Abstract
Position-dependent cell fate determination and pattern formation are unique aspects of the development of plant structures. The establishment of single-celled leaf hairs (trichomes) from pluripotent epidermal (protodermal) cells in Arabidopsis provides a powerful system to determine the gene regulatory networks involved in cell fate determination. To obtain a holistic view of the regulatory events associated with the differentiation of Arabidopsis epidermal cells into trichomes, we combined expression and genome-wide location analyses (ChIP-chip) on the trichome developmental selectors GLABRA3 (GL3) and GLABRA1 (GL1), encoding basic helix-loop-helix (bHLH) and MYB transcription factors, respectively. Meta-analysis was used to integrate genome-wide expression results contrasting wild type and gl3 or gl1 mutants with changes in gene expression over time using inducible versions of GL3 and GL1. This resulted in the identification of a minimal set of genes associated with the differentiation of epidermal cells into trichomes. ChIP-chip experiments, complemented by the targeted examination of factors known to participate in trichome initiation or patterning, identified about 20 novel GL3/GL1 direct targets. In addition to genes involved in the control of gene expression, such as the transcription factors SCL8 and MYC1, we identified SIM (SIAMESE), encoding a cyclin-dependent kinase inhibitor, and RBR1 (RETINOBLASTOMA RELATED1), corresponding to a negative regulator of the cell cycle transcription factor E2F, as GL3/GL1 immediate targets, directly implicating these trichome regulators in the control of the endocycle. The expression of many of the identified GL3/GL1 direct targets was specific to very early stages of trichome initiation, suggesting that they participate in some of the earliest known processes associated with protodermal cell differentiation. By combining this knowledge with the analysis of genes associated with trichome formation, our results reveal the architecture of the top tiers of the hierarchical structure of the regulatory network involved in epidermal cell differentiation and trichome formation.
Highlights
Position-dependent cell fate determination and pattern formation are unique aspects of the development of plant structures
We describe the identification of a set of genes directly regulated by GL3 and GL1. These GL3/GL1 direct targets, identified by a combination of chromatin immunoprecipitation (ChIP) methods coupled with the hybridization of whole-genome Arabidopsis tiling arrays (ChIP-chip) and ChIP analyses on factors known to be involved in trichome formation, include genes involved in the regulation of gene expression, in the control of endoreduplication, in metabolic functions as well as several genes with previously unknown functions
We adapted chromatin immunoprecipitation (ChIP) methods coupled with the hybridization of whole-genome Arabidopsis tiling arrays (ChIP-chip) using antibodies against GFP to immunoprecipitate the chromatin fragments associated with the GL3-YFP and GL1-YFP-MYC regulators obtained from formaldehyde cross-linked green tissues of three-week old Arabidopsis plants
Summary
Position-dependent cell fate determination and pattern formation are unique aspects of the development of plant structures. The establishment of single-celled leaf hairs (trichomes) from pluripotent epidermal (protodermal) cells provides a powerful system to determine the genetic networks and positional cues involved in cell fate determination [1,2,3,4]. In the Arabidopsis leaf, trichomes constitute the first differentiated cell type. While the number is variable between different leaves and ecotypes, trichomes represent 1–2% of the roughly 1.26104 cells that constitute the Arabidopsis leaf adaxial epidermis. Because of the ease to score mutants, ,70 genes involved in various aspects of trichome initiation, spacing, size and morphology have been identified [3] (Table S1)
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