title: In vivo Rac/Rop localization as well as interaction with RhoGAP and RhoGDI in tobacco pollen tubes: analysis by low-level expression of fluorescent fusion proteins and bimolecular fluorescence complementation.
abstract: Polarized Rac/Rop GTPase signaling plays a key role in polar cell growth, which is essential for plant morphogenesis. The molecular and cellular mechanisms responsible for the polarization of Rac/Rop signaling during polar cell growth are only partially understood. Mutant variants of Rac/Rop GTPases lacking specific functions are important tools to investigate these mechanisms, and have been employed to develop a model suggesting that RhoGAP (GTPase activating protein) and RhoGDI (Guanine Nucleotide Dissociation Inhibitor) mediated recycling of Rac/Rop GTPases maintains apical polarization of Rac/Rop activity in pollen tubes, which elongate by 'tip growth' (an extreme form of polar cell growth). Despite the importance of these mutant variants for Rac/Rop functional characterization, their distinct intracellular distributions have not been thoroughly comparatively and quantitatively analyzed. Furthermore, support for the proposed RhoGAP and RhoGDI functions in apical polarization of Rac/Rop activity based on the analysis of in vivo interactions between these proteins and Rac/Rop GTPases has been missing. Here, extensive fluorescent protein tagging and bimolecular fluorescence complementation (BiFC) analyses are described of the intracellular distributions of wild type and mutant variants of the tobacco pollen tube Rac/Rop GTPase Nt-Rac5, as well as of interactions of these Nt-Rac5 variants with RhoGAP and RhoGDI proteins, in normally growing transiently transformed pollen tubes. Presented results substantially enhance our understanding of apical dynamics of pollen tube Rac/Rop signaling proteins, confirm previously proposed RhoGAP and RhoGDI functions in Rac/Rop polarization and provide important technical insights facilitating future in vivo protein localization and BiFC experiments in pollen tubes.
authors.affiliation: Department of Plant Biology, Swedish University of Agricultural Sciences, Box 7080, SE-750 07, Uppsala, Sweden.
authors.forename: Jia
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Plant Biology, Swedish University of Agricultural Sciences, Box 7080, SE-750 07, Uppsala, Sweden.
authors.forename: D Magnus
authors.initials: DM
authors.lastname: Eklund
authors.affiliation: Cell Biology, Department of Biology, University of Erlangen-Nuremberg, 91058, Erlangen, Germany.
authors.forename: Adriana
authors.initials: A
authors.lastname: Montes-Rodriguez
authors.affiliation: Cell Biology, Department of Biology, University of Erlangen-Nuremberg, 91058, Erlangen, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Kost
mesh_terms: D020690:GTPase-Activating Proteins / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D053205:Pollen Tube / Q000378:metabolism*; D011485:Protein Binding; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; D010940:Plant Proteins; C085379:rho GTPase-activating protein; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020744:rac GTP-Binding Proteins
keywords: Nicotiana tabacum; Rac/Rop GTPases; RhoGAP; RhoGDI; bimolecular fluorescence complementation; pollen tube; tip growth