Physiological adaptation and molecular regulatory networks of Salix linearistipularis under alkaline stress

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Alkaline stress is a common abiotic factor that greatly hinders crop growth and industrial productivity. Salix linearistipularis is a multi-purpose industrial crop (timber, wickerwork, and bioenergy) that exhibits comparatively high tolerance to alkaline conditions. Accordingly, we focus on elucidating the regulatory mechanisms that underpin its alkaline-stress responses. Understanding these mechanisms will inform the development and genetic improvement of alkaline-tolerance crops. To systematically unravel the molecular regulatory network of alkaline responses in S. linearistipularis , we integrated physiological experiments, RNA-seq technology, and a time-ordered gene co-expression network (TO-GCN) analysis to elucidate the underlying regulatory mechanisms. Physiological indicators showed the dynamic process of photosynthesis and reactive oxygen species (ROS) levels under NaHCO 3 treatment. RNA-seq identified differentially expressed genes (DEGs) that we organized into 10 temporal levels (L1-L10). GO enrichment and TO-GCN revealed coordinated modules, including the photosynthetic system, antioxidant system, mitogen-activated protein kinase (MAPK) signaling pathways, calcium ions (Ca 2+ ) signaling pathways, and cytochrome P450 monooxygenase (CYP) system. Yeast one-hybrid (Y1H) assays indicated that bHLH, bZIP, and LOB transcription factors bind the SlPIP2 promoter, supporting TF-promoter interactions relevant to alkaline stress. Collectively, these results systematically elucidate a dynamic regulatory network underlying alkaline adaptation in S. linearistipularis , providing candidate genes and TF-target relationships for breeding alkaline-tolerant crop germplasm. • Multi-pathway analysis reveals alkaline network regulation in S. linearistipularis . • First application of TO-GCN reveals gene expression patterns under alkaline stress. • Hierarchical modeling predicts potential interactions between TF and non-TF. • Provides valuable insights and hypotheses on plant alkaline stress mechanisms.