Regulation of stem cell maintenance and cell differentiation states in Arabidopsis root development

The experiments presented in this thesis topic the role of transcription factor family members in regulating growth, development, and maintenance of the Arabidopsis root. We demonstrate a conserved homeobox transcription factor regulates distal stem cell maintenance and expand the notion that the PLETHORA (PLT) family of transcription factors specifically regulates stem cell properties to a significantly broader role. In addition, we show that members of the PLT gene family can activate transcriptional targets in a dose-dependent fashion to establish a feed-forward network that regulates root growth and development. Chapter one reviews the molecular genetical approaches that have been adopted to address Arabidopsis root development. Taking embryogenesis as a starting point, we describe the genes and mechanisms involved in root meristem and stem cell patterning and maintenance. In chapter two we demonstrate a role for the homeobox gene WOX5 (WUSCHEL-RELATED 5), a homologue of WUS, in regulating distal stem cell maintenance in a non-cell autonomous fashion downstream from the SHR/SCR signaling pathway. While loss-of-function leads to stem cell differentiation, overexpression of WOX5 results in the accumulation of distal stem cells that operate independent from quiescent centre (QC) signaling. By reciprocal expression experiments we show that WOX5 and WUS are functionally equivalent in stem cell control, indicating that the organizers of both stem cell systems employ related regulators to provide stem cell maintenance signaling. Chapter three describes the functional analysis of four PLT family members that are redundantly required for embryonic root development and post-embryonic root growth in a dose-dependent fashion. Transcriptional and translational studies of the PLT proteins reveal their graded expression spanning the root meristem with highest expression in the stem cell area, intermediate levels in the transit amplifying zone, and low levels in the differentiation zone. By changing the shape of the PLT expression patterns we show that cell fate within the root meristem depends on the amount of PLT cells are subjected to; high PLT levels maintain stem cell fate, intermediate levels control cell division, and low levels allow cell elongation. Chapter four elaborates on the PLT dosage-dependent control of root cell fate by demonstrating that direct targets of PLT2 are expressed in non-overlapping expression domains within the root meristem. By manipulating PLT2 protein levels in the root meristem, we demonstrate a corresponding shift in the target expression domain; indicating PLT2 can regulate its transcriptional targets in a concentration-dependent manner. In addition, the varied identity of downstream targets suggests that PLT2 controls diversified developmental pathways associated with cell proliferation and growth. Interestingly, the identification of auxin transport facilitators, biosynthetic regulators and signaling components as direct PLT2 targets indicate a multi-level regulatory feedback into the auxin gradient that specifies root patterning. In chapter five the results presented in the previous chapters will be discussed in the light of recent insights in stem cell maintenance and specification, morphogen theory and transcriptional networks to give directions to future research