LAUSR

Plant development and shape formation is dependent on the establishment and maintenance of polar cell growth. Central to the regulation of cell polarity are Rho of plants (ROP) small GTPase family. ROPs are membrane-anchored molecular switches, which exist in a GTP ‘ON’ or GDP ‘OFF’ state. They are activated by ROP guanine nucleotide exchange factors (GEFs), which facilitate GDP release and are rapidly inactivated by ROP GTPase-activating proteins (GAPs)1. Rho superfamily GTPases-based polarity is generally thought to take place by highly restricted GEF distribution and more widely dispersed GAP distribution, a mechanism often referred to as ‘local activation and global inhibition’. In budding yeast, the Bem1 scaffold mediated restriction of Cdc42 and its GEF Cdc24 has been established as a paradigm for localized activation and polarity generation by Rho GTPases2. In plants, local accumulation of GEFs during root hairs and trichome development as well as in secondary cell wall pits formation has been associated with local ROP activation and regulation of cell polarity3–5. In this issue, Kulich et al. describe a novel scaffold-associated mechanism that locally restricts GAP distribution6, highlighting the importance of ROP inactivation in regulation of cell polarity. Two types of ROPGAPs have been identified in plants: the Cdc42and Rac-interacting binding (CRIB) domain containing ROPGAPs and the pleckstrin homology (PH) domain-containing ROP1 enhancer (REN, also known as RENGAPs or PHGAPs). In pollen tubes, ROPGAPs localized to the shank7, while the RENs localize to subapical cytoplasmic vesicles, restricting ROP activity to the tip8. Kulich et al.6 also report that three Arabidopsis ARMADILLO REPEAT ONLY (ARO) proteins, ARO2/3/4, regulate root hair and trichome development by functioning as plasma membrane-associated scaffolds, which recruit RENs and GTP-bound ROPs to specific sites in the plasma membrane (Fig. 1). The AROs form an evolutionarily conserved family of proteins found in bryophytes and higher plants that contain two separate ARMADILLO (ARM) repeat motifs. Complementation assays with a Marachantia polymorpha ARO homologue suggest that ARO function is conserved and present an ancient mechanism for the regulation of ROP signalling in plants. First, the expression pattern of ARO2, ARO3 and ARO4 was examined using a fluorescent reporter gene controlled by the promoter sequence of each of the three genes. This analysis showed that the AROs are expressed in bulging trichoblasts (root-hair-forming cells) and developing trichomes. Examination of transfer DNA (T-DNA) insertion mutants followed. While single mutants in each of the AROs did not display a visible phenotype, the combined mutation in aro2/3 and the triple aro2/3/4 mutants developed irregularly shaped, split or burst root hairs. The aro2/3/4 triple mutants also developed fewer fully matured trichomes and a large proportion of burst trichomes. To obtain further insight into the function of AROs, their subcellular distribution was studied in developing trichomes, root hair and root epidermal cells using combination of confocal and total internal reflection fluorescence/ variable angle epifluorescence microscopy (TIRF/VAEM) microscopies as well as biochemical fractionations. The combined analysis revealed that AROs are localized in plasma membrane-associated punctate structures at the apex of new trichome branches and apical membrane of root hair bulges. Membrane localization and complementation assays demonstrated that AROs interact with anionic lipids in the Trichomes a b