Identification of novel regulators required for early development of vein pattern in the cotyledons by single‐cell RNA‐sequencing

Abstract

SUMMARY The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3‐day‐old cotyledons using single‐cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3‐day‐old cotyledons based on single‐cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein.

Identification of the Regulators of Epidermis Development under Drought- and Salt-Stressed Conditions by Single-Cell RNA-Seq

“…Histochemical GUS staining was performed as described previously [ 64 ]. Samples were fixed in 90% acetone at −20 °C, rinsed four times with 0.1 M sodium phosphate buffer (pH 7.4), and then incubated in X-Gluc solution (0.1 M sodium phosphate (pH 7.4), 3 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.5 g L −1 5-bromo-4-chloro-3-indolyl-β-d-glucuronide cyclohexilammonium salt) at 37 °C.…”

“…Histochemical GUS staining was performed as described previously [64]. Samples were fixed in 90% acetone at −20 • C, rinsed four times with 0.1 M sodium phosphate buffer (pH 7.4), and then incubated in X-Gluc solution (0.1 M sodium phosphate (pH 7.4), 3 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.5 g L −1 5-bromo-4-chloro-3-indolyl-β-d-glucuronide cyclohexilammonium salt) at 37 • C. After staining, samples were incubated in methanol to remove chlorophyll and then mounted in the clearing solution (a mixture of chloral hydrate, water, and glycerol in a ratio of 8:2:1).…”

Identification of two bZIP transcription factors that regulate development of pavement and trichome cells in Arabidopsis thaliana by single-cell RNA-sequencing

“…Genes with the greatest variable expression amongst single cells were identified using the method described by (Macosko et al, 2015). The tSNE analysis, UMAP analysis, and DEG identification were performed as previously described (Liu et al, 2020; Liu et al, 2022).…”

“…Histochemical GUS staining was performed with a G3061 GUS staining Kit (Solarbio Co., Beijing, China) according to the manufacturer’s instructions as previously described (Liu et al, 2022).…”

“…The precise underlying molecular mechanisms responsible for the fate determination of TCs and PCs, however, are still unclear. Single-cell RNA-sequencing (scRNA-seq) technology allows the analysis of transcriptional profiles of different types of cells and to identify genes that are specifically expressed at different developmental and morphogenetic stages (Zhang et al, 2019; Liu et al, 2020; Wendrich et al, 2020; Kim et al, 2021; Liu et al, 2021; Serrano-Ron et al, 2021; Liu et al, 2022). Therefore, we conducted a scRNA-seq analysis of 3-day-old true leaves of Arabidopsis wild-type (WT) to elucidate the mechanisms that regulate the fate and development of TCs and PCs.…”