Root hairs, long tubular-shaped outgrowths from root epidermal cells, are an excellent model for the study of cell specification and differentiation. Their wide distribution among all major groups of vascular plants provides evidence for a long evolutionary history and their importance to the success of diverse plant groups in the adaptation to changing environmental conditions (Peterson & Farquhar, 1996). Root hairs increase the root surface area to aid plants in nutrient acquisition, anchorage and interactions with microbes (Grierson & Schiefelbein, 2002). In Arabidopsis, the specification of epidermal cells is biased by an as yet unidentified positional signal derived from the cortex that is perceived by the leucine-rich repeat (LRR) kinase SCRAMBLED (Kwak & Schiefelbein, 2008). The signal, which is supposedly stronger at epidermal cells that lie over the clefts of two underlying cortical cells, represses the expression of WEREWOLF (WER) and MYB23, which negatively regulate the root hair cell fate. A homeodomain protein, GLABRA2 (GL2), represses the hair cell fate by altering the cell developmental program in epidermal cells that occupy nonhair positions (i.e. that are located over tangential cell walls of cortical cells). After cell specification, root hair development is initiated by the formation of a cell wall bulge at the basal end of the cell. The third stage of root hair formation is characterized by the rapid elongation of the hair via tip growth. Tip growth is associated with the establishment of tip-focused Ca2+ gradients, extensive cytoskeleton reorganization, and the transport and deposition of cell wall materials. The restriction of growth at the hair tip is controlled by the reactive oxygen species (ROS)-generating NADPH oxidase ROOT HAIR DEFECTIVE2 (RHD2; AtrbohC) by activating Ca2+ channels (Foreman et al., 2003). The Rho GTPase GDP dissociation inhibitor SCN1/AtrhoGDI1 has been identified as a component that focuses the RHD2/AtrbohC-catalyzed generation of ROS to hair tips (Carol et al., 2005).
Cell wall expansion is a fundamental process in the morphogenesis of plant cells. Several cell wall-related proteins, such as CELLULOSE SYNTHASE LIKE D2 (CSLD2), CSLD3/RHD7, LEUCINE-RICH REPEAT EXTENSIN1 (LRX1) and PROLINE-RICH PROTEIN3 (PRP3), promote root hair formation (Bernhardt & Tierney, 2000; Baumberger et al., 2001; Favery et al., 2001; Bernal et al., 2008). CSLD3 is required for the organization of both cellulose and xyloglucans on root hair cell walls (Galway et al., 2011). Oxidative cross-linking of tyrosine (Tyr) residues in the extensin (EXT) domain is crucial for LRX1 function in the cell wall (Baumberger et al., 2001; Ringli, 2010). Peroxidases are known to mediate such cross-linking during plant defense reactions, and have also been predicted to form cross-links with EXTs during root hair formation (Almagro et al., 2009; Velasquez et al., 2011).
The redox state of the cell is crucial for cell cycle progression and is critical for the transition from cell proliferation to differentiation. In mammals, ROS levels have been found to dictate the fate and differentiation of cells (Owusu-Ansah & Banerjee, 2009). Decreased ROS levels in rhd2 root tip cells interfered with microtubule organization and induced macrotubule assembly in Arabidopsis (Livanos et al., 2012). In addition, disturbed ROS homeostasis caused atypical tubulin formation and affected mitosis in root tip cells of Triticum turgidum and Arabidopsis (Livanos et al., 2012). A molecular link between ROS distribution and transcription was revealed in a study on the UPBEAT1 (UPB1) transcription factor in Arabidopsis. UPB1 negatively regulates peroxidases that control the hydrogen peroxide (H2O2) and superoxide () balance, which are crucial for the transition from proliferation to differentiation in the Arabidopsis root (Tsukagoshi et al., 2010).
Transcriptional Mediators, central co-regulators of transcription, have been identified as large protein complexes in eukaryotes. They form a bridge between RNA polymerase II (RNA Pol II) and transcription factors to initiate transcription. The Mediator complex plays important roles in several developmental processes in yeast, Drosophila, mice and humans (Hentges, 2011). However, the function of Mediator subunits in plant development has only recently begun to be explored. Biochemical purification of plant Mediators identified 21 conserved and six Arabidopsis-specific Mediator subunits plus six paralogs (Backstrom et al., 2007). In Arabidopsis, Mediator subunits play diverse roles, including plant growth and development. For instance, STRUWWELPETER (SWP)/MED14 controls the cell number during primordia initiation and also regulates the duration of cell proliferation in aerial organs (Autran et al., 2002). The Mediator subunits MED12-MED13 have been shown to regulate developmental timing during embryo patterning (Gillmor et al., 2010). PHYTOCHROME AND FLOWERING TIME1 (PFT1)/MED25 has been shown to restrict cell growth, whereas MED8 acts independent of PFT1 to control organ growth (Xu & Li, 2011, 2012). Transcriptional Mediators and ROS regulate the transcription during growth and development in both plants and animals. However, no direct relationship has been implicated at the transcriptional level in either plants or animals.
In an attempt to uncover as yet unexplored roles of Mediators in plant developmental processes, we set out to explore a possible involvement of Mediator subunits in the morphogenesis of root hairs, a prime model for plant cell differentiation. We report that the transcriptional Mediator complex subunit PFT1 controls ROS balance in roots and is critical for root hair differentiation and elongation. From transcriptional profiling experiments, we conclude that PFT1 regulates peroxidase-mediated H2O2 formation to maintain the balance between H2O2 and during root hair formation. Another Mediator subunit, MED8, is also involved in root hair formation, but acts independently of PFT1. Our results provide evidence for a control of root epidermal cell differentiation by the spatial distribution of ROS along the root.