Alternation of antioxidative enzyme gene expression in rice seedlings exposed to methylene blue

Hydroponic experiments were conducted with rice seedlings (Oryza sativa L. cv. ND1) exposed to methylene blue (MB) to investigate the accumulation of hydrogen peroxide (H2O2) and activities and gene expression of antioxidant enzymes. Results showed that a linear decrease in relative growth rate and water use efficiency was observed with rice seedlings with increasing MB concentrations. MB-induced accumulation of H2O2 was evident in both roots and shoots. The activities of antioxidative enzymes were analyzed and found to be different at different MB treatment concentrations. The activities of enzymes related to the ascorbate-glutathione cycle were more sensitive to MB treatments than other antioxidative enzymes. Transcript level, by real-time quantitative PCR, of antioxidative enzymes showed that the analyzed genes were differently expressed during different MB concentrations in both roots and shoots. The isoform analysis of superoxide dismutase (SOD) gene showed that the expressions of Cu/ZnSOD and MnSOD were relatively constant, where significant upregulation of FeSOD was observed with rice seedlings exposed to high MB concentrations. Furthermore, the expression of CAT, POD, and MDHAR genes responded biphasically to MB treatments by showing negligible changes at 1.56-15.63 μM MB and significant induction at 31.36-62.52 μM MB. The expression of GR, APX, and DHAR genes showed a remarkable induction to MB. Our results suggest that on transcription level, and in accordance with enzymatic responses, enzymes of GR, APX, and DHAR play central role in the H2O2 detoxification in rice seedlings under MB exposure.