LlHsfA5‐LIHsfA4 antagonism balances thermotolerance via LlAPX2‐mediated reactive oxygen homeostasis in Lilium longiflorum

The lily is a world famous cut flower that grows well in cool climates, and its production is affected by high temperatures during the summer in China. Heat shock transcription factors (HSFs) participate in the heat stress response (HSR) in many plants. In this study, LlHsfA5 was identified in the lily transcriptome, and its expression was shown to be upregulated under heat stress. Specifically, this factor exhibits weak transactivation activity and binds to the heat stress element (HSE) as well as interacts with LlHsfA4, impairing its HSE binding capacity and suppressing its transcriptional activity. Silencing LlHsfA5 enhanced thermotolerance, whereas silencing LlHsfA4 decreased heat tolerance. Co-silencing the two genes restored the phenotype to that of the control group, indicating their antagonistic regulatory roles in the HSR. Reactive oxygen species (ROS) were significantly reduced in lilies with silenced LlHsfA5 but elevated in those with silenced LlHsfA4 compared with the control. The co-silencing of these two genes normalized ROS levels, indicating their joint role in maintaining ROS homeostasis. The expression of ascorbate peroxidases (APX) genes was upregulated and downregulated in LlHsfA5-silenced and in LlHsfA4-silenced lilies, respectively. LlAPX2-silenced lilies were shown to be hypersensitive to heat stress. LlHsfA4 directly bound to the promoter of LlAPX2, while LlHsfA5 inhibited this binding. This study revealed a sophisticated regulatory network where LlHsfA4 functions as a positive regulator of thermotolerance by activating LlAPX2 to mitigate oxidative damage, while LlHsfA5 inhibits the activity of LlHsfA4, acting as a negative regulator. The LlHsfA5-LlHsfA4 complex modulates ROS homeostasis as well as the HSR in lilies. These findings offer valuable insights into the mechanisms through which HSF complexes regulate ROS pathways in plants under heat stress.