Spermidine treatment to rice seedlings recovers salinity stress-induced damage of plasma membrane and PM-bound H+-ATPase in salt-tolerant and salt-sensitive rice cultivars

Abstract

Due to their polycationic nature, Spermidine (Spd(3+)) and Spermine (SPM4+) are known to interact with polyanionic compounds, e.g. negatively charged head group of phospholipid membrane components, thereby stabilizing salinity stress-induced damage of plasma membrane (PM). But to what extent polyamine-mediated restoration of activities of PM-bound enzymes occurs and differs within salt-sensitive and salt-tolerant rice cultivars is totally unknown. Therefore, PM was isolated from the roots of 3-day-old rice seedlings from two salt-tolerant (Nonabokra and Pokkali) and two salt-sensitive (M-1-48 and IR8) cultivars treated with none (control) or with NaCl (150 mM, 16 h) alone or with Spd (I mM, 16 h). Vanadium sensitive but K+ stimulated H+-ATPase activity from equal amount of PM was measured by estimating released Pi. Results showed that nine-fold higher level of H+-ATPase (100% vanadium sensitive) was detected from PM of Nonabokra roots in comparison to M-1-48 roots. Salinity stress alone to the seedlings significantly reduces the activity of PM-bound H(+-)ATPase. The activity of H+-ATPase was restored to some extent in the roots treated with NaCl stress in presence of I mM Spd. Analysis of PM-bound polyamine from untreated control roots showed only Putrescine from M-1-48 and IR8 cultivars, whereas roots of salt-tolerant plants, Nonabokra and Pokkali, have only Spermidine and Spermine. PM-bound H+-ATPase activity of control and treated plants, when measured by NADH oxidation (coupled reaction), 2.5-3.0-fold higher activity was detected from salt-tolerant cultivars. Salinity stress to the plants severely inhibits H+-ATPase activity and Spermidine co-treatment significantly recovers its activity in all four cultivars. Western Blot with equal amount of 5% SDS extracted protein from roots when analyzed by the polyclonal antibody raised against H+-ATPase (PM-bound) of Arabidopsis thaliana showed NaCl stress-induced decrease and Spermidine-induced recovery of 100 kDa polypeptide (known MW of 100 kDa H+-ATPase from rice). These results clearly demonstrate for the first time that the deficit of salt-sensitive rice cultivars, e.g. high accumulation of Na+, loss of K+ ion, salinity stress-induced sharp inhibition of PM-bound H+-ATPase activity, could be overcome by supplying Spermidine exogenously.