Single-cell sequencing reveals dynamic cell development trajectories in two kiwifruit (Actinidia chinensis) genotypes with contrasting cold resistance

Abstract Kiwifruit ( Actinidia Lindl.) is a valuable fruit tree species, with its cold resistance being influenced by leaf structure and various cell types. Despite significant progress in understanding the cold stress response during the last decade, the mechanisms underlying the formation of distinct cold-resistant cell types in kiwifruit, particularly at the single-cell level, remain poorly understood. In this study, single-cell RNA sequencing (scRNA-seq) of leaves from ‘Hongyang’ (HY, A. chinensis, cold sensitive) and its transgenic line with overexpressed AaBAM3.1 (designated as HT, cold resistant) was employed to construct a single-cell transcriptional atlas to investigate the cell heterogeneity of two kiwifruit genotypes exhibiting different cold resistance capacities. A total of 5,611 and 13,466 single cells were obtained from HY and HT, respectively. The cells were classified into eight clusters based on gene expression patterns, and key genes associated with specific cell types in both genotypes were identified. Notably, pseudotime trajectory analysis revealed distinct developmental paths for guard cells and mesophyll cells. Additionally, a transcription factor, AaTIFY, exhibited genotype-specific expression and negatively regulated cold resistance. In summary, these results demonstrate that scRNA-seq offers valuable insights into cell differentiation and development in kiwifruit with varying cold resistance at the single-cell level and highlights novel genes related to cold tolerance in kiwifruit.