2.3 BES1 accumulates during the memory phase
To explore the expression pattern of BES1 in the memory phase, we carried out a time course towards seedling collected at different time points indicated in Fig. 2A. The transcriptional level of BES1remained high until 48 h into the thermomemory phase, and still more than 6 fold compared with the control in 48 h (Fig. 2B). Consistent with this, GUS analysis also demonstrates that heat priming inducible expression of BES1 (Fig. 2C). Next, we assessed whether the increased mRNA level in the memory phase leads to the accumulation of BES1. We investigated the abundance of BES1 during thermomemory assay and observed more than 2-fold accumulation in the memory phase compared with control (prior priming), and BES1 protein remains abundant until 48 h into the thermomemory phase (Fig. 2D). To test whether the sustained high level of BES1 protein at later memory time points are due to the rise of transcriptional level and requires de novo protein synthesis, we applied cycloheximide (CHX) to inhibit protein translation. Col-0 seedlings were treated with CHX after priming and BES1 protein level was determined by immune blotting at 12 h, 24 h and 48 h into the memory phase. Compared with mock treatment (DMSO), BES1 protein level declined in the memory phase (Fig. 2E), supporting our suspect that increases in BES1 mRNA contribute to the accumulation of BES1 protein during the memory phase. In addition, we also concerned about the effect caused by protein degradation, since the increased BES1 may also come from the contribution of weakening degradation. Compared with mock treatment, treatment with MG132 resulted in an increase of BES1 within the memory phase (Fig. 2F), indicating that the degradation of BES1 weakened during that time. Furthermore, we carried out a combined treatment with Col-0 seedling using both CHX and MG132. Not surprisingly, BES1 abundance in these seedlings was slightly higher than that of CHX treatment but obvious lower than MG132 treatment (Fig. 2G). These results demonstrate that de novo synthesis and weakened degradation mutually contribute to the accumulation of BES1 during the memory phase.