ROS metabolism are affected in OEmiR399 transgenic Arabidopsis plants under freezing stress
To verify the effect of tae-miR399 on oxidative tolerance induced by freezing in Arabidopsis plants, we determined ROS including hydrogen peroxide (H2O2) and super-oxygen ion (O2.−) levels in different Arabidopsis lines after freezing stress by histochemical staining and ROS content determination. Nitroblue tetrazolium (NBT) staining for O2.− and 3, 3′-diaminobenzidine (DAB) staining for H2O2 showed ROS accumulated to higher levels in WT than that in OEmiR399 transgenic Arabidopsis plants under freezing stress (Fig. 11). These results indicated that OEmiR399 transgenic Arabidopsis plants exhibited stronger ability to scavenge ROS and lesser symptoms in response to freezing stress. The activities of SOD, POD and CAT in OEmiR399 transgenic Arabidopsis plants were higher than that in WT under freezing stress (Fig. 12). However, the expression of SOD, POD and CAT encoding genes in OEmiR399 transgenic Arabidopsis were not up-regulated under freezing stress. Moreover, the expression of some SOD, POD and CAT encoding genes in OEmiR399 transgenic Arabidopsis was significantly lower than that in WT (Fig. 12).