Dynamic Localization Of ROP2 GTPase In Root Hairs Requires The Trans-Golgi Network/Early Endosome- Mediated Vesicle Trafficking In Parallel To GDI1

Root hair is a tubular-shaped outgrowth from root epidermal cells, which is the main part to absorb water and inorganic salts. The morphology of root hairs has important significance for the development of plants and the response to environmental stress. In addition, root hair is an excellent system to study cell polarity. It has been extensively studied that plant tip growth is regulated by spatiotemporally restricted localization of activated ROPs at a restricted region of the plasma membrane. ROP GTPases (Rho-related GTPases from plant) are key molecular switches in plants, playing important roles in plant growth and development, stress responses, disease resistance and hormone signaling and so on. As molecular switches, ROPs have inactive GDP-bound and activated GTP-bound forms, whose rapid interconvertion is catalyzed by guanine nucleotide exchange factors (RopGEFs) and GTPase activating proteins (RopGAPs). GEFs stimulate the exchange of GDP for GTP to activate ROPs whereas GAPs stimulate the GTPase activity of ROPs to inactivate ROPs. The third class of regulators for ROPs is guanine nucleotide dissociation inhibitors (RhoGDIs). Roles of RhoGDIs on membrane extraction and cytoplasmic sequestration of ROPs were well demonstrated in pollen tubes, indicating negative roles of GDIs on ROP signaling. However, mutants of Arabidopsis GDI1, supercentipede 1 (scnl), grew root hairs with multiple initiation sites, demonstrating that GDI1 is essential to ensure a single and unique polarization site during tip growth. Indeed, studies in yeast and metazoans have demonstrated that GDIs play important roles in polarized cell growth by shuttling Rho GTPases between different membrane compartments. What’s more, vesicle trafficking plays an important role in Rho recycling in parallel to RhoGDIs in yeast and animal cells. Although plant cells contain a complex endomembrane system within which proteins are dynamically transported and plenty have been achieved in the past decade in deciphering the regulatory roles of plant vesicular trafficking, it was still obscure whether and through what trafficking routes ROPs are sorted and what components are involved in their targeting.In this study, we examined the potential roles of GDI1 and vesicle trafficking on the dynamic targeting of ROPs during Arabidopsis root hair growth by combining genetic interference and pharmacological approaches.The main results and conclusions presented are as follows:(1) GDI1 is crucial for dynamic ROP2 recycling at the apical PM of root hairs.Functional loss of GDI1, scnl-1, displayed multiple hair initiation in one trichoblast and ectopic localization of ROP2 along the entire root hair plasma membrane. To determine whether the multiple hair initiation in scnl-1 correlated with ectopic distribution of polarity clusters, we introduced the Prou BQ10:CITRINE-TUBBY-C fluorescence marker line P15Y to label the dynamic distribution of phosphatidyl inositol 4,5-bisphosphate (PIP2) in scnl-1. During root hair initiation, PIP2 was distributed at the apical PM of all bulges within one scn1-1 trichoblast compared to a single site in wild type, suggesting ectopic distribution of polarity clusters in scn1-1. To determine whether GDI1 played a positive role in delivery of ROP2 to the apical PM, we applied FRAP(Fluorescence Recovery After Photobleaching) assay to wild type or scn1-1 expressing GFP-ROP2 in root hairs. We found that the recovery of ROP2 at the apical PM was significantly reduced in scn1-1 compared to that in wild type, suggesting that GDI1 was critical for the dynamic targeting of ROP2 targeting to the apical PM in root hairs.(2) TGN/EE(trans-Golgi network/early endosomes)-mediated vesicle trafficking plays an important role on the dynamic targeting of ROP2To determine whether the dynamic localization of ROP2 at the apical PM of root hairs also requires vesicular trafficking, we blocked specific vesicular trafficking routes by combining pharmacological and genetic approaches and analyzed the dynamic localization of ROP2. We found that the dynamic targeting of ROP2 relies on brefeldin A (BFA)-sensitive, ADP-ribosylation factorl (ARF1)-mediated post-Golgi trafficking. Specifically, ROP2 recycling depends on Adaptin Protein 1 (AP1)-regulated protein sorting at the trans-Golgi network/early endosomes (TGN/EE), but not on RabA4b-mediated post-Golgi secretion or RAB5-mediated vacuolar trafficking.(3) GDI1 and vesicle trafficking synergistically regulate ROP2 recycling in root hairsBy using the expression of a dominant-negative ARF1 (ARF1-DN) or BFA to inhibit post-Golgi secretion, we found that the recovery of ROP2 at the apical PM of root hairs was not significantly reduced. However, the expression of ARF1-DN in scnl-1 or BFA treatment of scnl-1 resulted in a significant delay of ROP2 FRAP, suggesting that GDI plays an crucial role in ROP2 recycling whereas vesicle trafficking-mediated ROP2 recycling is influenced by the GDI1 pathway.Conclusion:GDI1 and vesicle trafficking synergistically mediated the polar growth of root hairs.We report here that ROP2 targeting relies on the TGN/EE-centered protein sorting in parallel to GDI1 in root hairs. GDI1 is critical for establishing and maintaining a single polarization site, while TGN/EE-mediated ROP recycling contributes to the robustness of polarization. Results presented here demonstrated an evolutionarily conserved regulatory mechanism underlying the establishment and maintenance of a single and unique polarization site during tip growth.