AEC resistant rice mutants induced by gamma-ray irradiation may include both elevated lysine production and increased activity of stress related enzymes

Abstract

To increase the contents of specific free amino acids in the rice (Oryza sativa L.) cultivar “Donganbyeo”, mutant cell lines resistant to growth inhibition by S-(2-aminoethyl)-cysteine (AEC) were selected from calli irradiated with gamma-rays through embryo culture. In the four AEC resistant M3 lines, LR3–69, LR5–132, LR7–2 and LR9–48, the amino acids contents were 26, 7, 29 and 37%, respectively, greater than in the “Donganbyeo”. The most significant amino acid increases in the resistant lines were in lysine and tryptophan levels. The lysine levels of the four resistant lines were 2.66, 1.58, 2.37 and 2.29 times, higher than in the “Donganbyeo”. Total proteins extracted and resolved by SDS–PAGE, showed few differences in the protein profiles of the control and mutant lines. In glutelins, SDS–PAGE protein patterns showed two typical mutant protein bands at 24 kDa. The SDS–PAGE profiles of mutant prolamines revealed two distinct bands of different intensities, at ca. 22 and 18 kDa. AEC may inhibit cell growth irreversibly by competing with lysine for incorporation into protein. This incorporation presumably leads to an altered protein conformation and function, and accounts for the cytotoxic effects of AEC. Therefore, AEC creates stresses in vivo much like other environmental stresses. Changes in gene expression in response to AEC were analyzed by 2-dimensional gel electrophoresis (2-DE). The number of new protein spots found in resistant mutants of LR3–69, LR5–132 and LR9–48 were 4, 11 and 16, respectively. De novo synthesized and enhanced proteins in these mutant lines after induction with AEC were 13, 11 and 9, and 7, 12 and 13 spots, respectively. The five proteins (L1, L2, L5, L6 and L7) were identified as a 33 kDa oxygen-evolving protein of photosystem II, ribulose-1,5-bisphosphate carboxylase (Rubisco) large subunit, ascorbate peroxidase (APX), glutamine synthetase (GS) and Cu/Zn superoxide dismutase (Cu/Zn SOD). Based on the 2-DE results, the AEC stress-mediated responses of two antioxidant enzymes, SOD and APX, were examined. Compared with a 50.3% increase in the control, increases in the rates of SOD activity of LR3–69, LR7–2 and LR9–48 were 136.5, 177.1 and 184.8%, respectively. After AEC treatment, the APX activity level in the control increased by 5.4%, whereas those of the mutant lines, LR3–69, LR5–132, LR7–2 and LR9–48, increased to 1.6, 1.4, 2.4 and 4.0 times that of the untreated control, respectively.