CBF signaling pathway are affected in OEmiR399 transgenic Arabidopsis plants under freezing stress
Based on the important role of CBF signaling pathway in plant response to freezing stress, the expression of CBFs genes (AtCBF1 ,AtCBF2 and AtCBF3 ) and CORs genes (AtCOR15A ,AtCOR15B , AtCOR47 and AtCOR413IM ) in Arabidopsis were analyzed. The qRT-PCR results of CBFs showed that before treatment the expressions of AtCBF2 and AtCBF3 in OEmiR399 lines were significantly higher than that in WT (Fig. 6B and C). At 4°C, there was no obvious difference in the expression of AtCBFs in all Arabidopsis lines (Fig. 6 A, B and C). At -10℃, the expressions ofAtCBFs in all Arabidopsis lines increased rapidly, and the expressions of AtCBFs in OEmiR399 transgenic Arabidopsis plants were significantly higher than that of WT (Fig. 6 A, B and C). The qRT-PCR results of CORs showed that the expression of most CORs genes selected in all Arabidopsis lines increased with different degrees (Fig. 6 D, E, F and G). At low temperature, the expressions of AtCOR15A andAtCOR15B in all Arabidopsis lines increased substantially, while the expression of AtCOR15A was no obvious difference in all Arabidopsis lines and the expression of AtCOR15B in OEmiR399 transgenic Arabidopsis plants was lower than that in WT (Fig. 6D and E). The expressions of AtCOR47 and AtCOR413IM were greatly affected by OEmiR399 (Fig. 6 F and G). Before the cold treatment, the expression of AtCOR47 in OEmiR399 transgenic Arabidopsis plants was always significantly higher than that in WT. At low temperature, the expressions of AtCOR47 and AtCOR413IM in OEmiR399 transgenic Arabidopsis were higher than that of WT (Fig. 6F and G).
AtICE1 is an upstream regulator of the CBF signaling pathway, the previous study documented that CsUBC24 could inhibit CsICE1 in citrus(R. Wang et al., 2020). To verify whether tae-miR399 affected the CBF signal pathway by regulating AtICE1 at low temperature, we used Western Blot technology to detect the AtICE1 content of all Arabidopsis lines at low temperature. The results showed that with the decrease of temperature, the AtICE1 contents of OEmiR399 transgenic Arabidopsis lines were always higher than that of WT (Fig. 7A and B). The AtICE1 transcription levels of all Arabidopsis lines were detected. The results showed that only at 4℃, the AtICE1 transcription levels of OEmiR399 transgenic Arabidopsis lines were higher than that of WT (Fig. 7C).