Heterotrimeric G-protein complex and G-protein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C

Abstract

Heterotrimeric G-proteins transduce signals from activated G-protein-coupled receptors (GPCR) to appropriate downstream effectors and thereby play an important role in signaling. A role of G-proteins in salinity and heat stress tolerance has not heretofore been described. We report isolation of cDNAs of two isoforms of G (Gα1, 1152 bp; Gα2, 1152 bp), one Gβ (1134 bp), two isoforms of Gγ (Gγ1, 345 bp; Gγ2, 303 bp) and a GPCR (1008 bp) from Pisum sativum, and purification of all the encoded recombinant proteins (Gα, 44 kDa; Gβ, 41 kDa; Gγ, 14 kDa; GPCR, 35 kDa). The transcript levels of Gα and Gβ were upregulated following NaCl, heat and H₂O₂ treatments. Protein-protein interaction studies using an in vitro yeast two-hybrid system and in planta co-immunoprecipitation showed that the Gα subunit interacted with the pea Gβ subunit and pea phospholipase C (PLCδ) at the calcium-binding domain (fn1). The GTPase activity of the G subunit increased after interaction with PLCδ. The GPCR protein interacted with all the subunits of G-proteins and with itself. Transgenic tobacco plants (T₀ and T₁) constitutively over-expressing G showed tolerance to salinity and heat, while Gβ-over-expressing plants showed only heat tolerance, as tested by leaf disk senescence assay and germination/growth of T₁ seeds/seedlings. These findings provide direct evidence for a novel role of Gα and Gβ subunits in abiotic stress tolerance and possible cross-talk between PLC- and G-protein-mediated signaling pathways.