Abstract
Main conclusion
NO and H2 synergistically promote cucumber adventitious rooting by regulating hormone, photosynthesis and antioxidant pathways. Adventitious root formation is essential for plant propagation and is modulated by nitric oxide (NO) and hydrogen gas (H2). This study used transcriptome sequencing to investigate cucumber explants treated with sodium nitroprusside (SNP, an NO donor) and hydrogen-rich water (HRW, an H2 donor). In total, 2716 differentially expressed genes (DEGs) were identified under SNP treatment and 1443 under HRW treatment, with 570 genes co-regulated by both signals. KEGG enrichment analysis revealed significant enrichment in pathways involved in phytohormone signaling, photosynthesis-antenna proteins, and glutathione metabolism. Functional characterization of the 570 common genes showed enrichment of protein kinases, cytochrome P450, and transcription factor families (AP2/ERF, MYB, bHLH, NAC), and protein-protein interaction network analysis identified five hub genes (CsAUX1, CsCYCD3-3, CsGSTU8, CsXTH3, CsERFC3). Physiological assays verified that SNP and HRW coordinately regulated phytohormone homeostasis: they decreased zeatin content while increasing brassinosteroids and ethylene levels. Additionally, both treatments improved photosynthetic parameters and enhanced glutathione antioxidant activity. Time-course qPCR revealed that HRW induced faster gene expression changes (peaks at 12-24 h) than SNP (peaks at 24-48 h), and NO scavenger (cPTIO) experiments confirmed that SNP effects are strictly NO-dependent while HRW acts through both NO-dependent and NO-independent pathways. Oxidative stress markers (MDA, H2O2, O2-) were reduced, and antioxidant enzymes (SOD, POD, CAT, APX, GR, GPX, GSTs) were elevated, indicating enhanced redox buffering capacity rather than oxidative stress. Cis-regulatory analysis further detected transcription factor binding sites related to light, hormone, and stress responses. Collectively, these findings systematically reveal a synergistic molecular network by which NO and H2 promote adventitious rooting. This study enhances our understanding of the regulatory mechanisms underlying adventitious root development and offers a theoretical foundation for optimizing propagation techniques in horticultural crops.