Improved oxidative tolerance in suspension-cultured cells of C4-pepctransgenic rice by H2O2 and Ca2+ under PEG-6000

To understand the molecular responses of PC (Overexpressing the maize C-4-pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC)), to drought stress at cell level, we analyzed changes in the levels of signaling molecules (hydrogen peroxide (H2O2), calcium ion (Ca2+), and nitric oxide (NO)) in suspension-cultured PC and wild-type (WT) rice (Oryza sativa L.) cell under drought stress induced by 20% polyethylene glycol 6000 (PEG-6000). Results demonstrated that PC improved drought tolerance by enhancing antioxidant defense, retaining higher relative water content, survival percentages, and dry weight of cells. In addition, PEPC activity in PC under PEG treatment was strengthened by addition of H2O2 inhibitor, dimethylthiourea (DMTU) and NO synthesis inhibitor, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), respectively, while that in PC was weakened by addition of free calcium chelator, ethylene glycol-bis(b-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA)+calcium channel outflow inhibitor, ruthenium red (RR)+plasma membrane channel blocker La(NO3)(3), but EGTA+RR did not. Results also showed that NO and Ca2+ was lying downstream of H2O2 in drought-induced signaling. Calcium ion was also involved in the expression of C-4-pepc in PC. These results suggested that PC could improve oxidative tolerance in suspension-cultured cells and the acquisition of this tolerance required downregulation of H2O2 and the entry of extracellular Ca2+ into cells across the plasma membrane for regulation of PEPC activity and C-4-pepc expression.