Rice heterotrimeric G-protein gamma subunits (RGG1 and RGG2) are differentially regulated under abiotic stress

Abstract

Heterotrimeric G-proteins (α, β and γ subunits) are primarily involved in diverse signaling processes by transducing signals from an activated transmembrane G-protein coupled receptor (GPCR) to appropriate downstream effectors within cells. The role of α and β G-protein subunits in salinity and heat stress has been reported but the regulation of γ subunit of plant G-proteins in response to abiotic stress has not heretofore been described. In the present study we report the isolation of full-length cDNAs of two isoforms of Gγ [RGG1(I), 282 bp and RGG2(I), 453 bp] from rice (Oryza sativa cv Indica group Swarna) and described their transcript regulation in response to abiotic stresses. Protein sequence alignment and pairwise comparison of γ subunits of Indica rice [RGG(I)] with other known plant G-protein γ subunits demonstrated high homology to barley (HvGs) while soybean (GmG2) and Arabidopsis (AGG1) were least related. The numbers of the exons and introns were found to be similar between RGG1(I) and RGG2(I), but their sizes were different. Analyses of promoter sequences of RGG(I) confirmed the presence of stress-related cis-regulatory signature motifs suggesting their active and possible independent roles in abiotic stress signaling. The transcript levels of RGG1(I) and RGG2(I) were upregulated following NaCl, cold, heat and ABA treatments. However, in drought stress only RGG1(I) was upregulated. Strong support by transcript profiling suggests that γ subunits play a critical role via cross talk in signaling pathways. These findings provide first direct evidence for roles of Gγ subunits of rice G-proteins in regulation of abiotic stresses. These findings suggest the possible exploitation of γ subunits of G-protein machinery for promoting stress tolerance in plants.

Figure 1. Amino acid sequence alignment of rice G-protein gamma subunits using ClustalW program (www.ebi.ac.uk/clustalw). Gaps were inserted to optimize the alignment are indicated by dashes. (A) RGG1(I) protein aligned with Japonica rice [RGG1(J); AK241226.1], maize (ZmG1; NP_001152725), barley (HvG1, AK359503), sorghum (SbG1, XP_002464204), Arabidopsis (AGG1; NP_567147.1) and soybean (GmG1; Glyma10 g03610). (B) RGG2(I) protein with Japonica rice (RGG2; NM_001052368.1), maize (ZmG2; NP_001151842), barley (HvG2, AK367089), sorghum (SbG2, XP_002451511), Arabidopsis (AGG2; AT3G22942.1) and soybean (GmG2; Glyma02 g16190). (C) RGG1(I) and RGG2(I) aligned together. Identical residues denoted by asterisk. Different motifs and patterns identified using Expasy PROSITE database.

Figure 2. In silico analysis of RGG1(I) and RGG2(I). (A--B) Dendrogram showing evolutionary relationship of RGG1(I) (A) and RGG2(I) (B) with related proteins. The evolutionary history was inferred using the Neighbor-Joining method and evolutionary distances were computed using the Poisson correction method. These phylogenetic analyses were conducted in MEGA5. (C--D) The schematic representation of genomic organization (exon–intron organization) of the genomic sequence of RGG1(I) (C) and RGG2(I) (D) genes. Closed boxes represent exons, and lines between closed boxes represent introns. The dark boxes represent the UTRs. The position of ATG and TAA are marked. The numbers below the lines and the above boxes indicate the sizes (bp) of introns, UTR and exons, respectively. (E--F) Stress-responsive cis-elements and phytohormones responsive elements in the 2 kb 5′-upstream regions of RGG1(I) (E) and RGG2(I) (F). The lines represent 5′-upstream regions of RGG(I) genes. The elements located in the positive strand are above the lines, while those in the reverse strand are indicated below the line. ABRE, abscisic acid responsive element; ARE, auxin responsive factor (TGA-box; MeJAE, methyl jasmonate responsive element; GARE, gibberellic acid-responsive element; DASR, defense and stress responsive element; GT1-Box; SAR, salicylic acid responsive element; HSRE, heat stress responsive element; LTR, Low temperature responsive element.

Figure 3. Quantitative real-time PCR analyses showing expression profile of RGG1(I) and RGG2(I) in total RNA isolated from three-week-old rice seedling leaf blades samples collected at different time intervals, treated under different abiotic stress conditions (A) 200 mM NaCl; (B) 200 mM KCl; (C) Cold (4°C); (D) Heat (42°C); (E) drought condition and (F) 100 μM ABA. Error bars are SD.