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Isolation, in silico characterization, localization and expression analysis of abiotic stress-responsive rice Gprotein β subunit (RGB1) a
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Dinesh K Yadav , Devesh Shukla & Narendra Tuteja
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Plant Molecular Biology Group; International Centre for Genetic Engineering and Biotechnology; Aruna Asaf Ali Marg, New Delhi, India Published online: 16 Apr 2014.
Click for updates To cite this article: Dinesh K Yadav, Devesh Shukla & Narendra Tuteja (2014) Isolation, in silico characterization, localization and expression analysis of abiotic stress-responsive rice G-protein β subunit (RGB1), Plant Signaling & Behavior, 9:5, e28890, DOI: 10.4161/psb.28890 To link to this article: http://dx.doi.org/10.4161/psb.28890
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Research Paper
Research Paper
Plant Signaling & Behavior 9, e28890; April; © 2014 Landes Bioscience
Isolation, in silico characterization, localization and expression analysis of abiotic stressresponsive rice G-protein β subunit (RGB1) Dinesh K Yadav, Devesh Shukla†, and Narendra Tuteja* Plant Molecular Biology Group; International Centre for Genetic Engineering and Biotechnology; Aruna Asaf Ali Marg, New Delhi, India †
Current address: Department of Biology; Western Kentucky University; Bowling Green, KY USA
Keywords: Abiotic stress; heterotrimeric G-proteins; metals; rice G-protein beta-subunits; signal transduction, sub-cellular localization
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Abbreviations: ABA, abscisic acid; ARE, auxin responsive elements; GPCR, G-Protein coupled receptor; RGB1(I), β-subunit of Indica rice; RGB1(J), β-subunit of Japonica rice
Heterotrimeric G-proteins constitute the classical signaling paradigm along with their cognate G-protein coupled receptors (GPCRs) and appropriate downstream effectors. G-protein complex is composed of highly conserved Gα, Gβ, and Gγ subunits. In the present study, we have characterized the cis-regulatory elements of the promoter, signature motifs, transcript profile in response to abiotic stresses, and sub-cellular localization of G-protein β subunit RGB1(I) from Indica rice. The RGB1(I) promoter sequence has various stress-related cis-regulatory elements suggesting its role in abiotic stress signaling. Presence of six WD-40 repeat signature motifs in RGB1(I) suggest its role in exchange of GDP by GTP in Gα subunit and receptor recognition. Presence of multiple N-myristoylation consensus sites in RGB1(I) protein sequence, which is necessary for membrane localization of protein, confirms the association of RGB1(I) in plasma membrane. Extrinsic association of RGB1(I) with plasma membrane seems essential for its role in regulation of signaling pathways and adaptation to high salt stress. We report the sub-cellular localization of RGB1(I) in plasma membrane, cytosol and nucleus. The localization of RGB1(I) in nucleus supports its possible interaction with transcription factors regulating the expression of salt stress responsive genes. The RGB1(I) transcript was upregulated under KCl, cold, dehydration and micronutrient (Mn2+ and Zn2+) stress. However, transcript variation under elevated temperature, ABA, NaCl, and toxic heavy metals (viz. arsenite, arsenate, cadmium and lead) was not encouraging. These evidences indicate an active and significant role of RGB1(I) in the regulation of abiotic stresses in rice and propound its possible exploitation in the development of abiotic stress tolerance in crops.
Introduction The ability of multi-cellular organisms to sense the environmental stimuli and respond appropriately is an utmost requirement for synchronized development, ordered growth, maintenance and successful reproduction. The evolution of an efficient cellular communication system allows cells to coordinated response to environmental stimuli as well as to each other by integrating the wide array of extracellular signals to intracellular downstream effectors.1 Heterotrimeric guanine nucleotide binding proteins (G-proteins) are well characterized signaling molecules that interact with plasma membrane localized G-protein coupled receptors (GPCRs) and transduce majority (~80%) of extracellular signals across the cell membranes. However, these functions are believed to be performed by regulator of G-protein signaling (RGS) in plants.2
Heterotrimeric G-protein complex consists of α (Gα), β (Gβ), and γ (Gγ) subunits. According to classical paradigm, G-protein signaling is initiated by the association of extracellular ligand with GPCR (or RGS) and subsequently inducing a conformational change which promotes the exchange of GDP for GTP associated to Gα subunit. GTP bound Gαβγ heterotrimeric complex tends to dissociate into Gα-GTP monomer and Gβγ heterodimer. The dissociated units of Gα-GTP monomer and Gβγ heterodimer can interact with a wide array of specific downstream effectors of distinct signaling pathways. The whole genome sequencing efforts have established that the heterotrimeric G-protein mediated signaling mechanism is highly complex. Unlike human proteome that contains 1300 odd theoretical heterotrimeric complexes, plants have very small repertoire of G-protein subunit genes.3 The fully sequenced Arabidopsis genome contains only one gene for each canonical Gα
*Correspondence to: Narendra Tuteja, Email: [email protected] Submitted: 03/21/2014; Revised: 04/14/2014; Accepted: 04/14/2014; Published Online: 04/16/2014 Citation: Yadav DK, Shukla D, Tuteja N. Isolation, in silico characterization, localization and expression analysis of abiotic stress-responsive rice G-protein β subunit (RGB1). Plant Signaling & Behavior 2014; 9:e28890; PMID: 24739238; http://dx.doi.org/10.4161/psb.28890
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Figure 1A. See next page for legend.
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Figure 1A (See previous page for figure). Multiple sequence alignment and phylogenetic tree of β sub-units of G protein sequences. Different amino acid sequences of β sub-unit of G protein aligned using Clustal Omega, are AtGβ (Arabidopsis thaliana), GmGβ (Glycine max), NtGβ (Nicotiana tabacum), PsGβ (Pisum sativum), RcGβ (Ricinus communis), SbGβ (Sorghum bicolor), ZmGβ (Zea mays) andal RGB(I) (Rice Gβ, Indica).
(GPA1),4 Gβ (AGB1)5 and three genes for Gγ subunits, AGG1, AGG2 and AGG3.6,7 G-protein γ subunits provide functional selectivity in G-βγ dimer signaling in Arabidopsis and some new elements exist in the heterotrimeric G-protein signaling complex.8 Two Gα subunit genes in pea (PGA1 and PGA2)9 and four Gα subunit genes (GmGα 1–4) in soybean10 were reported. Fully sequenced rice genome contains only one conventional Gα and Gβ, but three Gγ subunits (RGG1, RGG2 and RGG3), and a GPCR.11 RGG3 is a homolog of AGG3 and is known as DEP1.7 However, several GPCR-type candidates were initially proposed in plant kingdom but later discredited.2,12,13 Despite the limited number of the components, G-protein complexes in plants regulate diverse signaling pathways including hormone signaling, environmental sensing, ion channel regulation, and disease response and cell death.14,15 Recently, Pandey16 identified an elaborate network of G-proteins in soybean. In Arabidopsis, AGB1 was reported to play an important role in the development of leaf, flower, and fruit.17 Utsunomyia et al.18 reported that RGB1 knockdown lines generated in RGA1 deficient d1–5 lines caused dwarfism and browning of internodes and lamina joint regions. These observations suggest the predominant role of RGB1 in G-protein mediated signaling that regulates cellular proliferation and seed fertility through functional cooperation with RGA1. In the present study, we have isolated and characterized Gβ subunit from Indica rice. The deduced protein sequence was used to study its structural features and phylogenetic relatedness. Furthermore, the genomic organization and promoter analysis was also done using in silico methods. The sub-cellular localization and transcript abundance of RGB1(I) was studied under different abiotic stress conditions including high salt, cold, heat, dehydration, ABA and heavy metal(loids).
Results Isolation and in silico analysis of RGB1(I) The full-length coding region of RGB1(I) was PCR amplified with gene specific primers using first-strand cDNA as the template. The analysis of amplified and sequenced DNA fragment revealed 1143 bp complete transcript of RGB1(I). The deduced protein sequence from full-length cDNA sequence showed a putative protein comprising of 380 amino acids. The putative protein showed computed molecular mass and iso-electric point (pI) as 41.7 kDa and 7.13, respectively. The multiple sequence alignment of RGB1(I) subunit at amino acid level with other Gβ subunits from related plant genera is shown in Figure 1A. Phylogenetic analysis of RGB1 duly reflects the taxonomic grouping (Fig. 1B). The phylogenetic tree displayed separate clades for eudicots, monocots and lower plants including bryophytes and pteridophytes. Interestingly, all the members of monocots belong to the poaceae family. Pairwise amino acid sequence comparison of RGB1(I) showed 99%
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identity with RGB1(J) followed by maize (ZmGB1) and sorghum (SbGB1) with 94% identity, either. It showed 92% and 91% identity with wheat and oat, respectively (Table 1). The observed phylogenetic relatedness suggest that evolution of Gβ subunit in rice is similar to closely related grass family members (Fig. 1B). Genomic organization of RGB1(I) Comparison of full-length cDNA sequence of RGB1(I) with its genomic DNA sequence identified six exons (109, 95, 425, 363, 113 and 38 bp) and five introns (128, 4326, 608, 607 and 518 bp). The sequence contains 5′-UTR of 75 bp and 458 bp at its 3′-end (Fig. 2A). Computational analysis of promoter elements of RGB1(I) The occurrence and distribution of regulatory cis-elements in 1.5 kb upstream promoter region of RGB1(I) was analyzed as shown in Figure 2B. Stress-responsive regulatory cis-elements identified in the promoter region of RGB1(I) contained several phytohormones responsive elements like, three abscisic acid responsive element (ABRE) reportedly associated with abscisic acid responsiveness and drought tolerance. In addition one gibberellic acid responsive, one ethylene responsive, one jasmonate responsive and one salicylic acid responsive elements were also identified. Besides these, it also has salt induced responsive element (GT-1 motif), circadian regulating cis-elements, low temperature responsive element (LTR) and one W-box domain. W-box domain is especially involved in response to the WRKY transcription factor family, and express under the abiotic and biotic stresses. Various significant motifs, patterns and biological sites were identified in RGB1(I) using the Expasy PROSITE database of protein domains, families and functional sites (Fig. 2C). It showed the six WD-40 repeats, viz. 62–103: PQGHSGKVYS LDWTPEKNWI VSASQDGRLI VWNALTSQKT HA,153– 195: LTGHKGYVSS CQYVPDQETR LITSSGDQTC VLWDVTTGQR ISI, 201–243: PSGHTADVLS LSINSSISNM FVSGSCDATV RLWDIRIASR AVR, 245286: YHGHEGDINS VKFFPDGQRF GTGSDDGTCR LFDVRTGHQL QV, 290–333: PDRNDNELPT VTSIAFSISG RLLFAGYSNG DCYVWDTLLA EVV, and 340–380: QNSHEGRISC LGLSSDGSAL CTGSWDKNLK IWAFSGHRKIV, are involved in the formation of β propeller sheet. It showed the presence of nine N-myristoylation sites (viz. 120 – 125; GQsvAC, 126 – 131; GGldSA, 127 – 132; GLdsAC, 190 – 195; GQriSI, 224 – 229; GScdAT, 261 – 266; GQrfGT, 267 – 272; GSddGT, 337 – 342; GNlqNS, 356 - 361; GSalC) six casein kinase II phosphorylation site, (viz. 3 - 6: SvaE; 20 23: SlrE; 35 - 38: TdvE; 139 - 142: SqaD; 266 - 269: TgsD; 318 - 321: SngD); seven protein kinase C phosphorylation site (viz. 20 - 22: SLR; 66 - 68: SGK; 98 - 100: SqK; 229 - 231: TvR; 254 - 256: SvK; 272 - 274: TcR; 309 - 311: SgR) and one potential glycosylation site at position 214 (NSS). The constituent amino acid residues of identified significant motifs, patterns and biological sites in RGB1(I) showed a high conserved score values (Fig. 2C).
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Table 1. Pairwise amino acid sequence identity of G-protein β subunit among economically important taxa of grass family *
Oryza sativa Indica
Oryza sativa Japonica
Triticum aestivum GB1
Zea mays gbp2
Avena fatua Gb1
Sorghum bicolor Gb
Oryza sativa Indica
*
99%
92%
94%
91%
94%
*
93%
95%
92%
95%
*
92%
92%
92%
*
91%
98%
*
91%
Oryza sativa Japonica Triticum aestivum GB1 Zea mays gbp2 Avena fatua Gb1
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Sorghum bicolor Gb
*
Transcript profile of RGB1(I) by real-time quantitative PCR The transcript profile of RGB1(I) under 200 mM NaCl treatment showed no significant difference unlike to the response under treatment of 200 mM KCl, as compared with control (Fig. 3A). Exposure to 200 mM KCl (Fig. 3A) upregulated the RGB1(I) mRNA abundance by ~9.0 and ~7.0-fold after 2h and 3h, respectively and eventually reached to its basal levels. The transcript profile of RGB1(I) showed no significant change under the exposure of 100 µM ABA level. Cold stress treatment increased the RGB1(I) transcript by ~12-fold after 1h, followed by a drop of ca. ~4-fold after 6h. It significantly increased again by ca. 35-fold after 12h (Fig. 3B). The change RGB1(I) mRNA abundance due to heat exposure was insignificant except for 1h (Fig. 3B). The expression of RGB1(I) during the dehydration stress condition showed a significant and intense increase in transcript level. It rapidly increased ~30 fold after 1h and was maintained > 34-fold up to 6h and subsequently reduced to basal level (Fig. 3B). The exposure to 300 µM Zn2+ upregulated the expression of RGB1(I) by ca. 3-fold at 1h and ca. 5-fold at 12h in comparison to non-treated control samples (Fig. 3C). Treatment of seedlings with 500 µM Mn2+ induced the expression by 6-fold at 1h and 6h (Fig. 3C). Exposure to 100 µM Cd 2+ induced the reduction in mRNA abundance of RGB1(I) at 1h, 2h and 12h in a range of ca. 0.3–0.5-fold (Fig. 3D). Exposure to 250 µM As(V) treatment caused a downregulation during 2h to 6h. An increase was observed in transcript level by ~2.5-fold after 12h (Fig. 3D). Under the exposure of As(III), the expression initially decreased by 0.7-fold up to 2h, followed by an increase by ~3.5-fold at 3h. This increment in expression was prolonged by ≥1.3-fold up to 12h. (Fig. 3E). The treatment with 700 μM Pb2+ caused a slight but rhythmic change in abundance of transcript and maximum increase was observed at 6h. RGB1(I) localizes to plasma membrane, cytosol and nucleus The study of sub-cellular localization was performed in onion peels bombarded with gold particles coated with plasmid construct 35S:RGB1-sGFP. Transient expression of RGB1(I)sGFP in epidermal cells of onion showed the prominent GFP signals from periphery and nucleus of the cells suggesting plasmamembrane and nuclear localization of fusion protein (Fig. 4). The thick level of fluorescence at the periphery indicates that the RGB1 may be localized to cytosol. This observation was also confirmed by the plasmolysis of cells expressing RGB1(I)-sGFP fusion protein. On the other hand, the cells expressing only GFP
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(empty vector) showed faint signal throughout the cell. Hence, the images explicitly suggest that RGB1(I) resides on the plasma membrane, cytosol as well as in the nucleus.
Discussion Global climatic changes in rice cultivating areas like, frequent drought, increasing atmospheric temperature and soil salinity, heavy metal(loid)s pollution, oxidative stress and various other abiotic stresses affect its production. It warrants the need to identify suitable candidate genes and annotate their role in abiotic stress tolerance/ resistance in plants to achieve sustainable crop improvement. Heterotrimeric G-proteins are major group of signaling molecules responsible for a variety of cellular response to external stimuli as inferred from pharmacological studies. These signaling proteins represent a potential genetic tool for modification of plants to overcome various abiotic stresses. However, unlike animal kingdom, plants have a limited repertoire of classical G-proteins, suggesting a mechanistically different signaling network as compared with mammals. Presence of four Gα, four Gβ and two Gγ gene sequences in soybean suggests 32 possible combinations of G protein heterotrimer. The Gα subunit in soybean has two distinct mode of GTP-hydrolysis indicating a complex and much diversified G-protein signaling network among the plants.10 Accomplishing this diversity we have isolated RGB1(I) to comprehend its structural features, subcellular localization and significance in abiotic stress response. Multiple sequence alignment of RGB1(I) subunit and its comparison with other known Gβ subunit in monocots showed a high level of similarity (Fig. 1A). All the significant structural and functional motifs required for its function were conserved. The RGB1(I) amino acid sequence showed the presence of six most important signature motif/sequences of Gβ subunit, the WD-40 repeats (Fig. 2C) which are involved in the formation of the seven propeller blades and contain the typical feature of Gβ subunits proteins. In these signature motifs, highly conserved aspartic acid is present in between strand b and c of WD repeats rather the Aspartic acid present in WD itself.19,20 These highly conserved aspartic acids are involved in the formation of an interand intra-blade hydrogen bond triad involving histidine in GH motif and a Ser/Thr in the blade b strand.21 RGB1(I) showed the presence of nine N-myristoylation sites, six casein kinase-II phosphorylation sites and six protein kinase C
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Figure 1B. A rooted phylogenetic tree was constructed using MEGA5 showing evolutionary relatedness of G protein β subunits in plants. The Bootstrap values from 1000 replicates are shown for selected branches.
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Figure 2. See next page for legend.
phosphorylation sites. N-myristoylation is a co/post-translational covalent protein modification that promotes association of
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modified proteins with lipid bilayer membrane. The association of protein with lipid bilayer is crucial for its role in regulating
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Figure 2 (See previous page for figure). In silico analysis of RGB1(I). (A) The schematic representation of genomic organization (exon–intron organization) of the genomic sequence of RGB1(I) genes. Closed boxes represent exons, and lines between closed boxes represent introns. The dark boxes represent the UTRs. The position of start (ATG) and stop codons (TGA) are marked. The numbers below the lines and the above boxes indicate the sizes (bp) of introns, UTR and exons, respectively. (B) Stress-responsive cis-regulatory elements and phytohormones responsive elements in the 1.5 kb 5′-upstream regions of RGB1(I) gene. The elements located in the (+) strand are above the lines, while those in the (-) strand are indicated below the line. ABRE = Abscisic acid responsive element; ARE = Auxin responsive factor (TGA-box); MeJAE = Methyl jasmonate responsive element; GT1Box; LTR = Low Temperature responsive element and anoxia specific element. (C) Different motifs, important active site and patterns identified using Expasy PROSITE database.
signal transduction and is critical to accustom plants against high salt stress.22 Thus, multiple N-myristoylation sites in RGB1(I) and increased transient transcript profile due to increased KCl level suggest its possible role in maintaining K+ homeostasis in rice plant.23 The suggested role of RGB1(I) in ion homeostasis is further supported by its topographic association with plasma membrane, cytosol and nucleus as shown in Figure 4. The presence of multiple casein kinase-II phosphorylation sites suggests its critical role in the circadian rhythm response in rice, similar to Arabidopsis.24 RGB1(I) amino acid sequence showed a high degree of conservation at N- and C-terminus including all important signature motifs (Fig. 2C). The phylogenetic tree was grouped into three taxonomical clusters such as lower plants (bryophytes/pteridophytes), monocots and eudicots. The cluster of monocot is represented by the members of poaceae family including Indica rice. The existence of stress responsive regulatory cis-elements in the promoter region of RGB1(I) indicate its involvement in plant immunity against abiotic stress management. However, the expression of RGB1(1) was not induced in presence of ABA, NaCl and toxic heavy metals (Cd 2+, As(V), As(III), and Pb2+). Studies on physiological responses of plants against heavy metal(loid)s suggest the existence of diverse mechanisms to combat with aforesaid stresses. However, the detailed mechanisms of cellular signaling under heavy metal(loid)s stress in plants remain obscured. Upregulated transcript level of RGB1(I) under Zn2+ and Mn2+ exposure can be correlated due to the presence of circadian regulatory ciselements in its promoter sequence (Fig. 2B). Zn2+ and Mn2+ are reportedly used as micro-nutrients. They are required by many metabolic enzymes as cofactors to participate in critical cellular metabolism. Critical metabolic interactions between Zn2+ and Ca 2+ translate into molecular interactions. Therefore, Zn2+ deficiency can be attributed to defected uptake of Ca 2+ and extracellular Zn2+ concentration affect intracellular Ca 2+.25 Smith et al.26 propounded the mechanism involving heavy metal(loid) s ions signaling through an “orphan” G-protein-coupled receptor mediated signaling via Phospholipase C, and subsequent Inositol triphosphate (IP3) activated pumping of Ca 2+ from ER. The results of present study supports the active role of RGB1(I) following the similar mechanism since it showed a significant upregulation of RGB1(I) expression with time in presence of elevated concentration of Zn2+ and Mn2+ (Fig. 3C). The G-β subunit of Arabidopsis, AGB1, have been shown to interact with few cold and drought stress regulated glycine-rich RNA binding proteins27 and our result showing the rhythmic regulation of RGB1(I) further proves this finding. Rhythmic responses of RGB1 have also been found during drought stress. Several cold and drought regulated glycine-rich RNA binding proteins have
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been reported to show the rhythmic responses under cold and drought treatments.28 Evidences for the interaction of heterotrimeric G-proteins, particularly, β subunit with transcription factors were reported.27,29 Similar to tobacco and Arabidopsis, RGB1(I) is also found to be localized on the plasma membrane, cytosol and nucleus.30,31 In the present study, sub-cellular localization of RGB1(I) using transient expression of RGB1(I)-sGFP fusion in onion peel was demonstrated. The observation of nuclear localization of RGB1 supports the earlier reports showing interaction of G-protein subunits with transcription factors.29 The present study suggests that the RGB1(I) not only transduce extracellular signals to their downstream effector molecules, but also transduce the extracellular stimuli, alone or with interacting transcription factors inside the nucleus to regulate the expression of target genes. This type of transcriptional regulation might be similar to the regulation of glucocorticoid receptor (GR) protein whose transcriptional activity is suppressed in nucleus after interaction with G-protein β subunit.32 Besides, there are several reports in animal system in which G-protein β subunit localization was reported in the nucleus, cytoplasm as well as in the membrane fraction.33 However, the mechanism and function of plant Gβ in the nucleus is yet to be elucidated. In conclusion, the complete RGB1(I) coding sequence was isolated and its expression profile in under various abiotic stress conditios was characterized. The transcript profile of RGB1(I) and presence of plant hormone stress responsive regulatory ciselements suggest that unknown transcription factors might be interacting with these regulatory elements and controlling the signaling via RGB1(I) under various abiotic stresses. The subcellular localization studies revealed that the RGB1(I) might be involved in relaying the extracellular signal and play a wider role in regulating the gene expression inside the nucleus. Our study extends the evidences for G-protein mediated signaling in stress related physiology of plants. It proposes a new tool for modulation of plants for abiotic stress tolerance. Overall, the characterization of RGB1(I) described in the present study will help in better understanding of the role of G-protein β subunit in abiotic stress signal transduction pathways.
Materials and Methods Plant material and stress treatment Rice (Oryza sativa cv Indica group Swarna) seeds were grown in vermiculite in transgenic greenhouse under 16/8 h day light condition. Abiotic stress conditions were simulated by transferring the seedlings into 1 × MS medium supplemented with 200 mM NaCl, 200 mM KCl; toxic heavy metal(loid)s salts
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Figure 3. Quantitative real-time PCR analyses showing transcript profile of RGB1(I) under different abiotic stress. Total RNA was isolated from three week old rice seedling leaf blade collected at different time intervals after treatment under different abiotic stress conditions. (A) 100 µM ABA; 200 mM KCl; and 200 mM NaCl; (B) Cold (4 °C) and Heat (42 °C); and dehydration (C) 300 µM Zn2+ and 500 µM Mn2+; and (D) 100 µM Cd2+, 250 µM As(V), 25 µM As(III) and 500 µM Pb2+. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s pairwise comparison test to compare the mean differences in fold change expression of OsGβ during different time points of exposure with respect to control (0h). Data are expressed as mean ± SD of three independent experiments, each experiment consisting of three technical replicates. *, ** indicate values that differ significantly at P < 0.05 and P > 0.01, respectively according to Tukey’s pairwise comparison test.
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Figure 4. Sub-cellular localization of rice RGB1(I)-sGFP fusion protein using transient expression in onion epidermal peel cells. (A), Upper panel is showing the sGFP signals from cell expressing RGB1-sGFP and signal is located at the periphery and the nucleus of the cell. Lower panel is showing signals from plasmolysed cells as treated with 30% sucrose to reconfirm the localization at the periphery and nucleus. (B) Upper panel is showing sGFP signal from cells bombarded with empty vector for the purpose of control and lower panel is showing signals from plasmolysed control cells. GFP signal from control cells is faint and spread throughout the cell. Left panel is showing overlay image; middle panel is showing only GFP signal and right panel is showing corresponding bright field image. Image bars are mentioned in respective panel.
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(100 µM Cd 2+ as CdCl2, 25 µM As(III) as Na 2AsO2, 250 µM As(V) as Na 2HAsO4, 500 µM Pb2+ as PbCl2); essential heavy metals (300 µM Zn2+ as ZnCl2 and 500 µM Mn2+ as MnCl2)34 and 100 μM abscisic acid.34 Each stress treatment was performed in magenta boxes and incubated at room temperature for defined time intervals. For cold (4 °C) and heat (42 °C) treatment, seedlings in 1 × MS medium were kept in incubators at defined temperatures. Dehydration stress condition was simulated by uprooting the seedlings and keeping them on blotting paper for the mentioned period of time. Leaf blades of the respective stress treated seedlings were harvested at different time intervals (viz. 1h, 2h, 3h, 6h, and 12h). Seedlings grown in 1 × MS medium in transgenic greenhouse under similar condition without any treatment were taken as a control. After sampling, the leaf blades (10 seedlings per treatment) were snap frozen in liquid nitrogen and stored at -80 °C until use. Isolation of RNA and cDNA Preparation Total RNA was isolated from different stress treated and control leaf blades of rice seedlings sampled at different time interval, in TRIZOL LS reagent (Invitrogen, Life Technologies, USA) following manufacturer’s instructions. Genomic DNA contamination was digested by RNase-free DNaseI treatment. The first strand cDNA was synthesized from 1 μg of total RNA using Superscript II Reverse Transcriptase (Invitrogen, Life Technologies USA) using oligo(dT)18 primer according to the manufacturer’s instructions. Experiments were repeated thrice, independently. Isolation and sequence analysis of RGB1(I) RGB1(I) was amplified by PCR using first strand cDNA as template and forward primer5′-CTCGAGCATA TGGCGTCCGT GGCGGAGCTC-3′ and reverse primer 5′GGATTCTCAA ACTATTTTCC GGTGTCC-3′, respectively, using annealing temperature of 60 °C (Ta = 60 °C). Amplified fragment was cloned into the pGEMTeasy vector (Promega). The putative recombinant colonies of E. coli DH5α, showing desired amplicon, were used for isolation of plasmid DNA using QIAprep Spin Miniprep kit (Qiagen). The insertion of RGB1(I) gene was confirmed by restriction digestion using with NdeI and EcoRI restriction enzymes and positive clone was subjected to nucleotide sequencing using T7 forward and SP6 reverse primers. The RGB1(I) sequence was submitted to GenBank as accession number HM768320.1. In Silico analysis of promoters and gene sequence of RGB1(I) In order to analyze the putative cis-elements in the promoters, 1.5 kb genomic sequence upstream of the translation initiation codon of RGB1(I) gene was searched on cis-element database (http://bioinformatics.psb.ugent.be /webtools /plantcare / html/).35 BLAST search in rice genome annotation project (http://rice.plantbiology.msu.edu/) was used to identify RGB1(I) genomic DNA sequences including 5′- and 3′-UTR, exon and intron sequences. In Silico analysis of RGB1(I) protein The deduced amino acid sequence of RGB1(I), was compared with the Gβ subunits of important model plants viz. Arabidopsis thaliana, Glycine max, Nicotiana tabacum, Pisum sativum, Ricinus communis, Sorghum bicolor, Zea mays by multiple amino acid
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sequence alignment using Clustal Omega multiple sequence alignment program (http://www.ebi.ac.uk/Tools/msa/clustalO/) and Jal view was used to show the alignment. To decipher the evolutionary history of G-protein β subunits from plant kingdom, a rooted phylogenetic tree was constructed employing MEGA version 5 using Maximum Parsimony method.36 Tree #1 out of 4 most parsimonious trees (length = 1315) is shown. The tree was obtained using the Close-Neighbor-Interchange algorithm37 with search level 1 in which the initial trees were obtained with the random addition of sequences (10 replicates). The analysis involved nucleotide sequences from 38 different genera. All positions containing gaps and missing data were eliminated. The Bootstrap values from 1000 replicates are shown for selected branches. The deduced amino acid sequence of RGB1(I), was compared with the Gβ subunits of Japonica rice, maize, barley, Sorghum, wheat and similarity was calculated using Clustal Omega. The functional motifs, patterns and biologically significant sites in RGB1(I) amino acid sequence were located by ExPASy Proteomics Server ScanPro site (http://www.expasy.org/tools/scanprosite/). Quantitative Real-Time PCR The transcript profile of RGB1(I) in leaf blades under different stress conditions were determined by quantitative real-time PCR using StepOne Real Time PCR system (Applied BioSystems), essentially as described earlier.38 In brief, Power SyberGreen PCR master mix (Applied BioSystems) was used in a 20 µl reaction mixture containing 10 pico mole of each gene specific primer pair (α-tubulin forward 5′-GGTGGAGGTG ATGATGCTTT-3′ and reverse 5′-ACCACGGGCA AAGTTGTTAG-3′ and RGB1(I) forward 5′-GGGAAACAGG AGGTTGAACA-3′ and reverse 5′-GCGTCTCATG CTCTCATCAA-3′) and 1 µl of stress treatment specific cDNA. PCR reaction conditions were defined as one cycle of 10 min for 95 °C for initial denaturation followed by 40 cycles of 15s at 95 °C, 1 min at 59 °C. Optical data were collected after every cycle. The melt curve of PCR products was generated by heating them at temperatures starting from 55 °C to 95 °C, with a gradual increase by 0.5 °C at every step. Rice α-tubulin gene was used as internal reference. Experiments were repeated three times independently with three technical replicates in each experiment for each time point of treatment. Relative gene expression was calculated using the 2-ΔΔCT values following Livaks’ method.39 Preparation of construct (35S:RGB1-sGFP) and localization of RGB1(I) To study the sub-cellular localization of RGB1(I), the translational fusion cassette of RGB1(I) for transient expression was developed using pUC-based p326-sGFP vector containing CaMV35S-sGFP-(S65T) and NosT.40 The expression construct (CaMV35S:RGB-sGFP) was prepared by sub-cloning of XbaI/ BamHI digested fragment amplified from plasmid pGEMTRGB1 using RGBXbaIF, 5′-GATCTAGAAT GGCGTCCGTG GCGGAGCT-3′ and RGBBamHIR, 5′-TCAGGATCCA ACTATTTTCC GGTGTCC-3′ primers. XbaI/BamHI digested PCR amplicon was inserted into XbaI/BamHI sites of plasmid p326-sGFP to create chimeric RGB1(I)-GFP-fusion construct under the control of the 35S promoter. Two microgram of
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purified plasmid (RGB1-sGFP) was coated on gold particles (1.0 µm) and bombarded on to the onion epidermis kept on 0.5 x MS petri-plate using particle delivery system (PDS-1000/He, Bio-Rad, USA) set at 1100 psi from a distance of 6 cm. GFP fluorescence was examined using confocal microscope (Nikon A-1R) after 16 h incubation in dark with high humidity. Onion epidermal peels were detached from agar plate and mounted in 10% glycerol for microscopic observations. Epidermal peels were plasmolysed by immersing in 30% sucrose solution for 30 min. Excitation wavelength was set at 488 nm and emission wavelength was recorded between 505–530 nm BP filters. Statistical analysis The quantitative real-time gene expression data were analyzed by one way analysis of variance (ANOVA), and significant differences between individual means determined using Tukey’s pairwise comparison test at the 5% and 1% (P < 0.05 and P < 0.01) confidence level. References 1. Jones AM, Assmann SM. Plants: the latest model system for G-protein research. EMBO Rep 2004; 5:572-8; PMID:15170476; http://dx.doi. org/10.1038/sj.embor.7400174 2. Urano D, Chen JG, Botella JR, Jones AM. Heterotrimeric G protein signalling in the plant kingdom. Open Biol 2013; 3:120186; PMID:23536550; http://dx.doi.org/10.1098/ rsob.120186 3. Assmann SM. Heterotrimeric and unconventional GTP binding proteins in plant cell signaling. Plant Cell 2002; 14:S355-73; PMID:12045288 4. Ma H, Yanofsky MF, Meyerowitz EM. Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc Natl Acad Sci U S A 1990; 87:3821-5; PMID:2111018; http://dx.doi.org/10.1073/ pnas.87.10.3821 5. Weiss CA, Garnaat CW, Mukai K, Hu Y, Ma H. Isolation of cDNAs encoding guanine nucleotidebinding protein beta-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc Natl Acad Sci U S A 1994; 91:9554-8; PMID:7937804; http://dx.doi.org/10.1073/pnas.91.20.9554 6. Mason MG, Botella JR. Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with Gbeta. Biochim Biophys Acta 2001; 1520:147-53; PMID:11513956; http://dx.doi.org/10.1016/S0167-4781(01)00262-7 7. Chakravorty D, Trusov Y, Zhang W, Acharya BR, Sheahan MB, McCurdy DW, Assmann SM, Botella JR. An atypical heterotrimeric G-protein γ-subunit is involved in guard cell K+ -channel regulation and morphological development in Arabidopsis thaliana. Plant J 2011; 67:840-51; PMID:21575088; http:// dx.doi.org/10.1111/j.1365-313X.2011.04638.x 8. Trusov Y, Zhang W, Assmann SM, Botella JR. Ggamma1 + Ggamma2 not equal to Gbeta: heterotrimeric G protein Ggamma-deficient mutants do not recapitulate all phenotypes of Gbetadeficient mutants. Plant Physiol 2008; 147:63649; PMID:18441222; http://dx.doi.org/10.1104/ pp.108.117655 9. Marsh JF 3rd, Kaufman LS. Cloning and characterisation of PGA1 and PGA2: two G protein alpha-subunits from pea that promote growth in the yeast Saccharomyces cerevisiae. Plant J 1999; 19:237-47; PMID:10476071; http://dx.doi. org/10.1046/j.1365-313X.1999.00516.x
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
Work on plant abiotic stress tolerance and G-protein signaling in NT’s laboratory is supported by Department of Biotechnology (DBT), and Department of Science and Technology (DST), Government of India. We thank to Mrs. Poornima for technical help in confocal microscopy. We thank Dr. Renu Tuteja and Mr. Deepak Bhardwaj for their help in corrections of the manuscript. Author Contributions
DKY performed the research, analyzed data, and wrote the manuscript; DS performed the experiments and helped in writing the manuscript; NT designed research, analyzed the data, and wrote the manuscript.
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34. Yadav DK, Shukla D, Tuteja N. Rice heterotrimeric G-protein alpha subunit (RGA1): in silico analysis of the gene and promoter and its upregulation under abiotic stress. Plant Physiol Biochem 2013; 63:26271; PMID:23313793; http://dx.doi.org/10.1016/j. plaphy.2012.11.031 35. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 2002; 30:325-7; PMID:11752327; http://dx.doi.org/10.1093/ nar/30.1.325 36. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011; 28:2731-9; PMID:21546353; http://dx.doi.org/10.1093/molbev/msr121 37. Nei M, Kumar S. Molecular Evolution and Phylogenetics. 2000; Oxford University Press, New York. 38. Yadav DK, Islam SM, Tuteja N. Rice heterotrimeric G-protein gamma subunits (RGG1 and RGG2) are differentially regulated under abiotic stress. Plant Signal Behav 2012; 7:733-40; PMID:22751322; http://dx.doi.org/10.4161/psb.20356
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Isolation, in silico characterization, localization and expression analysis of abiotic stress-responsive rice Gprotein β subunit (RGB1) a
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Dinesh K Yadav , Devesh Shukla & Narendra Tuteja
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Plant Molecular Biology Group; International Centre for Genetic Engineering and Biotechnology; Aruna Asaf Ali Marg, New Delhi, India Published online: 16 Apr 2014.
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Research Paper
Research Paper
Plant Signaling & Behavior 9, e28890; April; © 2014 Landes Bioscience
Isolation, in silico characterization, localization and expression analysis of abiotic stressresponsive rice G-protein β subunit (RGB1) Dinesh K Yadav, Devesh Shukla†, and Narendra Tuteja* Plant Molecular Biology Group; International Centre for Genetic Engineering and Biotechnology; Aruna Asaf Ali Marg, New Delhi, India †
Current address: Department of Biology; Western Kentucky University; Bowling Green, KY USA
Keywords: Abiotic stress; heterotrimeric G-proteins; metals; rice G-protein beta-subunits; signal transduction, sub-cellular localization
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Abbreviations: ABA, abscisic acid; ARE, auxin responsive elements; GPCR, G-Protein coupled receptor; RGB1(I), β-subunit of Indica rice; RGB1(J), β-subunit of Japonica rice
Heterotrimeric G-proteins constitute the classical signaling paradigm along with their cognate G-protein coupled receptors (GPCRs) and appropriate downstream effectors. G-protein complex is composed of highly conserved Gα, Gβ, and Gγ subunits. In the present study, we have characterized the cis-regulatory elements of the promoter, signature motifs, transcript profile in response to abiotic stresses, and sub-cellular localization of G-protein β subunit RGB1(I) from Indica rice. The RGB1(I) promoter sequence has various stress-related cis-regulatory elements suggesting its role in abiotic stress signaling. Presence of six WD-40 repeat signature motifs in RGB1(I) suggest its role in exchange of GDP by GTP in Gα subunit and receptor recognition. Presence of multiple N-myristoylation consensus sites in RGB1(I) protein sequence, which is necessary for membrane localization of protein, confirms the association of RGB1(I) in plasma membrane. Extrinsic association of RGB1(I) with plasma membrane seems essential for its role in regulation of signaling pathways and adaptation to high salt stress. We report the sub-cellular localization of RGB1(I) in plasma membrane, cytosol and nucleus. The localization of RGB1(I) in nucleus supports its possible interaction with transcription factors regulating the expression of salt stress responsive genes. The RGB1(I) transcript was upregulated under KCl, cold, dehydration and micronutrient (Mn2+ and Zn2+) stress. However, transcript variation under elevated temperature, ABA, NaCl, and toxic heavy metals (viz. arsenite, arsenate, cadmium and lead) was not encouraging. These evidences indicate an active and significant role of RGB1(I) in the regulation of abiotic stresses in rice and propound its possible exploitation in the development of abiotic stress tolerance in crops.
Introduction The ability of multi-cellular organisms to sense the environmental stimuli and respond appropriately is an utmost requirement for synchronized development, ordered growth, maintenance and successful reproduction. The evolution of an efficient cellular communication system allows cells to coordinated response to environmental stimuli as well as to each other by integrating the wide array of extracellular signals to intracellular downstream effectors.1 Heterotrimeric guanine nucleotide binding proteins (G-proteins) are well characterized signaling molecules that interact with plasma membrane localized G-protein coupled receptors (GPCRs) and transduce majority (~80%) of extracellular signals across the cell membranes. However, these functions are believed to be performed by regulator of G-protein signaling (RGS) in plants.2
Heterotrimeric G-protein complex consists of α (Gα), β (Gβ), and γ (Gγ) subunits. According to classical paradigm, G-protein signaling is initiated by the association of extracellular ligand with GPCR (or RGS) and subsequently inducing a conformational change which promotes the exchange of GDP for GTP associated to Gα subunit. GTP bound Gαβγ heterotrimeric complex tends to dissociate into Gα-GTP monomer and Gβγ heterodimer. The dissociated units of Gα-GTP monomer and Gβγ heterodimer can interact with a wide array of specific downstream effectors of distinct signaling pathways. The whole genome sequencing efforts have established that the heterotrimeric G-protein mediated signaling mechanism is highly complex. Unlike human proteome that contains 1300 odd theoretical heterotrimeric complexes, plants have very small repertoire of G-protein subunit genes.3 The fully sequenced Arabidopsis genome contains only one gene for each canonical Gα
*Correspondence to: Narendra Tuteja, Email: [email protected] Submitted: 03/21/2014; Revised: 04/14/2014; Accepted: 04/14/2014; Published Online: 04/16/2014 Citation: Yadav DK, Shukla D, Tuteja N. Isolation, in silico characterization, localization and expression analysis of abiotic stress-responsive rice G-protein β subunit (RGB1). Plant Signaling & Behavior 2014; 9:e28890; PMID: 24739238; http://dx.doi.org/10.4161/psb.28890
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Figure 1A. See next page for legend.
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Figure 1A (See previous page for figure). Multiple sequence alignment and phylogenetic tree of β sub-units of G protein sequences. Different amino acid sequences of β sub-unit of G protein aligned using Clustal Omega, are AtGβ (Arabidopsis thaliana), GmGβ (Glycine max), NtGβ (Nicotiana tabacum), PsGβ (Pisum sativum), RcGβ (Ricinus communis), SbGβ (Sorghum bicolor), ZmGβ (Zea mays) andal RGB(I) (Rice Gβ, Indica).
(GPA1),4 Gβ (AGB1)5 and three genes for Gγ subunits, AGG1, AGG2 and AGG3.6,7 G-protein γ subunits provide functional selectivity in G-βγ dimer signaling in Arabidopsis and some new elements exist in the heterotrimeric G-protein signaling complex.8 Two Gα subunit genes in pea (PGA1 and PGA2)9 and four Gα subunit genes (GmGα 1–4) in soybean10 were reported. Fully sequenced rice genome contains only one conventional Gα and Gβ, but three Gγ subunits (RGG1, RGG2 and RGG3), and a GPCR.11 RGG3 is a homolog of AGG3 and is known as DEP1.7 However, several GPCR-type candidates were initially proposed in plant kingdom but later discredited.2,12,13 Despite the limited number of the components, G-protein complexes in plants regulate diverse signaling pathways including hormone signaling, environmental sensing, ion channel regulation, and disease response and cell death.14,15 Recently, Pandey16 identified an elaborate network of G-proteins in soybean. In Arabidopsis, AGB1 was reported to play an important role in the development of leaf, flower, and fruit.17 Utsunomyia et al.18 reported that RGB1 knockdown lines generated in RGA1 deficient d1–5 lines caused dwarfism and browning of internodes and lamina joint regions. These observations suggest the predominant role of RGB1 in G-protein mediated signaling that regulates cellular proliferation and seed fertility through functional cooperation with RGA1. In the present study, we have isolated and characterized Gβ subunit from Indica rice. The deduced protein sequence was used to study its structural features and phylogenetic relatedness. Furthermore, the genomic organization and promoter analysis was also done using in silico methods. The sub-cellular localization and transcript abundance of RGB1(I) was studied under different abiotic stress conditions including high salt, cold, heat, dehydration, ABA and heavy metal(loids).
Results Isolation and in silico analysis of RGB1(I) The full-length coding region of RGB1(I) was PCR amplified with gene specific primers using first-strand cDNA as the template. The analysis of amplified and sequenced DNA fragment revealed 1143 bp complete transcript of RGB1(I). The deduced protein sequence from full-length cDNA sequence showed a putative protein comprising of 380 amino acids. The putative protein showed computed molecular mass and iso-electric point (pI) as 41.7 kDa and 7.13, respectively. The multiple sequence alignment of RGB1(I) subunit at amino acid level with other Gβ subunits from related plant genera is shown in Figure 1A. Phylogenetic analysis of RGB1 duly reflects the taxonomic grouping (Fig. 1B). The phylogenetic tree displayed separate clades for eudicots, monocots and lower plants including bryophytes and pteridophytes. Interestingly, all the members of monocots belong to the poaceae family. Pairwise amino acid sequence comparison of RGB1(I) showed 99%
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identity with RGB1(J) followed by maize (ZmGB1) and sorghum (SbGB1) with 94% identity, either. It showed 92% and 91% identity with wheat and oat, respectively (Table 1). The observed phylogenetic relatedness suggest that evolution of Gβ subunit in rice is similar to closely related grass family members (Fig. 1B). Genomic organization of RGB1(I) Comparison of full-length cDNA sequence of RGB1(I) with its genomic DNA sequence identified six exons (109, 95, 425, 363, 113 and 38 bp) and five introns (128, 4326, 608, 607 and 518 bp). The sequence contains 5′-UTR of 75 bp and 458 bp at its 3′-end (Fig. 2A). Computational analysis of promoter elements of RGB1(I) The occurrence and distribution of regulatory cis-elements in 1.5 kb upstream promoter region of RGB1(I) was analyzed as shown in Figure 2B. Stress-responsive regulatory cis-elements identified in the promoter region of RGB1(I) contained several phytohormones responsive elements like, three abscisic acid responsive element (ABRE) reportedly associated with abscisic acid responsiveness and drought tolerance. In addition one gibberellic acid responsive, one ethylene responsive, one jasmonate responsive and one salicylic acid responsive elements were also identified. Besides these, it also has salt induced responsive element (GT-1 motif), circadian regulating cis-elements, low temperature responsive element (LTR) and one W-box domain. W-box domain is especially involved in response to the WRKY transcription factor family, and express under the abiotic and biotic stresses. Various significant motifs, patterns and biological sites were identified in RGB1(I) using the Expasy PROSITE database of protein domains, families and functional sites (Fig. 2C). It showed the six WD-40 repeats, viz. 62–103: PQGHSGKVYS LDWTPEKNWI VSASQDGRLI VWNALTSQKT HA,153– 195: LTGHKGYVSS CQYVPDQETR LITSSGDQTC VLWDVTTGQR ISI, 201–243: PSGHTADVLS LSINSSISNM FVSGSCDATV RLWDIRIASR AVR, 245286: YHGHEGDINS VKFFPDGQRF GTGSDDGTCR LFDVRTGHQL QV, 290–333: PDRNDNELPT VTSIAFSISG RLLFAGYSNG DCYVWDTLLA EVV, and 340–380: QNSHEGRISC LGLSSDGSAL CTGSWDKNLK IWAFSGHRKIV, are involved in the formation of β propeller sheet. It showed the presence of nine N-myristoylation sites (viz. 120 – 125; GQsvAC, 126 – 131; GGldSA, 127 – 132; GLdsAC, 190 – 195; GQriSI, 224 – 229; GScdAT, 261 – 266; GQrfGT, 267 – 272; GSddGT, 337 – 342; GNlqNS, 356 - 361; GSalC) six casein kinase II phosphorylation site, (viz. 3 - 6: SvaE; 20 23: SlrE; 35 - 38: TdvE; 139 - 142: SqaD; 266 - 269: TgsD; 318 - 321: SngD); seven protein kinase C phosphorylation site (viz. 20 - 22: SLR; 66 - 68: SGK; 98 - 100: SqK; 229 - 231: TvR; 254 - 256: SvK; 272 - 274: TcR; 309 - 311: SgR) and one potential glycosylation site at position 214 (NSS). The constituent amino acid residues of identified significant motifs, patterns and biological sites in RGB1(I) showed a high conserved score values (Fig. 2C).
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Table 1. Pairwise amino acid sequence identity of G-protein β subunit among economically important taxa of grass family *
Oryza sativa Indica
Oryza sativa Japonica
Triticum aestivum GB1
Zea mays gbp2
Avena fatua Gb1
Sorghum bicolor Gb
Oryza sativa Indica
*
99%
92%
94%
91%
94%
*
93%
95%
92%
95%
*
92%
92%
92%
*
91%
98%
*
91%
Oryza sativa Japonica Triticum aestivum GB1 Zea mays gbp2 Avena fatua Gb1
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Sorghum bicolor Gb
*
Transcript profile of RGB1(I) by real-time quantitative PCR The transcript profile of RGB1(I) under 200 mM NaCl treatment showed no significant difference unlike to the response under treatment of 200 mM KCl, as compared with control (Fig. 3A). Exposure to 200 mM KCl (Fig. 3A) upregulated the RGB1(I) mRNA abundance by ~9.0 and ~7.0-fold after 2h and 3h, respectively and eventually reached to its basal levels. The transcript profile of RGB1(I) showed no significant change under the exposure of 100 µM ABA level. Cold stress treatment increased the RGB1(I) transcript by ~12-fold after 1h, followed by a drop of ca. ~4-fold after 6h. It significantly increased again by ca. 35-fold after 12h (Fig. 3B). The change RGB1(I) mRNA abundance due to heat exposure was insignificant except for 1h (Fig. 3B). The expression of RGB1(I) during the dehydration stress condition showed a significant and intense increase in transcript level. It rapidly increased ~30 fold after 1h and was maintained > 34-fold up to 6h and subsequently reduced to basal level (Fig. 3B). The exposure to 300 µM Zn2+ upregulated the expression of RGB1(I) by ca. 3-fold at 1h and ca. 5-fold at 12h in comparison to non-treated control samples (Fig. 3C). Treatment of seedlings with 500 µM Mn2+ induced the expression by 6-fold at 1h and 6h (Fig. 3C). Exposure to 100 µM Cd 2+ induced the reduction in mRNA abundance of RGB1(I) at 1h, 2h and 12h in a range of ca. 0.3–0.5-fold (Fig. 3D). Exposure to 250 µM As(V) treatment caused a downregulation during 2h to 6h. An increase was observed in transcript level by ~2.5-fold after 12h (Fig. 3D). Under the exposure of As(III), the expression initially decreased by 0.7-fold up to 2h, followed by an increase by ~3.5-fold at 3h. This increment in expression was prolonged by ≥1.3-fold up to 12h. (Fig. 3E). The treatment with 700 μM Pb2+ caused a slight but rhythmic change in abundance of transcript and maximum increase was observed at 6h. RGB1(I) localizes to plasma membrane, cytosol and nucleus The study of sub-cellular localization was performed in onion peels bombarded with gold particles coated with plasmid construct 35S:RGB1-sGFP. Transient expression of RGB1(I)sGFP in epidermal cells of onion showed the prominent GFP signals from periphery and nucleus of the cells suggesting plasmamembrane and nuclear localization of fusion protein (Fig. 4). The thick level of fluorescence at the periphery indicates that the RGB1 may be localized to cytosol. This observation was also confirmed by the plasmolysis of cells expressing RGB1(I)-sGFP fusion protein. On the other hand, the cells expressing only GFP
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(empty vector) showed faint signal throughout the cell. Hence, the images explicitly suggest that RGB1(I) resides on the plasma membrane, cytosol as well as in the nucleus.
Discussion Global climatic changes in rice cultivating areas like, frequent drought, increasing atmospheric temperature and soil salinity, heavy metal(loid)s pollution, oxidative stress and various other abiotic stresses affect its production. It warrants the need to identify suitable candidate genes and annotate their role in abiotic stress tolerance/ resistance in plants to achieve sustainable crop improvement. Heterotrimeric G-proteins are major group of signaling molecules responsible for a variety of cellular response to external stimuli as inferred from pharmacological studies. These signaling proteins represent a potential genetic tool for modification of plants to overcome various abiotic stresses. However, unlike animal kingdom, plants have a limited repertoire of classical G-proteins, suggesting a mechanistically different signaling network as compared with mammals. Presence of four Gα, four Gβ and two Gγ gene sequences in soybean suggests 32 possible combinations of G protein heterotrimer. The Gα subunit in soybean has two distinct mode of GTP-hydrolysis indicating a complex and much diversified G-protein signaling network among the plants.10 Accomplishing this diversity we have isolated RGB1(I) to comprehend its structural features, subcellular localization and significance in abiotic stress response. Multiple sequence alignment of RGB1(I) subunit and its comparison with other known Gβ subunit in monocots showed a high level of similarity (Fig. 1A). All the significant structural and functional motifs required for its function were conserved. The RGB1(I) amino acid sequence showed the presence of six most important signature motif/sequences of Gβ subunit, the WD-40 repeats (Fig. 2C) which are involved in the formation of the seven propeller blades and contain the typical feature of Gβ subunits proteins. In these signature motifs, highly conserved aspartic acid is present in between strand b and c of WD repeats rather the Aspartic acid present in WD itself.19,20 These highly conserved aspartic acids are involved in the formation of an interand intra-blade hydrogen bond triad involving histidine in GH motif and a Ser/Thr in the blade b strand.21 RGB1(I) showed the presence of nine N-myristoylation sites, six casein kinase-II phosphorylation sites and six protein kinase C
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Figure 1B. A rooted phylogenetic tree was constructed using MEGA5 showing evolutionary relatedness of G protein β subunits in plants. The Bootstrap values from 1000 replicates are shown for selected branches.
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Figure 2. See next page for legend.
phosphorylation sites. N-myristoylation is a co/post-translational covalent protein modification that promotes association of
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modified proteins with lipid bilayer membrane. The association of protein with lipid bilayer is crucial for its role in regulating
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Figure 2 (See previous page for figure). In silico analysis of RGB1(I). (A) The schematic representation of genomic organization (exon–intron organization) of the genomic sequence of RGB1(I) genes. Closed boxes represent exons, and lines between closed boxes represent introns. The dark boxes represent the UTRs. The position of start (ATG) and stop codons (TGA) are marked. The numbers below the lines and the above boxes indicate the sizes (bp) of introns, UTR and exons, respectively. (B) Stress-responsive cis-regulatory elements and phytohormones responsive elements in the 1.5 kb 5′-upstream regions of RGB1(I) gene. The elements located in the (+) strand are above the lines, while those in the (-) strand are indicated below the line. ABRE = Abscisic acid responsive element; ARE = Auxin responsive factor (TGA-box); MeJAE = Methyl jasmonate responsive element; GT1Box; LTR = Low Temperature responsive element and anoxia specific element. (C) Different motifs, important active site and patterns identified using Expasy PROSITE database.
signal transduction and is critical to accustom plants against high salt stress.22 Thus, multiple N-myristoylation sites in RGB1(I) and increased transient transcript profile due to increased KCl level suggest its possible role in maintaining K+ homeostasis in rice plant.23 The suggested role of RGB1(I) in ion homeostasis is further supported by its topographic association with plasma membrane, cytosol and nucleus as shown in Figure 4. The presence of multiple casein kinase-II phosphorylation sites suggests its critical role in the circadian rhythm response in rice, similar to Arabidopsis.24 RGB1(I) amino acid sequence showed a high degree of conservation at N- and C-terminus including all important signature motifs (Fig. 2C). The phylogenetic tree was grouped into three taxonomical clusters such as lower plants (bryophytes/pteridophytes), monocots and eudicots. The cluster of monocot is represented by the members of poaceae family including Indica rice. The existence of stress responsive regulatory cis-elements in the promoter region of RGB1(I) indicate its involvement in plant immunity against abiotic stress management. However, the expression of RGB1(1) was not induced in presence of ABA, NaCl and toxic heavy metals (Cd 2+, As(V), As(III), and Pb2+). Studies on physiological responses of plants against heavy metal(loid)s suggest the existence of diverse mechanisms to combat with aforesaid stresses. However, the detailed mechanisms of cellular signaling under heavy metal(loid)s stress in plants remain obscured. Upregulated transcript level of RGB1(I) under Zn2+ and Mn2+ exposure can be correlated due to the presence of circadian regulatory ciselements in its promoter sequence (Fig. 2B). Zn2+ and Mn2+ are reportedly used as micro-nutrients. They are required by many metabolic enzymes as cofactors to participate in critical cellular metabolism. Critical metabolic interactions between Zn2+ and Ca 2+ translate into molecular interactions. Therefore, Zn2+ deficiency can be attributed to defected uptake of Ca 2+ and extracellular Zn2+ concentration affect intracellular Ca 2+.25 Smith et al.26 propounded the mechanism involving heavy metal(loid) s ions signaling through an “orphan” G-protein-coupled receptor mediated signaling via Phospholipase C, and subsequent Inositol triphosphate (IP3) activated pumping of Ca 2+ from ER. The results of present study supports the active role of RGB1(I) following the similar mechanism since it showed a significant upregulation of RGB1(I) expression with time in presence of elevated concentration of Zn2+ and Mn2+ (Fig. 3C). The G-β subunit of Arabidopsis, AGB1, have been shown to interact with few cold and drought stress regulated glycine-rich RNA binding proteins27 and our result showing the rhythmic regulation of RGB1(I) further proves this finding. Rhythmic responses of RGB1 have also been found during drought stress. Several cold and drought regulated glycine-rich RNA binding proteins have
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been reported to show the rhythmic responses under cold and drought treatments.28 Evidences for the interaction of heterotrimeric G-proteins, particularly, β subunit with transcription factors were reported.27,29 Similar to tobacco and Arabidopsis, RGB1(I) is also found to be localized on the plasma membrane, cytosol and nucleus.30,31 In the present study, sub-cellular localization of RGB1(I) using transient expression of RGB1(I)-sGFP fusion in onion peel was demonstrated. The observation of nuclear localization of RGB1 supports the earlier reports showing interaction of G-protein subunits with transcription factors.29 The present study suggests that the RGB1(I) not only transduce extracellular signals to their downstream effector molecules, but also transduce the extracellular stimuli, alone or with interacting transcription factors inside the nucleus to regulate the expression of target genes. This type of transcriptional regulation might be similar to the regulation of glucocorticoid receptor (GR) protein whose transcriptional activity is suppressed in nucleus after interaction with G-protein β subunit.32 Besides, there are several reports in animal system in which G-protein β subunit localization was reported in the nucleus, cytoplasm as well as in the membrane fraction.33 However, the mechanism and function of plant Gβ in the nucleus is yet to be elucidated. In conclusion, the complete RGB1(I) coding sequence was isolated and its expression profile in under various abiotic stress conditios was characterized. The transcript profile of RGB1(I) and presence of plant hormone stress responsive regulatory ciselements suggest that unknown transcription factors might be interacting with these regulatory elements and controlling the signaling via RGB1(I) under various abiotic stresses. The subcellular localization studies revealed that the RGB1(I) might be involved in relaying the extracellular signal and play a wider role in regulating the gene expression inside the nucleus. Our study extends the evidences for G-protein mediated signaling in stress related physiology of plants. It proposes a new tool for modulation of plants for abiotic stress tolerance. Overall, the characterization of RGB1(I) described in the present study will help in better understanding of the role of G-protein β subunit in abiotic stress signal transduction pathways.
Materials and Methods Plant material and stress treatment Rice (Oryza sativa cv Indica group Swarna) seeds were grown in vermiculite in transgenic greenhouse under 16/8 h day light condition. Abiotic stress conditions were simulated by transferring the seedlings into 1 × MS medium supplemented with 200 mM NaCl, 200 mM KCl; toxic heavy metal(loid)s salts
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Figure 3. Quantitative real-time PCR analyses showing transcript profile of RGB1(I) under different abiotic stress. Total RNA was isolated from three week old rice seedling leaf blade collected at different time intervals after treatment under different abiotic stress conditions. (A) 100 µM ABA; 200 mM KCl; and 200 mM NaCl; (B) Cold (4 °C) and Heat (42 °C); and dehydration (C) 300 µM Zn2+ and 500 µM Mn2+; and (D) 100 µM Cd2+, 250 µM As(V), 25 µM As(III) and 500 µM Pb2+. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s pairwise comparison test to compare the mean differences in fold change expression of OsGβ during different time points of exposure with respect to control (0h). Data are expressed as mean ± SD of three independent experiments, each experiment consisting of three technical replicates. *, ** indicate values that differ significantly at P < 0.05 and P > 0.01, respectively according to Tukey’s pairwise comparison test.
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Figure 4. Sub-cellular localization of rice RGB1(I)-sGFP fusion protein using transient expression in onion epidermal peel cells. (A), Upper panel is showing the sGFP signals from cell expressing RGB1-sGFP and signal is located at the periphery and the nucleus of the cell. Lower panel is showing signals from plasmolysed cells as treated with 30% sucrose to reconfirm the localization at the periphery and nucleus. (B) Upper panel is showing sGFP signal from cells bombarded with empty vector for the purpose of control and lower panel is showing signals from plasmolysed control cells. GFP signal from control cells is faint and spread throughout the cell. Left panel is showing overlay image; middle panel is showing only GFP signal and right panel is showing corresponding bright field image. Image bars are mentioned in respective panel.
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(100 µM Cd 2+ as CdCl2, 25 µM As(III) as Na 2AsO2, 250 µM As(V) as Na 2HAsO4, 500 µM Pb2+ as PbCl2); essential heavy metals (300 µM Zn2+ as ZnCl2 and 500 µM Mn2+ as MnCl2)34 and 100 μM abscisic acid.34 Each stress treatment was performed in magenta boxes and incubated at room temperature for defined time intervals. For cold (4 °C) and heat (42 °C) treatment, seedlings in 1 × MS medium were kept in incubators at defined temperatures. Dehydration stress condition was simulated by uprooting the seedlings and keeping them on blotting paper for the mentioned period of time. Leaf blades of the respective stress treated seedlings were harvested at different time intervals (viz. 1h, 2h, 3h, 6h, and 12h). Seedlings grown in 1 × MS medium in transgenic greenhouse under similar condition without any treatment were taken as a control. After sampling, the leaf blades (10 seedlings per treatment) were snap frozen in liquid nitrogen and stored at -80 °C until use. Isolation of RNA and cDNA Preparation Total RNA was isolated from different stress treated and control leaf blades of rice seedlings sampled at different time interval, in TRIZOL LS reagent (Invitrogen, Life Technologies, USA) following manufacturer’s instructions. Genomic DNA contamination was digested by RNase-free DNaseI treatment. The first strand cDNA was synthesized from 1 μg of total RNA using Superscript II Reverse Transcriptase (Invitrogen, Life Technologies USA) using oligo(dT)18 primer according to the manufacturer’s instructions. Experiments were repeated thrice, independently. Isolation and sequence analysis of RGB1(I) RGB1(I) was amplified by PCR using first strand cDNA as template and forward primer5′-CTCGAGCATA TGGCGTCCGT GGCGGAGCTC-3′ and reverse primer 5′GGATTCTCAA ACTATTTTCC GGTGTCC-3′, respectively, using annealing temperature of 60 °C (Ta = 60 °C). Amplified fragment was cloned into the pGEMTeasy vector (Promega). The putative recombinant colonies of E. coli DH5α, showing desired amplicon, were used for isolation of plasmid DNA using QIAprep Spin Miniprep kit (Qiagen). The insertion of RGB1(I) gene was confirmed by restriction digestion using with NdeI and EcoRI restriction enzymes and positive clone was subjected to nucleotide sequencing using T7 forward and SP6 reverse primers. The RGB1(I) sequence was submitted to GenBank as accession number HM768320.1. In Silico analysis of promoters and gene sequence of RGB1(I) In order to analyze the putative cis-elements in the promoters, 1.5 kb genomic sequence upstream of the translation initiation codon of RGB1(I) gene was searched on cis-element database (http://bioinformatics.psb.ugent.be /webtools /plantcare / html/).35 BLAST search in rice genome annotation project (http://rice.plantbiology.msu.edu/) was used to identify RGB1(I) genomic DNA sequences including 5′- and 3′-UTR, exon and intron sequences. In Silico analysis of RGB1(I) protein The deduced amino acid sequence of RGB1(I), was compared with the Gβ subunits of important model plants viz. Arabidopsis thaliana, Glycine max, Nicotiana tabacum, Pisum sativum, Ricinus communis, Sorghum bicolor, Zea mays by multiple amino acid
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sequence alignment using Clustal Omega multiple sequence alignment program (http://www.ebi.ac.uk/Tools/msa/clustalO/) and Jal view was used to show the alignment. To decipher the evolutionary history of G-protein β subunits from plant kingdom, a rooted phylogenetic tree was constructed employing MEGA version 5 using Maximum Parsimony method.36 Tree #1 out of 4 most parsimonious trees (length = 1315) is shown. The tree was obtained using the Close-Neighbor-Interchange algorithm37 with search level 1 in which the initial trees were obtained with the random addition of sequences (10 replicates). The analysis involved nucleotide sequences from 38 different genera. All positions containing gaps and missing data were eliminated. The Bootstrap values from 1000 replicates are shown for selected branches. The deduced amino acid sequence of RGB1(I), was compared with the Gβ subunits of Japonica rice, maize, barley, Sorghum, wheat and similarity was calculated using Clustal Omega. The functional motifs, patterns and biologically significant sites in RGB1(I) amino acid sequence were located by ExPASy Proteomics Server ScanPro site (http://www.expasy.org/tools/scanprosite/). Quantitative Real-Time PCR The transcript profile of RGB1(I) in leaf blades under different stress conditions were determined by quantitative real-time PCR using StepOne Real Time PCR system (Applied BioSystems), essentially as described earlier.38 In brief, Power SyberGreen PCR master mix (Applied BioSystems) was used in a 20 µl reaction mixture containing 10 pico mole of each gene specific primer pair (α-tubulin forward 5′-GGTGGAGGTG ATGATGCTTT-3′ and reverse 5′-ACCACGGGCA AAGTTGTTAG-3′ and RGB1(I) forward 5′-GGGAAACAGG AGGTTGAACA-3′ and reverse 5′-GCGTCTCATG CTCTCATCAA-3′) and 1 µl of stress treatment specific cDNA. PCR reaction conditions were defined as one cycle of 10 min for 95 °C for initial denaturation followed by 40 cycles of 15s at 95 °C, 1 min at 59 °C. Optical data were collected after every cycle. The melt curve of PCR products was generated by heating them at temperatures starting from 55 °C to 95 °C, with a gradual increase by 0.5 °C at every step. Rice α-tubulin gene was used as internal reference. Experiments were repeated three times independently with three technical replicates in each experiment for each time point of treatment. Relative gene expression was calculated using the 2-ΔΔCT values following Livaks’ method.39 Preparation of construct (35S:RGB1-sGFP) and localization of RGB1(I) To study the sub-cellular localization of RGB1(I), the translational fusion cassette of RGB1(I) for transient expression was developed using pUC-based p326-sGFP vector containing CaMV35S-sGFP-(S65T) and NosT.40 The expression construct (CaMV35S:RGB-sGFP) was prepared by sub-cloning of XbaI/ BamHI digested fragment amplified from plasmid pGEMTRGB1 using RGBXbaIF, 5′-GATCTAGAAT GGCGTCCGTG GCGGAGCT-3′ and RGBBamHIR, 5′-TCAGGATCCA ACTATTTTCC GGTGTCC-3′ primers. XbaI/BamHI digested PCR amplicon was inserted into XbaI/BamHI sites of plasmid p326-sGFP to create chimeric RGB1(I)-GFP-fusion construct under the control of the 35S promoter. Two microgram of
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purified plasmid (RGB1-sGFP) was coated on gold particles (1.0 µm) and bombarded on to the onion epidermis kept on 0.5 x MS petri-plate using particle delivery system (PDS-1000/He, Bio-Rad, USA) set at 1100 psi from a distance of 6 cm. GFP fluorescence was examined using confocal microscope (Nikon A-1R) after 16 h incubation in dark with high humidity. Onion epidermal peels were detached from agar plate and mounted in 10% glycerol for microscopic observations. Epidermal peels were plasmolysed by immersing in 30% sucrose solution for 30 min. Excitation wavelength was set at 488 nm and emission wavelength was recorded between 505–530 nm BP filters. Statistical analysis The quantitative real-time gene expression data were analyzed by one way analysis of variance (ANOVA), and significant differences between individual means determined using Tukey’s pairwise comparison test at the 5% and 1% (P < 0.05 and P < 0.01) confidence level. References 1. Jones AM, Assmann SM. Plants: the latest model system for G-protein research. EMBO Rep 2004; 5:572-8; PMID:15170476; http://dx.doi. org/10.1038/sj.embor.7400174 2. Urano D, Chen JG, Botella JR, Jones AM. Heterotrimeric G protein signalling in the plant kingdom. Open Biol 2013; 3:120186; PMID:23536550; http://dx.doi.org/10.1098/ rsob.120186 3. Assmann SM. Heterotrimeric and unconventional GTP binding proteins in plant cell signaling. Plant Cell 2002; 14:S355-73; PMID:12045288 4. Ma H, Yanofsky MF, Meyerowitz EM. Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc Natl Acad Sci U S A 1990; 87:3821-5; PMID:2111018; http://dx.doi.org/10.1073/ pnas.87.10.3821 5. Weiss CA, Garnaat CW, Mukai K, Hu Y, Ma H. Isolation of cDNAs encoding guanine nucleotidebinding protein beta-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc Natl Acad Sci U S A 1994; 91:9554-8; PMID:7937804; http://dx.doi.org/10.1073/pnas.91.20.9554 6. Mason MG, Botella JR. Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with Gbeta. Biochim Biophys Acta 2001; 1520:147-53; PMID:11513956; http://dx.doi.org/10.1016/S0167-4781(01)00262-7 7. Chakravorty D, Trusov Y, Zhang W, Acharya BR, Sheahan MB, McCurdy DW, Assmann SM, Botella JR. An atypical heterotrimeric G-protein γ-subunit is involved in guard cell K+ -channel regulation and morphological development in Arabidopsis thaliana. Plant J 2011; 67:840-51; PMID:21575088; http:// dx.doi.org/10.1111/j.1365-313X.2011.04638.x 8. Trusov Y, Zhang W, Assmann SM, Botella JR. Ggamma1 + Ggamma2 not equal to Gbeta: heterotrimeric G protein Ggamma-deficient mutants do not recapitulate all phenotypes of Gbetadeficient mutants. Plant Physiol 2008; 147:63649; PMID:18441222; http://dx.doi.org/10.1104/ pp.108.117655 9. Marsh JF 3rd, Kaufman LS. Cloning and characterisation of PGA1 and PGA2: two G protein alpha-subunits from pea that promote growth in the yeast Saccharomyces cerevisiae. Plant J 1999; 19:237-47; PMID:10476071; http://dx.doi. org/10.1046/j.1365-313X.1999.00516.x
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
Work on plant abiotic stress tolerance and G-protein signaling in NT’s laboratory is supported by Department of Biotechnology (DBT), and Department of Science and Technology (DST), Government of India. We thank to Mrs. Poornima for technical help in confocal microscopy. We thank Dr. Renu Tuteja and Mr. Deepak Bhardwaj for their help in corrections of the manuscript. Author Contributions
DKY performed the research, analyzed data, and wrote the manuscript; DS performed the experiments and helped in writing the manuscript; NT designed research, analyzed the data, and wrote the manuscript.
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