Short CommuniCAtion
Plant Signaling & Behavior 6:8, 1079-1086; August 2011; ©2011 Landes Bioscience
Rice G-protein coupled receptor (GPCR)
In silico analysis and transcription regulation
under abiotic stress
Dinesh Kumar Yadav and narendra tuteja*
international Center for Genetic Engineering and Biotechnology; Aruna Asaf Ali marg; new Delhi, india
Key words: abiotic stress, G-protein coupled receptor, myristoylation, protein kinase C, real-time PCR, rice, signal transduction
Abbreviations: GPCR, G-protein coupled receptor; OsGPCR, GPCR of rice; PsGPCR, GPCR of pea; ZmGPCR, GPCR of maize;
GCR1, GPCR of Arabidopsis
the majority of transmembrane signal transduction in response to diverse external stimuli is mediated by G-protein
coupled receptors (GPCrs) and are the principal signal transducers. GPCrs are characterized by seven membranespanning domains with an extracellular n-terminus and a cytoplasmic C-terminus which functions along with GtPbinding protein in a highly coordinated fashion. role of heterotrimeric G-proteins in abiotic stresses has been reported,
but the response of GPCr is not yet well characterized. in the present study we report the isolation of one putative GPCr
(966 bp) from indica rice (Oryza sativa cv. indica group Swarna) and described its transcriptional regulation under abiotic
stresses. Amino acid sequence analyses shows the presence of typical heptahelical transmembrane spanning domains
with extracellular n-terminus involved in ligand binding and cytoplasm facing C-terminus that binds with heterotrimeric
G-protein. Sequence analysis also conirmed the presence of all signature motifs required for functional GPCr. Domain
and site prediction shows the presence of myristoylation sites for membrane association and protein kinase C sites
for its desensitization. the transcript levels of rice GPCr was induced following naCl and ABA treatments. however, in
drought condition the expression proile of GPCr upregulated during early exposure which subsequently decreased.
on the other hand it seems no signiicant efect due to cold and heat stress. these indings provide a direct evidence
for transcriptional regulation of rice GPCr under abiotic stress conditions. these indings also suggest that GPCr can be
exploited for promoting stress tolerance in plants.
Introduction
A prerequisite for the maintenance of homeostasis in a living
organism is fine tuned communication between cell and environment. It helps the cells to sustain in unfavorable environment and
develop tolerance against stress conditions.1-3 One of the primary
sensing mechanisms used by metazoans involves GPCR signaling
cascades. Basic cell signaling machinery is composed of a signaling triad (receptor/transducer/effector). These cascades are composed of, at the most simplistic level, a plasma membrane localized
stimulus-sensing GPCR that transduces the extra-cellular signal
to an intracellular heterotrimeric G-protein complex, thereby
activating downstream signaling cascades. GPCRs interact with
a complex containing a GTPase, called heterotrimeric G-proteins
(Gαβγ) that form classical signal transduction complexes conserved in all eukaryotes.4-6 The heterotrimeric G-protein mediate the coupling of signal transduction from activated GPCR to
appropriate downstream effectors and thereby play an important
role in signaling.7 Binding of diverse ligand to their cognate
GPCR activates the heterotrimeric G-protein-mediated signaling
pathway by promoting the exchange of Gα-bound GDP for
GTP dissociating Gβγ dimer from the Gα. The GTP-bound
activated Gα and the freely released Gβγ dimer activate downstream effectors protein thus transducing the extra-cellular signal
to intra-cellular downstream cascades. Regulator of G-protein
Signaling (RGS) proteins, which preferentially bind to activated
Gα and accelerate its intrinsic GTPase activity,8 thus, initiates
deactivation of the G-protein signaling. GPCR sequence conservation even within a single GPCR family of an organism can be
lower than 25%,9 GPCRs are identified not by sequence homology but rather by their ability to couple with an intracellular
heterotrimeric G-protein complex and by their two-dimensional
topology, which classically consists of an extracellular amino terminus, seven membrane spanning domains connected by three
intracellular and three extracellular loops, and an intracellularly
located carboxy-terminal tail.
Whole genome sequencing efforts have shown that heterotrimeric G-protein signaling can be highly complex. GPCRs in
plants are not well characterized as compared to GPCRs from
animal system. Till to date there has been only one putative
*Correspondence to: Narendra Tuteja; E-mail:
[email protected]
Submitted: 04/06/11; Accepted: 04/07/11
DOI:10.4161/psb.6.8.15771
www.landesbioscience.com
Plant Signaling & Behavior
1079
Figure 1. For igure legend, see following page.
1080
Plant Signaling & Behavior
Volume 6 issue 8
Figure 1 (See previous page). Amino acid sequence alignment of indica rice using ClustalW program (www.ebi.ac.uk/clustalw) and its transmembrane regions. (A) Amino acid alignment of osGPCr with other plants Japonica rice (osGPCr; nm_001063604.1), maize (ZmGPCr; nm_001153424.1),
Arabidopsis (GCr1; nm103724) and pea (PsGPCr; DQ010316.2). it shows most of identity in the transmembrane regions. Asterisk shows identical amino
acids. Gaps inserted to optimize the alignment are indicated by dashes. (B) inter ProScan (www.ebi.ac.uk/interProScan); an integrated documentation
resource was used for family identiication of osGPCr.
GPCR (GCR1) identified and experimentally investigated in
Arabidopsis and rice model plants.10-13 Their signaling role in
stress conditions are still under investigation (Table 2). In the
present study we have studied its role under different abiotic
stresses in rice.
Results
Cloning of OsGPCR cDNA. The OsGPCR (GPCR from Indica
rice) gene was amplified by PCR using rice first-stranded cDNA
as template. Sequence analysis of the OsGPCR showed that the
amplified fragment encodes a full-length transcript, which is 966
bp in size. The deduced amino acid sequence revealed a protein
consisting of 321 amino acid residues with a predicted molecular
mass of about 36.06 kDa and pI 9.15.
In silico analyses of OsGPCR. Amino acid sequence alignment of GPCR from Indica rice with corresponding GPCRs
from Arabidopsis, Japonica rice, maize and pea are shown in
Figure 1A. The amino acid sequence alignment of OsGPCR with
GPCR of Japonica rice, Arabidopsis (GCR1), maize and pea is
shown in Figure 1A, which shows that it possess the conserved
and typical seven transmembrane regions. Most of the homology
shared between these sequences is in the seven transmembrane
regions. The presence of seven transmembrane regions was further confirmed by the transmembrane hidden Markov model
(TMHMM2) (Fig. 1B). Sequence comparison of OsGPCR with
GPCRdB using PREDGPCR shows that OsGPCR is a member
of the class A Rhodpsin-like receptor family with signature pattern similar to that of Prostanoid/Thromboxane rec. ScanProsite
results together with ProRule-based predicted intra-domain features. Expasy PROSITE database of protein families and domains
revealed different motifs, patterns and biologically significant
sites (Fig. 2A). It predicted six potent N-myristoylation sites, viz
25–30: GTsaAV; 75–80: GLsnAF; 131–136: GTslAT; 143–148:
GSdyGR; 264–269: GLfnSI; 272–277: GLnsSV, one cAMP- and
cGMP-dependent protein kinase phosphorylation site, 50–53:
RKfS, three protein kinase C phosphorylation site viz 53–55: SfK;
202–204: SdR; 276–278: SvR; and four casein kinase II phosphorylation site viz 71–74: TimE; 116–119: TdvE; 253–256: SilD; and
305–308: SqqE. Two potential N-glycosylation sites were located
at IL3 and IL4 loops at positions 193–196: NATR and 274–277:
NSSV. The phylogenetic tree constructed by ClustalW aligned
amino acid sequences of GPCR from Indica rice with corresponding GPCRs from Arabidopsis, Japonica rice, maize and pea using
Neighbor-Joining method showed that OsGPCR was closely
related to ZmGPCR while distantly related to PsGPCR (Fig. 2D).
The OsGPCR shares 98% identity with GPCR of Japonica rice
followed by 84% identity with maize GPCR (ZmGPCR), 62%
with Arabidopsis GPCR (AtGCR1) while showing least homology of 47% with pea (PsGPCR) (Table 1).
www.landesbioscience.com
Quantitative real-time PCR. The salt treatment showed a
significant increase in the expression level of GPCR. The 200
mM NaCl treatment induced the elevated expression of GPCR
by ~9-fold as early as 1 h and this elevation was maintained
up to 6 h. It appears as an early as well as prolong and strong
response against NaCl exposure (Fig. 3A). However, the same
effect was not observed with KCl treatment (Fig. 3B) suggesting
that increased expression of OsGPCR was due to the exposure
to high level of Na+ ion. Exposure to heat and cold stress showed
no significant change in the expression of OsGPCR up to 12 h
(Fig. 3C and E). During the drought-stress period, expression of
OsGPCR rapidly increased (24-fold) by 1 h, whereas it decreased
down ~600-fold after 12 h (Fig. 3F).
Expression of OsGPCR under ABA treatment appears as significant and early response. In this case a significant increase of
~8-fold was observed in expression of OsGPCR at as early as 1 h
that still increased to ~13-fold at 6 h before decreasing to ~4-fold
at 12 h (Fig. 3D).
Discussion
Rice cultivating areas, worldwide, are frequently exposed to many
abiotic stresses like drought, salinity, extreme temperature, oxidative stress, heavy metal to impede rice growth and production.
It promotes to elucidate the mechanisms of plant tolerance or
resistance to a variety of stresses and improve the ability of crops
to cope with the stresses. Responses of plants to stress conditions
include alteration in gene expression that lead to alterations in
protein synthesis.
Heterotrimeric G-protein complex and related GPCR(s) are
reported to play an important role in abiotic stresses (Table 2).14
GPCR transduce the extra-cellular signal to an intracellular
heterotrimeric G-protein complex, thereby activating downstream signaling cascades. The presence of GPCR(s) in plants
has only been indirectly implicated. GPCRs are characterized
by their two-dimensional topology, which classically consists
of an extracellular ligand binding amino terminus, seven membrane spanning domains connected by three intracellular and
three extracellular loops, and an intracellularly located carboxyterminal tail. In the present study the structural predictions of
OsGPCR showed the hydrophobic domains that form seven
transmembrane spanning (7TMs) α-helices which are linked by
alternate intra- and extra-cellular hydrophilic regions (Fig. 2A
and B). The ubiquitous and inevitable seven TM structure place
the N- and C-terminal segments at opposite surfaces of the
membrane allowing ligand binding at the N-terminal segment
and phosphorylation at the C-terminal segment for desensitization.16 The increased expression level of OsGPCR in presence of
ABA (Fig. 3D), suggest its role in ABA signaling pathway by
activating down stream effectors through binding with Gβγ
Plant Signaling & Behavior
1081
1082
Plant Signaling & Behavior
Volume 6 issue 8
Figure 2 (See previous page). (A) the motifs, patterns and biologically signiicant sites in osGPCr sequence were identiied using Expasy ProSitE
database of protein families and domains. (B) the seven transmembrane α-helical regions of osGPCr were predicted using the transmembrane hidden markov model (tmhmm version 2.0, www.cbs.dtu.dk/services/tmhmm) program. (C) Diagrammatic presentation of osGPCr showing topographic locations of biologically signiicant sites. (D) the dendrogram showing evolutionary history of osGPCr protein. Phylogenetic analyses were
conducted using the neighbor-Joining method with pair wise deletion of alignment gaps, Poisson correction for amino acid substitutions in mEGA 4.
the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches.
subunits. OsGPCR protein sequence showed the presence of six
N-myristoylation sites (Fig. 2A) which is a co-translational or
post-translational covalent modifier of proteins that can promote
its association with membrane lipid. It is essential for the proper
functioning of proteins in regulating the signaling pathways and
involved in adaptation to high salt stress in plants.16 Presence of
the six potential N-myristoylation sites seems very important for
membrane localization17 and multi-spanning of OsGPCR that
might lead to initiate compartmentalization of extracellular
signals.
The presence of two protein kinase C phosphorylation site
at ICL1/TM2 junction and TM7/ICL4 junction and three
casein kinase II phosphorylation site (Fig. 2A) might be playing very crucial role in the regulation of many cellular processes
by desensitizing GPCRs via feedback regulation by the second
messenger-stimulated kinases. Phosphorylation of OsGPCR by
specific serine18,19 residues located in the third cytoplasmic loop
or C-terminal tail of the receptors serine, theronine and tyrosine
residues participate in desensitization.20 Phosphorylation directly
alters receptor conformation such that interaction with the
G-protein is impaired. This type of receptor regulation generally
mediates “heterologous” or non-“agonist-specific” desensitization because any stimulant that elevates cAMP (or diacylglycerol
in the case of PKC) has the potential to cause the phosphorylation and desensitization of any GPCR containing an appropriate
PKA and/or PKC consensus phosphorylation site.21 The presence of three casein kinase II phosphorylation sites consolidate
the unidirectional deactivation of OsGPCR after transducing
the signal to downstream G-protein mediated signal channelization, since casein kinase II has been shown to participate in
hierarchical phosphorylation reactions.22 Stress responsive genes
are known be expressed either through an ABA-dependent or
ABA-independent pathway.23 The expression profile of OsGPCR
under high NaCl treatment suggests that GPCR induces ABAdependent pathway.
Furthermore, osmotic and ionic stresses induce secondary
cellular perturbations that arise from ROS which initiate signal
transduction pathways that modulate plant defensive processes.24
A population of unique salt regulated ESTs were identified
that detected salt regulated transcripts defining transcriptional
response to salt stress in Arabidopsis.25 In the present study, we
found an intense increase in the mRNA abundance of OsGPCR
under Na+ (Fig. 3A) salt stress unlike to K+ (Fig. 3B), similar to
Arabidopsis as high K+/Na+ concentration is a requisite in view of
plant nutrition.3 These results suggest that GPCR gene is strongly
induced by Na+ salt stress as soon as by 1 h. Since, plant survival
in severe stress condition likely requires very immediate cellular
responses, whereas transcriptional regulation may be sufficient for
stress recovery and adaptation. hence, the intense early response of
www.landesbioscience.com
OsGPCR under salt, ABA and drought stress seems to be involved
in early cellular response signaling and might be leading to transcriptional regulation through G-protein signaling pathway.
The present study identifies the active participation of
OsGPCR in abiotic stress response. Though its role appears as
an immediate cellular response, the successive transcriptional
regulation, stress recovery and adaptation needs to be studied
in details. Taken together, the observations reported in this
study present a direct evidence for the regulation of transcript of
OsGPCR in response to abiotic stress. These studies could also
provide new insight into the novel role of OsGPCR in abiotic
stresses, thus suggesting an important molecule for manipulating
stress tolerance in plants. These findings also provide an excellent starting point to investigate its potential roles in rice plant
stress tolerance. Overall, this study will contribute to our better
understanding of G-proteins signaling under stress conditions in
higher plants.
Materials and Methods
Plant material and stress treatment. Rice (Oryza sativa cv.
Indica group Swarna) seeds were grown in vermiculite in transgenic house under 16/8 h day light condition. For abiotic stress
treatment, the 3-wk-old seedlings were treated to salt (200 mM
NaCl, 200 mM KCl), abscisic acid (100 μM ABA), cold (4°C),
heat (42°C) and drought conditions. Samples were aliquoted at
different time intervals (viz. 1 h, 2 h, 3 h, 6 h, 12 h and nontreated samples). After sampling, the tissues were snap frozen in
liquid nitrogen and stored at -72°C until use.
Isolation of RNA and cDNA preparation. Total RNA was isolated from 100 mg of samples with TriZOL LS reagent (Invitrogen
Life Technologies USA). The contaminating genomic DNA was
removed by DNaseI treatment. The total RNA obtained was
used as template for cDNA synthesis. The first strand cDNA
was synthesized from 5 μg of total RNA using Superscript II
Reverse Transcriptase (Invitrogen Life Technologies USA) with
oligo(dT)18 primer according to the manufacturer’s instructions.
Cloning of OsGPCR gene of indica rice. For cloning of rice
G-protein coupled receptor, the known sequence of GPCR gene
were first aligned and primers were designed from the 5'-UTR
and 3'-UTR regions of the most conserved areas. For the amplification of G-protein coupled receptor (OsGPCR), the primer
pair 5'-CTC GAG CAT ATG GCG GCA TCG GCG GCG G-3'
(Oligo-1, forward) (XhoI and NdeI sites italicized) and 5'-GAA
TTC CTA TGT GTT ACT CGC ATC GAC AAT AAG AG-3'
(Oligo-2, reverse) (EcoRI site italicized) was used for PCR. In
PCR reactions, using the respective primers and Indica rice firststranded cDNAs as template, the DNA fragments of 966 bp was
amplified representing OsGPCR. The full-length rice G-protein
Plant Signaling & Behavior
1083
of Indica rice was compared with that of
Japonica rice, Arabidopsis, maize and pea
by multiple amino acid sequence alignment using clustalw 2.0 program (www.
ebi.ac.uk/clustalw).26 The pair wise amino
acid sequence identity between GPCRs of
Indica rice with Japonica rice, Arabidopsis,
maize and pea was calculated using software DiAlign version 2.1 (Genomatix).
The clustalW aligned amino acid sequences
of OsGPCR of Indica rice, Japonica rice,
Arabidopsis, maize and pea were used to
infer the evolutionary relationship among
them using using the Neighbor-Joining
method with pairwise deletion of alignment gaps, Poisson correction for amino
acid substitutions and bootstrap test (1,000
replicates). The phylogenetic analyses were
done using MEGA4.27 PREDGPCR (bioinformatics.biol.uoa.gr/PRED-GPCR), 27
was used for the recognition and classification at the family level by comparison with
GPCRdB. The presence of seven transmembrane regions was further confirmed
by the transmembrane hidden Markov
model (TMHMM2).29 Further, OsGPCR
sequence was analysed with InterPro, an
integrated documentation resource for
protein families, domains, regions and
sites.30 Expasy PROSITE database of protein families and domains was used to find
different motifs, patterns and biologically
significant sites in OsGPCR amino acid
sequence.
Quantitative real-time PCR. The
expression levels of GPCR under different
stress conditions in rice plant leaves were
determined by real time PCR. Quantitative
Figure 3. Quantitative real-time PCr analyses of osGPCr in diferent abiotic stress conditions
real-time PCR reactions were performed on
((A) 200 mm naCl; (B) 200 mm KCl; (C) heat 42°C; (D) 100 μm ABA; (E) Cold 4°C and (F) Drought)
StepOne Real-Time PCR system (Applied
using cDnA prepared from 3-wk-old seedling leaf blades. total rnA was isolated from samples
collected at diferent time intervals. Error bars are SD.
Biosystems). Using Power SyberGreen
PCR master mix (Applied BioSystems), a
coupled receptor gene amplified was cloned into the pGEMT 20 μl reaction mixture containing 10 pM of each gene specific
easy vector. The positive colonies of E. coli DH5α cells show- primer pair (α-tublin forward 5'-GGT GGA GGT GAT GAT
ing desired amplification were used for isolation of plasmid DNA GCT TT-3' and reverse 5'-ACC ACG GGC AAA GTT GTT
using QIAprep Spin Miniprep kit (Qiagen) following manufac- AG-3'; rice G-protein coupled receptor forward 5'-GGA TGG
turer’s instructions. The plasmid DNA was confirmed for the CTG TTG GCA TAA GT-3' and reverse 5'-GAC GAG TTG
gene insertion by restriction digestion using with NdeI and EcoRI AGC CCA TAA GC-3') and 1 μl of stress treatment specific
enzymes. The potential positive clone of OsGPCR was subjected cDNA was used for the PCR reaction. Cycling conditions conto nucleotide sequence determination and the sequence was sub- sisted of one cycle of 10 min for 95°C, and 40 cycles of 15 s at
mitted to Genbank (accession number HQ676132.1).
95°C and 20 s at 59°C. Fluorescence intensity was measured after
In silico analysis of OsGPCR. A homology search was per- every cycle. PCR products were melted by gradually increasing
formed using BLAST (NCBI, www.ncbi.nlm.nih.gov/BLAST) the temperature from 55–95°C in 0.5°C increments at every step.
using the deduced amino acid sequences of the OsGPCR. The Rice α-tubilin gene was used as internal reference. Raw expresDNA sequence of OsGPCR genes were used to deduce the sion values were calculated in Microsoft Excel using the average
amino acid sequence using translate tool at Expasy. The GPCR C T values following Livaks’ method.31
1084
Plant Signaling & Behavior
Volume 6 issue 8
Table 1. Amino acid sequence identity (percent) of GPCr from indica rice (osGPCr) with corresponding proteins from japonica rice (osGPCr), arabidopsis (GCr1), pea (PsGPCr) and maize (ZmGPCr)
OsIGPCR
OsIGPCR
OsJGPCR
ZmGPCR
AtGPCR
PsGPCR
***
98
84
62
47
85
63
48
***
61
46
***
OsJGPCR
ZmGPCR
***
AtGPCR
62
***
PsGPCR
Table 2. List of reported G-protein coupled receptor-like genes from Arabidopsis thaliana, which have been reported as connected to abiotic stress
responses
SN
Gene name
Locus/Accession No.
Function
1
GCr1 (G-protein-coupled
receptor1)
At1G48270
A protein similar to G-coupled receptor with seven transmembrane
regions. involved in dormancy and flowering. reduction of expression
results in decreased sensitivity to cytokinin
2
G-protein coupled receptor
(GCr2)
At1G52920
An ABA receptor, activate downstream ABA effectors and to trigger the
ABA responses.
3
GCL1 (GCr2 like-1)
At5G65280
Encodes a protein similar to GCr2 a putative G protein coupled receptor
ABA receptor. Loss of function mutations in GCL1 show no ABA response.
GCL1 is a homolog of LAnCL1 and LAnCL2, in human bacterial lanthionine synthetase.
4
GCL2 (GCr2 like-2)
At2G20770
Encodes a protein similar to GCr2 a putative G protein coupled receptor
thought to be an ABA receptor. GCL2 also has similarity to LAnCL1 and
LAnCL2, human homologs of bacterial lanthionine synthetase.
Acknowledgments
Work on signal transduction and plant stress signaling in N.T.’s
laboratory is partially supported by Department of Science and
Technology (DST), Government of India.
References
1.
Redhead CR, Palme K. The genes of plant signal transduction. Crit Rev Plant Sci 1996; 15:425-54.
2. Mahajan S, Tuteja N. Cold, salinity and drought
stresses: An overview. Arch Biochem Biophys 2005;
444:139-58.
3. Tuteja N. Mechanisms of high salinity tolerance in
plants. Meth Enzymol 2007; 428:419-38.
4. Bockaert J, Pin JP. Molecular tinkering of G proteincoupled receptors: an evolutionary success. EMBO J
1999; 18:1723-9.
5. Fredriksson R, Schioth HB. The repertoire of G-protein
coupled receptors in fully sequenced genomes. Mol
Pharmacol 2005; 67:1414-25.
6. Tuteja N. Signaling through G protein coupled receptors. Plant Sig Behav 2009; 4:942-7.
7. Tuteja N, Sopory SK. Plant signaling in stress:
G-protein coupled receptors, heterotrimeric G-proteins
and signal coupling via phospholipases. Plant Sig Behav
2008; 3:79-86.
8. Neubig RR, Siderovski DP. Regulators of G protein
signaling as new central nervous system drug targets.
Nat Rev Drug Discov 2002; 1:187-97.
9. Oliveira L, Paiva AC, Vriend G. A low resolution
model for the interaction of G proteins with G proteincoupled receptors. Protein Eng 1999; 12:1087-95.
10. Plakidou-Dymock S, Dymock D, Hooley R. A higher
plant seventransmembrane receptor that influences
sensitivity to cytokinins. Curr Biol 1998; 8:315-24.
11. Colucci G, Apone F, Alyeshmerni N, Chalmers D,
Chrispeels MJ. GCR1, the putative Arabidopsis G
protein-coupled receptor gene is cell cycleregulated,
and its overexpression abolishes seed dormancy and
shortens time to flowering. Proc Natl Acad Sci USA
2002; 99:4736-41.
www.landesbioscience.com
12. Apone F, Alyeshmerni N, Wiens K, Chalmers D,
Chrispeels MJ, Colucci G. The G-protein-coupled
receptor GCR1 regulates DNA synthesis through activation of phosphatidylinositol-specific phospholipase
C. Plant Physiol 2003; 133:571-9.
13. Warpeha KM, Upadhyay S, Yeh J, Adamiak J, Hawkins
SI, Lapik YR. The GCR1, GPA1, PRN1, NF-Y
signal chain mediates both blue light and abscisic
acid responses in Arabidopsis. Plant Physiol 2007;
143:1590-600.
14. Misra S, Wu Y, Venkataraman G, Sopory SK, Tuteja
N. Heterotrimeric G-protein complex and G-proteincoupled receptor from a legume Pisum sativum: role in
salinity and heat stress and crosstalk with phospholipase
C. Plant J 2007; 51:656-69.
15. Lefkowitz R. G Protein-coupled Receptors III. New
roles for receptor kinases and β-arrestins in receptor
signaling and desensitization. J Biol Chem 1998;
273:18677-80.
16. de Jonge HR, Hogema B, Tilly BC. Protein
N-myristoylation: critical role in apoptosis and salt
tolerance. Science STKE 2000; 63:1.
17. Sessa WC, Barber CM, Lynch KR. Mutation of
N-myristoylation site converts endothelial cell nitric
oxide synthase from a membrane to a cytosolic protein.
Circ Res 1993; 72:921-4.
18. Bouvier M, Hausdorff WP, De Blasi A, O’Dowd BF,
Kobilka BK, Caron MG, Lefkowitz RJ. Removal of
phosphorylation sites from the β2-adrenergic receptor delays onset of agonist-promoted desensitization
Nature 1988; 333:370-3.
19. Hausdorff WP, Bouvier M, O’Dowd BF, Irons GP,
Caron MG, Lefkowitz RJ. Phosphorylation sites on
two domains of the beta 2-adrenergic receptor are
involved in distinct pathways of receptor desensitization J Biol Chem 1989; 264:12657-65.
Plant Signaling & Behavior
20. Kemp BE, Pearson RB. Protein kinase recognition
sequence motifs. Trends Biochem Sci 1990; 15:342-6.
21. Lefkowitz RJ. G protein-coupled receptors III. new
roles for receptor kinases and β-arrestins in receptor
signaling and desensitization. J Biol Chem 1998;
273:18677-80.
22. Mauxion F, Borgne RL, Munier-Lehmann H, oflack B.
A casein kinase II phosphorylation site in the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor determines the high affinity interaction
of the AP-1 golgi assembly proteins with membranes.
J Biol Chem 1996; 271:2171-8.
23. Tuteja N. Abscisic acid and abiotic stress. Plant Sig
Behav 2007; 2:135-8.
24. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ. Plant
cellular and molecular responses to high salinity. Annu
Rev Plant Physiol Plant Mol Biol 2000; 51:463-99.
25. Gong Z, Koiwa H, Cushman MA, Ray A, Bufford D,
Kore-eda S, et al. Genes that are uniquely stress regulated in salt overly sensitive (sos) mutants. Plant Physiol
2001; 126:363-75.
26. Thompson JD, Higgins DJ, Gibson TJ. CLUSTALW:
Improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice.
Nucleic Acids Res 1994; 22:4673-80.
27. Tamura K, Dudley J, Nei M, Kumar S. MEGA4:
Molecular Evolutionary Genetics Analysis (MEGA)
software version 4.0. Mol Biol Evol 2007; 24:1596-9.
28. Papasaikas PK, Bagos PG, Litou ZI, Promponas VJ,
Hamodrakas SJ. PRED-GPCR: GPCR recognition
and family classification server. Nucleic Acids Res
2004; 32:380-2.
1085
29.
Krogh A, Larsson B, von Heijne G, Sonnhammer EL.
Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J
Mol Biol 2001; 305:567-80.
30. Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder
N, Apweiler R, Lopez R. InterProScan: protein
domains identifier. Nucleic Acids Res 2005; 33:116-20.
31. Livak KJ, Schmittgen TD. Analysis of relative gene
expression data using real-time quantitative PCR and
the 2-ΔΔCT method. Methods 2001; 25:402-8.
1086
Plant Signaling & Behavior
Volume 6 issue 8