Stage-specific transcriptomic responses of wheat to heat, drought, and combined stress: AP2/ERF regulatory networks and functional characterization of TaEREBP1-L.

Wheat (Triticum aestivum L.) production is increasingly constrained by drought, heat, and their combination, especially in arid regions like Xinjiang, China. While previous studies have focused on single-stress responses, the molecular mechanisms underlying wheat adaptation to combined drought and heat stress (HD) remain elusive. Here, we performed transcriptome profiling at three key spike developmental stages-Jointing Stage (JS), Stamen and Pistil Primordial Differentiation Stage (SPDS), and Tetrad Stage (TS)-under drought (DS), heat (HS), and combined HD conditions. Comparative analysis revealed that HD triggered the most extensive transcriptional reprogramming, with 3,884, 5,230, and 2,793 stage-specific upregulated genes across JS, SPDS, and TS, respectively. Notably, the overlap between HS- and HD-responsive genes was higher than that between DS and HD during early stages, indicating stress-specific transcriptional shifts. Functional enrichment highlighted pathways related to osmotic adjustment, oxidative defense, and phytohormone signaling, particularly ABA and JA biosynthesis. Among stress-responsive transcription factors, the AP2/ERF family showed prominent enrichment, with TaEREBP1-L (TraesCS5A02G215900) identified as a central regulator. Dual-luciferase reporter assays confirmed that TaEREBP1-L directly activated the promoters of ABA- and JA-pathway genes (AAO3, AOC2). Co-expression analysis further revealed its coordination with membrane transporters (e.g., ABC and MATE families), suggesting an integrative regulatory function under HD conditions. Transgenic Arabidopsis overexpressing TaEREBP1-L displayed significantly enhanced drought and heat tolerance, evidenced by increased proline content, antioxidant enzyme activity (SOD, CAT), and elevated ABA/JA levels. These results demonstrate that TaEREBP1-L confers broad-spectrum stress tolerance by orchestrating hormonal and physiological pathways. Our findings provide valuable insights into combinatorial stress adaptation and identify TaEREBP1-L as a promising target for improving wheat resilience under climate-related challenges.