2.5 The abundance of dephosphorylated BES1 is responsible for BR-enhanced thermomemory
To elucidate the critical function of BES1 accumulated during thermal memory phase for BR signal, we detected BES1 protein by immunoblot analysis in det2 and BES1-RNAi seedlings at 48 h of memory phase. As shown in Supplementary Fig. 2, the abundance of dephosphorylated BES1 was higher in det2 , whereas it was almost undetectable in BES1-RNAi . This finding provides further evidence for BES1 involved in the regulation of thermomemory, increased BES1 level in det2 could explain the enhanced thermotolerance indet2 mutant (Supplementary Fig. 1) and a lack of BES1 could be responsible for the thermomemory function-loss in BES1-RNAi (Fig. 2).
We next subjected 5-day-old bes1-D mutant and Col-0 seedlings to our priming and triggering stimulus protocol, since the amino acid mutation in bes1-D results in an increase in the total amount of BES1 protein. Not surprisingly, dephosphorylated BES1 in bes1-Dis comparably more abundant than that of Col-0 during the whole memory phase. Consistent with this observation, bes1-D seedlings revealed better thermotolerance than Col-0 in both unprimed and primed conditions, as the survival rate was comparably higher than Col-0 (Fig. 4C), accompanied with lower electrolyte leakage in bes1-D (Fig. 4D), underscoring the contribution of bio activated BES1 to elevating the thermolerance and enhance thermomemory.