Striking differences exist in the conservation of several members of the small GTPase family in plants, indicating that some of the basic signalling pathways are fundamentally differently organized. This may be particularly relevant for the pathways controlling cellular polarity. The absence of homologues of Cdc42 in plants and the relative abundance of a Rho-related plant-specific family, named Rop, raises questions as to their precise functions. In this paper we show for the first time that two members of this family, AtRop4 and AtRop6, are involved in cell polarity control in Arabidopsis roots.
Cellular localization of Rop GTPases
In root meristem, endogenous Arabidopsis Rop GTPases localized predominantly to the plasma membranes, as was also true for GFP-tagged AtRop4 in BY-2 cells. N-terminal GFP tagging preserves the function and accurately reflects the intracellular localization of Rho GTPases (e.g. Larochelle et al., 1997; Kost et al., 1999; Bischoff et al., 2000; Michaelson et al., 2001), although overexpression of GFP–Rop may result in apolar localization in growing pollen tubes (Li et al., 1999). We also noticed that tip localization in growing root hairs detected with immunolocalization was much more pronounced than with GFP-tagged Rop, probably as a result of overexpression of the GFP fusion protein using the GVG system. The apolar plasma membrane localization of overexpressed Rop GTPases might be caused by insufficient levels of RhoGDI, needed for recycling of Rop to defined plasma membrane locations. In dividing tobacco BY-2 cells, GFP–AtRop4 strongly labelled the cell plate membrane. This could also be observed in cells induced for shorter periods, containing lower levels of GFP fusion protein just visible by epifluorescence microscopy, suggesting that cell plate localization was not a result of overexpression (data not shown). The cell plate is a site of actin assembly (Endlé et al., 1998), which might suggest a possible regulatory role for Rop GTPases in actin dynamics at this location. However, expression of wild-type or constitutively active GFP–AtRop4 in tobacco BY-2 cells did not visibly affect cell plate formation (data not shown). Our localization data raise the question of whether AtRop proteins would function similarly in cell plate formation and in tip growth, acting on the same effectors.
Involvement in root hair development
Genetic analysis indicates the existence of discrete steps in root hair development in Arabidopsis, namely bud site selection, budding and tip growth (as reviewed by Hülskamp et al., 1998; Schiefelbein, 2000). Pollen tube and root hair tip growth are highly similar with respect to the presence of a vesicle-rich apical region, a reverse-fountain type of cytoplasmic streaming and a tip-high Ca2+ gradient (Pierson et al., 1996; Wymer et al., 1997; de Ruijter et al., 1998; Yang, 1998). In elongating pollen tubes Rop GTPase is associated with the apical plasma membrane and has an important regulatory role in polar pollen tube growth (Lin et al., 1996; Lin and Yang, 1997; Kost et al., 1999; Li et al., 1999). The apical localization of Rop in tip-growing root hairs indicated a more general role for Rop GTPases in plant tip growth. This was verified here by the expression of constitutively active AtRop4 and AtRop6 in growing root hairs leading to loss of polarity. Our data correlate well with previous observations when constitutively active pollen Rop genes AtRac2 and AtRop1 were expressed in pollen tubes (Kost et al., 1999; Li et al., 1999), thus demonstrating a close conservation of Rop function in tip growth in sporophyte and gametophyte alike.
Pollen Rop proteins have been implicated in mediating a Ca2+-dependent pathway leading to exocytosis (Lin and Yang, 1997; Li et al., 1999). Whether pollen Rop proteins directly regulate Ca2+ influx or whether the effect is mediated by reorganization of cortical actin at the tube apex remains an important open question (Li et al., 1999; Zheng and Yang, 2000). The recently reported association of pollen Rop with PtdIns kinase activity, and the localization of PtdIns (4,5)P2 at the apical plasma membrane of pollen tubes, have provided evidence for an essential function of apical PtdIns (4,5)P2 in pollen tube elongation (Kost et al., 1999). In addition to its effect on actin organization, PtdIns (4,5)P2 was also suggested to be important as a substrate in inositol trisphosphate production and, consequently, in the generation of the tip-high Ca2+ gradient (Kost et al., 1999; Zheng and Yang, 2000). Our observation that polar localization of Arabidopsis Rop proteins precedes visible bulging on the trichoblast indicates a role for Rop proteins in root hair initiation as well as in tip growth. The early bulging of the trichoblast, unlike tip growth, is not accompanied by a tip-high Ca2+ gradient and is insensitive to Ca2+ channel blocker verapamil, suggesting that Ca2+ does not trigger the initiation of root hairs (Wymer et al., 1997). This indicates that polar localization of Rop proteins is independent of a localized Ca2+ influx, and might suggest that Rop proteins are not sufficient to create the tip-high Ca2+ gradient observed during the later stage of tip growth.
Root hair formation requires a local reorganization of the actin cytoskeleton. The axial actin bundles in growing root hairs are perpendicular to their orientation in the trichoblast (Baluška et al., 2000b). In the subapical region of growing root hairs these thick bundles branch off into ever thinner bundles. The apical vesicle-rich region is enriched with dense F-actin meshwork (Baluška et al., 2000b). This dynamic configuration of the actin cytoskeleton is thought to be required for delivery of secretory vesicles to the growth site. Bulging of the trichoblast, unlike tip growth, is insensitive to actin drugs (Miller et al., 1999; Ovecka et al., 2000). However, untreated bulges contain stabilizing actin filaments and F-actin-devoid bulges are mechanically unstable (Baluška et al., 2000b). Although it is not clear whether Rop GTPase regulates actin during trichoblast bulging, it might regulate the actin cytoskeleton during and after the transition from bulging to tip growth, which is F-actin dependent and coincides with a severing and rearrangement of actin filaments (Braun et al., 1999; Miller et al., 1999; Baluška et al., 2000b). The localization of Rop GTPase at the bud site was reminiscent of the polar localization of Cdc42 or Rho1p in yeast bud formation (Ziman et al., 1993; Yamochi et al., 1994).
Rop proteins in root meristem were present in transverse plasma membranes. Since the corresponding cross-walls are non-growing (Baluška et al., 2000a), this indicated that Rop GTPases are not exclusively associated with sites of high secretion. The cross-wall domains in root meristem are enriched in myosin VIII and actin, and act as putative F-actin organizing centres during mitosis and elongation (Reichelt et al., 1999; Baluška et al., 2000a). The corresponding localization of putative F-actin organizing centres and Rop GTPases in root meristem further suggested a regulatory role for Rop in actin dynamics.
Actin-disrupting drugs did not interfere with Rop localization to the root hair bud site. In contrast, BFA inhibited the early localization of Rop at the bud site, implying an essential role for Arf GTPase in the establishment of polarity in plant cells. BFA inhibits the enzyme activity of Arabidopsis GNOM Arf GEF, and causes internalization of the auxin efflux carrier PIN1 from its usual polar localization on the plasma membrane (Gälweiler et al., 1998; Müller et al., 1998; Steinmann et al., 1999). However, inhibition of polar auxin transport by NPA does not affect root hair initiation (Masucci and Schiefelbein, 1994), indicating that BFA does not inhibit polar localization of Rop by blocking polar auxin transport. An attractive possibility is that root hair initiation begins with an Arf-dependent, actin-independent process, such as secretion at an internal cue, and is then followed by polar localization of Rop.
The isotropic growth phenotype of root hairs was characterized by the presence of multiple Ca2+ foci that marked the sites where growth was occurring. In this respect, the swellings resembled root hairs treated with tubulin drugs (Bibikova et al., 1999). Since tubulin and actin drugs, exocytosis inhibitors and phytohormones may all cause root hair branching and swelling (Ovecka et al., 2000), this indicates that tip growth can be inhibited in various ways resulting in isotropic growth. In growing root hairs, new membrane is continuously added at the tip by secretory vesicle fusion, and the polar localization of Rop GTPase probably relies on inactivation and recycling from the flank to the tip. Overexpression of constitutively active Rop GTPase caused isotropic growth, indicating that inactivation or retrieval of Rop GTPase from the root hair flanks is required to restrict secretory growth at the tip. Since tip growth relies on the integrity of fine actin filament bundles and a dense F-actin meshwork at the root hair tips (Baluška et al., 2000b; Esseling et al., 2000), it is possible that the phenotype results primarily from an effect of constitutively active Rop GTPase on actin dynamics. The large increase in cell volume and surface compared with wild-type root hairs might result from persistent oversecretion of expansins (Baluška et al., 2000b), or else, be driven primarily by excessive vacuolar expansion. Cell elongation in the hypocotyl is an example of polar diffuse growth in plants and is dependent on the integrity of both the microtubule and actin cytoskeletons (Kropf et al., 1998; Baluška et al., 2000b). Swelling in elongating epidermal cells of the hypocotyl might thus result from a disorientation of the cytoskeleton in cells containing constitutively active Rop GTPase, but the precise steps of this process remain to be determined. The root hair and hypocotyl phenotypes indicated a central role for Rop proteins in both ways of polar growth.
In Arabidopsis root meristem, Rop GTPase was predominantly localized to transverse plasma membranes, and in trichoblasts was polarly localized during root hair formation, implying a role for Rop proteins in root hair budding as well as in tip growth. The inhibition of polar localization of Arabidopsis Rop proteins by BFA indicated an early role for Arf GTPase in root hair formation and also implied that AtRop function is closely connected to Arf-dependent vesicle trafficking. The cell swelling in the hypocotyl of transgenic seedlings expressing constitutively active AtRop4 implied a role for this particular Rop protein in elongation growth. The isotropic growth phenotypes of constitutively active AtRop4 and AtRop6 in root hairs demonstrated a similar role for different Rop proteins in both pollen and root hair tip growth. Moreover, our results show a general function of AtRop GTPases in polarized cell growth in plants.