High amino acid accumulating 5‐methyltryptophan‐resistant rice mutants may include an increased antioxidative response system

Abstract

In a previous study, we developed 5‐methyltryptophan (5MT)‐resistant rice (Oryza sativa L.) mutant lines via in vitro mutagenesis. These mutant lines exhibited elevated free amino acid content, in addition to a marked tolerance to a 5MT inhibition. In this study, we verified these increased protein and amino acid contents in the advanced mutant lines, and discovered that the anthranilate synthase (AS, EC 4.1.3.27) activity of the mutant plants was 2.2–3 times as high as that of the control. In all four tested 5MT‐resistant mutant lines, AS activity proved to be less sensitive to tryptophan inhibition than that of the control. Proteins produced, either in elevated amounts or de novo in response to 5MT were studied by comparison of silver‐stained two‐dimensional gels of leaf proteins, between the control and two 5MT‐resistant mutant lines. At least 20 proteins exhibited either elevated expression or de novo generation following exposure to growth‐inhibitory concentrations of 5MT in MRI‐40. We assessed the 5MT stress‐mediated responses of the four antioxidant enzymes; catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1) and aspartate peroxidase (APX, EC 1.11.1.11). We found that the activity levels of all four enzymes were increased as a result of 5MT treatment, in both the control and the 5MT‐resistant mutant lines. However, the mutant lines exhibited more pronounced increases in the antioxidant enzymes than did the control. Significant differences in these activity increases were observed between the control and the mutant lines in the SOD and APX activity assays. Native PAGE confirmed these differences in SOD and APX activity, with the separation patterns of the isoforms of SOD and APX. These results mean that the 5MT‐resistant mutants might possess active antioxidant systems which protect the cell from 5MT stress that may induce the production of reactive oxygen species.