Fine localization and functional differentiation of exocarp cell clusters during fruit senescence revealed by single-cell and spatial transcriptomics

The differentiation trajectories of 13 cell clusters in H. undatus during senescence have been revealed by single-cell transcriptome (scRNA-seq) analysis, and the five cell clusters in the exocarp have been found to induce resistance mainly. However, the precise localization and functional differentiation of these five cell clusters remain unclear. In this study, phenotypic changes in the exocarp were recorded, and the ROS production and flavonoid biosynthesis in the exocarp were elucidated. The trajectories of the cell clusters involved in exocarp function were further revealed in scRNA-seq profiles. Through integrated analysis of scRNA-seq and spatial RNA-seq data at resolutions of 0.5, 0.6, and 1.5, using algorithms SingleR, SciBet, CARD, and RCTD, the fine localization of these clusters from the outer to inner layers was achieved. RNA-FISH was used to validate the specific expression of marker genes HuSP2 and HuCEL2 in different locations of the exocarp. Finally, the differentiation trajectories of the five cell clusters were analyzed by selecting highly correlated cells with specific functions for each cluster. The core roles of HuPRP and HuCRRSP55 in the gene regulatory networks of exocarp clusters were revealed. A hypothesis that the five clusters in the exocarp participate in the induction of resistance was proposed.