Download The a-subunit of a heterotrimeric G-protein from

Journal of Experimental Botany, Vol. 50, No. 330, pp. 53–61, January 1999
The a-subunit of a heterotrimeric G-protein from
tobacco, NtGPa1, functions in K+ channel regulation
in mesophyll cells
Gerhard Saalbach1,4, Gabriel Natura2, Wolfgang Lein1, Peter Buschmann2, Ingo Dahse2,5,
Mathias Rohrbeck1 and Ference Nagy3
1 Institute of Plant Genetics and Crop Plant Research, IPK, Corrensstrasse 3, D-06466 Gatersleben, Germany
2 Institute of Biochemistry and Biophysics, Friedrich-Schiller-University Jena, Philosophenweg 12,
D-07743 Jena, Germany
3 Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, PO Box 521,
H-6701 Szeged, Hungary
Received 8 April 1998; Accepted 14 August 1998
Abstract
Introduction
Heterotrimeric GTPases (G-proteins) are implicated in
many cellular signalling processes. In plants, a function of a specific G-protein has only recently been
characterized. A cDNA clone encoding a G-protein asubunit was isolated from tobacco (Nicotiana tabacum
L.). The deduced amino acid sequence of this asubunit (NtGPa1) has 91% homology to GPa1 from
Arabidopsis thaliana. Sequence comparisons with
other plant G-proteins show that these two a-subunits
belong to the only class of plant G-proteins known to
date. The NtGPa1 cDNA was placed under the control
of the CaMV 35S promoter both in sense and antisense orientation. These constructs were stably
transformed into tobacco plants. As shown by patchclamp experiments, mesophyll protoplasts of transformed tobacco plants over-expressing NtGPa1 sense
or antisense RNA exhibited enhanced plasmalemma
K+ conductances compared to the wild type.
By contrast, mesophyll protoplasts of transformed
tobacco plants expressing the cholera toxin A1subunit, a G-protein activator, exhibited a reduced
plasmalemma K+ conductance. These results indicate
for the first time a role of a specific G-protein in the
regulation of K+ channels.
The function of heterotrimeric GTPases (G-proteins) has
been well characterized in animal systems and a number
of reviews on various aspects of G-protein function are
available (Bourne et al., 1990; Hepler and Gilman, 1992;
Clapham and Neer, 1993; Dickey and Birnbaumer, 1993).
G-proteins are composed of three different subunits, the
a-, b-, and c-subunits. They are among the most important intracellular molecular switches used in the transduction of signals from the cell surface into the cell. They
are activated by receptors with seven transmembranespans (7TMS ), and this activation involves the release of
bound GDP, binding of GTP by the a-subunit (Ga), and
dissociation of the a-subunit from the heterotrimeric
complex. Both the GTP-loaded a-subunit and the bccomplex can transduce the signal to a variety of effectors
such as adenylylcyclase, phospholipases and ion channels.
The activated state of the G-protein is terminated when
the bound GTP is hydrolysed by the GTPase activity of
the a-subunit.
In plants, several approaches have demonstrated a
similar role of G-proteins in different signal transduction
pathways (for reviews see Ma, 1994; Millner and Causier,
1996; Palme, 1996). However, these results were mainly
obtained from the use of general modulators of G-protein
activity which do not allow the identification of the
specific G-protein(s) involved. For example, single-cell
microinjection experiments with phytochrome-deficient
tomato cells demonstrated a role for G-proteins in phyto-
Key words: G-protein, cloning, antisense, K+ channel
regulation, Nicotiana tabacum.
4 Present address: Risø National Laboratory, Plant Biology and Biogeochemistry Department, Building 330, PO Box 49, DK-4000 Roskilde, Denmark.
5 To whom correspondence should be addressed. Fax: +49 3641 949352. E-mail: [email protected]
© Oxford University Press 1999
54
Saalbach et al.
chrome signalling (Neuhaus et al., 1993). Upon injection
of GTPcS or cholera toxin (CTX ) (both are activators
of G-proteins) responses normally dependent on phytochrome were initiated in the absence of phytochrome in
these mutant cells. Accordingly, injection of GDPbS or
pertussis toxin (inhibitors of G-protein activity) prevented
the effects of co-injected phytochrome. G-proteins also
play a role in the pathogen defence response. This has
been shown by the expression of the gene encoding the
CTX A1-subunit in tobacco plants (Beffa et al., 1995).
These plants exhibited a strongly enhanced level of salicylic acid and a constitutive expression of some PR
proteins leading to an increased resistance to bacterial
infection.
Several results also suggest that plant hormones such
as gibberellin (Jones et al., 1998), cytokinin (PlackidouDymock et al., 1998), and the stress hormone, abscisic
acid (ABA), act via a G-protein pathway (for review see
Assmann, 1996). The latter data were mainly obtained
from studies of K+ channel activities in stomatal guard
cells. The use of modulators of G-protein activity clearly
showed a role of G-proteins in the regulation of K+
channels in these cells. For example, GTPcS inhibited the
inward-rectifying K+ channel in guard cells (FairleyGrenot and Assmann, 1991) and the outward-rectifying
K+ channel in mesophyll cells (Li and Assmann, 1993).
Effects on K+ channels were also observed when CTX,
pertussis toxin, or mas7 were used. The mas7 peptide (a
homologue of mastoparan) mimics a portion of the 7TMS
receptor and activates G-proteins. Application of mas7
to Vicia faba guard cells strongly inhibited inwardly
directed K+ channels (Armstrong and Blatt, 1995). In
other cases, contradictory results were obtained for
GTPcS (Lee et al., 1993) and mas7 ( Kelly et al., 1995).
This seems to depend on the conditions, in particular
on the cytosolic Ca2+ concentration, and might point
towards the existence of more than one G-protein in
guard cells (for review see Assmann, 1996).
Efforts to clone plant G-protein a-subunits led to the
isolation of several cDNA and genomic clones from
different plant species. Sequences have been reported for
Arabidopsis thaliana (Ma et al., 1990), Lycopersicon esculentum (Ma et al., 1991), Oryza sativa (Seo et al., 1995),
and other plant species. The comparison of the deduced
amino acid sequences shows a very high degree of similarity among all reported sequences. Thus only one class of
G-protein genes has been cloned in plants so far. This is
different from mammalian cells where G-protein subunits
are encoded by large gene families. In A. thaliana the
expression of GPa1 has been studied by RNA hybridization and immunolocalization experiments. Expression
could be detected in all organs and cell types examined,
but the level of this G-protein varied between different
cell types ( Weiss et al., 1993; Huang et al., 1994). All
meristems and organ primordia had much higher levels
than differentiated cells of mature organs. GPa1 has been
localized not only to the plasma membrane but also to
the endoplasmic reticulum ( Weiss et al., 1997). The
function of this plant G-protein is still unclear. Only in
one case has a specific function in the regulation of a
plant plasma membrane Ca2+ channel by bacterially
expressed GPa1 from L. esculentum been demonstrated
(Aharon et al., 1998).
In the present paper, the isolation and functional
characterization of a cDNA clone encoding a G-protein
a-subunit (NtGPa1) from Nicotiana tabacum (L.) is
reported. The deduced amino acid sequence shares high
homology with the other known plant G-protein asubunits. This cDNA clone was transformed into tobacco
plants and expressed both in sense and antisense orientation. Patch-clamp studies on mesophyll protoplasts indicated a function of NtGPa1 in K+ channel regulation.
Materials and methods
Cloning of NtGPa1
NtGPa1 cDNA clones were isolated from a Nicotiana tabacum
(L.) SR-1 leaf cDNA library (constructed in ZAPII vector and
purchased from Stratagene) by the plaque hybridization method
using as probe a full-length cDNA fragment coding for the
Arabidopsis thaliana (L.) GPA1 protein. The A. thaliana GPA1
cDNA was isolated by Ma et al. (1990) and the pKS plasmid
containing the AtGPa1 cDNA insert was kindly provided by
Dr Ma. Labelling of the probe, hybridization and washing of
the filters was performed as described by Merkle et al. (1994).
Nucleotide sequences of both strands of the longest isolated
cDNA (1592 bp) were determined by the dideoxy chaintermination method and the sequence comparison was performed with the PALIGN program of the PC/GENE sequence
analysis package (Intelligenetics).
Plant transformation, RNA-blot hybridization and plant
characterization
A DNA fragment comprising the entire coding region of the
NtGPa1 cDNA was generated by using the polymerase chain
reaction. A fragment encoding a truncated form of NtGPa1
lacking the last 15 C-terminal amino acids (NtGPa1 ) was
DEL
produced in the same way. Both fragments were inserted into
the plant transformation vector BinAr (Höfgen and Willmitzer,
1990) where they are under the control of the CaMV 35S
promoter. The fragment encoding the unmodified NtGPa1 was
inserted in sense and antisense orientation, respectively. These
constructs were stably transformed into tobacco plants (N.
tabacum L. cv. Samsun NN ) by using Agrobacterium tumefaciens
as described previously (Saalbach et al., 1996). Putative
transformants were selected on medium with kanamycin
(50 mg l−1). Total RNA was isolated from young leaves by
using the ‘RNeasy’ RNA isolation kit from Qiagen. Expression
of the NtGPa1 sense or antisense RNA was analysed by RNA
gel-blot hybridization according to standard procedures.
Transformed tobacco plants expressing NtGPa1 antisense
RNA were analysed for altered sensitivity to pathogens, for
salicylic acid formation and for PR-protein expression as
described by Beffa et al. (1995). The activation of a woundinducible MAP kinase was determined by using an in-gel
protein kinase assay with myelin basic protein according to
K+ channel regulation 55
Usami et al. (1995). Basic activities of phospholipases C and
D (PLC, PLD) were measured on isolated microsomal fractions
from tobacco leaves. PLC activity was analysed according to
Hirayama et al. (1995), and PLD activity was analysed
according to Munnik et al. (1995) or Pappan et al. (1997).
Patch-clamp analysis of mesophyll protoplasts
Patch-clamp measurements were performed as described previously (Saalbach et al., 1997). Briefly, protoplasts isolated from
leaves of in vitro-grown tobacco plants were transferred to the
bath solution containing 30 mM KCl, 4 mM CaCl , 2 mM
2
MgCl , 1 mM K H- and KH PO buffer, 10 mM 2-[N-morphol2
2
2 4
ino]-ethanesulphonic acid/1,3-bis[tris(hydroxymethyl )methylamino]-propane (MES/BTP; Sigma), pH 5.8, adjusted to 500
mOsm with mannitol. The pipette solution contained 180 mM
KCl, 6.7 mM EGTA, 2 mM MgCl , 3.5 mM CaCl , 4 mM
2
2
MgATP (Sigma), 10 mM MES/BTP, pH 7.2, adjusted to 550
mOsm with mannitol. The membrane potential and currents
across the entire surface of the plasmalemma were measured in
the whole-cell configuration by standard patch-clamp techniques
(Hamill et al., 1981). I–V curves for protoplasts were obtained
from steady-state currents induced by 1.8 s voltage pulses
repeated in increments of 20 mV at intervals of 5 s. Recordings
were performed and low-pass-filtered with an Axopatch-1D
amplifier (Axon Instruments, Foster City, USA). The software
package pCLAMP 5.5 (Axon) was used for the generation of
sequences of test voltage potentials, data recording and
data storage.
Results
Cloning of NtGPa1, a G-protein a-subunit from tobacco
A cDNA-clone encoding a G-protein a-subunit from a
tobacco cDNA library was isolated by screening with
GPA1 clone from A. thaliana. The deduced amino acid
sequence of the NtGPa1 tobacco clone ( EMBL Acc. No.
Y08154) is highly homologous to other known plant
G-protein a-subunits ( Table 1). The homology of the
Table 1. Homologies (identity plus similarity) of the deduced
amino acid sequence of NtGPa1 from tobacco shared with
G-protein a-subunit peptide sequences from different plant species
The homologies to mammalian G-protein a-subunits are given in the
lower part of the table.
GPa from:
NtGPa1
Nicotiana plumbaginifolia1
Lycopersicon esculentum2
Arabidopsis thaliana3
Glycine max (SAG1)4
Glycine max (SGA2)5
Lotus japonicus6
Oryza sativa7
97.6%
96.6%
91.4%
89.4%
93.0%
93.8%
84.2%
GBA1 (G ) from Bos taurus8
0
GB12 (G ) from Mus musculus9
i
GBAS (G ) from Mus musculus9
s
40.7%
38.9%
34.8%
References: 1Kaydamov et al., EMBL Acc. No. Z72389); 2Ma et al.,
1991; 3Ma et al., 1990; 4Kim et al., 1995; 5Gotor et al.., EMBL Acc.
No. X95582; 6Poulsen et al., 1994; 7Seo et al., 1995; 8Van Meurs et al.,
1987; 9Sullivan et al., 1986). Sequence alignments were performed by
using the PALIGN program (PC/GENE, Intelligenetics).
amino acid sequences is generally around 90%. For
example, it shares 91% homology with AtGPa1 from A.
thaliana. If these plant sequences are compared with
sequences from mammals, a slightly higher homology to
members of the Ga-subfamily 1 (involving G and G )
i
0
than to members of the subfamily 2 (involving G ) can
s
be observed (see last three lines of Table 1).
Antisense and over-expression of NtGPa1 in transformed
tobacco plants
A PCR-fragment comprising the entire coding region of
the NtGPa1 cDNA was inserted into the plant transformation vector BinAR (Höfgen and Willmitzer, 1990)
either in normal or inverted orientation. In this vector,
the insert is under the control of the CaMV 35S promoter,
and normal orientation should result in over-expression
of NtGPa1 while inverted orientation should lead to high
level expression of antisense RNA in transformed plants.
A truncated form of NtGPa1 lacking the last 15
C-terminal amino acids (NtGPa1 ) was used as well.
DEL
Transformation of tobacco plants (N. tabacum SNN ) was
carried out with Agrobacterium tumefaciens. The expression of the transformed NtGPa1 constructs was demonstrated by RNA gel-blotting (Fig. 1). As can be seen on
the blot, the level of the antisense RNA ( Fig. 1A) varied
considerably among the different transformants, but the
wild-type sense RNA could not be detected in any of
these transformants. Similar experiments revealed the
over-expression of sense RNA in the transformed plants
( Fig. 1B). In this way, transformed plants were selected
over-expressing either sense or antisense RNA of
NtGPa1, as well as plants over-expressing NtGPa1
DEL
sense RNA (Fig. 1C ). Transformed plants were grown
to maturity and seeds were harvested. The phenotype of
all transformed plants did not show any abnormalities
during any stage of the development.
Patch-clamp analysis reveals enhanced plasmalemma K+
conductance in tobacco plants transformed with NtGPa1
To study the regulation of ion channels whole-cell patchclamp recordings were performed with isolated tobacco
mesophyll protoplasts. As described previously (Saalbach
et al., 1997), whole-cell recording of wild-type protoplasts
revealed the predominant activity of outward-rectifying
K+ channels (Fig. 2) since the outwardly directed K+
currents could always be induced to much higher levels
than the inwardly directed K+ currents. Therefore, attention was focused on changes of the K+ conductance at
positive voltages. It was demonstrated that the K+ conductance of tobacco mesophyll protoplasts (wild type) is
generally sensitive to modulators of G-protein activity.
For example, the non-hydrolysable G-protein inactivator
GDPbS enhanced the K+ conductance of the
plasmalemma (Fig. 2).
56
Saalbach et al.
Fig. 1. RNA gel-blot demonstrating the expression of NtGPa1 antisense
(A) or sense (B, C ) RNA in transformed tobacco plants. Untransformed
tobacco plants (wt) were used as control. (A) In the control a weak
signal representing the wild-type sense RNA of NtGPa1 could be
detected ( lane 7). This band was not present in any of the other lanes
representing a number of transformed plants expressing the NtGPa1
antisense RNA; lane 5: antisense transformant ‘T7 ’, lane 8: ‘T9 ’; the
as
as
arrowheads at the left indicate the positions of the 28S and 18S rRNA
as a size marker. (B, C ): RNA gel-blots showing the over-expression
of NtGPa1 and NtGPa1
sense RNA, respectively, in a number of
DEL
transformed plants; lane 9 in (B): over-expression transformant ‘T8 ’;
ov
lane 9 in (C ) ): over-expression transformant ‘T8 ’.
DEL
Fig. 2. Averaged steady-state I–V curves of the whole-cell K+ currents
of mesophyll protoplasts from N. tabacum SNN without GDPbS
(control, squares) or with 2 mM GDPbS (circles) in the pipette.
The comparison of the maximum outward-current density (at +90 mV ) of wild-type protoplasts and of transformed protoplasts expressing NtGPa1 antisense RNA
indicated that in transformed protoplasts the K+ outward-rectifier is more activated than in the control. These
results were obtained with several transformed plants.
Data are shown in Fig. 3 for the antisense transformant
T7 expressing a high level of antisense RNA (see
as
Fig. 1A). From the I–V curves shown in Fig. 3 it was
calculated that the current density observed in mesophyll
protoplasts from T7 was significantly enhanced (71%)
as
over that of the wild type. At +90 mV the current density
in T7 mesophyll protoplasts reached 60.1±4.0 pA pF−1
as
(n=19) versus 35.0±1.7 pA pF−1 (n=29) in the wild
type ( Fig. 3; Table 2). A similar result was obtained with
T9 , another NtGPa1 antisense transformant (see lane 8
as
in Fig. 1 and row 6 in Table 2). Though the inwardly
directed currents are relatively small in tobacco mesophyll
protoplasts they may also be enhanced in the transformed
plants (Fig. 3).
The comparison of the maximum outward-current
density (at +90 mV ) of wild-type protoplasts and of
transformed protoplasts expressing NtGPa1 sense RNA
indicates that in transformed protoplasts the K+ outwardrectifier is also more activated than in the control. This
was the case both with NtGPa1 and NtGPa1 . As
DEL
shown in Table 2 for T8 (an NtGPa1 sense transov
formant) and for T8
(an NtGPa1
sense transDEL
DEL
formant), the current density at +90 mV in these
transformed plants reached approximately 60 pA pF−1
versus 35.0±1.7 pA pF−1 (n=29) in the wild type. The
increase of outward-rectifying K+ channel activity in
these plants is very similar to that in the antisense plants.
These results suggest that both antisense and sense RNA
Fig. 3. Averaged steady-state I–V curves of the whole-cell K+ currents
of mesophyll protoplasts from N. tabacum (SNN ) obtained under
standard conditions. Comparison between the wild type (wt, n=29)
and transformed plant T7 (n=19) expressing high level of NtGPa1
antisense RNA (see Fig. 1A). Bars represent the standard error of
the mean.
K+ channel regulation 57
Table 2. K+ channel activity given as averaged (±standard error) K+ outward-current density at +90 mV (right column) in wholecell patch-clamp measurements of mesophyll protoplasts from wild-type and different transformed tobacco plants
Since NtGPa1 transformants and CTX transformants were generated with different tobacco wild-type cultivars, both N. tabacum cv. Samsun NN
(N. t. SNN ) and N. tabacum cv. Petit Havana SR-1 (N. t. SR-1) were analysed for comparison with the different transformants. Rows 5 to 8:
transformed tobacco plants generated in this study (see Fig. 1 and text for details); row 3: 2S albumin (storage protein) from Brazil nut (Saalbach
et al., 1996); row 4: rab-homologous protein from V. faba (Saalbach and Thielmann, 1995); row 9: CTX transformants (Beffa et al., 1995); row
10: 14-3-3-like protein from V. faba (Saalbach et al., 1997). Inserts are under the control of the CaMV 35S promoter in both plant transformation
vectors used in this study (BinAr: Höfgen and Willmitzer, 1990; pGA471: An, 1987).
Tobacco genotype
1
2
3
4
5
6
7
8
9
10
N.
N.
N.
N.
N.
N.
N.
N.
N.
N.
t.
t.
t.
t.
t.
t.
t.
t.
t.
t.
SNN
SR-1
SNN
SNN
SNN
SNN
SNN
SNN
SR-1
SNN
Vector
Insert (transferred gene)
Current density (pA pF−1)
—
—
BinAr
pGA471
BinAr
BinAr
BinAr
BinAr
pMON501
pGA471
—
—
2S albumin
Vfypt3
NtGPa1 (antisense) T7
as
NtGPa1 (antisense) T9
as
NtGPa1 (sense, ori ) T8
ov
NtGPa1
(sense, del ) T8
DEL
DEL
CTX A1-subunit
14-3-3
35.0±1.7 (n=29)
33.6±3.8 (n=16)
37.4±1.6 (n=18)
35.1±1.9 (n=17)
60.1±4.0 (n=19)
62.6±8.1 (n=9)
59.6±4.4 (n=7)
62.3±2.3 (n=12)
13.9±1.5 (n=9)
60.2±2.4 (n=27)
over-expression had the similar effect on the function of
NtGPa1 in the transformed plants.
To ensure that the observed effects of NtGPa1 RNA
expression on K+ channel activity were specifically produced by the NtGPa1 expression, several control measurements were carried out with tobacco wild-type protoplasts
and with protoplasts from plants transformed with different unrelated genes. The data given in Table 2 show that
the current densities of protoplasts from two wild-type
cultivars (N. tabacum SNN and SR-1) were in the same
range (approximately 35 pA pF−1). Similar values were
obtained with protoplasts from transformed tobacco
plants over-expressing a rab11-homologous GTPase from
V. faba presumably involved in vesicular transport
(Saalbach and Thielmann, 1995). The over-expression of
a 2S albumin (Saalbach et al., 1996), a seed storage
protein from Brazil nut (Bertholletia excelsa H.B.K.), had
also no effect on the K+ channel activity. The data in
Table 2 also show that the K+ conductance was indifferent
to the two plant transformation vectors used. As described
previously (Saalbach et al., 1997), the over-expression of
14-3-3 proteins, a family of proteins known to play
an important role in the regulation of key enzymes in
signal transduction, caused an increase of K+ channel
activity comparable to that observed in the NtGPa1
transformants.
Pseudomonas tabaci, accumulated high levels of salicylic
acid, and constitutively expressed certain pathogenesisrelated (PR) protein genes (Beffa et al., 1995).
CTX can activate signalling pathways dependent on
G-proteins by ADP-ribosylation of the G-protein asubunit. Therefore, it was obvious to assume that K+
channel activities might also be affected in the transformed
plants. Whole-cell patch-clamp recordings were performed with mesophyll protoplasts of such plants and of
wild-type plants of N. tabacum L. (SR-1) for comparison.
As shown in Fig. 4, the outward-rectifying K+ channel
activity in mesophyll protoplasts of CTX plants was
greatly reduced. The maximum outward-current density
at +90 mV was 13.9±1.5 pA pF−1 (n=9). This represents a significant reduction by 59% compared to the wild
type. Contrary to the NtGPa1 transformants, K+ inward
Reduced K+ channel activity in tobacco plants transformed
with the CTX A1-subunit
Tobacco plants (N. tabacum L. cv. Petit Havana SR-1)
transformed with a chimeric gene encoding the cholera
toxin (CTX ) A1-subunit under the control of the lightinducible Cab-1 promoter were produced and described
by Beffa et al. (1995). The transformed plants developed
more slowly than the wild type and showed greatly
reduced susceptibility to the bacterial pathogen,
Fig. 4. Averaged steady-state I–V curves of the whole-cell K+ currents
of mesophyll protoplasts from N. tabacum (SR-1). Comparison between
wild-type (wt, n=16) and transformed plants (Beffa et al., 1995)
expressing the CTX A1-subunit (n=9). Bars represent the standard
error of the mean.
58
Saalbach et al.
currents seem
transformants.
not
to
be
affected
in
the
CTX
Discussion
A cDNA clone encoding an a-subunit of a heterotrimeric
G-protein was isolated from tobacco (N. tabacum). The
deduced amino acid sequence shares a high degree of
homology with all other known plant G-protein a-subunits. This means that (contrary to animals) only one
class of Ga has been cloned from plants so far. Only
recently, a partial PCR clone encoding GPa1 from Avena
fatua sharing only 40% homolgy with AtGPa1 has been
reported (Jones et al., 1998). A similar situation seems
to be true for the G-protein b-subunits (Gb) ( Weiss et al.,
1994; Palme, 1996). Two cDNA clones encoding Gb from
tobacco sharing very high homology with all known plant
Gbs were also isolated in our laboratory ( W Lein, unpublished results). Taken together, plants seem to have only
one class of the typical heterotrimeric G-proteins. To
date, the specific function of this G-protein is still unclear.
Several signalling pathways in plants have been shown
to involve (a) G-protein(s) (Millner and Causier, 1996).
This includes the phytochrome signalling pathway
(Neuhaus et al., 1993; Romero and Lam, 1993), different
reactions to pathogens such as induction of PR proteins
(Beffa et al., 1995) and fungal elicitor-induced dephosphorylation of plasma membrane H+-ATPase ( Xing
et al., 1997), and the regulation of K+ channels in
mesophyll cells and guard cells (Assmann, 1996). It
remains an open question which specific G-protein(s)
is/are involved in these different pathways. A particularly
intriguing part of this problem is whether all these functions are covered by the only G-protein class known so
far or whether there are more G-proteins with specific
functions in plants.
Another interesting characteristic of the plant
G-protein(s) becomes obvious regarding the factors activating them. Phytochrome is cytosolic and receptors for
pathogen elicitors (Bent, 1996) are also not related to the
7TMS receptors coupled to mammalian G-proteins
(Dohlmann, 1991; Watson and Arkinstall, 1994). GPa1
from A. thaliana has been localized both to the plasma
membrane and to the endoplasmic reticulum ( ER) ( Weiss
et al., 1997). At the ER it could be involved in vesicle
transport as known from animal cells. In relation to the
receptor systems known to activate plant G-proteins (see
above) one could also speculate that signalling could be
via cytosolic factors to the ER-localized G-protein and
not across a membrane (Nürnberg and Ahnert-Hilger,
1996; Weiss et al., 1997). On the other hand, biochemical
methods and EST database scanning provided indications
for the presence of 7TMS receptor homologues in plants
(Millner and Causier, 1996). Recently, a putative 7TMS
receptor homologue has been cloned from A. thaliana
which seems to be involved in cytokinin signal transduction (Plackidou-Dymock et al., 1998).
In an attempt to study the function of the cloned Ga
(NtGPa1), transformed tobacco plants over-expressing
either sense or antisense RNA of NtGPa1 were generated.
The RNA expression was demonstrated by RNA gelblotting. In the case of the antisense plants, no wild-type
sense RNA could be detected on the blots. However,
since immunological detection of the NtGPa1 protein in
microsomal fractions from tobacco plants proved difficult,
it has not been possible so far to demonstrate any change
of the NtGPa1 protein level in the transformed plants
(data not shown). The phenotype of all transformed
plants did not show any abnormalities during any stage
of the development, indicating that severe disturbances
of important signalling pathways did not occur. The
NtGPa1 antisense plants were tested for several possible
effects, such as PR-protein induction, activation of phospholipases C and D, and a wound-inducible MAP-kinase,
but by using the methods mentioned under Materials and
methods in all these cases no clear alterations could be
observed (data not shown).
Eventually, patch-clamp analysis on isolated mesophyll
protoplasts of the transformed plants was performed to
study the behaviour of K+ channels. As described previously (Saalbach et al., 1997) these studies showed the
predominant presence of outward-rectifying K+ channels
in the plasma membrane of tobacco mesophyll cells,
which can be activated by positive membrane potentials.
In wild-type cells, the outward-current density could,
on average, be induced to values around 35 pA pF−1
at +90 mV. Analysis of several transformed plants
expressing either sense or antisense RNA of NtGPa1
revealed that the K+ channels were more activated than
in the wild type. In both cases, a significant increase
(70%) of K+ channel activity was generated in the
transformed plants. By analysis of several control plants
it was shown that this activation was specific for the
expression of the NtGPa1 sense or antisense RNA. Plants
transformed with different vectors and with different
genes unrelated to signal transduction processes showed
K+ channel activities comparable to that of the wild type.
A specific effect on G-proteins by the expression of
NtGPa1 in the transformed plants is also supported by
the observation that GTPcS has different effects on K+
channel activities in wild-type, antisense, and overexpression plants (G Natura et al., unpublished results).
The phenomenon that both antisense plants and plants
over-expressing NtGPa1 sense RNA exhibited the same
effect on K+ channels activity could be explained by the
known fact that over-expression of sense RNA can mimic
the action of antisense expression and may thus also
reduce the level of the corresponding protein (co-sense
suppression) (Mol et al., 1990). In this respect it has to
be considered that bc-subunits can also regulate effector
K+ channel regulation 59
proteins such as ion channels and phospholipases
(Clapham, 1996; Exton, 1997). A reduction of the Ga
level in the sense and antisense plants might lead to a
higher level of free bc-subunits able to interact with
potential effectors. Alternatively, both reduced and
enhanced levels of NtGPa1 in antisense and sense transformants, respectively, might affect the stoichiometry of
the G-protein subunits disturbing their function in a
similar way.
Furthermore, it was found that the activity of outwardrectifying K+ channels was significantly reduced (59%)
in mesophyll cells of transformed plants expressing a
CTX A1-subunit. CTX activates some G-proteins by
ADP-ribosylation of the a-subunit. NtGPa1 can be activated by CTX because of the presence of an Arg residue
in the G2 domain. Provided NtGPa1 is the only G-protein
present in tobacco mesophyll cells (or the only one which
can be activated by CTX ), the results would be in
accordance with the effect that antisense/sense RNA overexpression leads to inactivation of NtGPa1 causing
activation of the K+ channels while CTX-activation of
NtGPa1 inactivates the K+ channels.
The activity of inward-rectifying K+ channels was also
enhanced in the NtGPa1 transformants while the activity
of these channels was not affected in the CTX transformants. This could be attributed to the fact that CTX
could activate several G-proteins which might be present
in the plants. Activation of both a stimulatory and
an inhibitory G-protein could compensate each other
in the regulation of the inward-rectifying K+ channels.
However, the lack of effect on the inward current after
CTX-transformation, on the one hand, and the response
to unspecific G-protein modulators ( Fig. 2), on the other
hand, rather suggests the existence of a G-protein specifically involved in the regulation of the inward-rectifying
channels being insensitive to CTX. In any case, these
assumptions would imply that inward- and outwardrectifying K+ channels are regulated differently, but
NtGPa1 seems to be important in both cases.
Similarly to the data reported here on the regulation
of K+ channels by NtGPa1, the homologue from
L. esculentum (LeGPa1) was found to regulate a
plasmalemma Ca2+ channel (Aharon et al., 1998). These
results indicate that the plant GPa1 G-proteins are
involved in the regulation of different ion channels.
It remains open how NtGPa1 regulates the ion channels. In animal cells, Gbc directly binds and regulates the
GIRK family of inward-rectifying K+ channels (Inanobe
et al., 1995). A membrane-delimited pathway of
G-protein regulation has been demonstrated for the
inwardly directed K+ channels of V. faba guard cells
( Wu and Assmann, 1994) suggesting a possible direct
G-protein interaction of the channel. Both in animal and
in plant cells, K+ channels can be regulated by
phosphorylation, Ca2+, and lipid second messengers.
Phosphorylation also plays a role in the K+ channel
activity of tobacco mesophyll protoplasts as it is decreased
by kinase inhibitors (G Natura et al., unpublished results).
The elevation of cytosolic Ca2+ inhibits inward-rectifying
K+ channels ( Kelly et al., 1995), and cytosolic release of
Ca2+ or inositol-1,4,5-trisphosphate (IP ) from their
3
caged forms also inhibited these K+ channels in guard
cells (Blatt et al., 1990). Outwardly directed K+ channels
in mesophyll cells of V. faba were also inhibited by
elevated Ca2+ (Li and Assmann, 1993). Recently, an
outward-rectifying K+ channel with a new structure and
a steep Ca2+ dependency has been cloned from A. thaliana
(Czempinski et al., 1997).
These data are suggestive of the involvement of phospholipases (mainly PLC ) in the G-protein regulation of
K+ channels (Millner and Causier, 1996). G-proteinmediated activation of PLC has been demonstrated by
the use modulators of G-protein activity. Mastoparan
stimulated IP formation in Chlamydomonas reinhardtii
3
(Quarmby et al., 1992) and in Daucus carota cells (Drøbak
and Watkins, 1994; Cho et al., 1995), and exposure of
Chlamydomonas eugametus cells to mastoparan efficiently
induced PLD activity (Munnik et al., 1995). In animal
cells, members of the G family of G-protein a-subunits
q
directly bind and activate PLCb isozymes ( Exton, 1997).
To characterize the signalling pathway of NtGPa1 further,
a PLC assay with recombinant PLC from A. thaliana and
with recombinant NtGPa1 is being used in this laboratory. Results indicate a direct interaction of NtGPa1 with
the PLC ( W Lein, unpublished results). Although no
alteration of total PLC activity could be found in the
NtGPa1 transformants (see above) that could be due to
the existence of many PLC isoforms and to the insensitivity of the methods used, the observed interaction of
NtGPa1 with a plant PLC points towards PLC as the
link between NtGPa1 and the K+ channel activation
reported in this paper.
Acknowledgements
We want to thank Ingrid Otto, Sylvia Swetik, Heidi Traber,
and Holger Sack for excellent technical assistance and Birgit
Schäfer for photographic work. Annette Kaiser (Braunschweig)
was partly involved in plasmid construction and plant transformation work. We thank Dr Hong Ma (Cold Spring Harbor
Laboratory) for kindly providing the A. thaliana GPa1 clone.
This work was partly supported by a grant from the DFG (Sa
564/7–1 and Da 266/8–1) and by a grant from the
Kultusministerium des Landes Sachsen-Anhalt (861A/8284).
References
Aharon GS, Gelli A, Snedden WA, Blumwald E. 1998. Activation
of a plant plasma membrane Ca2+ channel by TGa1, a
heterotrimeric G protein a-subunit homologue. FEBS Letters
424, 17–21.
60
Saalbach et al.
An G. 1987. Binary Ti vectors for plant transformation and
promoter analysis. Methods in Enzymology 153, 292–305.
Armstrong F, Blatt MR. 1995. Evidence for K+ channel control
in Vicia guard cells coupled by G-proteins to a 7TMS
receptor mimetic. The Plant Journal 8, 187–198.
Assmann SM. 1996. Guard cell G proteins. Trends in Plant
Science 1, 73–74.
Beffa R, Szell M, Meuwly P, Pay A, Vogelilange R, Metraux
JP, Neuhaus G, Meins F, Nagy F. 1995. Cholera toxin
elevates pathogen resistance and induces pathogenesis-related
gene expression in tobacco. The EMBO Journal 14,
5753–5761.
Bent AF. 1996. Plant disease resistance genes: function meets
structure. The Plant Cell 8, 1757–1771.
Blatt MR, Thiel G, Trentham DR. 1990. Reversible inactivation
of K+ channels of Vicia stomatal guard cells following
the photolysis of caged inositol 1,4,5-trisphosphate. Nature
346, 766–769.
Bourne HR, Sanders DA, McCormick F. 1990. The GTPase
superfamily: conserved structure and molecular mechanism.
Nature 349, 117–127.
Cho MH, Tan Z, Erneux C, Shears SB, Boss WF. 1995. The
effects of mastoparan on the carrot cell plasma membrane
polyphosphoinositide phospholipase C. Plant Physiology
107, 845–856.
Clapham DE. 1996. The G-protein nanomachine. Nature
379, 297–299.
Clapham DE, Neer EJ. 1993. New roles for G-protein bcdimers in transmembrane signalling. Nature 365, 403–406.
Czempinski K, Zimmermann S, Ehrhardt T, Müller-Röber B.
1997. New structure and function in plant K+ channels:
KCO1, an outward rectifier with a steep Ca2+ dependency.
The EMBO Journal 16, 2565–2575.
Dickey BF, Birnbaumer L. 1993. GTPases in biology. Heidelberg:
Springer-Verlag.
Dohlmann HG, Thorner J, Caron MG, Leftowitz RJ. 1991.
Model systems for the study of seven-transmembrane-segment
receptors. Annual Review of Biochemistry 60, 653–688.
Drøbak BK, Watkins PAC. 1994. Inositol (1,4,5)-trisphosphate
production in plant cells: stimulation by the venom peptides,
melittin and mastoparan. Biochemical and Biophysical
Research Communications 205, 739–745.
Exton JH. 1997. Cell signalling through guanine-nucleotidebinding regulatory proteins (G proteins) and phospholipases.
European Journal of Biochemistry 243, 10–20.
Fairley-Grenot KA, Assmann SM. 1991. Evidence for G-protein
regulation of inward K+ channel current in guard cells of
fava bean. The Plant Cell 3, 1037–1044.
Hamill O, Marty A, Neher E, Sakmann B, Sigworth F. 1981.
Improved patch-clamp techniques for high-resolution current
recording from cells and cell-free membrane patches. Pflügers
Archiv 391, 85–100.
Hepler GR, Gilman AG. 1992. G proteins. Trends in Biochemical
Sciences 17, 383–387.
Hirayama T, Ohto C, Mizoguchi T, Shinozaki K. 1995. A gene
encoding a phosphatidylinositol-specific phospholipase C is
induced by dehydration and salt stress in Arabidopsis thaliana.
Proceedings of the National Academy of Sciences, USA 92,
3903–3907.
Höfgen R, Willmitzer L. 1990. Biochemical and genetic analysis
of different patatin isoforms expressed in various organs of
potato (Solanum tuberosum). Plant Science 66, 221–230.
Huang H, Weiss CA, Ma H. 1994. Regulated expression of the
Arabidopsis G protein a subunit gene GPA1. International
Journal of Plant Science 155, 3–14.
Inanobe A, Morishige KI, Takahashi N, Ito H, Yamada M,
Takumi T, Nishina H, Takahashi K, Kanaho Y, Katada T.
1995. G beta gamma directly binds to the carboxyl terminus
of the G protein-gated muscarinic K+ channel, GIRK1.
Biochemical and Biophysical Research Communications 212,
1022–1028.
Jones HD, Smith SJ, Desikan R, Plackidou-Dymock S,
Lovegrove A, Hooley R. 1998. Heterotrimeric G proteins are
implicated in gibberellin induction of a-amylase gene expression in wild oat aleurone. The Plant Cell 10, 245–253.
Kelly WB, Esser JE, Schroeder JI. 1995. Effects of cytosolic
calcium and limited, possible dual, effects of G protein
modulators on guard cell inward potassium channels. The
Plant Journal 8, 479–489.
Kim WY, Cheong NE, Lee DC, Je DY, Bahk JD, Cho MJ, Lee
SY. 1995. Cloning and sequencing analysis of a full-length
cDNA encoding a G protein alpha subunit, SGA1, from
soybean. Plant Physiology 108, 1315–1316.
Lee HJ, Tucker EB, Crain RC, Lee Y. 1993. Stomatal opening
is induced in epidermal peels of Commelina communis L. by
GTP analogs or pertussis toxin. Plant Physiology 102, 95–100.
Li W, Assmann SM. 1993. Characterization of a G-proteinregulated outward K+ current in mesophyll cells of Vicia
faba L. Proceedings of the National Academy of Sciences,
USA 90, 262–266.
Ma H. 1994. GTP-binding proteins in plants—new members of
an old family. Plant Molecular Biology 26, 1611–1636.
Ma H, Yanofsky MF, Huang H. 1991. Isolation and sequence
analysis of TGA1 cDNAs encoding a tomato G protein
asubunit. Gene 107, 189–195.
Ma H, Yanofsky MF, Meyerowitz EM. 1990. Molecular cloning
and charaterization of GPA1, a G protein a subunit gene
from Arabidopsis thaliana. Proceedings of the National
Academy of Sciences, USA 87, 3821–3825.
Merkle T, Haizel T, Matsumoto T, Harter K, Dallmann G,
Nagy F. 1994. Phenotype of the fission yeast cell cycle
regulatory mutant pim1–46 is suppressed by a tobacco cDNA
encoding a small, ran-like GTP-binding protein. The Plant
Journal 6, 555–565.
Millner PA, Causier BE. 1996. G-protein coupled receptors in
plant cells. Journal of Experimental Botany 47, 983–992.
Mol JN, van der Krol AR, van Tunen AJ, van Blokland R, de
Lange P, Stuitje AR. 1990. Regulation of plant gene
expression by antisense RNA. FEBS Letters 268, 427–430.
Munnik T, Arisz SA, De Vrije T, Musgrave A. 1995. G protein
activation stimulates phospholipase D signaling in plants.
The Plant Cell 7, 2197–2210.
Neuhaus G, Bowler C, Kern R, Chua N-H. 1993. Calcium/
calmodulin-dependent and independent phytochrome signal
transduction pathways. Cell 73, 937–952.
Nürnberg B, Ahnert-Hilger G. 1996. Potential roles of heterotrimeric G proteins of the endomembrane system. FEBS
Letters 389, 61–65.
Palme K. 1996. A role for the heterotrimeric G-protein switch
in higher plants. In: Smallwood M, Knox JP, Bowles DJ,
eds. Membranes: specialized functions in plants. Oxford:
BIOS Scientific Publishers Limited, 151–163.
Pappan K, Zheng S, Wang X. 1997. Identification and
characterization of a novel plant phospholipase D that
requires polyphosphoinositides and submicromolar calcium
for activity in Arabidopsis. Journal of Biological Chemistry
272, 7048–7054.
Plackidou-Dymock S, Dymock D, Hooley R. 1998. A higher
plant seven-transmembrane receptor that influences sensitivity
to cytokinins. Current Biology 8, 315–324.
Poulsen C, Mai XM, Borg S. 1994. A Lotus japonicus cDNA
K+ channel regulation 61
encoding an a-subunit of a heterotrimeric G-protein. Plant
Physiology 105, 1453–1454.
Quarmby LM, Yueh YG, Cheshire JL, Keller LR, Snell WJ,
Crain RC. 1992. Inositol phospholipid metabolism may
trigger flagellar excision in Chlamydomonas reinhardtii.
Journal of Cell Biology 116, 737–744.
Romero LC, Lam E. 1993. Guanine nucleotide binding protein
involment in early steps of phytochrome-regulated gene
expression. Proceedings of the National Academy of Sciences,
USA 90, 1465–1469.
Saalbach G, Rosso M, Schumann U. 1996. The vacuolar
targeting signal of the 2S albumin from Bertholletia excelsa
(H.B.K.) resides at the C-terminus and involves the
C-terminal propeptide as an essential element. Plant
Physiology 112, 975–985.
Saalbach G, Schwerdel M, Natura G, Buschmann P, Christov V,
Dahse I. 1997. Over-expression of plant 14–3-3 proteins in
tobacco: enhancement of the plasmalemma K+ conductance
of mesophyll cells. FEBS Letters 413, 294–298.
Saalbach G, Thielmann J. 1995. Isolation and characterization
of five cDNA-clones encoding small GTP-binding proteins
from field bean (Vicia faba). Journal of Plant Physiology
145, 665–673.
Seo HS, Kim HY, Jeong JY, Lee SY, Cho MJ, Bahk JD. 1995.
Molecular cloning and characterization of RGA1 encoding a
G protein alpha subunit from rice (Oryza sativa L. ir-36).
Plant Molecular Biology 27, 1119–1131.
Sullivan KA, Liao YC, Alborzi A, Beiderman B, Chang FH,
Masters SB, Levinson AD, Bourne HR. 1986. Inhibitory and
stimulatory G proteins of adenylate cyclase: cDNA and
amino acid sequences of the alpha chains. Proceedings of the
National Academy of Sciences, USA 83, 6687–6691.
Usami S, Banno H, Ito Y, Nishihama R, Machida Y. 1995.
Cutting activates a 46-kilodalton protein kinase in plants.
Proceedings of the National Academy of Sciences, USA 92,
8660–8664.
Van Meurs KP, Angus CW, Lavu S, Kung HF, Czarnecki SK,
Moss J, Vaughan M. 1987. Deduced amino acid sequence of
bovine retinal G alpha: similarities to other guanine
o
nucleotide-binding proteins. Proceedings of the National
Academy of Sciences, USA 84, 3107–3111.
Watson S, Arkinstall S. 1994. The G-protein linked receptor
facts book. London: Academic Press Limited.
Weiss CA, Huang H, Ma H. 1993. Immunolocalization of the
G protein alpha subunit encoded by the GPA1 gene in
Arabidopsis. The Plant Cell 5, 1513–1528.
Weiss CA, Garnaat CW, Mukai K, Hu Y, Ma H. 1994. Isolation
of cDNAs encoding guanine nucleotide-binding protein betasubunit homologues from maize (ZGB1) and Arabidopsis
(AGB1). Proceedings of the National Academy of Sciences,
USA 91, 9554–9558.
Weiss CA, White E, Huang H, Ma H. 1997. The G protein a
subunit (GPa1) is associated with the ER and the plasma
membrane in meristematic cells of Arabidopsis and cauliflower. FEBS Letters 407, 361–367.
Wu WH, Assmann SM. 1994. A membrane-delimited pathway
of G-protein regulation of the guard-cell inward K+ channel.
Proceedings of the National Academy of Sciences, USA 91,
6310–6314.
Xing T, Higgins VJ, Blumwald E. 1997. Identification of G
proteins mediating fungal elicitor-induced dephosphorylation
of host plasma membrane H+-ATPase. Journal of
Experimental Botany 48, 229–237.