Global Dynamic Molecular Profiles of Stomatal Lineage Cell Development by Single-Cell RNA Sequencing

Abstract

30The regulation of stomatal lineage cell development has been extensively investigated. 31 However a comprehensive characterization of this biological process based on 32 single-cell transcriptome analysis has not yet been reported. Here, we performed 33 RNA-seq on over 12,844 individual cells from the cotyledons of five-day-old 34 Arabidopsis seedlings. We identified 11 cell clusters corresponding mostly to cells at 35 specific stomatal developmental stages with a series of new marker genes. 36 Comparative analysis of genes with the highest variable expression in these cell 37 clusters revealed three transcriptional networks that regulate the development of 38 mesophyll and guard cells, as well as the differentiation from protodermal to guard 39 mother cells. We investigated the developmental dynamics of marker genes via 40 pseudo-time analysis which revealed potential interactions between them. The 41 identification of several novel marker genes suggests new regulatory mechanisms 42 during development of stomatal cell lineage. 43 44 45 46 47 48 49 50 51 52 3 / 37 53 54 55 75 MacAlister et al., 2007; Pillitteri et al., 2007). To specify each cell state differentiation, 76 SPCH, MUTE, and FAMA form heterodimers with two paralogous bHLH-leucine 77 zipper (bHLH-LZ) transcription factors, SCREAM (SCRM) and SCRM2 (Kanaoka et 78 al., 2008). In addition, two partially redundant R2R3 MYB transcription factors, 79 FOUR LIPS (FLP) and MYB88, control stomatal terminal differentiation 80 independently of FAMA (GMC to GCs) (Lai et al., 2005; Ohashi-Ito and Bergmann, 81 2006). Two secreted cysteine-rich peptides, EPIDERMAL PATTERNING FACTOR1 82 4 / 37 (EPF1) and EPF2, are expressed at later and earlier stages of stomatal development, 83 respectively. These peptides are perceived by the cell-surface receptors, ERECTA 84 (ER)-family leucine-rich repeat receptor kinases (LRR-RKs), ER-LIKE1 (ERL1) and 85 ERL2, resulting in inhibition of stomatal development (Shpak et al., 2005; Hara et al., 86 2007; Hunt and Gray, 2009b; Lee et al., 2012). The receptor-like protein TOO 87 MANY MOUTHS (TMM) modulates the signaling strength of ER-family receptor 88kinases in a domain-specific manner (Nadeau and Sack, 2002; Lee et al., 2012). 89 Genetic evidence suggests that these signals are mediated via a mitogen-activated 90 protein kinase (MAPK) cascade, which eventually downregulates the transcription 91 factors responsible for initiating stomatal lineage via direct phosphorylation 92 (Bergmann et al., 2004; Lampard et al., 2008; Lampard et al., 2009; Kim et al., 2012). 93 Stomagen (also known as EPF-LIKE9) peptide promotes stomatal development by 94 competing with EPF2 for binding to ER (Sugano et al., 2010; Zhang et al., 2014; 95 Hronkova et al., 2015). One homeodomain-leucine zipper IV (HD-ZIP IV) protein, 96 HOMEODOMAIN GLABROUS2 (HDG2), acts as a key epidermal component 97 promoting stomatal differentiation (Peterson et al., 2013). It is highly expressed in 98 meristemoids, and a hdg2 mutant exhibits delayed meristemoid-to-GM...

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

“…We could not identify TCs by the expression of the TC marker gene GL2 because the size of TCs was too large to pass through the cell strainer. In our previous study, we found that some marker genes were detected in several cell types but at different levels of expression (Liu et al, 2020). The top 10 marker genes for each of the studied cell types other than TCs were specifically expressed in the corresponding cell types, except for the markers of MPCs, GMCs, and GCs.…”

“…Pseudo-time trajectory analysis of single-cell transcriptomes was conducted using Monocle 2 (Trapnell et al, 2014) as previously described (Liu et al, 2020).…”

“…Thus, leaf epidermal cells are comprised of trichome cells (TCs), guard cells (GCs), and pavement cells (PCs) (Marks, 1997). Previous studies have systematically and comprehensively characterized the developmental dynamics of the transcriptomes of stomatal lineage cells (Liu et al, 2020). It is now important to examine the processes underlying the fates and development of PCs and TCs.…”

“…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.…”

Crafting a blueprint for single-cell RNA sequencing

“…These studies demonstrated the remarkable power of scRNA-Seq to detect rare cell types/states and to resolve developmental trajectories in a complex tissue. Subsequent studies have diversified to applying this technique to examine the development of stomata [15,16], leaf vasculature [17,18], shoot apices [19][20][21], inflorescences [22], and lateral root primordia [23], in species such as arabidopsis, maize (Zea mays), rice (Oryza sativa), and tomato (Solanum lycopersicum) (Table 1). Together, these early studies have provided unique insights into the processes of differentiation [4,6,9], tissue-specific abiotic stress responses [5,24], cell-type-specific responses to genetic perturbations [4,6,8,19], and cell-cycle regulator dynamics [16,21], thus providing a strong examination of the virtues of utilizing the technology in plants (Table 1).…”