Single-cell and spatial transcriptomics reveals the spatiotemporal trajectory of the small peptide TAP4 in delaying...

Trypsin (Try) has been reported to possess specific superoxide anion-scavenging activity and to exert pronounced anti-senescence effects in Hylocereus undatus fruit; however, the mechanisms underlying its anti-senescence function remain unclear. This study identified TAP4 as the most bioactive peptide among Try autolysis peptides (TAPs). Single-cell transcriptomic analysis revealed that TAP4 alters pericarp cell differentiation trajectories during senescence, particularly in endocarp (EN) cells. It activates the transcription factor (TF) HuWRKY21-1, specifically inducing immune responses in inner pericarp cells and upregulating 44 immune-related genes, including HuIGP4 and HuLYK6. Downstream factors, such as HuFULL and ethylene response factors (ERFs), promote the biosynthesis of antioxidants, such as antheraxanthin and hyperin. Functional validation via virus-induced gene silencing (VIGS), callose deposition, disease resistance assays, and quantitative reverse-transcription PCR confirmed the roles of key TFs, including HuWRKY21-1, HuFULL, and HuERF30-1, in immune activation and resistance reconstruction. This work provides evidence for the potential of the EN as a defensive tissue in fruit and opens new avenues for understanding the mechanisms underlying the establishment of plant resistance.