Project description:FLP and MYB88 are two paralogous MYB proteins, regulating the symmetric division of guard mother cell during Arabidopsis stomatal development. To understand their molecular functions, we performed genome-wide identification of FLP/MYB88 binding targets using ChIP-chip with FLP/MYB88 antibody. By comparing ChIP-chip between wild-type and flp-1 myb88 lines, a total genes were identified as putative direct targets for FLP/MYB88.
Project description:Stomata are highly specialized organs which consist of pairs of guard cells and regulate gas and water vapor exchange in plants. While early stages of guard cell differentiation have been described and were interpreted in analogy to processes of cell type differentiation in animals, the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, scap1 (stomatal carpenter 1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as a K+ channel protein, MYB60 transcription factor, and pectin methyl esterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation.
Project description:Stomata are pores in the epidermis of plants that can open and close, allowing for gas exchange with the environment, vital for photosynthesis, and regulating transpiration. They are formed from protodermal cells by a series of asymmetric and symmetric divisions and differentiation steps. Initiation into and progression within the stomatal lineage are driven by the consecutive expression and action of three sets of bHLH transcription factor dimers (SPCH, MUTE, FAMA plus their heterodimer partners SCRM or SCRM2) and is accompanied by massive changes in the transcriptome as well as the chromatin landscape. Strong shifts in chromatin accessibility have been observed specifically at the initiation stage and at the transition from division to differentiation. In line with this, FAMA, which promotes the differentiation of stomata, has been shown to interact with RBR, which can recruit the repressive PRC2 complex, to shut down the early stomatal lineage transcriptional program. Interestingly, several of the stomatal lineage bHLHs bind chromatin regions that are closed/inaccessible in a primordial state (the shoot apical meristem). This suggests that the stomatal lineage bHLHs could be able to access closed chromatin and that they might also be directly involved in opening (and closing) of chromatin at a subset of their target regions. In order to test this hypothesis and identify chromatin modifiers that associate with the stomatal lineage bHLH dimers, we did proximity labeling with TurboID fused to SCRM, which is expressed throughout the stomatal lineage and is the main heterodimerization partner of SPCH, MUTE and FAMA. We identified a number of proteins acting as chromatin modifiers and remodelers, including the histone acetyl transferase HAC1, which deposits activating marks on nucleosomes, and multiple subunits of the ATPase-dependent chromatin remodeling SWI/SNF complex. Together, these data support a role of SCRM and its partners in regulating target gene accessibility to impose lasting changes in gene expression within the stomatal lineage.
Project description:Plant cells are totipotent and hence can dedifferentiate and re-differentiate, making it possible to clone entire plant parts from a single cell. It is hence critical for plant cells to maintain specific cell-states during and after differentiation. Stomata, microscopic valves on the plant epidermis required for efficient gas exchange and water management, have emerged as a powerful model system for understanding how de novo lineage-specific stem cell initiate, proliferate and differentiate into specialized cell types during their development of the plant epidermis. The stomatal lineage emerged from a subpopulation of protodermal cells as meristemoid mother cells (MMCs) that undergoes an asymmetric entry division to give a rise meristemoid and its sister cell called the stomatal-lineage ground cell (SLGC). After several rounds of asymmetric cell division, meristemoids differentiate into round guard mother cells (GMC), which divide symmetrically and terminally differentiate into paired guard cells (GCs). We have adapted the INTACT (Isolation of Nuclei TAgged in Specific Cell Types) system to isolate each stomatal lineage-specific nuclei followed by ATACseq (Assay for Transposase-Accessible Chromatin). We showed that chromatin accessibility is dynamic throughout the stomatal lineage progression. Further, the analysis of TF binding sites in differentially accessible regions led to discover that combinatorial cis-regulatory elements and transcription factor circuits controls lineage specific cell state transition during stomatal development.
Project description:Identifying interaction partners and protein complex compositions for transcription factors (TFs) can produce valuable information on the mechanisms by which they regulate gene expression or are themselves regulated by signaling pathways in the cell. FAMA is a TF that is specifically expressed in the last stages of stomatal guard cell development in the epidermis of young leaves and other aerial tissues of higher plants. It is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. How this is achieved mechanistically is still unclear. We therefore isolated putative FAMA interaction partners from young Arabidopsis seedlings expressing FAMA-CFP under the FAMA promoter using proteasome inhibitor treatment and formaldehyde crosslinking, followed by a classical co-immunoprecipitation mass spectrometry approach. Plants expressing nuclear GFP under a stomatal lineage specific promoter were used as controls. In two independent experiments with different crosslinking and immunoprecipitation conditions, we found ICE1, a known heterodimerization partner of FAMA, to be enriched in FAMA-CFP versus control samples. Other proteins with known transcriptional regulator or co-regulator function were not identified, presumably due to the low abundance of the bait protein. The interaction of FAMA with BRXL2, another regulator of guard cell development, which was detected in one experiment, is likely an artifact since BRXL2 does not localize to the nucleus under normal conditions.
Project description:We initiated a study to investigate the transcriptional profiles associated with cell states of the stomatal lineage. A stem-cell like precursor of stomata, a meristemoid. reiterates asymmetric divisions and renews itself before differentiating into guard cells. The transient and asynchronous nature of the meristemoid has made it difficult to study its molecular characteristics. Through combinatorial use of genetic resources that either arrest or constitutively drive stomatal cell-state progressions due to loss- or gain-of-function mutations in the key transcription factor genes, SPEECHLESS, MUTE, and SCRM, we obtained seedlings highly enriched in pavement cells, meristemoids, or stomata. Here we present transcriptome and genome-wide trends in gene regulation associated with each cell state and identify molecular signatures associated with meristemoids. 12 samples are included in this study. Three biological replicates of 5-dag seedlings of speechless, scrm-D and scrm-D;mute were compared wild type seedlings for changes in gene expression.
Project description:Stomata are highly specialized organs which consist of pairs of guard cells and regulate gas and water vapor exchange in plants. While early stages of guard cell differentiation have been described and were interpreted in analogy to processes of cell type differentiation in animals, the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, scap1 (stomatal carpenter 1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as a K+ channel protein, MYB60 transcription factor, and pectin methyl esterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation. We isolated guard cell protoplasts from 4-week-old WT(Col-0) and scap1 mutant plants and extracted RNA independently. Four biological replicates were performed for each experiment.
Project description:Dynamic cell identities underlie flexible developmental programs. The stomatal lineages in the Arabidopsis leaf epidermis feature asynchronous and indeterminate divisions that can be modulated by environmental cues. The products of these lineages, stomatal guard cells and pavement cells, regulate plant-atmosphere exchanges, and the epidermis as a whole influences overall leaf growth. How flexibility is encoded in development of the stomatal lineage, and how cell fates are coordinated in the leaf are open questions. Here, we offer single-cell transcriptomes to uncover models of cell differentiation within Arabidopsis leaf tissue.