Global dynamics and cytokinin participation of salt gland development trajectory in recretohalophyte Limonium bicolor

Abstract Salt gland is an epidermal Na+ secretory structure that enhances salt resistance in the recretohalophyte sea lavender (Limonium bicolor). To elucidate the salt gland development trajectory and related molecular mechanisms, we performed single-cell RNA sequencing of L. bicolor protoplasts from young leaves at salt gland initiation and differentiation stages. Dimensionality reduction analyses defined 19 transcriptionally distinct cell clusters, which were assigned into four broad populations—promeristem, epidermis, mesophyll, and vascular tissue—verified by in situ hybridization. Cytokinin was further proposed to participate in salt gland development by the expression patterns of related genes and cytological evidence. By comparison analyses of scRNA-seq with exogenous application of 6-benzylaminopurine, we delineated five salt gland development-associated sub-clusters and defined salt gland specific differentiation trajectories from sub-clusters 8, 4, or 6 to sub-cluster 3 and 1. Additionally, we validated the participation of TRIPTYCHON and the interacting protein Lb7G34824 in salt gland development, which regulated the expression of cytokinin metabolism and signaling related genes such as GLABROUS INFLORESCENCE STEMS 2 to maintain cytokinin homeostasis during salt gland development. Our results generated a gene expression map of young leaves at single-cell resolution for the comprehensive investigation of salt gland determinants and cytokinin participation that helps elucidate cell fate determination during epidermis formation and evolution in recretohalophytes.