Temporal dynamics of the HSF-HSP regulatory network and flavonoid metabolism coordinate physiological adaptation to heat stress in Iris

Heat stress threatens ornamental plant productivity. Iris tectorum ( Iris ), a prized ornamental species valued for its diverse flower colors and architectural forms, suffers from heat-induced physiological damage that diminishes its economic value. Here we conducted a time-series analysis (0, 1, 3, 6, 9, and 24 h at 40°C) integrating physiological assays, transcriptomics, and untargeted metabolomics, to elucidate the temporal dynamics of heat stress response in Iris tectorum 'Hot Spicy',. Physiologically, heat stress induced progressive membrane lipid peroxidation (MDA accumulation), transient activation of antioxidant enzymes (SOD and POD peaking at 6 h), and sustained proline accumulation, revealing a temporally phased stress response. Transcriptomic analysis identified a “Heat stress factors-Heat Shock Proteins” (HSF-HSP) network exhibited tightly coordinated temporal dynamics: small HSPs (such as HSP26.7 , HSP16.0 and HSP22.0 ) responded rapidly within 1–3 h (39.46-fold), broad HSP activation peaked at 6–9 h (56.50-fold), and sustained HSP70 -mediated protection persisted at 24 h (7.77-fold). HSFs showed hierarchical activation, with early-responsive HSFA5 (7.99-fold), mid-phase HSFA6b , and late-phase HSFA3/HSFA4a orchestrating the transcriptional cascade. The Abscisic acid signaling pathway was systematically activated, with progressive PYL receptor upregulation (5.87-fold) and late-stage ABF induction (4.30-fold), coinciding with activation of flavonoid biosynthesis genes. Metabolomic analysis confirmed coordinated accumulation of flavonoids (naringenin chalcone, (-)-epigallocatechin) from 6–24 h, mirroring transcriptional activation of PAL , CHS , and DFR . Weighted Gene Co-expression Network Analysis (WGCNA) revealed a core module (MEbrown4) enriched in phenylpropanoid biosynthesis and hormone signaling, with hub genes including KIN10 and HOP2 . This study provides the first time-resolved landscape of heat adaptation in Iris , revealing a phased cascade from early HSF-HSP activation to sustained flavonoid accumulation, and identifies key genetic targets for breeding thermotolerant ornamentals.