Temporal multi-omics analysis of HSF-HSP and flavonoid pathways under heat stress in Iris

Heat stress poses a significant threat to the productivity of ornamental plants. Iris tectorum (Iris) is a highly valued ornamental species known for its diverse flower colors and architectural forms. However, heat‑induced physiological damage can substantially reduce its economic value. To investigate the temporal dynamics of the heat stress response in I. tectorum 'Hot Spicy', we conducted a time-series experiment at 40 °C for 0, 1, 3, 6, 9, and 24 h, integrating physiological measurements, transcriptomics, and metabolomics. Physiologically analyses showed that heat stress induced progressive membrane lipid peroxidation, as indicated by MDA accumulation, transient activation of antioxidant enzymes, with SOD and POD peaking at 6 h, and sustained proline accumulation, reflecting a phased stress response. Transcriptomic analysis showed temporally coordinated expression of heat stress factors-heat shock proteins (HSF-HSP) co-expression networks. Small HSPs genes, including HSP26.7, HSP16.0 and HSP22.0 were rapidly induced within 1-3 h, with increases of up to 39.46-fold. Broad activation of HSP genes peaked at 6-9 h, reaching up to 56.50-fold, whereas HSP70 expression persisted at 24 h, with increases of up to 7.77-fold. HSFs displayed hierarchical activation, with HSFA5 responded early, HSFA6b in the mid-phase, and HSFA3/HSFA4a in the late phase, suggesting a layered transcriptional temporal sequence. During the late phase (6-24 h), the ABA signaling pathway and flavonoid biosynthesis genes were upregulated, accompanied by the accumulation of flavonoids such as naringenin chalcone and (-)-epigallocatechin. Weighted Gene Co-expression co-expression Analysis (WGCNA) identified a core module (MEbrown4) enriched in phenylpropanoid biosynthesis and hormone signaling, with KIN10 and HOP2 as highly connected candidate hub genes. Together these results provide a time-resolved multi-omics landscape of heat adaptation in Iris and suggests a phased temporal pattern response from early HSF-HSP activation to sustained flavonoid accumulation. The identified candidates, including HSFA5, HSP70-16, ABF3, KIN10, HOP2, provide candidate genetic resources for future functional validation in Iris.