Heterotrimeric G-protein complex and G-protein

Comments

Transcription

Heterotrimeric G-protein complex and G-protein
The Plant Journal (2007) 51, 656–669
doi: 10.1111/j.1365-313X.2007.03169.x
Heterotrimeric G-protein complex and G-protein-coupled
receptor from a legume (Pisum sativum): role in salinity and
heat stress and cross-talk with phospholipase C
Shikha Misra†, Yuliang Wu†, Gayatri Venkataraman, Sudhir K. Sopory and Narendra Tuteja*
Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi – 67, India
Received 19 March 2007; revised 7 April 2007; accepted 26 April 2007
*
For correspondence (fax +91 11 26162316; email [email protected]).
†
These authors contributed equally to this work.
Summary
Heterotrimeric G-proteins transduce signals from activated G-protein-coupled receptors (GPCR) to appropriate
downstream effectors and thereby play an important role in signaling. A role of G-proteins in salinity and heat
stress tolerance has not heretofore been described. We report isolation of cDNAs of two isoforms of Ga (Ga1,
1152 bp; Ga2, 1152 bp), one Gb (1134 bp), two isoforms of Gc (Gc1, 345 bp; Gc2, 303 bp) and a GPCR (1008 bp)
from Pisum sativum, and purification of all the encoded recombinant proteins (Ga, 44 kDa; Gb, 41 kDa; Gc,
14 kDa; GPCR, 35 kDa). The transcript levels of Ga and Gb were upregulated following NaCl, heat and H2O2
treatments. Protein–protein interaction studies using an in vitro yeast two-hybrid system and in planta
co-immunoprecipitation showed that the Ga subunit interacted with the pea Gb subunit and pea phospholipase C (PLCd) at the calcium-binding domain (C2). The GTPase activity of the Ga subunit increased after
interaction with PLCd. The GPCR protein interacted with all the subunits of G-proteins and with itself.
Transgenic tobacco plants (T0 and T1) constitutively over-expressing Ga showed tolerance to salinity and heat,
while Gb-over-expressing plants showed only heat tolerance, as tested by leaf disk senescence assay and
germination/growth of T1 seeds/seedlings. These findings provide direct evidence for a novel role of Ga and Gb
subunits in abiotic stress tolerance and possible cross-talk between PLC- and G-protein-mediated signaling
pathways.
Keywords: abiotic/environmental stress, heterotrimeric G-proteins, G-protein-coupled receptors, legume,
phospholipase C, signal transduction.
Introduction
Plant growth and development are mediated by a complex
array of signaling pathways, coordinated by exogenous
factors, that regulate all phases of growth including cell
division, differentiation and cell death. One of the most
ancient and evolutionary conserved mechanisms for transducing extracellular signals is the G-protein signaling pathway. Broadly, G-proteins can be classified as either
monomeric G-proteins (small G-protein family) (Yang,
2002), which include molecules such as Ras, Rho, Rab, Ran
and Arf (molecular weight 20–40 kDa), or heterotrimeric
G-proteins (large G-protein family) composed of a
(39–52 kDa), b (34–36 kDa) and c (7–10 kDa) subunits
(Gilman, 1987). The heterotrimeric G-proteins are among the
most important intracellular molecular switches, transducing
signals from an activated transmembrane G-protein-coupled
656
receptor (GPCR) to appropriate downstream effectors within
cells, thereby playing an important role in signal transduction (Gilman, 1987). The Ga subunit carries the binding site
for the nucleotide and GTPase activity. In mammals,
approximately 20 Ga, six Gb and at least 12 Gc subunit isoforms are present. In animal systems, the Ga subunits
exhibit considerable structural and functional diversity and
have been assigned to four major families (Gs, Gi/o, Gq and
G12) according to sequence homology (Simon et al., 1991).
G-protein-coupled receptors form a superfamily of integral membrane protein receptors that contain seven transmembrane a-helical regions. They bind to a vast variety of
ligands, and are involved in various signaling pathways.
GPCR is a guanine nucleotide exchange factor that promotes
the exchange of GDP/GTP associated with the Ga subunit.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd
Stress tolerance by pea heterotrimeric G-proteins 657
Upon activation by a wide range of stimuli, the GPCRs
interact with their cognate G-protein, inducing GDP release
with subsequent GTP binding to the Ga subunit (see Hamm,
1996). The exchange of GDP for GTP results in activation of
the Ga subunit (Ga-GTP), which leads to dissociation of the
Gb/Gc dimer from Ga. Both these moieties interact with
various downstream effector molecules and initiate unique
intracellular signaling responses. After signal propagation,
the GTP of Ga-GTP is hydrolyzed to GDP, and Ga becomes
inactive (Ga-GDP), which leads to its re-association with the
Gb/Gc dimer to form the inactive heterotrimeric complex
(see Jones and Assmann, 2004). Although the human
genome contains about 1000 GPCRs, only a single gene,
GCR1, encoding a putative GPCR has been identified in
Arabidopsis thaliana (Plakidou-Dymock et al., 1997). It was
reported to be cell-cycle-regulated (Colucci et al., 2002) and
is involved in ABA signaling in guard cells (Pandey and
Assmann, 2004).
In plants, the G-protein cascade has been studied to some
extent in Arabidopsis and rice but much of it has not yet been
deduced. The genomes of diploid angiosperms, such as that
of the model species A. thaliana, contain only a single
canonical Ga gene, GPA1 (Ma et al., 1990), one Gb gene,
AGB1 (Weiss et al., 1994), and two Gc genes, AGG1 and
AGG2 (Arabidopsis Genome Initiative, 2000; Mason and
Botella, 2001). Two Ga subunits (PGA1 and PGA2) have been
reported in pea (Marsh and Kaufman, 1999). In plants,
G-proteins have been reported to be involved in processes
such as ion channel signaling, abscisic acid signaling (Wang
et al., 2001), and modulation of cell proliferation (Ullah et al.,
2001) in Arabidopsis. However, a wide range of processes,
including seed germination, shoot and root growth, and
stomatal regulation, are altered in Arabidopsis and rice plants
with mutations in G-protein components (Ullah et al., 2001).
The role of G-proteins in salinity and heat stress tolerance
has not been well studied. In this work, we describe the
cloning and functional characterization of the three subunits
of heterotrimeric G-proteins and a GPCR from pea. We
identify a novel role for G-proteins in salinity and heat-stress
tolerance in plants, and possible cross-talk between the
phospholipase C (PLC)- and G-protein-mediated signaling
pathways.
Results
Cloning and sequence analysis of PsGa1, PsGa2, PsGb,
PsGc1, PsGc2 and PsGPCR cDNAs
The PsGa1 and PsGa2 cDNAs were amplified by PCR using
pea double-stranded cDNAs as a template. Sequence analysis of the PsGa1 and PsGa2 cDNAs shows that they encode
a full-length cDNA, which is 1.15 kb in size in both cases. The
deduced amino acid sequence revealed a protein consisting
of 384 amino acid residues with a predicted molecular mass
of about 44.5 kDa and pI 5.81 for PsGa1, and a protein consisting of 384 amino acid residues with a predicted
molecular mass of about 44.48 kDa and pI 5.70 for PsGa2.
The cDNA clone of PsGb was obtained after screening the
pea cDNA library using radiolabeled tobacco Gb cDNA as
a probe. Sequence analysis of PsGb cDNA shows that it is a
full-length cDNA, which is 1.13 kb in size and encodes
a protein consisting of 377 amino acid residues with
a predicted molecular mass of about 41 kDa and pI 7.04.
For cloning of PsGc1 and PsGc2, a far-Western technique
was used, utilizing PsGb protein and antibody against PsGb
protein. Sequence analysis of the PsGc1 and PsGc2 cDNAs
shows that they encode full-length cDNAs of 0.3 kb. The
deduced amino acid sequence revealed a protein consisting
of 114 amino acid residues with a predicted molecular mass
of about 12 kDa and pI 8.57 for PsGc1, and a protein
consisting of 100 amino acid residues with a predicted
molecular mass of about 10 kDa and pI 7.60 for PsGc2.
The cDNA clone of PsGPCR was obtained after screening
the pea cDNA library with radiolabeled Arabidopsis GCR1
cDNA as a probe. Sequence analysis of PsGPCR cDNA
shows that it encodes a full-length cDNA, which is 1.008 kb
in size. The deduced amino acid sequence revealed a protein
consisting of 335 amino acid residues with a predicted
molecular mass of about 35 kDa and pI 10.60.
Amino acid alignments of Ga1, Ga2, Gb, Gc1 and Gc2 from
pea with their corresponding subunits from Arabidopsis are
shown in Figure 1 (a–d). The PsGa1 and PsGa2 subunits are
86% identical, share 73–92% identity with Arabidopsis Ga
subunits (AtGPA1) and contain all the reported conserved
domains of the Ga subunit (Figure 1a). The PsGb subunit
shares 85% identity with the Gb subunit from Arabidopsis
and also contains seven conserved WD repeats (Figure 1b).
The PsGc1 and PsGc2 subunits are only 15% identical, share
30–50% identity with Arabidopsis Gc subunits (AGG1 and
AGG2) and contain a conserved isoprenylation site (Figure 1c,d). No significant homology of PsGc1 and PsGc2
subunits with the mammalian c-subunit was observed.
The amino acid sequence alignment of PsGPCR with
Arabidopsis GCR1 is shown in Figure 2(a), which shows that
it contains the conserved seven transmembrane regions.
There is 50% identity between these two sequences, and
most of it is in the seven transmembrane regions. The
presence of seven transmembrane regions was further
confirmed by the transmembrane hidden Markov model
(TMHMM) (Figure 2b). Sequence comparison of PsGPCR with
GPCRdB shows that PsGPCR is a member of the class B
secretin-like receptor family.
Expression and purification of PsGa1, PsGb, PsGc1 and
PsGPCR proteins
The pea cDNA encoding Ga1 was cloned into the expression
vector pET28a (pET28a-PsGa1) and the recombinant protein
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
658 Shikha Misra et al.
Figure 1. CLUSTALW amino acid sequence alignment of PsGa1, PsGa2, PsGb, PsGc1 and PsGc2
with corresponding subunits from Arabidopsis.
(a) CLUSTALW alignment of PsGa1 and PsGa2 with
GPA1 from Arabidopsis showing 86% identity.
Both Ga1 and Ga2 contain all the conserved
domains: (1) the MGXXXS sequence (myristylation site), containing glycine at position 2, at the
N-terminal of Ga1 (involved in binding of the bc
subunit, the effector and the receptor), (2) the
Gbc binding site (amino acids 1–36 at the Nterminal end), (3) the arginine residue at position 191 that serves as the site for ADP-ribosylation by cholera toxin (CTX), (4) conformational
switch regions I, II and III at positions 190–200,
219–238 and 250–261, respectively, (5) GTP-binding sites at positions 42–58, 219–224, 285–291
and 354–358, and (6) Receptor binding domains
present at 15–33, 347–364 and 375–384 positions.
(b) CLUSTALW alignment of PsGb with AGB1 from
Arabidopsis showing 80% identity. The sequence
contains seven WD repeats.
(c,d) CLUSTALW alignment of PsGc1and PsGc2
with Arabidopsis AGG1 and AGG2, respectively,
showing 50% identity of PsGc1with AGG1 and
40% identity of PsGc2 with AGG2. A prenyl group
binding site (CAAX box) was observed at the
C-termini of PsGc1 and PsGc2. The accession
numbers for the amino acid sequences are
NM128127
(GPA1),
AF537218
(PsGa1),
AF533438 (PsGa2), U12232 (AGB1), AF145976
(PsGb), AF283673 (AGG1), DQ010315 (PsGc1),
AF347077 (AGG2) and AY876935 (PsGc2).
(a)
(b)
(c)
(d)
from the soluble fraction was purified by Ni2+–NTA–agarose
column chromatography. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis (SDS–PAGE) analysis
showed an additional highly expressed 44 kDa polypeptide
for PsGa1 in an IPTG-induced fraction compared with an
uninduced fraction. Purified PsGa1 protein showed a 44 kDa
band on SDS–PAGE (Figure 3a, lane 4). A similar result was
obtained for the PsGa2 protein. The anti-PsGa1 antibody
cross-reacted with the PsGa2 protein in a Western blot
analysis (data not shown). Further detailed work was mainly
performed with PsGa1.
The pea cDNA encoding Gb was cloned in pET28a
(pET28a-PsGb) and the recombinant protein was purified
from the inclusion bodies by Ni2+–NTA–agarose column
chromatography. The purified denatured protein was
refolded on the column and eluted for functional analysis.
SDS–PAGE analysis showed a highly expressed 41 kDa
polypeptide for PsGb (Figure 3b, lane 4).
The pea cDNA encoding PsGc1 was cloned in pET28a
(pET28a-PsGc1) and the recombinant protein was purified
from the soluble fractions through Ni2+–NTA–agarose column chromatography. SDS–PAGE analysis showed a purified 14 kDa protein for PsGc1 (Figure 3c, lane 3). The pea
cDNA encoding PsGPCR was also cloned in pET28a (pET28aPsGPCR) and the recombinant protein was purified in
the soluble form through Ni2+–NTA–agarose column
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
Stress tolerance by pea heterotrimeric G-proteins 659
Figure 2. CLUSTALW amino acid sequence alignment of PsGPCR with Arabidopsis GCR1 and its
transmembrane regions.
(a) Amino acid sequence alignment of PsGPCR
with Arabidopsis GCR1. The sequence contains
seven transmembrane a-helical regions. There is
50% identity between these two sequences and
most of it is in the transmembrane regions.
(b) Transmembrane regions and topology of
PsGPCR predicted using the Simple Modular
Architecture Retrieval Tool (SMART version 3.5)
and transmembrane hidden Markov model
(TMHMM version 2.0, http://www.cbs.dtu.dk/
services/TMHMM) programs. The accession
numbers for the amino acid sequences are
NM103724 (GCR1) and DQ010316 (PsGPCR).
Figure 3. Protein expression, purification of
heterotrimeric G-protein subunits, GPCR, GTP
binding assay and GTPase activity of Ga.
(a,b) Expression of (a) PsGa1 and (b) PsGb in the
bacterial culture. Marker (lane 1), uninduced
protein (lane 2), induced protein (lane 3) and
purified protein (lane 4).
(c,d) Expression of (c) PsGc1 and (d) PsGPCR in
the bacterial culture. Uninduced protein (lane 1),
induced protein (lane 2) and purified protein
(lane 3).
(e) The GTP binding assay of PsGa1. Lane 1,
PsGa1 protein; lane 2, BSA (negative control).
(f) GTPase assay of PsGa1 protein. Lane 1, PsGb
protein; lane 2, pea PLC protein; lane 3, PsGa1
protein; lane 4, PsGa1 + PsGb protein; lane 5,
PsGa1 + pea PLC protein.
(g) Data from (f) expressed quantitatively as
percentage Pi released after GTP hydrolysis
(mean of three experiments). An equal amount
of Ga1 protein was used in the GTPase assay in
(f).
chromatography. SDS–PAGE analysis showed a purified
35 kDa protein for PsGPCR (Figure 3d, lane 3).
protein (44 kDa) binds GTP, while the negative control (BSA)
did not.
GTP binding assay
In vitro protein–protein interactions of PsGa1, PsGb, PsGc1,
PsPLCd and PsGPCR
To verify that recombinant PsGa1 protein is a functional
GTP-binding protein, we carried out an in vitro GTP-binding
assay. Autoradiography (Figure 3e) revealed that PsGa1
The interaction between PLC and the Ga subunit is shown in
Figure 4(a) (representative result). All the interaction images
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
660 Shikha Misra et al.
Figure 4. Interaction between various subunits of G-proteins, PsGPCR and PLC.
(a) In vitro protein–protein interaction between PLC (radiolabeled) and PsGa1 (unlabeled) proteins. Lane 1, radiolabeled PLC, lane 2, 100 mM KCl wash; lanes 3–5, the
three fractions eluted with 500 mM KCl in buffer. The other interactions identified through this procedure are not shown here.
(b) Summary of in vitro interactions between PsGa1, PsGb, PsGc1, PsGPCR, pea PLC and the C2 domain. (+) represents a positive interaction and (–) represents no
interaction.
(c) Yeast two-hybrid system-based interaction between PsGa1 and pea PLC: (i) template for panels (ii–vi), showing phenotypes on (ii) a YPD plate, (iii) on a synthetic
dextrose plate lacking tryptophan, (iv) on synthetic dextrose plate lacking leucine, (v) on a synthetic dextrose plate lacking leucine, tryptophan and histidine, and (vi)
a b-galactosidase filter lift assay. The other interactions identified through this procedure are not shown here.
(d) Summary of the interactions identified by the yeast two-hybrid system between PsGa1, PsGb, PsGc1, PsGPCR, pea PLC and the C2 domain. (+) represents a
positive interaction and (–) represents no interaction.
(e,f) In planta co-immunoprecipitation assays using PsGa1 and PsGb antibodies, respectively. Western blots of PsGa1 antibody-bound proteins using anti-PsGa1
antibodies (lane 1), PsGa1 antibody-bound proteins using anti-PsPLC antibodies (lane 2), PsGa1 antibody-bound proteins using anti-PsGb antibodies (lane 3), PsGb
antibody-bound proteins using anti-PsGa1 antibodies (lane 4), PsGb antibody-bound proteins using anti-PsPLC antibodies (lane 5) and PsGb antibody-bound
proteins using anti-PsGb antibodies (lane 6).
(g,h) Western blot analysis of cytosolic (lane 1), nuclear (lane 2) and microsomal (lane 3) fractions using antibody against PsGa (g) and PsGb (h).
looked the same; therefore, the results of the interactions are
summarized in Figure 4(b). Overall, the results clearly show
that PsGa1 interacts with Gb, PLCd, the C2 domain of PLCd
and PsGPCR, but not with PsGc1 or PsGa1 itself; PsGb
interacts with Ga1, PsGc1, PLCd and PsGPCR, but not with
the C2 domain of PLCd or PsGb itself; PsGc1 interacts with
PsGb and PsGPCR, but not with Ga1, PLCd, the C2 domain of
PLCd or PsGc1 itself. None of the subunits (PsGa1, PsGb or
PsGc1) forms a homodimer by self-interaction. The pea
GPCR protein was found to form oligomers by self-inter-
acting, and also interacted with all three subunits of G-proteins but not with PLCd or the C2 domain of PLCd (Figure 4b).
Interactions of PsGa1, PsGb, PsGc1, PsPLCd and PsGPCR by
the yeast two-hybrid system
For the yeast two-hybrid assay, the complete ORF of one
gene was cloned in a yeast AD vector (pGADT7) and the
complete ORF of the second gene was cloned in a yeast BD
vector (pGBKT7). The results obtained by interaction through
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
Stress tolerance by pea heterotrimeric G-proteins 661
the yeast two-hybrid system were similar to those obtained
through in vitro interactions. The results of interaction
between PLC and the Ga subunit are shown in Figure 4(c) as a
representative image. In Figure 4(c), panel (i) is a template for
panels (ii) to (vi) showing the streaked clones: (1,2) clones 1
and 2, yeast (AH109) cells containing two co-transformants
(from the same plate) of BD-PsPLCd and AD-PsGa1; (3) AD/
BD, co-transformants of empty AD and BD vectors; (4) BD,
co-transformants of empty BD vector alone; (5) AD,
co-transformants of empty AD vector alone; (6) Ga1/BD, cotransformants of AD-PsGa1 and empty BD vector; (7) PLCd/
AD, co-transformants of BD-PsPLCd and empty AD vector; (8)
yeast AH109 cells alone. All these transformants (1–7) and
AH109 cells (8) grew on the yeast extract/peptone/dextrose
(YPD) plate (non-selective medium) (Figure 4c, ii). Apart from
AH109 cells (8) and the cells containing AD vector (5 and 7), all
the co-transformants containing BD vector showed growth
on SDTrp- medium (single drop-out selection medium lacking Trp) (Figure 4c, iii). Apart from AH109 cells (8) and the
cells containing BD vector (4 and 6), all the co-transformants
containing AD vector showed growth on SDLeu- medium
(single drop-out selection medium lacking Leu) (Figure 4c,
iv). On selection medium lacking Leu, Trp and His, but containing 15 mM 3-AT (SDL-T-H- + 15 mM 3-AT), only selected
clones of co-transformants (BD-PsPLCd plus AD-PsGa1), in
which the HIS3 gene was transactivated, grew (Figure 4c, v).
This confirmed the interaction of PsPLCd and PsGa1 proteins.
The results from a b-galactosidase filter assay of colonies of
co-transformants (BD-PsPLCd plus AD-PsGa1) further confirmed the interaction between PsPLCd and proteins (Figure 4c, vi; blue colonies). The results clearly show that
PsPLCd interacts with PsGa1 in the yeast two-hybrid system.
All the other interaction images appeared the same, and
the results are summarized in Figure 4(d). The results
obtained were identical to those obtained in the in vitro assay.
In planta protein–protein interactions among PsGa1, PsGb
and PsPLC proteins by co-immunoprecipitation assay
The in vivo interactions between PsGa, PsGb and PsPLC
proteins were checked by an in planta co-immunoprecipitation assay. The results show that PsGa and PsGb proteins
interacted with each other as well as with the PsPLC protein
(Figure 4e,f). These results further confirmed that these
proteins interact in planta as well as in vitro.
Western analysis of various cell fractions using Ga and Gb
antibodies shows that pea Ga and Gb are present in cytosolic
fractions in addition to their presence in nuclear and
microsome fractions (Figure 4g,h).
GTPase activity of PsGa1 and its stimulation by PsPLC
Both the in vitro and ex vivo as well as in planta interactions
showed that PsGa and PsGb interact with PLC. Therefore, it
is worth checking the GTP hydrolysis (GTPase) activity of
PsGa1 protein, and whether the above interaction has any
effect on this activity. The results showed that PsGa1 protein
has GTPase activity (Figure 3f, lane 3), but, as expected,
recombinant PsGb protein (lane 1) did not show any such
activity. To check whether the interaction of PsPLCd with
PsGa1 has any effect on the GTPase activity of PsGa1 protein, PLCd protein was pre-incubated with PsGa1 protein in
the GTPase assay. The results showed that PsPLCd protein
(lane 2) has no GTPase activity itself, but that it stimulated
the GTPase activity of PsGa1 protein (lane 5), while the PsGb
protein has no effect on the GTPase activity of PsGa1 protein
(lane 4). Figure 3(g) shows the quantitative estimation of the
enhancement of GTPase activity of PsGa1 protein shown in
Figure 3(f). The stimulation of GTPase activity of PsGa1
protein by PLC protein was found to be specific and reproducible at a statistically significant level (P < 0.0005) (Figure 3g). As the PsGa1, PsGb and PsPLCd proteins were
purified using a similar method but only PsGa1 showed
GTPase activity, PsGb (lane 1) and PsPLCd (lane 2) proteins
serve as negative controls for this assay.
PsGa1 and PsGb are induced by salinity, heat and H2O2
stresses in pea
In order to study the effect of salinity, heat and H2O2 stresses
on expression of the PsGa1 and PsGb genes, 7- to 8-day-old
pea seedlings were exposed to 300 mM NaCl (for 3 and 6 h),
heat (37C and 42C for 6 h) or H2O2 (100 nM for 90 or
180 min), and the transcript levels in the leaf tissue were
analyzed. Significant induction of PsGa1 and PsGb mRNAs
was observed after 3 h exposure to 300 mM NaCl, 6 h
exposure to 42C temperature, and 180 min exposure to
H2O2 stresses (Figure 5a,b, lanes 2, 5 and 7, respectively),
compared to control (without any stress treatment) (lane 1).
No induction was seen after 6 h exposure to NaCl (lane 3)
and less induction was seen at 37C (lane 4). In case of H2O2,
at 90 min exposure only the PsGa1 transcript level was significantly induced, while that of PsGb was not (lane 6).
Functional validation of G-proteins by analyzing sense
transgenic plants
To establish the functional significance of PsGa1, PsGa2 and
PsGb genes, the complete ORFs of these genes were separately cloned in pBI–121 vector in the sense orientation, and
transformed into tobacco plants using Agrobacteriummediated transformation. The schematic representation of
the sense constructs, confirmation of transgenic plants (T0)
by GUS assay, and results of PCR, Southern and Western
analyses are shown in Supplementary Figure S1. From this
analysis, plants with a single copy of the transgene and
those over-expressing proteins were selected for further
studies. The wild-type (WT) tobacco plants did not show
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
662 Shikha Misra et al.
cross-reactivity with pea protein antibodies. This could be
due to the very low levels of native G proteins present in
tobacco. In sense plants, the gene integration was found to
be stable and hence protein expression was present in the T1
generation. In general, the morphological and growth
characteristics of T0 generation transgenic tobacco plants
were similar to those of untransformed plants (WT).
Tolerance of T0 transgenic plants to salinity stress
Figure 5. Transcript analysis of PsGa1 and PsGb genes in the presence of
abiotic stress.
(a,b) Transcript levels of (a) the PsGa1 gene and (b) the PsGb gene in control
tissue (lane 1), tissue treated with 300 mM NaCl for 3 h (lane 2), tissue treated
with 300 mM NaCl for 6 h (lane 3), tissue treated at 37C for 6 h (lane 4), tissue
treated at 42C for 6 h (lane 5), tissue treated with 100 nM H2O2 for 90 min
(lane 6) and tissue treated with 100 nM H2O2 for 180 min (lane 7).
To test for salinity tolerance, leaf disks from all three lines
(S1, S2 and S3) of T0 transgenic plants and WT tobacco
were floated separately on 0, 150 or 300 mM NaCl for 72 h.
Salinity-induced loss of chlorophyll was lower in Ga1- and
Ga2-over-expressing lines (sense) compared with that in
the WT (Figure 6a,b). However, Gb-over-expressing lines
(sense) showed no difference when compared to WT plants
(Figure 6c). The damage caused by stress was reflected in
the degree of bleaching (yellow color) observed in leaf
tissue after 72 h. It was evident that Ga1 and Ga2 transgenic tobacco plants have a better ability to tolerate salinity
stress, while Gb-over-expressing lines cannot tolerate the
salinity stress. Plants transformed with antisense constructs did not show altered salinity tolerance (data not
shown).
Figure 6. Leaf disk senescence assay for salinity and heat tolerance in transgenic plants.
(a–c) Chlorophyll content determined by leaf disk assay from wild-type (WT) and three different sense plants (S1, S2 and S3 lines) of (a) Ga1-over-expressing, (b)
Ga2-over-expressing and (c) Gb-over-expressing plants after induction in 0, 150 and 300 mM NaCl solutions.
(d–f) Chlorophyll content determined by leaf disk assay from WT and three different sense plants (S1, S2 and S3 lines) of (d) Ga1-over-expressing, (e) Ga2-overexpressing and Gb-over-expressing plants after induction in water at room temperature, 37 and 42C. In all the cases, the assay was performed in more than one
sample. Data are means SEM.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
Stress tolerance by pea heterotrimeric G-proteins 663
Tolerance of T0 transgenic plants to high temperature stress
To test for heat tolerance, leaf disks from all three lines (S1,
S2 and S3) of T0 transgenic and WT tobacco plants were
floated separately on water and incubated at 37C, 42C or
room temperature (RT). Heat-induced loss of chlorophyll
was lower in lines over-expressing Ga1, Ga2 and Gb compared with that from WT plants (Figure 6d–f). The damage
caused by stress was reflected in the degree of bleaching
(yellow color) observed in leaf tissue after 72 h. The results
clearly show that Ga1-, Ga2- and Gb-over-expressing lines
can tolerate the heat stress better than untransformed
plants. Plants transformed with antisense constructs did not
show altered heat tolerance (data not shown).
Analysis of T1 transgenic progeny
When the seeds from the T0 sense plants of Ga1, Ga2 and Gb
were plated onto kanamycin-containing medium, the segregation ratio was found to be in agreement with the
Mendelian ratio, i.e. 3:1 (Kanr/Kans). The presence of the
transgene on the T1 seedlings from each line was further
confirmed by PCR and the GUS assay (data not shown). As
mentioned above under heading ‘functional validation of
G-proteins’, the T1 transgenic plants were found to contain
the corresponding protein (Supplementary Figure S1). Leaf
disk senescence assays for salinity and heat tolerance of
T1 transgenic plants and WT tobacco were also performed.
Overall the results were similar to those for the T0 transgenic
plants, and clearly showed that over-expressing Ga1 and
Ga2 resulted in tolerance to both salinity and heat stresses,
while Gb-over-expressing transgenic plants showed tolerance to only high-temperature stress. Morphologically,
there was not much difference between the T1 generation of
Ga1, Ga2 and Gb tobacco transgenics and WT plants in
terms of height, chlorophyll content, flowering and seed
weight per pod.
Germination of T1 seeds and growth of the plants under
salinity and heat stress
The T1 Ga and Gb transgenic lines and WT tobacco seeds
germinated and grew normally in water (Figure 7, row 1). To
assess the effect of high salt and heat on the seed germination/growth of over-expressing Ga and Gb plants (T1) and
the kanamycin-positive T1 seedlings were characterized. In
the presence of salinity (150 or 300 mM NaCl), seeds of WT
and Gb-over-expressing plants showed no germination (or
only very slow germination), while seeds of Ga1 and Ga2over-expressing plants showed normal germination and the
plants did not develop any sign of stress (Figure 7, rows 2
and 3). In the presence of heat (37 or 42C), the WT showed
no germination (or only very slow germination), while seeds
from Ga1-, Ga2- and Gb-over-expressing plants showed
Figure 7. Germination pattern of T1 generation seeds. Germination of seeds
of transgenic tobacco sense plants over-expressing PsGa1 (a1S), PsGa2 (a2S)
and PsGb (bS) genes in water or NaCl (150 or 300 mM) or at high temperatures
(37 and 42C). Seeds from wild-type plants were also germinated under
similar conditions. Seeds were washed, spread on an autoclaved Whatman
disc number 1 and kept under the required conditions (http://www.vgdusa.
com/qualitative_filter_papers.htm). Statistically similar results were obtained
for the seven transgenic lines. These images are representative of one of the
lines for each gene. The water treatment was control for both salt and heat
stress. Salt stress was administered at room temperature, and water
treatment at room temperature was the control. Heat treatment was administered by germinating seeds in water at 37 or 42C, so water treatment at
room temperature was also the control for heat treatment.
normal germination and the plants did not develop any sign
of stress (Figure 7, rows 4 and 5). Statistically similar results
were obtained for the seven transgenic lines. Figure 7 shows
representative results for one of the lines. Quantitative
values as percentage germination for the experiment shown
in Figure 7 are shown in Supplementary Table S1.
Discussion
In higher plants, heterotrimeric G-proteins have been
implicated as involved in diverse signaling processes, but
the molecular mechanisms of their functions and their role
in abiotic stress signaling, especially as a result of salinity
and heat stress, are largely unknown. In this study, we have
isolated and characterized all three G-protein subunits and
the GPCR from a legume (pea), and show a novel role for
G-proteins in salinity and heat-stress signaling, and also
report possible cross-talk between G-proteins and PLC.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
664 Shikha Misra et al.
There are some changes in the sequence of Ga subunits
(PsGa1 and PsGa2) from pea as reported in this study
compared with those previously reported (PGA1 and PGA2;
Marsh and Kaufman, 1999). The difference between the pea
sequences was in eight amino acids, and this may be due to
presence of different isoforms of Ga in pea or may be an
ecotypic difference. Similar to the mammalian Ga, Gb and
Gc subunits and GPCR, the pea counterparts contained all
the conserved domains. The PsGa subunit also contained
Gly2, which could be used as another palmitoyl residue and
thereby may help it to anchor to the membrane as reported
for mammalian Gas (Kleuss and Krause, 2003). The significant homology of PsGa and PsGb with the reported
sequences from other plants suggests that the G-proteins
have been conserved during evolution. However, the PsGc1
and PsGc2 subunits of pea are unique and show less
homology with Arabidopsis (Mason and Botella, 2001) and
rice (Kato et al., 2004). In both Ga and Gb subunits, variable
regions can be seen in the N-termini, which are thought to
serve as the receptor binding and a/bc subunit binding
domains, and might lead to the transduction of signals via
G-proteins from various receptors to different targets.
Regarding GPCR, only one gene has been observed in pea,
similar to that reported in Arabidopsis (GCR1) (PlakidouDymock et al., 1997). The human genome contains about
1000 GPCRs, which are known to be involved in many signal
transduction pathways (Fredriksson and Schioth, 2005). The
fact that only one GPCR gene is found in pea and Arabidopsis suggests that, in plant evolution, the GPCR signaling
pathway has not diversified to the same extent as it has in
animals (Colucci et al., 2002). In fact, different evolutionary
lines have been shown in the GPCR family for Arabidopsis
and rice (Perez, 2005).
Interaction studies revealed that the pea GPCR protein
interacted with all three subunits of pea G-proteins as well as
forming an oligomer by self-interacting. The Ga and Gb
subunits, which are also present in the cytosol (Figure 2g,h),
may be responsible for interacting with GPCR. The Ga and
Gb subunits are also found to be present in the nucleus and
microsome fractions. The presence of Gb in the nucleus has
been shown previously (Peskan and Oelmuller, 2000). The
role of these proteins in the nucleus, if any, needs to be
determined in future studies. Previously, the Arabidopsis
GCR1 has also been reported to interact with Ga (GPA1) and
thereby regulate abscisic acid signaling (Pandey and
Assmann, 2004). The oligomeric nature of pea GPCR may
allow for a more complex ligand–receptor relationship (Park
and Palczewski, 2005). Our studies confirm that the Ga and
PLCd proteins from pea interact with each other; moreover,
this interaction was further mapped to the C-terminal
domain (C2 domain) of pea PLCd. In the animal system,
interaction between Gaq and PLCb has been reported
(Berstein et al., 1992) and has been mapped to its C-terminus
(Kim et al., 1996). Our results also show that the PsGb
protein interacts with PLCd protein but not with its C2
domain.
As an essential feature of the Ga subunit, the PsGa1
protein also contains GTPase and GTP-binding activities,
while other subunits and the GPCR show no such activities
(Iwasaki et al., 1997; Wise et al., 1997). Interestingly, we
found that pea PLCd protein stimulates the GTPase activity
of PsGa1, while PsGb protein had no such effect. This clearly
suggests that PLCd is one of the effector molecules of the Ga
subunit. In the animal system, it has been reported that PLCb
has the ability to activate the intrinsic GTPase activity of Gaq
(Berstein et al., 1992). In plants, only the PLCd isoform has
been cloned, and has been found to stimulate Ga GTPase
activity, suggesting the possibility of cross-talk between
them. In an earlier study, involvement of PLC in the GCR1and GPA1-mediated regulation of DNA synthesis was
reported in Arabidopsis (Apone et al., 2003). However, no
direct interaction was shown. Whether this interaction has
any effect on the activity of PLC still needs to be investigated.
The PsPLC that we have used in the study did not show
enzyme activity under the conditions tested (Venkataraman
et al., 2003). It is possible that this form may require
activation by G-proteins, and its role may also be to activate
the GTPase activity of Ga. Further work needs to be done to
determine the functional significance of this interaction in
plant cell signaling. It has been reported in plants that
G-protein activation stimulated phospholipase D (PLD) signaling (Munnik et al., 1995). However, it was found later that
Arabidopsis PLDa1 interacts with the G-protein a subunit
through a motif analogous to the DRY motif in GPCR (Zhao
and Wang, 2004). Thus, it seems that G-protein signaling in
plants can be transduced via both the PLC and PLD
pathways. In studying the regulation of Ga and Gb, we
observed the induction of pea Ga mRNA in response to
salinity and high temperature, while Gb mRNA was upregulated only in response to heat stress. The role of Arabidopsis G-proteins in abiotic stresses such as ABA and drought
tolerance (Chen et al., 2006) and in the oxidative stress
response to ozone (Joo et al., 2005) has been reported;
however, regulation of these genes in response to salinity
and heat stress has not been reported previously.
Interestingly, the pea Ga and Gb transcripts were also
upregulated by hydrogen peroxide (H2O2) treatment of pea
seedlings. H2O2, in addition to being a toxicant, is now
considered to be a signaling molecule and a regulator of the
expression of some genes encoding antioxidants, cell
rescue/defense proteins, and signaling proteins such as
kinase, phosphatase and transcription factors (Fedoroff,
2006). It has been shown that null mutations in the genes
encoding the a and b subunits of G-proteins, as compared to
wild type without any mutations, are more sensitive to
oxidative damage, and an early component of the oxidative
burst requires signaling via the G-protein heterotrimer (Joo
et al., 2005). It has also been shown that H2O2 signaling may
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
Stress tolerance by pea heterotrimeric G-proteins 665
regulate the functions of heat shock transcription factors,
thus inducing the expression of oxidative stress response
genes (Miller and Mittler, 2006). These studies show that
H2O2 could be one of the molecules used in the transduction
of stress signals for the alteration of expression profiles of
target genes (PsGa and PsGb).
To further evaluate the role of Ga and Gb in abiotic
stresses, particularly salinity and high temperature, we
determined that Ga-over-expressing plants showed tolerance to high salinity and high temperature. This was
indicated by the presence of higher chlorophyll content in
the leaf disks of salinity-stressed T0 and T1 transgenic plants,
whereas WT leaves become yellow. Similar results were
observed in transgenic tobacco plants (T0 and T1) overexpressing pea Gb, but only in response to heat stress. These
data suggest that tolerance in transgenics is conferred not
only by over-expression under the CaMV promoter but also
by regulating protein turnover under stress. These effects
seem to be related to over-expression of the proteins and are
not a result of the transformation event with the vector, as
the untransformed plants did not exhibit this effect. Interestingly, in pea, both PsGa and PsGb are induced in response
to salinity and higher temperature, but over-expression of
PsGb in tobacco plants confers only heat tolerance and not
salinity tolerance. This shows that, in addition to signaling
mediated via Gb, other pathways may be essential to confer
tolerance. It might be possible that calcium homeostasis
regulated via Ga/PLC is needed for salinity tolerance, a
process that may not be regulated via the Gb signaling
pathway. Moreover, the T1 seedlings were able to grow in
the continuous presence of salinity stress (over-expressing
Ga) and heat stress (over-expressing Ga and Gb). A similar
type of effect was not observed in plants with antisence
constructs of PsGa and PsGb genes (data not shown). These
results also indicate that the introduced trait is functional in
transgenic plants and that it is stable. The exact mechanism
of G-protein-mediated salinity and heat stress tolerance is
not understood. Whether the effect is due to salt (ionic) or
osmotic effects needs to be studied further. Recently, overexpression of a regulator of G-protein signaling has been
shown to confer drought tolerance (Chen et al., 2006).
In this paper, we provide direct evidence for the involvement of Ga and Gb subunits in conferring salinity and heatstress tolerance in transgenic tobacco plants, thus suggesting
a previously undescribed pathway for manipulating
salinity and heat-stress tolerance. Our results indicate that
the Ga-mediated pathway is responsible for conferring
salinity and high-temperature stress tolerance, whereas the
pathways triggered by Gb lead to heat tolerance. Abiotic
stress generates signals that are perceived by either GPCR or
osmotic sensors present in the cell membrane, and this leads
to activation of the G-protein (i.e. dissociation of the Ga and
Gbc dimers) and hence regulation of downstream effectors.
It is possible that Ga-mediated signaling involves the
activation/modulation of downstream effectors conferring
salinity and heat tolerance. One of the effector molecules
may be PLC, and this pathway may lead to an increase in
calcium in addition to the activation of other pathways. The
burst of calcium may lead to the activation of downstream
calcium-dependent pathways. In fact, the role of calcium in
salinity-stress tolerance has been elucidated via a number of
mechanisms (Mahajan et al., 2006). It is possible that PLC
could be involved in salinity tolerance. Thus it seems that
cross-talk between Ga and PLC may be an important step in
transducing signals that lead to salinity tolerance, and also in
regulating Ga-mediated pathways. PLC can turn the signal
off by activating the GTPase activity of Ga. The Gb-mediated
signal transduction leads only to high-temperature tolerance, and therefore it seems that pathways triggered by Gb
may not be involved in maintaining sodium homeostasis.
Further studies are needed to determine the exact mechanism by which Gb provides heat tolerance for plants. The set
of proteins that are regulated in the transgenic plants in
response to salinity and heat stress and their involvement in
tolerance require further study. Overall, the discovery of the
novel role of G-proteins in salinity and heat-stress tolerance
and the cross-talk between Ga and PLC makes an important
contribution to our better understanding of G-proteinmediated signaling pathways and abiotic stress signaling/
tolerance in plants.
Experimental procedures
Plant growth and treatment
Pea (Pisum sativum) seeds were surface-sterilized in a solution of
100% Clorox plus 0.05% Triton X–100 (Sigma, www.sigmaaldrich.
com) for 10 min, washed with sterilized water three times, and
incubated in sterilized water for at least 4 h. These pre-soaked seeds
were germinated in sterilized wet vermiculite under a 14 h/10 h
light/dark cycle at 25C for 7 days. After 7 days, leaves were frozen
in liquid nitrogen just before RNA was isolated.
Construction of P. sativum cDNA library
A cDNA library was constructed from 5 lg of poly(A)+ RNA (isolated
from the top four leaves of 7-day-old pea seedlings) in Uni-Zap XR
vector using a Zap cDNA synthesis kit (Stratagene; http://
www.stratagene.com/) according to the manufacturer’s protocol.
The resulting phage library contained 1 · 109 plaque-forming units
per ml. During library construction, some double-stranded cDNAs
were also synthesized from pea seedlings for use as a template for
real-time polymerase chain reaction cloning of cDNAs of G-protein
a1 and a2 subunits.
Cloning of cDNAs of G-protein a1, a2, b, c1 and c2 subunits
from pea
For cloning of pea G-protein a1 and a2 subunits, all the known
sequences of Ga genes were first aligned, and primers were
designed from the 5¢ UTR and 3¢ UTR regions of the most conserved
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
666 Shikha Misra et al.
areas. For the G-protein a1 subunit (PsGa1), the primer pair 5¢-ATGGGCTTACTCTGTAGCAAA-3¢ (Oligo-1, forward) and 5¢TCATTACAAGCCAGCCTCAAA-3¢ (Oligo-2, reverse) was used for
PCR. For the G-protein a2 subunit (PsGa2), the primer pair 5¢-ATGGGCTTAGTCTGTAGCAGA-3¢ (Oligo-3, forward) and 5¢TCATAATAATCCCGCTTCAAA-3¢ (Oligo-4, reverse) were used for
PCR. In PCR reactions, using the respective primer pairs and pea
double-stranded cDNAs as template, fragments of 1155 bp for the
G-protein a1 and a2 subunits were amplified and cloned into
pGEMT vector.
For cloning of the Gb subunit, the pea cDNA library was screened
using a radiolabeled tobacco Gb subunit as probe, kindly provided
by Dr R. Oelmuller (Ludwig-Maximilians-Universitat, Jena,
Germany). Various clones were obtained after tertiary screening
of the library. A clone of 1134 bp was used for further study.
For cloning of the Gc subunit, the pea cDNA library was screened
using a different approach (far-Western) as described below. After
induction of the pea cDNA library with IPTG, plaques were picked on
Hybond-C membrane (Amersham Biosciences, www.amersham.
com/business/Biosciences) and incubated with Gb subunit. The
membranes were then washed and incubated with Gb antibodies
followed by a standard Western blot procedure. Various interacting
plaques were selected in the primary screening, and plaque-purified
after secondary and tertiary screenings. Two clones, i.e. PsGc1
(345 bp) and PsGc2 (303 bp), were obtained, which showed some
homology with Arabidopsis AGG1 and AGG2, respectively.
cDNA sequencing was done by Macrogen, Korea (http://
www.macrogen.com/eng/sequencing/dua.jsp) using Sanger’s
method. Most of the routine sequence (DNA and amino acid)
analysis was performed using MACVECTOR (version 7; Oxford
Molecular Group). A homology search was performed using BLAST
(NCBI), http://www.ncbi.nlm.nih.gov/BLAST, and multiple sequence
alignment was performed using CLUSTALW alignment using
MACVECTOR software only.
Isolation of pea GPCR cDNA clone
For cloning of pea GPCR (PsGPCR), the pea cDNA library was
screened using heterologous, radiolabeled Arabidopsis GCR1 as
probe (kindly provided by Dr Sarah M. Assmann and Dr Sona
Pandey of Pennsylvania State University, Philadelphia, USA).
Various clones were obtained and plaque-purified after tertiary
screening of the library. A clone of 1008 bp in size was studied
further. After screening, the cDNA was sequenced and analysed as
described above.
Construction of plasmids for expression of PsGa1, PsGa2,
PsGb and PsGc1 subunits of G-Proteins and PsGPCR
The coding region of the PsGa1 subunit (1.15 kb) was amplified by
PCR and cloned in-frame into the NheI and XhoI sites of the pET28a
vector (+) (Novagen, www.emdbiosciences.com/html/NVG/
home.html). The primers for the Ga1 subunit were 5¢CTAGCTAGCATGGGCTTACTCTGTAGCAAA-3¢ (Oligo-5, forward)
containing an NheI site (underlined) and 5¢-GGCCTCGAGTCATTACAAGCCAGCCTCAAA-3¢ (Oligo-6, reverse) containing
an XhoI site (bold). This resulted in the construction of plasmid
pET28a-Ga1, and the sequence was verified before using for protein
expression.
The coding region of the PsGa2 subunit (1.15 kb) was amplified
by PCR and cloned in-frame into the NheI and XhoI sites of the
pET28a vector (+). The primers for the Ga2 subunit were 5¢CTAGCTAGCATGGGCTTAGTCTGTAGCAGA-3¢ (Oligo-7, forward)
(NheI site underlined) and 5¢-GGCCTCGAGTCATAATAATCCCGCTTCAAA-3¢ (Oligo-8, reverse) (XhoI site underlined). This
resulted in the construction of plasmid pET28a-Ga2, and the
sequence was verified before using for protein expression.
The coding region of the PsGb subunit (1134 bp) was amplified by
PCR and cloned in-frame into the NdeI and XhoI sites of the pET28a
vector (+). The primers were 5¢-GGAATTCCATATGTCCGTTGCGGACGTCAAA-3¢ (Oligo-9, forward) (NdeI site underlined) and
5¢-GGCCTCGAGTCAAATCACCTTCCTATGCCC-3¢ (Oligo-10, reverse)
(XhoI site underlined). This resulted in the construction of plasmid
pET28a-PsGb, and the sequence was verified before using for
protein expression.
The coding region of the PsGc1 subunit (342 bp) was amplified by
PCR and cloned in-frame into the NdeI and XhoI sites of the pET28a
vector (+). The primers were 5¢-GGAATTCCATATGATGCGCCTTTTCAATGTAGTGTAC-3¢ (Oligo-11, forward) (NdeI site underlined)
and 5¢-GGCCTCGAGTCACACGATCAGACAAACC-3¢ (Oligo-12,
reverse) (XhoI site underlined). This resulted in the construction of
plasmid pET28a-PGG1, and the sequence was verified before using
for protein expression.
The coding region of the PsGPCR (1008 bp) was restricted from
the pBSK vector using the EcoRI and NotI sites of the vector, and
cloned in-frame into the EcoRI and NotI sites of the pET28a vector
(+). This resulted in the construction of plasmid pET28a-PsGPCR,
and the sequence was verified before using for protein expression.
In all cases, the expressed proteins contain a His-tag at the
N-terminal region.
Expression and purification of Ga1, Ga2, Gb, Gc1 and GPGR
proteins
The plasmids pET28a-PsGa1, pET28a-PsGa2, pET28a-PsGb,
pET28a-PsGc1 and pET28a-PsGPCR were transformed separately
into Escherichia coli BL21 (DE3) plysS cells (Novagen). Fresh cultures of the E. coli containing the foreign gene were grown in LB
medium containing 50 lg ml–1 of kanamycin until the A600 reached
0.6, induced by IPTG (0.9 mM) and harvested by centrifugation
(2560 g). All the purification steps were performed at 4C. The
resulting pellet was resuspended in ice-cold Tris buffer, and lysed
by the freeze–thaw method according to the instructions provided
by the manufacturer. The cell lysate was centrifuged at 10 000 g for
10 min at 4C. As pET28a-PsGa1, pET28a-PsGa2, pET28a-PsGb,
pET28a-PGc1 and pET28a-PsGPCR were found to be present in the
soluble fraction, the recombinant proteins were purified to homogeneity from the resulting supernatant using nickel–NTA column
chromatography according to the manufacturer’s instructions
(Qiagen; http://www.qiagen.com/).
GTP-binding assay for PsGa1 protein
A 500 ng sample of Ga1 protein and BSA (as negative control),
together with pre-stained marker, were electrophoresed on a 12%
SDS–PAGE gel and transferred onto a nitrocellulose membrane.
The proteins on the membrane were denatured and renatured by
incubating in 6 M guanidine–HCl (made up in HSM buffer 1: 25 mM
HEPES–KOH, pH 7.7, 25 mM NaCl, 5 mM MgCl2) twice for 5 min at
4C. This was followed by incubation for six periods of 10 min in a
serial dilution (1:1) of denaturation buffer in HSM buffer 2 (containing 1 mM DTT) at 4C. The membrane was blocked in HSM
buffer 1 containing 1 mM DTT, 0.5% NP-40 and 5% milk for 60 min at
4C, followed by washing the membrane twice in the same solution
containing 1% milk. [c35S]GTP (DuPont NEN, http://www.las.
perkinelmer.com) was used for the GTP binding assay. ‘Cold’ GTP
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
Stress tolerance by pea heterotrimeric G-proteins 667
(50 lg, Sigma; http://www.sigmaaldrich.com/) and ‘hot’ [c35S] GTP
(50 lCi) were mixed in 5 ml of HSM buffer 2 and overlaid on the
membrane for 4 h at 4C with gentle shaking, followed by washing
the membrane in HSM buffer 2 for three periods of 10 min at 4C,
and exposed to film.
GTPase assay for PsGa 1 protein
[c32P]GTP (DuPont NEN) was used for GTPase assay. A 1 lg aliquot
of purified protein (Ga, Gb and PLC separately or combined) was
used in 10 ll reaction volume, which included c32pGTP (0.2 lCi),
5 mM GTP and reaction buffer (20 mM Tris–HCl pH 8.0, 1 mM MgCl2,
150 mM KCl, 8 mM DTT, 4% sucrose, 80 lg ml–1 BSA). The reaction
was incubated at 37C for 2 h, and 1 ll of the reaction mixture was
applied to the TLC plate. After drying, the TLC was chromatographed in buffer consisting of 1 M HCOOH (formic acid) with 0.5 M
LiCl. The chromatograph was stopped just before the solution
reached the top of TLC plate. The plate was dried in air, and exposed
to X-ray film overnight at room temperature.
In vitro protein–protein interactions among G-proteins, PLC
and GPCR
Briefly, one protein was immobilized on Ni+–NTA beads in the
native form. In the case of denatured proteins (Gb, PLC and the C2
domain of PLC), it was first refolded on the column by washing with
buffer with a gradually decreasing amount of urea until no urea was
present. The second protein was produced as 35S-labeled by transcription and translation with TnT wheatgerm extract. The radiolabeled second protein was incubated with the first protein (bound
to the Ni+–NTA beads) at 4C for 2 h, in 500 ll binding buffer containing 20 mM HEPES–KOH, pH 7.7, 75 mM KCl, 0.1 mM EDTA,
2.5 mM MgCl2, 1% milk, 1 mM PMSF, 1 mM DTT and 0.05% NP-40.
The beads were packed in a mini-column and the column was
washed three times with 1 ml of washing buffer (same as binding
buffer except it contained 100 mM KCl). One-tenth of the beads were
immediately boiled in 2· SDS loading buffer, and subjected to SDS–
PAGE. The second protein was eluted from the remainder of the
beads using binding buffer containing 500 mM KCl. The eluted
fractions were subjected to SDS–PAGE. The gel was fixed, dried and
exposed to X-ray film overnight at room temperature.
coding region of PLC (1785 bp) was amplified by PCR with primers
harboring restriction sites, and cloned in-frame into the EcoRI and
BglII sites of the binding domain vector pGBDC1. The coding
region of PsGPCR (1008 bp) was amplified by PCR with primers
harboring restriction sites, and cloned in-frame into the EcoRI–PstI
site of binding domain vector pGBKT7 and the EcoRI–XbaI site of
activation domain vector pGADT7.
To check the interactions among G-proteins, PsGPCR and PLC,
AD and BD vectors containing the desired plasmids were cotransformed into yeast strain AH109 harboring two reporter genes
(HIS3 and b-galactosidase) by the lithium acetate method. AH109
contains integrated copies of ADE2, HIS3 and lacZ (MAL1) reporter
genes under the control of distinct GAL4 upstream activating
sequences (UAS) and TATA boxes. These promoters yield strong
and very specific responses to GAL4. Yeast cells carrying both the
plasmids were selected on the synthetic medium lacking Leu and
Trp (SD-Leu–Trp-). The yeast cells were then streaked onto a SD
medium (Leu), Tryp), His)) containing 15 mM 3-AT to determine
expression of the HIS3 nutritional reporter. The b-galactosidase
expression of the His+ colonies was analyzed by filter-lift assays as
described by the manufacturer (Clontech).
In planta co-immunoprecipitation assay
The in vivo interactions between PsGa, PsGb and PsPLC were
checked by in planta co-immunoprecipitation assay using wholecell extracts isolated from pea leaves as described previously
(Yadav et al., 2002). The whole-cell extracts were incubated with IgG
isolated from polyclonal antibodies against PsGa and PsGb proteins
in two different tubes. The immune complex was pulled by protein A–Sepharose. The bound proteins were eluted using high-salt
(500 mM NaCl) buffer and subjected to SDS–PAGE (in triplicate).
Western blotting was performed using PsGa, PsGb and PsPLC
antibodies, respectively.
Northern analysis
Total RNA was isolated by a standard method as described in
Pham et al., (2000). About 30 lg of total RNA samples resolved on
formaldehyde–agarose gels (1.0%) were transferred onto Hybond-N
membranes (Amersham; http://www5.amershambiosciences.com/)
and hybridized with 32P-labeled PsGa or PsGb cDNA (ORF region)
probes as described previously (Mahajan et al., 2006).
Yeast two-hybrid assay to study the interaction of proteins
A Gal4-based two-hybrid system was used as described by the
manufacturer (Clontech; http://www.clontech.com/). The coding
region of the Ga subunit (1155 bp) was amplified by PCR with
primers harboring restriction sites, and cloned in-frame into the
SmaI and XbaI sites of the DNA activation domain vector pGADC1
(Clontech). This resulted in the vector pGADC1-PsGa, the
sequence of which was verified before using for yeast transformation. The coding region of the Gb subunit (1134 bp) was
amplified by PCR with primers harboring restriction sites, and
cloned in-frame into the EcoRI and BglII sites of the binding
domain vector pGBDC1. This resulted in the vector pGBDC1-PsGb,
whose sequence was verified before using for yeast transformation. The coding regions of the PsGc1 (345 bp) and PsGc2 (303 bp)
subunits was amplified by PCR with primers harboring restriction
sites, and cloned in-frame into the EcoRI and ClaI sites of the
activation domain vector pGADT7 ( Clontech). This resulted in the
vectors pGADT7-PsGc1 and pGADT7-PsGc2, the sequences of
which were verified before using for yeast transformation. The
Immunoblotting
Total soluble proteins from the tissue were separated by 12% SDS–
PAGE and analysed by Western blotting using antibodies against
PsGa or PsGb proteins (1:5000) as described previously (Pham et al.,
2000).
Raising of tobacco plants transgenic for PsGa1, PsGa2 and
PsGb genes
The complete ORFs of the PsGa1, PsGa2 and PsGb genes were
cloned into the XbaI site of the pBI-121 vector (Clontech) in the sense
orientation. This vector contains the gene of interest and the GUS
gene (uidA) under a single CaMV 35S promoter; however, a stop
codon has been inserted in between the inserted gene and the
reporter gene to avoid translational fusions. It also carries the NPTII
(kanamycin) gene as a selectable marker. Tobacco (Nicotiana
tabacum cv. Xanthi) leaf discs were transformed using a leaf disc
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
668 Shikha Misra et al.
transformation procedure with Agrobacterium tumefaciens
(LBA4404) containing the pBI-121 vector plus the gene of interest.
Putative T0 transgenic plants were regenerated from the callus in the
presence of kanamycin and further confirmed using PCR and GUS
assay (Jefferson et al., 1987). The seeds from these plants, i.e. T0
seeds, were germinated on kanamycin-containing medium to select
for transgenic T1 seedlings.
Stress treatments
Seven-day-old pea seedlings were transferred into NaCl (300 mM)
for 3 or 6 h, into H2O2 (100 nM) for 90 or 180 min, or to 37 or 42C
temperatures for 6 h. Young leaves of the stressed seedlings were
harvested after the indicated period. Seedlings grown in water
were used as a control. To monitor stress effects WT and kanamycin-positive T0 transgenic seeds were directly germinated on
agar plates prepared in MS medium (Sigma) supplemented with
150 or 300 mM NaCl for salinity stress, or on plates without supplemented NaCl for heat stress. For heat stress, the seeds were
allowed to germinate at 37 or 42C. The stressed seedlings were
later transferred to earthen pots and grown in a greenhouse (16 h
light/8 h dark, 25 2C), and watered fortnightly with a 200 mM
NaCl solution.
Leaf disk assay of Ga and Gb transgenic plants
Leaf disks of 1.0 cm diameter were excised from healthy and fully
expanded tobacco leaves of similar age from transgenic and WT
plants (60 days old). The disks were floated in a 6 ml solution of
NaCl (150 or 300 mM) or water (experimental control) for 72 h (Fan
et al., 1997). For heat-stress treatments, the leaf disks were floated in
6 ml of water and kept at 37 or 42C or room temperature (experimental control) for 72 h. The disks were then used for measuring
chlorophyll a and b spectrophotometrically after extraction in 80%
acetone. The NaCl and water treatments were carried out in continuous white light at 25 2C. The experiment was repeated at
least three times with the transgenic lines over-expressing Ga1, Ga2
and Gb.
Acknowledgements
We thank Dr Renu Tuteja and Dr Andre T. Jagendorf (Cornell
University, Ithaca, NY, USA) for helpful comments and scientific
corrections, and Ms Suzanne Kerbavcic (ICGEB, Trieste, Italy) for
grammatical corrections to the manuscript. Work in N.T.’s laboratory on G-proteins signaling is partially supported by Department
of Science and Technology, Government of India. We acknowledge the Council of Scientific and Industrial Research, New Delhi
for a fellowship to S.M. We also thank Vijaykanth, Sachin K. Gupta
for help with technical help in developing transgenic plants,
Gopal Krishna Pattanayak (JNU) for help with Northern blots,
Dr Tanushri Kaul for providing the PLC antibody and Mr Dang
Quang Hung for help with preparation of the illustrations.
Supplementary Material
The following supplementary material is available for this article
online:
Figure S1. Analysis of transgenic tobacco plants to confirm the
presence, integration and expression of the genes.
Table S1. Percentage germination of seedlings under stress
conditions.
References
Apone, F., Alyeshmerni, N., Wiens, K., Chalmers, D., Chrispeels,
M.J. and Colucci, G. (2003) The G-protein-coupled receptor GCR1
regulates DNA synthesis through activation of phosphatidylinositol-specific phospholipase C. Plant Physiol. 133, 571–579.
Arabidopsis Genome Initiative (2000) Analysis of the genome
sequence of the flowering plant Arabidopsis thaliana. Nature,
408, 796–815.
Berstein, G., Blank, J.L., Jhon, D.Y., Exton, J.H., Rhee, S.G. and
Ross, E.M. (1992) Phospholipase C-beta 1 is a GTPase-activating protein for Gq/11, its physiologic regulator. Cell, 70, 411–
418.
Chen, Y., Ji, F., Xie, H. and Liang, J. (2006) Overexpression of the
regulator of G- protein signaling protein enhances ABA-mediated
inhibition of root elongation and drought tolerance in Arabidopsis. J. Exp. Bot. 57, 2101–2110.
Colucci, G., Apone, F., Alyeshmerni, N., Chalmers, D. and
Chrispeels, M. J. (2002) GCR1, the putative Arabidopsis
G protein-coupled receptor gene is cell cycle-regulated, and its
overexpression abolishes seed dormancy and shortens time to
flowering. Proc. Natl Acad. Sci. USA, 96, 7575–7580.
Fan, L., Zheng, S. and Wang, X. (1997) Antisense suppression of
phospholipase D alpha retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves. Plant Cell,
9, 2183–2196.
Fedoroff, N. (2006) Redox regulatory mechanisms in cellular stress
responses. Ann. Bot. 98, 289–300.
Fredriksson, R. and Schioth, H.B. (2005) The repertoire of G-protein
coupled receptors in fully sequenced genomes. Mol. Pharmacol.
67, 1414–1425.
Gilman, A.G. (1987) G proteins: transducer of receptor-generated
signals. Annu. Rev. Biochem. 56, 615–649.
Hamm, H. (1996) The many faces of G-protein signaling. J. Biol.
Chem. 273, 669–672.
Iwasaki, Y., Kato, T., Kaidoh, T., Ishikawa, A. and Asahi, T. (1997)
Characterization of the putative a subunit of a heterotrimeric G
protein in rice. Plant Mol. Biol, 34. 563–572.
Jefferson, R.A., Kavanagh, T.A. and Bavan, M.W. (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion
marker in higher plants. EMBO J. 6, 3901–3907.
Jones, A. M. and Assmann, S. M. (2004) Plants: the latest model
system for G-protein research. EMBO Rep. 5, 572–578.
Joo, J.H., Wang, S., Chen, J.G., Jones, A.M. and Fedoroff, N.V.
(2005) Different signaling and cell death roles of heterotrimeric
G-protein a and b subunits in the Arabidopsis oxidative stress
response to ozone. Plant Cell, 17, 957–970.
Kato, C., Mizutani, T., Tamaki, H., Kumagai, H., Kamiya, T., Hirobe,
A., Fujisawa, Y., Kato, H. and Iwasaki, Y. (2004) Characterization
of heterotrimeric G protein complexes in rice plasma membrane.
Plant J. 38, 320–331.
Kim, H.Y., Cote, G.G. and Crain, R.C. (1996) Inositol 1,4,5-triphosphate may mediate closure of K+ channels by light and darkness
in Samanea saman motor cells. Planta, 198, 279–287.
Kleuss, C. and Krause, E. (2003) Gas is palmitoylated at the N-terminal glycine. EMBO J. 22, 826–832.
Ma, H., Yanofsky, M.F. and Meyerowitz, E.M. (1990) Molecular
cloning and characterization of GPA1, a G protein a subunit gene
from Arabidopsis thaliana. Proc. Natl Acad. Sci. USA, 87, 3821–
3825.
Mahajan, S., Sopory, S. and Tuteja, N. (2006) Cloning and characterization of CBL-CIPK signaling components from a legume
(Pisum sativum). FEBS J. 273, 907–925.
Marsh, J. F. and Kaufman, L. S. (1999) Cloning and characterization
of PGA1 and PGA2: two G protein a-subunits from pea that pro-
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669
Stress tolerance by pea heterotrimeric G-proteins 669
mote growth in the yeast Saccharomyces cerevisiae. Plant J. 19,
237–247.
Mason, M.G. and Botella, J.R. (2001) Isolation of a novel G-protein
c-subunit from Arabidopsis thaliana and its interaction with Gb.
Biochim. Biophys. Acta, 1520, 147–153.
Miller, G. and Mittler, R. (2006) Could heat shock transcription factors function as hydrogen peroxide sensors in plants? Ann. Bot.
98, 279–288.
Munnik, T., Arisz, S.A., Vrije, T.D. and Musgrave, A. (1995) G-protein
activation stimulates phospholipase D signaling in plants. Plant
Cell, 7, 2197–2210.
Pandey, S. and Assmann, S. A. (2004) The Arabidopsis putative G
protein-coupled receptor GCR1 interacts with the G protein a
subunit GPA1 and regulates abscisic acid signaling. Plant Cell, 16,
1616–1632.
Park, P.S-H. and Palczewski, K. (2005) Diversifying the repertoire of
G protein-coupled receptors through oligomerization. Proc. Natl
Acad. Sci. USA, 102, 8793–8794.
Perez, D.M. (2005) From plants to man: the GPCR ‘tree of life’. Mol.
Pharmacol. 67, 1383–1384.
Peskan, T. and Oelmuller, R. (2000) Heterotrimeric G-protein
b-subunit is localized in the plasma membrane and nuclei of
tobacco leaves. Plant Mol. Biol. 42, 915–922.
Pham, X.H., Reddy, M.K., Ehtesham, N.Z., Matta, B. and Tuteja, N.
(2000) A DNA helicase from Pisum sativum is homologous to
translation initiation factor and stimulates topoisomerase I
activity. Plant J. 24, 219–229.
Plakidou-Dymock, S., Dymock, D. and Hooley, R. (1997) A higher
plant seven-transmembrane receptor that influences sensitivity
to cytokinins. Curr. Biol. 8, 315–324.
Simon, M. I., Strathmann, M. P. and Gautam, N. (1991) Diversity of
G proteins in signal transduction. Science, 252, 802–808.
Ullah, H., Chen, J-G., Young, J., Im, K., Sussman, M. and Jones,
A.M. (2001) Modulation of cell proliferation by heterotrimeric G
protein in Arabidopsis. Science, 292, 2066–2069.
Venkataraman, G., Goswami, M., Tuteja, N., Reddy, M.K. and
Sopory, S.K. (2003) Isolation and characterization of a phospholipase C delta isoform from pea that is regulated by light
in a tissue specific manner. Mol. Genet. Genomics, 270, 378–
386.
Wang, X., Ullah, H., Jones, A. M. and Assmann, S.M. (2001) G
protein regulation of ion channel and abscisic acid signaling in
Arabidopsis guard cells. Science, 15, 2070–2072.
Weiss, C.A., Garnaat, C.W., Mukai, K., Huang, H. and Ma, H. (1994)
Isolation of cDNA encoding guanine nucleotide-binding protein
b-subunit homologues from maize (ZGB1) and Arabidopsis
(AGB1). Proc. Natl Acad. Sci. USA, 91, 9554–9558.
Wise, A., Thomas, P.G., Carr, T.H., Murphy, G.A. and Millner, P.A.
(1997) Expression of the Arabidopsis G-protein GPa1: purification
and characterization of the recombinant protein. Plant Mol. Biol.
33, 723–728.
Yadav, V., Kundu, S., Chattopadhyay, D., Negi, P., Wei, N., Deng,
X.-W. and Chattopadhyay, S. (2002) Light regulated modulation
of Z-box containing promoters by photoreceptors and downstream regulatory components, COP1 and HY5, in Arabidopsis.
Plant J. 31, 741–753.
Yang, Z. (2002) Small GTPases versatile signaling switches in
plants. Plant Cell, 14, S375–S388.
Zhao, J. and Wang, X. (2004) Arabidopsis phospholipase Da1
interacts with the heterotrimeric G protein a-subunit through a
motif analogous to the DRY motif in G protein-coupled receptors.
J. Biol. Chem. 279, 1794–1800.
The EMBL/GenBank accession numbers for the sequences isolated here are AF537218 (PsGa1 cDNA), AF533438 (PsGa2 cDNA),
AF145976 (PsGb cDNA), DQ010315 (PsGc1 cDNA), AY876935 (PsGc2 cDNA), DQ010316 (PsGPCR cDNA), AF533439 (PsGa2
genomic clone), AF533440 (PsGb genomic clone) and Y15253 (pea PLC).
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 656–669

Similar documents