Functional Analysis Of Arabidopsis RIC1in Pollen Tube Growth

After pollen grains germination on the stigma of a flowering plant, pollen tubes undergo rapid tip growth in the style and deliver two sperms to the ovule for fertilization. In pollen tubes, the establishment of cell polarity and the maintenance of polarized cell growth are precisely regulated spatially and temporally. Previous studies have demonstrated Ca2+, Rho-like GTPase of plant (ROP) small GTPases, and phospholipid-mediated pathways, are integrated into a complex network to regulate pollen tube growth, and the organization and dynamics of the actin cytoskeleton is a common target of these signaling pathways. In leaf pavement cells, Rop-interactive CRIB motif-containing protein1(RIC1), a microtubule-associated protein which is activated by ROP6, can promote the microtubule-severing activity of katanin P60subunit (KTN1), resulting in the microtubule reordering. The present study analyzed the mechanism of RIC1involved in regulation of pollen tube tip growth.RICl was prominently expressed in pollens and pollen tubes. Phenotype analyses demonstrated that RIC1is a negative regulator of pollen tube growth. Knockout of RIC1resulted in enhanced pollen tube elongation, while overexpression of RIC1dramatically inhibited pollen tube elongation. Previous studies demonstrated that RIC1is a microtubule-associated protein. However, no obvious difference was observed between ricl-1and wild type pollen tubes regarding the microtubules organization. Interestingly, pharmacological analysis suggested that the actin cytoskeleton was disturbed in ricl-1pollen tubes. F-actin in both apical and subapical region was increased in ricl-1pollen tubes, while a decrease of F-actin was observed in RIC1overexpression pollen tubes. Further investigation revealed that RIC1localized primarily to the apical plasma membrane (PM) of pollen tubes, and the appearance of this polarized PM localization was prior to pollen grain germination. Monitoring the dynamics of apical F-actin in growing pollen tubes indicated loss of RIC1resulted in an increase of apical F-actin, and the oscillation of F-actin exhibited a greater oscillatory amplitude. On the contrary, overexpression of RIC1resulted in a decrease in abundance and a lower oscillatory amplitude of apical F-actin. Quantification of the dynamics of single actin filament at apical PM demonstrated that the frequency of F-actin severing was decreased in ricl-1tubes, resulting in a longer lifetime and an increased maximum length of single actin filament that associated with the PM. In contrast, the F-actin severing frequency was increased when RIC1was overexpressed, leading to a shorter lifetime and decreased maximum length of PM-associated F-actin. The distribution of RIC1at the apical PM was highly dynamic and oscillated during pollen tube growth. However, its oscillation dramatically decreased upon suspension of tube growth, but could be recovered along with resumed pollen tube growth. Furthermore, RIC1H37H40D that lost PM localization, induced weaker growth inhibition compared to wild-type RIC1, suggesting the PM localization of RIC1is important for its function in pollen tube growth.In summary, the present study demonstrates that RIC1regulates F-actin dynamics at the apical PM by severing F-actin, establishing a novel mechanism that underlies regulation of pollen tube growth.