Rice G-protein coupled receptor (GPCR)

Abstract

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 membrane-spanning domains with an extracellular N-terminus and a cytoplasmic C-terminus which functions along with GTP-binding 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 confirmed 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 profile of GPCR upregulated during early exposure which subsequently decreased. On the other hand it seems no significant effect due to cold and heat stress. These findings provide a direct evidence for transcriptional regulation of rice GPCR under abiotic stress conditions. These findings 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 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.

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.

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).

Figure 1.

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 identification of OsGPCR.

Figure 2.

(A) The motifs, patterns and biologically significant sites in OsGPCR sequence were identified 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 significant 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.

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	OsJGPCR	ZmGPCR	AtGPCR	PsGPCR
OsIGPCR	***	98	84	62	47
OsJGPCR		***	85	63	48
ZmGPCR			***	61	46
AtGPCR				***	62
PsGPCR					***

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).

Figure 3.

Quantitative real-time PCR analyses of OsGPCR in different abiotic stress conditions ((A) 200 mM NaCl; (B) 200 mM KCl; (C) Heat 42°C; (D) 100 µM ABA; (E) Cold 4°C and (F) Drought) using cDNA prepared from 3-wk-old seedling leaf blades. Total RN A was isolated from samples collected at different time intervals. Error bars are SD.

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 carboxy-terminal 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 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 serine^18,19 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 ABA-dependent 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 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 non-treated 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 first-stranded cDNAs as template, the DNA fragments of 966 bp was amplified representing OsGPCR. The full-length rice G-protein coupled receptor gene amplified was cloned into the pGEMT easy vector. The positive colonies of E. coli DH5α cells showing desired amplification were used for isolation of plasmid DNA using QIAprep Spin Miniprep kit (Qiagen) following manufacturer's instructions. The plasmid DNA was confirmed for the gene insertion by restriction digestion using with NdeI and EcoRI enzymes. The potential positive clone of OsGPCR was subjected to nucleotide sequence determination and the sequence was submitted to Genbank (accession number HQ676132.1).

In silico analysis of OsGPCR.

A homology search was performed using BLAST (NCBI, www.ncbi.nlm.nih.gov/BLAST) using the deduced amino acid sequences of the OsGPCR. The DNA sequence of OsGPCR genes were used to deduce the amino acid sequence using translate tool at Expasy. The GPCR 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 real-time PCR reactions were performed on StepOne Real-Time PCR system (Applied Biosystems). Using Power SyberGreen PCR master mix (Applied BioSystems), a 20 µl reaction mixture containing 10 pM of each gene specific primer pair (α-tublin forward 5′-GGT GGA GGT GAT GAT GCT TT-3′ and reverse 5′-ACC ACG GGC AAA GTT GTT AG-3′; rice G-protein coupled receptor forward 5′-GGA TGG CTG TTG GCA TAA GT-3′ and reverse 5′-GAC GAG TTG AGC CCA TAA GC-3′) and 1 µl of stress treatment specific cDNA was used for the PCR reaction. Cycling conditions consisted of one cycle of 10 min for 95°C, and 40 cycles of 15 s at 95°C and 20 s at 59°C. Fluorescence intensity was measured after every cycle. PCR products were melted by gradually increasing the temperature from 55–95°C in 0.5°C increments at every step. Rice α-tubilin gene was used as internal reference. Raw expression values were calculated in Microsoft Excel using the average C_T values following Livaks' method.³¹

Abbreviations

GPCR

G-protein coupled receptor

OsGPCR

GPCR of rice

PsGPCR

GPCR of pea

ZmGPCR

GPCR of maize

GCR1

GPCR of Arabidopsis