Duplicated RGS (Regulator of G-protein signaling) proteins exhibit conserved biochemical but differential transcriptional regulation of heterotrimeric G-protein signaling in Brassica species

Abstract

G-alpha (Gα) and ‘Regulator of G-protein Signaling (RGS)’ proteins are the two key components primarily involved in regulation of heterotrimeric G-proteins signaling across phyla. Unlike Arabidopsis thaliana, our knowledge about G-protein regulation in polyploid Brassica species is sparse. In this study, we identified one Gα and two RGS genes each from three species of Brassica ‘U’ triangle and assessed the effects of whole genome triplication on the divergence of gene sequence and structure, protein-protein interaction, biochemical activities, and gene expression. Sequence and phylogenetic analysis revealed that the deduced Gα and RGS proteins are evolutionarily conserved across Brassica species. The duplicated RGS proteins of each Brassica species interacted with their cognate Gα but displayed varying levels of interaction strength. The Gα and the duplicated RGS proteins of Brassica species exhibited highly conserved G-protein activities when tested under in-vitro conditions. Expression analysis of the B. rapa RGS genes revealed a high degree of transcriptional differentiation across the tested tissue types and in response to various elicitors, particularly under D-glucose, salt and phytohormone treatments. Taken together, our results suggest that the RGS-mediated regulation of G-protein signaling in Brassica species is predominantly governed by stage and condition-specific expression differentiation of the duplicated RGS genes.

Interacting partners of Brassica juncea regulator of G-protein signaling protein suggest its role in cell wall metabolism and cellular signaling

“…The qRT-PCR analysis showed that all the members of B. juncea RGS genes were expressed although showing differential expression patterns ( Figure 1 C). The BjuA.RGS1 and BjuB.RGS1 were found to be highly expressed genes with profound expression observed in the root, flower, and siliques stages as also reported for the B. rapa ortholog, BraA.RGS1 [ 11 ]. The BjuB.RGS2 transcript had low abundance across all the examined tissue types.…”

“…Deduced RGS proteins of B. juncea shared 84.6–95.2% identity among themselves and 84–88.9% identity with the A. thaliana AtRGS1 (Supplementary Table S2). Amino acid sequence alignment showed that the B. juncea RGS proteins contain the N-terminal ‘7-TM domain’ and the C-terminal located ‘RGS domain’, and share high sequence conservation with orthologs from B. rapa and B. nigra ( Figure 1 A) [ 11 ]. Furthermore, a key residue (Glu320) responsible for the GAP activity of Arabidopsis RGS protein was also found to be highly conserved in BjuRGS proteins [ 9 ].…”

“…Gene-specific primers (Supplementary Table S1) were designed based on our previously reported RGS sequences from Brassica species [ 11 ], and the full-length RGS coding DNA sequences (CDS) were amplified from B. juncea . Following PCR amplification, the PCR products were cloned into a pENTR/D-TOPO vector (Invitrogen) and sequenced to determine the accuracy and identity of the clones.…”

Mulberry RGS negatively regulates salt stress response and tolerance

“…Signal transduction processes mediated by heterotrimeric guanine-nucleotide-binding protein (hereafter G-protein) complex consisting of Gα, Gβ and Gγ subunits play fundamental roles in regulating various cellular processes in all eukaryotes. 1,2 In animals, when G-protein-coupled receptors (GPCRs) bind their cognate ligands and the activated GPCRs serve as guanine nucleotide exchange factors that stimulate the exchange of GDP for GTP on Gα subunit. 3 And then, G-protein was dissociated into Gα-GTP and a Gβγ dimer, and both of these components trigger diverse signaling pathways through interacting with their respective downstream effectors.…”

“…8 Previous studies suggested that the interplay between RGS and Gα proteins is quite important in regulating G-protein mediated signaling pathways in plants, and RGS acts as a key point of ligand-dependent signal modulation of G-protein complex. 2,9 In Arabidopsis, D-glucose recruits the With No Lysine kinases (WNKs), AtWNK1, AtWNK8 and AtWNK10, to phosphorylate AtRGS1 and then the phosphorylated AtRGS1 was internalized, which uncouples AtGPA1 from AtRGS1 and causes the subsequent sustained activation of G-protein signaling. 10,11 It is suggested that autophagy pathway may play an important role in regulating AtRGS1 endocytosis after D-glucose stimulation, and the endocytosis of AtRGS1 was inhibited after the autophagy pathway was interrupted.…”