PlbHLH79 and PlbHLH31 antagonistically modulate alkane accumulation and high-temperature tolerance in herbaceous peony

Abstract High temperatures hinder plant growth and reduce agricultural productivity worldwide. Cuticular wax acts as a key physical barrier to mitigating this stress, yet its regulatory mechanisms remain poorly understood. Here, we demonstrated that cuticular wax content was closely associated with high-temperature tolerance among different herbaceous peony (Paeonia lactiflora Pall.) cultivars and that mechanical removal of epicuticular wax by gum arabic compromised this tolerance. Notably, under high-temperature stress (42℃ for 12 days), cuticular wax content increased by 10.40%, with alkane content rising by 3.98%, and the expression of the alkane biosynthetic gene ECERIFERUM1 (PlCER1) was upregulated by approximately 12-fold. Functional analysis showed that PlCER1 enhanced high-temperature tolerance by promoting alkane accumulation. Further analysis revealed that the basic helix-loop-helix (bHLH) transcription factors PlbHLH79 and PlbHLH31 regulated PlCER1 by competitively binding to the same E-box element in its promoter, with PlbHLH79 activating and PlbHLH31 repressing PlCER1 transcription. Moreover, we demonstrated that both PlbHLH79 and PlbHLH31 could form homodimers and heterodimers. Homodimerization enhanced the transcriptional activity of each factor, while heterodimerization reduced the activating function of PlbHLH79 and the repressive effect of PlbHLH31 on PlCER1 expression. Collectively, our findings reveal that PlbHLH79 and PlbHLH31 regulate PlCER1 through both competitive binding and dynamic dimer formation, controlling alkane accumulation and high-temperature tolerance in P. lactiflora. These results provide insights into the molecular mechanisms of plant adaptation to high-temperature stress and offer valuable resources for improving high-temperature tolerance in plants.