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

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 4 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 Single-cell RNA sequencing with exogenous application of 6-benzylaminopurine, we delineated 5 salt gland development-associated subclusters and defined salt gland-specific differentiation trajectories from Subclusters 8, 4, and 6 to Subcluster 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.

Global analysis of key post-transcriptional regulation in early leaf development of Limonium bicolor identifies a long non-coding RNA that promotes salt gland development and salt resistance.

Limonium bicolor, known horticulturally as sea lavender, is a typical recretohalophyte with salt glands in its leaf epidermis that secrete excess Na+ out of the plant. Although many genes have been proposed to contribute to salt gland initiation and development, a detailed analysis of alternative splicing, alternative polyadenylation patterns, and long non-coding RNAs (lncRNAs) has been lacking. Here, we applied single-molecule long-read mRNA isoform sequencing (Iso-seq) to explore the complexity of the L. bicolor transcriptome in leaves during salt gland initiation (stage A) and salt gland differentiation (stage B) based on the reference genome. We identified alternative splicing events and the use of alternative poly(A) sites unique to stage A or stage B, leading to the hypothesis that they might contribute to the differentiation of salt glands. Based on the Iso-seq data and RNA in situ hybridization of candidate genes, we selected the lncRNA Btranscript_153392 for gene editing and virus-induced gene silencing to dissect its function. In the absence of this transcript, we observed fewer salt glands on the leaf epidermis, leading to diminished salt secretion and salt tolerance. Our data provide abundant transcriptome resources for unraveling the mechanisms behind salt gland development and furthering crop transformation efforts towards enhanced survivability in saline soils.