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.