Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth

Abstract

Plants contain a novel unique subfamily of Rho GTPases, vital components of cellular signalling networks. Here we report a general role for some members of this family in polarized plant growth processes. We show that Arabidopsis AtRop4 and AtRop6 encode functional GTPases with similar intrinsic GTP hydrolysis rates. We localized AtRop proteins in root meristem cells to the cross-wall and cell plate membranes. Polar localization of AtRops in trichoblasts specifies the growth sites for emerging root hairs. These sites were visible before budding and elongation of the Arabidopsis root hair when AtRops accumulated at their tips. Expression of constitutively active AtRop4 and AtRop6 mutant proteins in root hairs of transgenic Arabidopsis plants abolished polarized growth and delocalized the tip-focused Ca2+ gradient. Polar localization of AtRops was inhibited by brefeldin A, but not by other drugs such as latrunculin B, cytochalasin D or caffeine. Our results demonstrate a general function of AtRop GTPases in tip growth and in polar diffuse growth.

Fig. 1. Alignment of AtRop4 and AtRop6 sequences. Added mutations G15V, Q64L (both constitutively active) and T17N (dominant-negative) are marked by arrows.

Fig. 2. Off rates (A) and intrinsic hydrolysis rates (B) for AtRab11c (diamond), AtRop6 (triangle), AtRop4 (circle) and a Rac-like GTPase (square) in high magnesium. Proteins were preloaded with either [α-³²P]GTP (A) or [γ-³²P]GTP (B), and then an excess of Mg²⁺ and cold GTP were added. Reaction aliquots were removed and the radioactivity bound to protein was determined by a nitrocellulose filter binding assay. The remaining GTP is expressed as log of the percentage of bound label at time zero.

Fig. 3. Localization of GFP–AtRop4 in transgenic tobacco BY-2 cells. GFP–AtRop4 (A–C) and GFP control (D). (B) Dividing GFP–AtRop4 cell with labelled cell plate membranes. Bar, 25 µm.

Fig. 4. Western analysis of GFP-tagged AtRop4 expressed in a transgenic BY-2 line. (A) Immunoprecipitation with anti-GFP monoclonal antibodies. Western analysis with anti-GFP polyclonals (lane 1) or anti-AtRop4 polyclonal antibody (lane 2). Molecular weight markers as indicated. (B) Immunoprecipitation with anti-AtRop4 antibody. Western with secondary reagent only (lane 1) or anti-AtRop4 polyclonal antibody (lane 2). A band corresponding to IgG heavy chain (50 kDa) is visible in both lanes. Molecular weight markers as indicated. (C) Immunoprecipitation of Rop proteins from whole Arabidopsis seedling extract with preimmune serum (lane 1) or anti-AtRop4 polyclonal antibody (lane 2). Western with anti-AtRop4 polyclonal antibody. Background Ig bands detected by secondary reagent are present in both lanes. The position of the Rop band is marked by an arrow.

Fig. 5. Immunolocalization of Rop proteins in Arabidopsis root tips using confocal microscopy. Epidermal meristem (A), close-up of epidermal meristem with newly completed cell plates (B), longitudinal section with central cylinder (C) and lateral root primordium (D). Images (A–C) are oriented with the root tip down. Bar, 25 µm.

Fig. 6. Immunolocalization of Rop proteins in the root hair-forming zone of Arabidopsis roots using confocal microscopy. (A–F) Sequence of root hair formation showing early establishment of a Rop-positive bud site (A and B), bulging (C), tip-growth (D and E) and Rop-negative finished root hair (F). Images are oriented with the root tip up. Bar, 20 µm. (G–J) Rop localization in trichoblast cell files of inhibitor-treated seedlings. Control (G), cytochalasin D (H), caffeine (I) and BFA (J). Images are oriented with the root tip left. Bar, 25 µm.

Fig. 7. Constitutively active GFP–AtRop causes root hair and epidermal cell swelling in transgenic Arabidopsis. (A and B) Root tip (A) and hypocotyl (B) of uninduced GFP–AtRop6 Q64L line. (C and D) Root tip of induced GFP–AtRop6 Q64L line (D, fluorescence image). (E and F) Hypocotyl of induced GFP–AtRop4 G15V line (F, fluorescence image). (G and H) Swollen root hair cell of induced GFP–Rop6 Q64L line (H, fluorescence image).

Fig. 8. Immunolocalization of the actin and microtubule cytoskeletons in swollen root hairs. (A) Actin, mature wild-type root hair, projection of 12 confocal planes (depth, 10 µm). (B) Actin, GFP–AtRop6 Q64L finished swelling, projection of 27 planes (depth, 50 µm). (C) Tubulin, mature wild-type root hair, projection of eight planes (depth, 8 µm). (D and E) Tubulin, GFP–AtRop6 Q64L finished swellings, projections of nine planes each (depth, 11 µm). Bar, 25 µm.

Fig. 9. Swelling root hairs contain multiple Ca²⁺ gradients. (A–F) Sequence of root hair formation in uninduced GFP–AtRop6 Q64L line. Early trichoblast (A) and early bulge formation (B) without high Ca²⁺ levels. (C) Start of tip growth with Ca²⁺ gradient in bulge. (D–F) Tip growth with tip-focused Ca²⁺ gradient. (G) Cortical cell (induced) with low Ca²⁺. (H–L) Sequence of root hair swelling in induced GFP–AtRop6 Q64L line. (H and I) Early trichoblasts with low Ca²⁺ levels. (J) Start of tip growth with slightly mislocalized Ca²⁺ gradient. (K and L) Isotropic growth with multiple Ca²⁺ foci. Ca²⁺ concentrations have been pseudocolour coded according to the inset scale. Bar, 25 µm.