rise in ATPase
concentration is confined to the epidermal tissue.
One interesting result of the present study was that
CHI evoked decay in IAA-enhanced elongation (e.g.,
Hager et al. t991) and decrease in VM (Fig. t) with a
strikingly similar kinetics. The close temporal corre-
lation of both parameters suggests that the CHI-
evoked depolarisation is related to the CHI-evoked
inhibition of coleoptile elongation. A plausible expla-
nation for such a link is that the antibiotic-evoked
depolarisation abolishes K + uptake through inward
rectifying K + channels, a process believed to be
essential for turgor driven growth (Thiel et al. 1996).
In addition, a more positive VMcould reverse the driv-
ing force for passive K + flux, resulting in the leakage
of osmolytes from CHI-treated maize coteoptiles
(Kutschera and Schopfer 1986). In this context the
inhibitory effect of CHI on protein synthesis (Edet-
mann et al. t989) may be viewed as complementary
to the inhibition of coleoptile elongation at least in the
short term.
The mechanism by which CHI causes membrane
depolarisation remains unknown. The similarity
between I~M values obtained from CHI-treated and
metabolically blocked ceils suggests that CHI inhibits
or short-circuits the contribution of H+-ATPase to ~v~.
Given that BFA had no short-term effect on VM, it can
be concluded that CHI-evoked depolarisation is not
caused by the inhibition of newly synthesised pro-
teins. The latter would not reach their site of action on
the plasma membrane in the presence of BFA. There-
fore, the data suggest that the antibiotic does not act
specifically as an inhibitor of protein synthesis but
produces significant side effects which have often
been reported in the past (Ellis and MacDonald 1970,
McMahon 1975).
G. Thiel et al.: Maintenance of polaxised membrane voltage
According to the "acid growth theory", cell elonga-
tion is achieved by auxin-stimulated proton extrusion
and consequent loosening of cell wall structures (e.g.,
Rayle and Cleland 1992). It is interesting to note that
COR and BFA, inhibitors which reduce IAA-
enhanced H + extrusion (Hager et al. 1991, Schindler
et al. 1994, Cho and Hong 1995), have no significant
impact on VM. Thus it must be concluded that IAA-
stimulated H + secretion is not achieved simply by
auxin-dependent stimulation of the H+-ATPase. There
is clearly an obligatory requirement for a functional
secretory system to guarantee complete IAA-promot-
ed H + extrusion (Schindler et al. 1994, Cho and Hong
1995). This correlation is reminiscent of a hypothesis
suggested by Ray (1977), namely that IAA-evoked
acidification of the cell wall is not ATPase-mediated
but results from secretion of ER-derived acidic
vesicles into the apoplast. Whether this putative pro-
ton secretion is indeed the mechanism which induces
growth or only an epiphenomenon needs further
investigation.
Acknowledgements
The work was supported by the Deutsche Forschungsgemeinschaft
(SFB 523). We are grateful to Dr. Julia Davies (University of York)
for hetp with the manuscript.
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