Stem cell regulation by the transcription factor WOX5 in the Arabidopsis thaliana root meristem

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The pluripotent stem cells located in plant meristems sustain the lifelong production of new tissues and organs such as roots, stems and leaves. Deciphering the mechanisms that regulate stem cells is therefore central to understanding plant growth and development. The columella stem cell niche of the root meristem has a simple organization that makes it an ideal model to study stem cell regulation: the quiescent center cells maintain a single layer of columella stem cells (CSCs), whose daughters differentiate directly into columella cells that sense gravity in the root cap. We have done extensive preparatory work for this proposal to address the mechanisms of CSC regulation in the model plant Arabidopsis at the genetic and molecular levels. We discovered that the mobile transcription factor WOX5 (WUSCHEL-RELATED HOMEOBOX 5) is a major regulator of CSC specification. Ectopic WOX5 expression generates induced columella stem cells (iCSCs) in sufficient amounts and purity for molecular analysis. Using a cell sorting strategy, we find that WOX5 directly regulates several hundred genes in iCSCs, suggesting that the stem cell state is the result of the balanced expression of a complex genetic network. Based on our preparatory data, we hypothesize that for the majority of its targets, WOX5 represses gene transcription by inducing repressive histone modifications, thus shaping the epigenetic landscape of the stem cells. In addition, WOX5 transcriptionally activates a group of genes by a yet unknown mechanism, including a novel, important regulator of CSC specification, the transcription factor HANABA TARANU (HAN). The goal of this proposal is to elucidate the WOX5-regulated transcriptional and signaling networks that underlie columella stem cell specification. First, using sorted cell-specific nuclei, we will investigate the genome-wide epigenetic modifications induced by WOX5 during reprogramming of differentiated cells into iCSCs and in genuine columella stem cells. Second, we will explore how two WOX5 target genes, coding for the stem cell inhibitor CDF4 and the positive stem cell regulator HAN, contribute to balancing stem cell maintenance and cell differentiation by genetic and molecular studies and by live imaging. All necessary methods and most materials have already been established, enabling us to start the experiments without delay. Combining genome-wide with gene specific functional studies provide a unique opportunity to achieve an integrative mechanistic picture of stem cell specification. Because WOX proteins are conserved regulators of stem cells also in other plant meristems, we expect that our results will provide insight into general mechanisms of plant stem cell regulation. Furthermore, this proposal will not only address fundamental questions in stem cell biology, but also will yield results of agronomic importance for sustained growth and productivity of crops.