Expression of abscisic acid-responsive element-binding protein in salt-tolerant indica rice (Oryza sativa L. cv. Pokkali)

Abstract

As the products of abiotic stress and ABA inducible genes are predicted to play an important role in the mechanism of salt tolerance, the expression of transcription factor that recognizes abscisic acid-responsive element (ABRE) is likely to be regulated when plants are exposed to abiotic stress. Northern analysis of total RNA from control and salt-treated 10-day-old Pokkali (salt tolerant) rice plants was performed to find out the level of transcripts homologous to wheat cDNA (GC19) for EmBP-1 (bZIP class factor), a transcription factor that recognizes ABRE. Salinity stress (72 h)-induced accumulation of two transcripts, of 2.0 kb (r2.0) and 1.5 kb (r1.5), in roots was detected. Both transcripts were detectable even after 6 h of salt or abscisic acid treatment, whereas sheath and lamina showed constitutive levels of r1.5 transcript. When 32P-labeled DNA containing ABRE was used in a gel mobility shift assay, a low level of complex formation by binding factor was detected from the nuclear extract of lamina of control rice plants. Quantitative enhancement of complex formation was found with the nuclear extract prepared from the lamina of plants treated with 200 mM NaCl for 26 h over control nuclear extract, suggesting a step of regulation of expression of ABRE-binding protein in response to salinity stress. South-western blot analysis of equal amounts of nuclear proteins of lamina showed binding of 32P-labeled ABRE-DNA with two polypeptides (22-28 kDa) present at constitutive levels in control or NaCl-treated plants. Preincubation of the laminar nuclear extract of control plants, with spermidine or proline at 5 mM concentration showed quantitative enhancement of ABRE binding activity. Kinetics of spermidine stimulation showed gradual increase of complex formation from 5 mM concentration. Similarly, addition of GTP to the control nuclear extract also showed quantitative enhancement of complex formation and heparin was found to inhibit GTP activated complex formation by about 25%. Results may suggest the presence of ABRE binding protein in presynthesized and inactive form in control plants and GTP mediated activation is probably one of the way to regulate the expression of ABRE-binding factor.