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.
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 Galpha (Galpha1, 1152 bp; Galpha2, 1152 bp), one Gbeta (1134 bp), two isoforms of Ggamma (Ggamma1, 345 bp; Ggamma2, 303 bp) and a GPCR (1008 bp) from Pisum sativum, and purification of all the encoded recombinant proteins (Galpha, 44 kDa; Gbeta, 41 kDa; Ggamma, 14 kDa; GPCR, 35 kDa). The transcript levels of Galpha and Gbeta were upregulated following NaCl, heat and H(2)O(2) treatments. Protein-protein interaction studies using an in vitro yeast two-hybrid system and in planta co-immunoprecipitation showed that the Galpha subunit interacted with the pea Gbeta subunit and pea phospholipase C (PLCdelta) at the calcium-binding domain (fn1). The GTPase activity of the Galpha subunit increased after interaction with PLCdelta. The GPCR protein interacted with all the subunits of G-proteins and with itself. Transgenic tobacco plants (T(0) and T(1)) constitutively over-expressing Galpha showed tolerance to salinity and heat, while Gbeta-over-expressing plants showed only heat tolerance, as tested by leaf disk senescence assay and germination/growth of T(1) seeds/seedlings. These findings provide direct evidence for a novel role of Galpha and Gbeta subunits in abiotic stress tolerance and possible cross-talk between PLC- and G-protein-mediated signaling pathways.
Misra
Shikha
S
Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi - 67, India.
Wu
Yuliang
Y
Venkataraman
Gayatri
G
Sopory
Sudhir K
SK
Tuteja
Narendra
N
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D018525:Germination / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D020033:Protein Isoforms; D021122:Protein Subunits; D043562:Receptors, G-Protein-Coupled; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D006861:Hydrogen Peroxide; D010738:Type C Phospholipases; D020962:Heterotrimeric GTP-Binding Proteins