Unknown,Transcriptomics,Genomics,Proteomics

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A transcriptional map following the developmental trajectory of the Arabidopsis stomatal lineage

ABSTRACT: Developmental transitions can be described in terms of morphology and individual genes expression patterns, but also in terms of global transcriptional and epigenetic changes. Most of the large-scale studies of such transitions, however, have only been possible in synchronized cell culture systems. Here we generate a cell type specific transcriptome of an adult stem-cell lineage in the Arabidopsis leaf using RNA sequencing and microarrays. RNA profiles of stomatal entry, commitment, and differentiating cells, as well as of mature stomata and the entire aerial epidermis give a comprehensive view of the developmental progression. To obtain pure cell populations corresponding to different stomatal lineage cell types we used Fluorescent Activated Cell Sorting (FACS) in combination with markers of early (SPCHp::SPCH-YFP, SSY), middle (MUTEp::nucGFP, MG), and late (FAMAp::GFP-FAMA, FGF) precursor stages, as well of mature stomata (enhancer trap E1728::GFP, E1728G) and a marker of the entire epidermis (ML1p::YFP-RCI2A, ML1Y). To minimize transcriptional differences due to age, all cell types were sorted from 10-day old aerial rosettes, and the specificity of expression pattern at this common time point confirmed via confocal microscopy. Total RNA was extracted from purified protoplasts (4,000 to 20,000 cells/replicate; 2 replicates/marker line; 3 replicates/SSY marker line) and transcript abundance measured using RNA sequencing.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Dominique Bergmann

PROVIDER: E-GEOD-58856 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population.

Adrian Jessika J Chang Jessica J Ballenger Catherine E CE Bargmann Bastiaan O R BO Alassimone Julien J Davies Kelli A KA Lau On Sun OS Matos Juliana L JL Hachez Charles C Lanctot Amy A Vatén Anne A Birnbaum Kenneth D KD Bergmann Dominique C DC

Developmental cell 20150401 1

Developmental transitions can be described in terms of morphology and the roles of individual genes, but also in terms of global transcriptional and epigenetic changes. Temporal dissections of transcriptome changes, however, are rare for intact, developing tissues. We used RNA sequencing and microarray platforms to quantify gene expression from labeled cells isolated by fluorescence-activated cell sorting to generate cell-type-specific transcriptomes during development of an adult stem-cell line ...[more]

PMID: 25850675

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Project description:Stomata are pores in the epidermis of plants that can open and close and allow for gas exchange vital for photosynthesis and regulate transpiration. Stomatal development is driven by a set of conserved bHLH transcription factors (SPCH, MUTE, FAMA, and their heterodimerization partners ICE1, SCRM2), that initiate and promote progression of cell fates in the stomatal lineage. Due to the shared ancestry of SPCH, MUTE and FAMA (subgroup Ia) and ICE1 and SCRM2 (subgroup IIIb) their DNA binding specificity is similar and there is some functional redundancy. However, individual bHLHs also have unique functions. For example, SPCH is required for initiation of the stomatal lineage, while FAMA is responsible for terminal differentiation of the guard cell pair. In grasses, the stomatal complex comprises the guard cell pair, and two flanking subsidiary cells. Recruitment of the latter from neighboring cell filed during development requires expression of MUTE. Remarkably, while MUTE is absolutely required for the promotion of guard mother cell fate in maize and rice, this is not the case in Brachypodium. This suggests that another TF can at least partially substitute for MUTE in this function. While different expression profiles of the bHLH dimers within the stomatal lineage may partially responsible for distinct functions of each pair, it is likely that each pair forms different transcriptional complexes and that interaction with other transcriptional regulators affects the dimer’s binding DNA binding and gene regulation properties. Given the differences in bHLH function between dicots and grasses and even within the grass family, we were interested in elucidating the protein interaction networks of the stomatal lineage regulators in Brachypodium. To this end, we performed co-immunoprecipitation coupled to LC-MS/MS of BdSPCH2-YFP, YFP-BdMUTE, YFP-BdFAMA, YFP-ICE1 and YFP-SCRM2 from the developmental zone of young B. distachyon leaves using GFP-Trap beads. All YFP-fusion proteins were expressed under the endogenous promoter in the Bd21-3 background. The only exception was BdICE1, which was expressed under the ZmUBI promoter, but was nevertheless mostly restricted to the stomatal lineage. As control lines we use the Bd21-3 wild type and a line expressing 3x YFPnls (nuclear YFP) under the MUTE promoter. Comparison of proteins enriched with the bHLH fusion proteins vs. the controls revealed overlapping and distinct putative interactors, which is in agreement with the assumption that these transcription factors have both shared and unique functions. Notably, in addition to the presumed hetero-dimerization partners, we found a number of other bHLH transcription factors that were identified with one or more of the bait proteins. This suggests the presence of a larger bHLH network acting in the stomatal lineage.

Dataset Information

A transcriptional map following the developmental trajectory of the Arabidopsis stomatal lineage

Publications

Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population.

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