RsHSFA3 of Rhododendron simsii confers plant heat tolerance by molecular regulation

Global warming severely restricts the cultivation and landscape application of heat-sensitive alpine rhododendrons. Rhododendron simsii, an important parent of hybrid azaleas, exhibits strong heat tolerance and wide environmental adaptability. Heat Shock transcription Factors (HSFs) act as core regulators governing plant heat tolerance. Screening and functional characterization of HSF genes in R. simsii can supply candidate genes for genetic improvement of heat-sensitive Rhododendron species. In this study, a total of 22 HSF family members were identified from the whole-genome sequence of R. simsii, one of which encodes a gene rapidly and markedly induced by heat stress, designated RsHSFA3. Subcellular localization assay verified that RsHSFA3 localizes to the cell nucleus. Heterologous overexpression of RsHSFA3 significantly elevated basal heat tolerance and acquired heat tolerance in transgenic Arabidopsis. CRISPR/Cas9-mediated gene-edited callus lines of R. simsii targeting RsHSFA3 were generated. Physiological measurements demonstrated that RsHSFA3 positively regulates heat tolerance by boosting reactive oxygen species (ROS) scavenging capacity and preserving cell membrane stability. Integrative analyses of DAP-seq, yeast one-hybrid and dual-luciferase reporter assay validated that RsHSFA3 binds to the promoters of heat-responsive genes including RsHSP70, RsHSP30, RsAPG2, RsXPO1 and RsHSP80 to activate their transcription. Yeast two-hybrid and bimolecular fluorescence complementation assays further revealed physical interactions between RsHSFA3 and RsHSP70 as well as RsHSP82. Collectively, RsHSFA3 synergistically enhances heat tolerance in R. simsii via activating the expression of heat shock proteins and facilitating ROS detoxification. The findings provide valuable genetic resource and theoretical foundation for directed heat-tolerance breeding of Rhododendron varieties.