The regulation of stomatal lineage cell development has been extensively investigated. However a comprehensive characterization of this biological process based on single-cell transcriptome analysis has not yet been reported. Here, we performed RNA-seq on over 12,844 individual cells from the cotyledons of five-day-old Arabidopsis seedlings. We identified 11 cell clusters corresponding mostly to cells at specific stomatal developmental stages with a series of new marker genes. Comparative analysis of genes with the highest variable expression in these cell clusters revealed three transcriptional networks that regulate the development of mesophyll and guard cells, as well as the differentiation from protodermal to guard mother cells. We investigated the developmental dynamics of marker genes via pseudo-time analysis which revealed potential interactions between them. The identification of several novel marker genes suggests new regulatory mechanisms during development of stomatal cell lineage. .

G-protein-coupled receptors (GPCRs) are key players in cell signaling, and their cell surface expression is tightly regulated. For many GPCRs such as β2-AR (β2-adrenergic receptor), receptor activation leads to downregulation of receptor surface expression, a phenomenon that has been extensively characterized. By contrast, some other GPCRs, such as GABA B receptor, remain relatively stable at the cell surface even after prolonged agonist treatment; however, the underlying mechanisms are unclear. Here, we identify the small GTPase Rap1 as a key regulator for promoting GABA B receptor surface expression. Agonist stimulation of GABA B receptor signals through Gαi/o to inhibit Rap1GAPII (also known as Rap1GAP1b, an isoform of Rap1GAP1), thereby activating Rap1 (which has two isoforms, Rap1a and Rap1b) in cultured cerebellar granule neurons (CGNs). The active form of Rap1 is then recruited to GABA B receptor through physical interactions in CGNs. This Rap1-dependent signaling cascade promotes GABA B receptor surface expression by stimulating receptor recycling. Our results uncover a new mechanism regulating GPCR surface expression and also provide a potential explanation for the slow, long-lasting inhibitory action of GABA neurotransmitter.

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Fragile X mental retardation protein (FMRP) is an RNA-binding protein important for the control of translation and synaptic function. The mutation or silencing of FMRP causes Fragile X syndrome (FXS), which leads to intellectual disability and social impairment. γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the mammalian central nervous system, and its metabotropic GABAB receptor has been implicated in various mental disorders. The GABAB receptor agonist baclofen has been shown to improve FXS symptoms in a mouse model and in human patients, but the signaling events linking the GABAB receptor and FMRP are unknown. In this study, we found that GABAB receptor activation upregulated cAMP response element binding protein-dependent Fmrp expression in cultured mouse cerebellar granule neurons via two distinct mechanisms: the transactivation of insulin-like growth factor-1 receptor and activation of protein kinase C. In addition, a positive allosteric modulator of the GABAB receptor, CGP7930, stimulated Fmrp expression in neurons. These results suggest a role for GABAB receptor in Fmrp regulation and a potential interest of GABAB receptor signaling in FXS improvement.

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...