A Systems Approach Reveals Regulatory Circuitry for Arabidopsis Trichome Initiation by the GL3 and GL1 Selectors

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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. The establishment of single-celled leaf hairs (trichomes) from pluripotent epidermal (protodermal) cells provides a powerful system to determine the gene regulatory networks involved in plant cell fate determination. Two transcription factors—GL1 and GL3—have been associated with the initiation of trichome formation; yet only a handful of GL1-GL3–regulated genes have previously been characterized. In this study, we combined expression analyses performed in a number of different genotypes to identify a minimal set of about 500 genes associated with trichome formation. We also used ChIP-chip to identify a set of about 20 genes that are immediate targets of GL3 and GL1. Many more genes are targeted by GL1 or by GL3, likely in cooperation with other bHLH of MYB partners, but not by both GL1 and GL3. As predicted for genes involved in the initiation of epidermal cell fate determination, several of the GL3/GL1 direct targets are expressed early during trichome formation, including the transcription factors MYC1 (bHLH), SCL8 (GRAS), and genes involved in the regulation of the endocycle (SIM and RBR1). Co-expression analyses permitted us to identify sets of target genes likely downstream of the GL3/GL1 regulated transcription factors, providing the first steps towards building the regulatory network associated with the differentiation of protodermal cells into trichomes.