Arabidopsis RIC1Regulates Plant Cell Tip Growth By Severing Microfilaments

Cell polarity plays a key role in cell differentiation, development and morphogenesis. Tip growth is an extreme form of polar cell growth and is wildly used as a model system to study the regulatory mechanisms for establishing and maintaining cell polarity. Filamentous actin cytoskeleton (F-actin) is involved in the cell tip growth. The organization and dynamics of F-actin are regulated by actin-binding proteins and many signaling pathways, such as small GTPases, Ca2+and phospholipid signaling.Previous studies have demonstrated that RIC1(Rop-interactive CRIB motif-containing protein1) can promote the microtubule-severing activity of katanin P60subunit (KTN1) in leaf pavement cells, resulting in the microtubule reordering. And a ROP6-RIC1pathway was found to inhibit PIN2endocytosis by stabilization of F-actin in root cells.The present study demonstrates that Arabidopsis RIC1is involved in regulation of tip growth by its F-actin-severing activity. Biochemical assay showed that RIC1had F-actin-severing and-capping activity in the presence of Ca2+. Analysis of GUS (β-glucuronidase) expression pattern showed that RIC1was prominently expressed in pollen tubes, indicating RIC1may play a role in pollen tube growth. Knockout of RICl resulted in increased pollen tube elongation, while RIC1overexpression dramatically inhibited pollen tube elongation. Using Lifeact-mEGFP to visualize F-actin organization, it was found that loss of RIC1led to an increase of F-actin, whereas overexpression of RIC1resulted in a decrease of F-actin in pollen tubes tip regions. RIC1localized mainly to pollen tube apical plasma membrane (PM), indicating that RIC1may function at apical PM. Quantification of actin dynamics at apical PM demonstrated that an increased severing frequency of F-actin in ricl-1pollen tubes, but overexpression of RIC1led to a decreased severing frequency of F-actin. We propose that RIC1severs F-actin at the apical PM to regulate pollen tube growth. Furthermore, overexpression of RIC1, a mutant form of RIC1which can not localize to the PM, still inhibited pollen tube growth. However, the growth inhibition was less severe compared to that caused by overexpression of wild-type form RIC1. Meanwhile, apical F-actin in RIC1H37D/H40D pollen tubes was less abundant compared to control, but was more abundant than that expressing wild-type RIC1. These results reveal that the PM localization of RIC1is important for its function, but RIC1can still functionon F-actinin the cytosol of pollen tube.Further study indicated that RIC1was also localized at apical PM in growing root hairs. Loss of RIC1function led to shorter root hairs, and overexpression of RIC1inhibited root hair elongation. The amount of F-actin at the apical region of ricl-1root hairs was notably increased. Overexpression of RICl resulted in axial actin cables protruding to the extreme apex. The result indicated that RIC1has a similar function in the root hair, which undergoes tip growth just like in the pollen tube.This study demonstrates RIC1may regulate tip growth by its F-actin-severing activity at apical PM as well as in cytosol, uncovering an unknown mechanism that underlying the regulation of tip growth.