http://192.168.1.40:8080/xmlui/handle/123456789/1137 2026-05-31T15:24:04Z http://192.168.1.40:8080/xmlui/handle/123456789/2349 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 Roy, P.; Niyogi, K.; Sengupta, Dibyendu N.; Ghosh, B. 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. DOI: 10.1016/j.plantsci.2004.08.014 2005-03-01T00:00:00Z http://192.168.1.40:8080/xmlui/handle/123456789/2347 Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta. Sarkar, S. N.; Bakshi, S.; Mokkapati, S. K.; Roy, S.; Sengupta, Dibyendu N. A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of. Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with P-32-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal. DOI: 10.1016/j.bbrc.2004.05.152 2004-07-16T00:00:00Z http://192.168.1.40:8080/xmlui/handle/123456789/2167 A dideoxynucleotide-sensitive DNA polymerase activity characterized from endoreduplicating cells of mungbean (Vigna radiata L.) during ontogeny of cotyledons Roy, Sujit; Sarkar, Sailendra Nath; Singh, Sanjay Kumar; SenGupta, Dibyendu Narayan Within this work we describe the purification and biochemical characterization of a ddNTP-sensitive DNA polymerase purified from mungbean (Vigna radiata cv B1, L.) seeds at 18 days after fertilization, when > 70% of the nuclei are reported to be in the endoreduplicated state. The purified enzyme is a single polypeptide of 62 kDa and many of its physicochemical properties are similar to those of mammalian DNA polymerase beta. Similar to the other X-family DNA polymerases, it lacks 3'-5' exonuclease activity and has short gap-filling and strand-displacement activity. The enzyme shows moderately processive DNA synthesis on a single-strand template. The determined N-terminal heptapeptide sequence of the enzyme showed clear homology with helix 1 of the N-terminal single strand DNA-binding domain (residues 32-41) of rat and human DNA polymerase beta. These results represent the first evidence for the identification and characterization of a ddNTP-sensitive DNA polymerase expressed during the endoreduplication cycle that shares biochemical and immunological similarity with mammalian DNA polymerase beta. DOI: 10.1111/j.1742-4658.2007.05744.x 2007-04-01T00:00:00Z http://192.168.1.40:8080/xmlui/handle/123456789/2166 An ABRE-binding factor, OSBZ8, is highly expressed in salt tolerant cultivars than in salt sensitive cultivars of indica rice Mukherjee, Kakali; Choudhury, Aryadeep Roy; Gupta, Bhaskar; Gupta, Sudhiranjan; SenGupta, Dibyendu Narayan Background: The bZIP class Abscisic acid Responsive Element (ABRE)-binding factor, OSBZ8 (38.5 kD) has been considered to regulate ABA-mediated transcription in the suspension cultured cells of japonica rice. Still, nothing is known about the expression of OSBZ8 at protein level in vegetative tissue of salt sensitive and salt tolerant rice plants. In our previous study, Electrophoretic Mobility Shift Assay (EMSA) of [P-32] ABRE-DNA and nuclear extracts prepared from the lamina of Pokkali rice plants has detected the presence of an ABRE-binding factor. Northern analysis has also detected salinity stress induced accumulation of transcripts for bZIP class of factor. Therefore, OSBZ8 was considered to play an important role in the regulation of transcription in the vegetative tissue of rice. The aim of this study is to find out whether OSBZ8 has any role in regulating the NaCl-stress induced gene expression in vegetative tissue and whether the expression of OSBZ8 factor directly correlates with the stress tolerance of different varieties of indica type rice. Results: Northern analysis of total RNA from roots and lamina of salt-sensitive M-I-48 and salt-tolerant Nonabokra, when probed with the N-terminal unique region of OSBZ8 (OSBZ8p, without the highly conserved basic region), a transcript of 1.3 kb hybridized and its level was much higher in tolerant cultivar. EMSA with Em Ia, the strongest ABA Responsive Element till reported from the upstream of EmBPI, and the nuclear extracts from laminar tissue of untreated and salt-treated seedlings of three salt sensitive, one moderately sensitive and two salt tolerant indica rice cultivars showed specific binding of nuclear factor to ABRE element. Intensity of binding was low and inducible in salt sensitive rice cultivars while high and constitutive in salt tolerant cultivars. EMSA with 300 bp 5' upstream region of Rab16A gene, a well known salt stress and ABA-inducible gene of rice, showed formation of two complexes, again very weak in salt sensitive and strong in salt tolerant rice cultivar. Conclusion: The bZIP factor OSBZ8 was found to be present in the ABRE-DNA: protein complex as shown by the supershift of the complex by the purified antiserum raised against OSBZ8p. Treatment of the seedlings with NaCl was found to enhance the complex formation, suggesting the regulation of OSBZ8 gene at both transcriptional and post-translational steps. Comparative EMSA with different varieties of rice suggests a positive correlation with the expression pattern of OSBZ8 and salt tolerance in rice cultivars. DOI: 10.1186/1471-2229-6-18 2006-08-30T00:00:00Z