tae-miR399-UBC24 module regulates the ROS metabolism response to freezing stress in Arabidopsis
Low temperature stress can significantly disturb ROS homeostasis in plants, leading to lipid peroxidation, damage to membranes (Ning, Li, Hicks, & Xiong, 2010) and increasing MDA content (J. J. Liu, Wei, & Li, 2014). In this study, under low temperature stress, OEmiR399 Arabidopsis had lower MDA content, indicating that it suffered less oxidative damage than WT. Further histochemical staining and physiological experiment methods to detect ROS content, the results showed that the H2O2 content and O2.- production of OEmiR399 Arabidopsis plants were visibly lower than that of WT under freezing stress (Fig. 11). Also, under freezing stress, the SOD, POD and CAT enzyme activities of OEmiR399 Arabidopsis plants were significantly higher than those of WT (Fig. 12, 13 and 14). These implies that OEmiR399 Arabidopsis plants have a strong ROS scavenging ability. Interestingly, the expression changes of the genes encoding SOD, POD and CAT in OEmiR399 Arabidopsis plants were not consistent with the enzyme activities. The expression of the most genes encoding SOD, POD and CAT in OEmiR399 Arabidopsis plants was significantly up-regulated at 24°C and 4°C, but not significantly changed or down-regulated at -10°C (Fig. 12, 13 and 14). Similary, the expression of genes encoding SOD and APX in bluegrass are significantly down-regulated under drought stress, while their enzyme activities are significantly up-regulated (Xu, Han, & Huang, 2011). Based on previous studies, we speculate that the changes in the enzyme activity of OEmiR399 Arabidopsis plants under freezing stress may not be caused by mRNA levels, but are regulated at the post-transcriptional level, which is due to the inhibition of enzyme inactivation/activation or degradation/synthesis caused by freezing stress. Homologous or heterologous overexpressing ICE1 in plants such as tomato and tobacco can increase its antioxidant enzyme activity to enhance the cold tolerance of plants (Feng et al., 2013; Luo et al., 2020). Therefore, in this study, under low temperature stress, the high ICE1 protein levels of OEmiR399 Arabidopsis plants may also be one of the reasons for its stronger ROS scavenging ability.
Combining with our phenotypic data, we can conclude that miR399-UBC24 module inhibit the degradation of ICE1 and promote the CBF signalling pathway and ROS clearance to initiate plant response to freezing stress. On the other hand, miR399-UBC24 module can accelerate starch degradation to accumulate a large amount of soluble sugar by promoting the Pi absorption and utilization efficiency, and then enhance freezing tolerance in plants (Fig. 14). These findings clarify the function of miR399-UBC24 module and suggest that tae-miR399-TaUBC24 can potentially be used to improve freezing tolerance in crops.