Intercellular protein movement plays a critical role in animal and plant development. SHORTROOT (SHR) is a moving transcription factor essential for endodermis specification in the Arabidopsis root. Unlike diffusible animal morphogens, which form a gradient across multiple cell layers, SHR movement is limited to essentially one cell layer. However, the molecular mechanism is unknown. We show that SCARECROW (SCR) blocks SHR movement by sequestering it into the nucleus through protein-protein interaction and a safeguard mechanism that relies on a SHR/SCR-dependent positive feedback loop for SCR transcription. Our studies with SHR and SCR homologs from rice suggest that this mechanism is evolutionarily conserved, providing a plausible explanation why nearly all plants have a single layer of endodermis.

Stem cell function during organogenesis is a key issue in developmental biology. The transcription factor SHORT-ROOT (SHR) is a critical component in a developmental pathway regulating both the specification of the root stem cell niche and the differentiation potential of a subset of stem cells in the Arabidopsis root. To obtain a comprehensive view of the SHR pathway, we used a statistical method called meta-analysis to combine the results of several microarray experiments measuring the changes in global expression profiles after modulating SHR activity. Meta-analysis was first used to identify the direct targets of SHR by combining results from an inducible form of SHR driven by its endogenous promoter, ectopic expression, followed by cell sorting and comparisons of mutant to wild-type roots. Eight putative direct targets of SHR were identified, all with expression patterns encompassing subsets of the native SHR expression domain. Further evidence for direct regulation by SHR came from binding of SHR in vivo to the promoter regions of four of the eight putative targets. A new role for SHR in the vascular cylinder was predicted from the expression pattern of several direct targets and confirmed with independent markers. The meta-analysis approach was then used to perform a global survey of the SHR indirect targets. Our analysis suggests that the SHR pathway regulates root development not only through a large transcription regulatory network but also through hormonal pathways and signaling pathways using receptor-like kinases. Taken together, our results not only identify the first nodes in the SHR pathway and a new function for SHR in the development of the vascular tissue but also reveal the global architecture of this developmental pathway.

Summary: Regulated cell division is critical for the development of multi‐cellular organisms. In the Arabidopsis root, SCARECROW (SCR) is required for the first cell division, but represses the subsequent, longitudinal asymmetric cell divisions that generate the two cell types of the ground tissue – cortex and endodermis. To elucidate the molecular basis of the role of SCR in ground tissue patterning, we screened for SCR‐interacting proteins using the yeast two‐hybrid method. A number of putative SCR‐interacting proteins were identified, among them LIKE HETEROCHROMATIN PROTEIN 1 (LHP1). In lhp1 mutants, a second longitudinal asymmetric cell division occurs in the ground tissue earlier than in wild‐type plants. Similar to the scr mutant, this premature middle cortex phenotype is suppressed by the phytohormone gibberellin (GA). We provide evidence that the N‐terminal domain of SCR is required for the interaction between SCR and LHP1 as well as with other interacting partners, and that this domain is essential for repression of asymmetric cell divisions. Consistent with a role for GA in cortex proliferation, mutants of key GA signaling components produce a middle cortex precociously. Intriguingly, we found that the spindly (spy) mutant has a similar middle cortex phenotype. As SPY homologs in animals physically interact with histone deacetylase, we examined the role of histone deacetylation in middle cortex formation. We show that inhibition of histone deacetylase activity causes premature middle cortex formation in wild‐type roots. Together, these results suggest that epigenetic regulation is probably the common basis for SCR and GA activity in cortex cell proliferation.