RHO GTPase in plants

Abstract

Plants possess a single subfamily of Rho GTPases, ROP, which does usual things as do Rho-family GTPases in animal and fungal systems, namely participating in the spatial control of cellular processes by signaling to the cytoskeleton and vesicular trafficking. As one would expect, ROPs are modulated by conserved regulators such as DHR2-type GEFs, RhoGAPs, and Rho GDIs. What is surprising is that plants have invented new regulators such as PRONE-type GEFs (known as RopGEFs) and effectors such as RICs and ICRs/RIPs in the regulation of the cytoskeleton and vesicular trafficking. This review will discuss recent work on characterizing ROP regulators and effectors as well as addressing why and how a mixture of conserved and novel Rho signaling mechanisms is utilized to modulate fundamental cellular processes such as cytoskeletal dynamics/reorganization and vesicular trafficking.

Introduction

Rho-family small GTPases are highly conserved molecular switches that typically function as “hubs” in signaling networks that control fundamental cellular processes depending on cytoskeletal reorganization/dynamics and/or polarized vesicular trafficking in eukaryotes, such as cell polarity generation, cell morphogenesis, cell movement, etc. In spite of the overall conserved cellular function, members of the Rho family have evolved to diverge to Rho, CDC42 and Rac subfamilies in animals and filamentous fungi and CDC42 and Rho in yeasts. Interestingly, plants possess ROP (Rho-like GTPases from plants) as a sole counterpart of the Rho family. ROPs sometimes are also referred to as RACs, because they share highest identity at the amino acid sequence level.1–3 However, phylogenetic analysis suggests that all ROPs evolved from the common ancestor of Rho, CDC42 and Rac proteins (Fig. 1). For this purpose, plants Rho-family small GTPases are referred to as ROPs in this review.

Since plants lack the Ras family of small GTPases, which participates in transmission of extracellular signals along with Rho GTPases in yeast and animals, ROPs are the sole signaling small GTPases in plants. Thus, it is expected that ROPs participate in wide variety of signaling events in plants. As their counterparts in yeast and animals, ROPs participate in signaling pathways that regulate cytoskeletal organization/dynamics and vesicular trafficking, consequently impacting cell polarization, polar growth and cell morphogenesis in plants.4–18 Moreover, much of our knowledge of ROP GTPase signaling can be linked to the study of the mechanisms underlying tip growth in pollen tubes and cell morphogenesis in leaf epidermal pavement cells (Fig. 2).19–24 Another area of intensive study concerning ROP GTPases is plant defense responses.2,25–36 ROPs have also been reported to regulate gene expression involved in hormone responses and responses to abiotic stresses.37–41 Finally ROP signaling has been implicated in light-mediated guard cell movement.42 Given the functional conservation for plant Rho GTPases, it is surprising to discover plant-specific downstream effectors that plant use to modulate these fundamental cellular processes. Plants do retain conserved regulators such as DHR2-type guanine nucleotide exchange factors (GEFs), conventional Rho GTPase-activiting proteins (RhoGAPs) and guanine nucleotide dissociation inhibitors (GDIs). Yet plants also invented their own regulators such as a novel family of GEFs termed RopGEFs. The comparison and contrast of ROP GTPase effectors (Table 1) and regulators with the counterparts from fungi and animals, and the implication of these new functional partners in the capability of ROP GTPases to orchestrate fundamental cellular processes in the plant cell environment will be discussed.

ROP Effector Proteins

A wide variety of Rho GTPase effector proteins is known in fungi and animals, ranging from protein kinases (e.g., PAKs) through cytoskeleton associated proteins (formins) to scaffold proteins like WAVE/SCAR.43,44 The majority of those effector proteins from other systems are missing in plants, and in those cases where homologs of animal or fungal Rho effectors are present in plants, e.g., formins, no evidence is available to support their function as direct effectors of ROPs. Instead, several families of plant-specific effector proteins for ROPs have been identified.

CRIB motif containing proteins.

The first example of ROP effector proteins is a class of plant-specific proteins containing a CRIB motif, named as ROP INTERACTIVE CRIB MOTIF-CONTAINING PROTEINS (RICs).45 The CRIB (Cdc42/Rac-interactive binding) motif is commonly found in Cdc42/Rac effector proteins such as WASPs and p21^PAK, and is responsible for their interactions with the GTP-bound form of Cdc42 and Rac GTPases. As in CRIB-containing Cdc42 and Rac effectors, the CRIB motif allows RICs to interact with activated ROPs. Although there are several residues that show variation among RICs, residues within CRIB domain are highly conserved as shown in Figure 3. Although RICs are highly variable, residues within CRIB motifs are highly conserved as shown in Figure 3. Another family of plant ROP-interacting proteins containing the CRIB motif is the RopGAP proteins.46 Interestingly these CRIB motifs found in ROP effectors and regulators, respectively, were probably originated from the same plant ancestor, because all plant CRIB motifs are show higher similarities with each other than with CRIB motifs in proteins from other kingdoms. Outside of the CRIB motif, which is localized in the central region of RICs, those proteins do not have any similarity to known proteins. The Arabidopsis genome encodes 11 RICs that are highly divergent, implying that they could have evolved to modulate a wide range of downstream signaling pathways. Subsequent functional analysis of RICs in Arabidopsis and other plants demonstrated that different RICs indeed exhibit distinct functions.11,13,45,47 Here we focus on three RICs that have been extensively characterized and are all linked to the regulation of cytoskeletal organization and dynamics.

Regulation of Actin Dynamics by Counteractive RIC4 and RIC3: Roles in Tip Growth

The tip-growing pollen tube turns out to be an excellent system for investigating the function and regulation of ROP-dependent actin dynamics (Fig. 2). Three closely related members of Arabidopsis ROP (ROP1, ROP3, ROP5) are expressed in pollen, and are functionally redundant in the regulation of tip growth in pollen tubes.48 Using a live F-actin marker, a highly dynamic form of F-actin localized to the apex of pollen tubes was observed and was shown to be regulated by ROP1 localized to the apical plasma membrane.5 The ROP-dependent dynamics of the apical F-actin is implicated in the modulation of tip-targeted exocytosis required for pollen tube tip growth.14

Actin dynamics involves the assembly and the subsequent disassembly of F-actin, which are traditionally thought to be activated by two independent signaling pathways, respectively. The dilemma is how could a single tip-localized ROP GTPase modulate the dynamics of F-actin at the tip of pollen tubes. The investigation of RICs reveals a mechanism by which a single ROP controls the tip actin dynamics. When overexpressed (OX), two structurally unrelated RICs, RIC3 and RIC4, both induced depolarized growth in tobacco pollen tubes associated with distinct changes in the dynamic tip F-actin; RIC3 OX caused disappearance of this actin, whereas RIC4 OX led to its stabilization.13,45 Their co-overxpression recovered normal polarized tip growth and the dynamics of the tip F-actin. These observations prompted Gu and his colleagues to test the hypothesis that these two ROP effectors act together coordinately to control actin dynamics.13 Additional experimentation by Gu et al. show that whether RIC4 promotes the assembly of F-actin by acting as a ROP1 effector in pollen tube tips. It was further found that RIC4 acts as an effector of ROP2 in promoting the formation of diffuse cortical F-actin also in the lobe of jigsaw-puzzle-shaped leaf epidermal pavement cells.11 Thus RIC4 appears to have a conserved function in the promotion of actin assembly in different cell types, but the mode of action for RIC4 regulation of actin assembly remains mysterious. Interestingly RIC4-dependent cortical F-actin is different from ARP2/3-dependent F-actin, and RIC4 might regulate a new type of actin nucleation.11

Gu et al. found that overexpression of RIC3 in pollen tubes induced changes in the actin cytoskeleton similar to those induced by high levels of cytosolic Ca²⁺. It turned out that RIC3 promotes the level of cytosolic Ca²⁺ at the tip of the tubes. Moreover they found that RIC3-dependent cytosolic Ca²⁺ activates the disassembly of the apical F-actin in pollen tubes. Furthermore, they showed that RIC3 also acts as an ROP1 effector in its action to regulate cytosolic Ca²⁺ levels and subsequently actin disassembly. Therefore, both acting downstream of ROP1, the RIC3 and RIC4 pathways counteract coordinately to control actin dynamics at the tip of pollen tubes.13

Being the first example of two counteracting pathways acting downstream of a single Rho GTPase to modulate actin dynamics, this finding raises some interesting questions. First, could this be a common mechanism for the regulation of actin dynamics in different systems? Although studies directly showing similar mode of action are yet to be obtained, several observations hint its generality in other systems accompanying tip growth such as root hair tip growth in plants, neurogenetic processes in animals and fungal hyphal growth.13 In those processes, Rho GTPases as well as F-actin dynamics and calcium gradient also play an essential role. As there are examples of promotion of both entry and release of calcium into and out of animal cells by Rho GTPases,49,50 coordination of polymerization and calcium-mediated depolymerization of F-actin may be achieved by single upstream Rho GTPase as in pollen tubes. Second, what is the significance of this mechanism in the regulation of polarized tip growth? A recent study generated some insights into the mechanism by which ROP1-dependent F-actin dynamics participates in the tip growth of pollen tubes.14 By using FRAP method with an RLK-GFP as a tracer, Lee et al. found that both the RIC4-dependent actin assembly and RIC3-dependent disassembly promotes transport and docking/fusion of exocytic vesicles to the apical PM, respectively, spatiotemporally coordinating targeted exocytosis for tip growth.14 Furthermore, these two pathways are counteractive in influencing the targeting and docking/fusion of exocytic vesicles, consistent with the observation that RIC3-mediated calcium accumulation and actin disassembly lag behind RIC4-mediated actin assembly. This temporal coordination of the two counteractive pathways can also explain F-actin's and calcium's participation of positive and negative feedback regulations of the apical ROP1 activity, respectively.51

RIC1 Regulation of Microtubule Organization

RIC1 has been shown to regulate microtubule organization important for the morphogenesis of pavement cells with interdigitated lobes and indentations11,12 (Fig. 2). This process also requires two coordinated counteractive pathways: The ROP2-RIC4 pathway localizes to lobe tips and activates the accumulation of F-actin there and the ROP6-RIC1 pathway that organize cortical microtubules (MTs) at the indenting regions.11,12 RIC1 is MT associated protein that promotes parallel arrangements of MTs, as overexpression of RIC1 caused dense parallel cortical MTs and consequently inhibition of lobe formation, whereas loss of function of RIC1 resulted in randomized cortical MTs.11 RIC1's association with MTs and function in MT organization is dependent on the activation of ROP6, which directly binds and activates RIC1. In the absence of ROP6, RIC1 is dissociated from MTs.12 How ROP6 activation induces RIC1 association and how RIC1 promotes MT organization once it is associated with MTs are interesting questions yet to be addressed.

ICR/RIP Family Members Act as Scaffold Protein Linking ROPs to Exocyst

Another family of ROP effector proteins in Arabidopsis, represented by Interactor of Constitutively Active ROP 1 (ICR1)/ROP-interactive Partner 1 (RIP1), was found by two independent studies involving yeast 2 hybrid screen for proteins interacting with an activated form of ROPs.15,52 The ICR/RIP family, composed of 5 members in Arabidopsis, is conserved in the plant kingdom, but does not exist in other eukaryotes. Moreover, unlike RICs ICRs/RIPs do not contain any motifs known to interact with other Rho GTPases. However, motif invariable among ICR/RIP members near the c-terminus of those proteins was shown to be necessary for binding to active ROP15 (Fig. 4). This plant-specific ROP-interactive motif suggests that plants have invented a completely new ICR/RIP family of Rho effectors. This is particularly interesting given the evidence that ICR1/RIP1 acts as a scaffold protein linking ROPs to exocyst, an exocytic vesicle-tethering complex highly conserved in eukaryotes.

Lavy et al. found that ICR1/RIP1 binds AtSEC3A, a subunit of the exocyst complex.15,53 In yeast, CDC42 directly interacts with SEC3 to recruit the exocyst complex to the site of CDC42 activation54 (Fig. 5), and thus yeast SEC3 contains a Rho GTPase binding motif. However, in plants and animals, SEC3 homologs lack the Rho interaction domain. In mammalian cells, CDC42/RhoA failed to bind SEC3 homolog, while the CDC42/RhoA effector protein IQGAP1 was shown to interact with Sec3/Sec8.55 Therefore, it appears that plants and mammals have evolved parallel mechanisms by adopting scaffolding proteins to link Rho-family GTPases to the exocyst.

Lavy et al. also found that T-DNA insertional mutant of ICR1 (icr1) show defects in plant morphogenesis. In the mutant, pavement cells became cubical, suggesting that these cells fail to expand, which is consistent with the essential role for the exocyst in cell expansion in plants.56,57 Interestingly, ICR1 is implicated in polar transport of auxin by regulating polar localization of PIN proteins that export auxin out of cells. PIN polarization is required for the establishment of auxin gradients that regulate various plant developmental processes and pattern formation.58,59–63 Root meristem in icr1 mutant collapses, a phenotype resembling root patterning defect induced by a block in polar auxin transport.15 Furthermore, ROPs contribute to polar PIN targeting64 as well as vesicle trafficking that is essential for polar PIN targeting.65 Indeed, recent work by the same group showed that ICR1/RIP1 contributes to the regulation of polar localization of PIN auxin efflux transporters to produce sites of auxin maxima in roots and embryos.17 Polarization of PINs involves both localized inhibition of PIN endocytosis and localized recycling.66 Interestingly ICR1 is polarly localized to the site of PIN and appears to be required for endocytic PIN recycling to the same site.17 This is consistent with the notion that ICR1 is involved in tethering of endocytic recycling vesicles to the ICR1 site. PIN protein expression is induced by auxin, suggesting that ICR1/RIP1 pathway may contribute to a positive feedback loop consisting of auxin and accumulation of PIN proteins at the sites of high auxin in roots. Evidence also suggests auxin inhibits PIN endocytosis to form feedback regulation of PIN at the sites of auxin maxima in roots.67,68 Thus, it seems that there are at least two pathways regulating PIN trafficking to produce the polar distribution of PIN proteins: one that inhibits their endocytosis and the other that promotes their recycling mediated by ICR1/RIP1. Interestingly a recent study suggests that extracellular auxin activates lobe-localized ROP2, which in turn is required for PIN1 polarization to the lobes in pavement cells, suggesting that auxin activation of ROP2 and PIN1 polarization form a positive feedback loop.69 Therefore it is reasonable to speculate that auxin activation of ROP2 promotes PIN1 polarization via coordination of two downstream pathways: an unknown pathway that inhibits PIN1 endocytosis and the ICR1/RIP1 pathway that promotes its recycling (Fig. 6). Clearly future work should test this possibility and determine whether this could be a common mechanism for the polarization of PIN proteins in various cell types.

The work by Li et al. also supports the notion that ICR1/RIP1 is involved in the exocytic process. They showed that ICR1/RIP1 localizes to the cell cortex of future germination sites and to the apical cortex of growing pollen tubes where exocytic activity in an active ROP1-dependent manner.52 Furthermore, overexpression of ICR1/RIP1 cause depolarization of pollen tubes, and enhance accumulation of GFP-ROP1 to the PM. Thus, ROP1 and ICR1/RIP1 could form another positive feedback loop to maintain high ROP activity at the tip of growing pollen tubes. Since the exocyst complex is required for tip growth in pollen tubes,56 ICR1/RIP1 may also act as scaffold protein to recruit exocyst to the apical PM where ROP1 is activated.

Other ROP Effector Proteins

Recently, a family of receptor-like cytoplasmic kinases and a cysteine-rich receptor kinase are identified as ROP interactors.70,71 Also, several proteins were identified as ROP effector proteins from studies of ROP proteins from Rice, named OsRAC1. OsRAC1 is involved in defense signaling in rice through multiple pathways including the regulation of NADPH oxidase dependent ROS production and the regulation of MAP kinase signaling.2,18,34,72

Of these, respiratory burst oxidase homolog (Rboh), catalytic subunit of NADPH oxidase involved in ROS production, is particularly interesting. Rboh proteins were shown to bind OsRAC1 directly.32 In mammalian cells, NADPH oxidase exists as the multi-protein complex including Rac. Activated Rac activates the NADPH oxidase complex by translocating p67 subunit to the complex.73 However, plant lacks subunits other than homologs to the catalytic subunit protein (Rboh) and OsRac1. Rboh possess N-terminal extension containing the EF-hand motifs that is absent from animal homologs. Wong et al. tested bindings between Rboh and OsRac1, and showed that OsRac1 binds Rboh at the N-terminal region in a GTP dependent manner. Moreover, they found that increase in cytosolic Ca²⁺ inhibits interaction between Rboh and OsRac1, and the EF-hand motif may contribute to the suppression mediated by Ca²⁺.

ROP Regulators: Tuning in Spatial Signals to ROPs

A key hallmark of Rho GTPase signaling is its spatial control of cellular processes. In this capacity, Rho GTPases are typically activated in local domains of the plasma membrane, such that they are capable of regulating the polarization of the cytoskeleton and of vesicular trafficking as discussed above for ROP effectors. Given the conservation of this spatial control in all eukaryotic cells, it is not surprising that some Rho regulators such as RhoGAPs (Rho GTPase activating proteins) and RhoGDIs (Rho guanine nucleotide dissociation inhibitors) are conserved for the spatial regulation of ROPs in plants. Investigation of these regulators in plant cellular systems may reveal paradigms for the spatial control of Rho GTPases (e.g., see REN1 below). Since types of cues that determine spatial activation of ROPs are likely plant cell-specific, one might expect that unique ROP activators could have evolved for the perception and transmission of these cues. Indeed as discussed below a plant-specific class of RhoGEFs (Rho guanine nucleotide exchange factors).

In addition to ROP regulators, posttranslational lipid modification of ROP proteins that affect their membrane attachment has also been implicated in the regulation of ROP activity. ROPs are categorized to 2 groups according to the type of lipid modifications on hyper-variable C-terminal regions. Type I ROPs are thought to be prenylated primarily by geranylgeranyltransferase, while type II ROPs are subject to S-acylation.74–76 A recent exciting study suggests that activated ROPs undergo S-acetylation at G-domain, which appears to be important for their association with lipid rafts.77,78 In-depth analysis of lipid modification of ROPs can be found in recent reviews.78–80

RhoGEFs

Animal and fungal RhoGEFs fall into two types: one containing a DBL homology (DH) domain and plestrin homology (PH) domain, the other containing Dock homology region (DHR) region.81 In mammals, each type of RhoGEFs is encoded by a large gene family. However plants lack DH-type RhoGEFs, which are often activated by receptor tyrosine kinase (RTK) or G protein coupled receptor (GPCR) signaling in mammalian systems.82–85 This is consistent with the fact that RTK is missing in plants and that a single Gα in Arabidopsis only has a minimal effect on plant growth and development.86 Furthermore, only a single DHR-type RhoGEF, named SPIKE1 (SPK1) is found in Arabidopisis.87 Knocking out SPK1 in Arabidopsis caused adult lethality and defects in the morphogenesis of leaf epidermal cells.87 Some of these changes such as the loss of jigsaw puzzle piece shape in pavement cells are similar to those induced by downregulation of ROPs.11,87,88 Recent work showed SPIKE1 could bind to ROPs in a DHR2-dependent manner and act as a RhoGEF.89 SPIKE1 may have a function not observed in animals, as it seems to act in the WAVE and ARP2/3 complexes to control the actin cytoskeleton.89 SPK1 does not affect other ROP-dependent processes such as tip growth in pollen tubes and root hairs, suggesting the existence of other types of RhoGEFs in plants.

Indeed a novel and plant-specific RhoGEF family, termed RopGEF, was identified.19,90 The RopGEF family contains 14 members in Arabidopsis which share a conserved PRONE (plant-specific Rop nucleotide exchanger) domain with all RopGEFs from other plant species that functions as the GEF catalytic domain.19,90 Although they do not share amino acid sequence similarity with animal and fungal RhoGEFs, the crystal structure analysis shows that their 3-D structure resembles other RhoGEFs, and they function as a dimer like other RhoGEFs.91 In vitro experiments suggest that the N- and C-termini of RhoGEF1 contains structural domains that interact with and inhibit the central PRONE domain, a regulatory feature that appears to be common among various RhoGEFs.19

The cellular function of the RhoGEF family is largely uncharacterized. Overexpression of RopGEF1 or RopGEF12 induced depolarization of pollen tube growth, similar to that induced by ROP1 overexpression, suggesting that these RopGEFs may act as an activator of ROP1 in controlling polar growth in pollen tubes.19,22 Growth depolarization is similarly induced by the overexpression of the full-length RopGEF1 and the PRONE domain, from which the autoinhibitory domains were removed, implying the existence of cellular mechanism for releasing the autoinhibition.19 A possible mechanism for removing C-terminal autoinhibiton was also suggested for RopGEF12.22 Interestingly, a tomato RopGEF homolog was independently identified from a yeast two-hybrid screen for proteins that interact with the kinase domain of pollen-expressed receptor-like protein kinases, LePRK1 and LePRK2.22,92,93 This led to testing a physical and functional interaction between the pollen-specific Arabidopsis RopGEF12 and PRK2a, a homolog of LePRK2.22 Indeed the C-terminal region of RopGEF12 was shown to interact with the kinase domain of the Arabidopsis PRK2a. A series of overexpression experiments suggest that this interaction recruits RopGEF12 to the plasma membrane and releases the inhibition of the C-terminal region on the GEF activity of the PRONE domain.22 A subclass of RopGEFs, all of which are expressed in pollen, contains conserved phosphorylation site at the C-terminal region, and S510D mutation has an impact on the function of RopGEF12 in pollen tubes.22 Thus PRK2a may regulate RopGEF12 via PRK2a-mediated phosphorylation of the C-terminal region.

These observations suggest that plant-specific RopGEFs might have evolved to transmit plant-specific signals that are perceived by the RLK superfamily (>400 members in Arabidopsis), which is the predominant cell-surface receptor ser/thr kinases in plants, given lack of RTKs and scarcity of GPCRs (if any) in plants. Thus it is anticipated that RLKs could be a primary mechanism by which extracellular signals activate ROPs in plants. Another RLK candidate for ROP activation has been reported in maize.94 The PAN1 RLK is required for the accumulation of a membrane-associated phosphoprotein, and also the accumulation of actin patches that is essential to polarize subsidiary mother cell (SMC) for asymmetric cell division toward guard mother cell (GMC).94 A recent study suggests that exracellular auxin activates both ROP2 and ROP6, respectively localized to the complementary lobing and indenting regions (see Fig. 2).69 Auxin activation of these two ROP signaling pathways requires auxin-binding protein 1 (ABP1), which apparently acts in the cell surface and does not contain a transmembrane domain. Presumably a cell surface protein such as an RLK could act as with ABP1 as auxin coreceptor to transmit extracellular auxin signals to ROPs. ROP GTPase signaling in this system has been suggested to coordinate cell polarization and cell morphogenesis between adjacent cells.69 Having a RLK cell surface receptor to perceive a ROP activating signal from adjacent cells could be a common mechanism for cell-cell coordination for the spatial control of cellular processes in multicellular plant tissues consisting of walled cells. Therefore we predict that identification of RLKs that regulate RopGEFs will be a fertile field for future studies of ROP signaling in connection to developmental mechanisms in plants.

RhoGAP and RhoGDI are Critical for Spatial and Temporal Control of Rho GTPase

Two types of RhoGAPs, both of which contain conserved RhoGAP domain, are known in plants, and both of them have been implicated in the spatial control of ROP activity.21,95 One family of Arabidopsis RhoGAPs with five members, termed RopGAPs, was initially identified in a yeast two-hybrid system for proteins interacting with constitutive active ROP1 (G15V).46 These RopGAPs contain a conserved GAP-like domain and a N-terminal CRIB motif that enhances their affinity for the GTP-bound form of ROP and the GAP activity.46 In tobacco pollen tubes, NtRhoGAP1 overexpression inhibited pollen tube elongation and caused narrower tubes, and GFP-tagged NtRhoGAP1 was localized preferentially in the shoulder of the tube apex.21 These observations led to the proposal that RopGAPs restrict ROP signaling to the apex of pollen tubes by lateral inhibition,21 as shown for several RhoGAPs in animal systems.96–99

Another type of RhoGAPs with three members in Arabidopsis was identified from genetic screen for ren mutations that enhance pollen tube tip swelling induced by ROP1 overexpression in Arabidopsis. REN1 contains an N-terminal PH domain and a RhoGAP domain, and two coiled-coil (CC) domains at the C-terminus (Fig. 7). This novel RhoGAP doesn't contain CRIB domain and is distinct from RhoGAPs in its cellular function.95 Knocking out REN1 causes swollen pollen tubes that are reminiscent of those induced by the constitutively active form of ROP1. The ren1 phenotype together with biochemical data indicates that REN1 acts as ROP1 RhoGAP to negatively regulate tip-localized ROP1. REN1 is localized to exocytic vesicles in the pollen-tube apex and the apical PM region where ROP1 is activated. These results suggest that REN1 act as a global inhibitor to spatially restrict ROP1 activity to the apical PM.95 Analysis of sptiotemporal dynamics of ROP1 activity in tobacco pollen tube tips also supports the existence of a global inhibitor in the restriction of ROP1 activity to the pollen tube apex.99 These findings reveal a new paradigm for the spatial regulation of Rho GTPase activity by RhoGAPs, which is distinct from the lateral inhibition known for several RhoGAPs found in animal systems.96–98

It was proposed that the global mode of action for the REN RhoGAP facilitates the self-regulation of Rho GTPases such as that found in pollen tube tips.99,100 Indeed, it was shown that the apical PM localization of REN1 oscillates behind ROP1 activation and ahead of ROP1 deactivation.95 Furthermore, REN1 function is dependent upon its targeting to the apical PM through its association with the exocytic vesicles, implying the presence of a negative feedback mechanism, in which ROP1-mediated polar exocytosis is important for REN1-mediated downregulation of ROP1.95 Thus the REN1 RhoGAP action appears to couple the temporal regulation with the spatial regulation of ROP activity, generating a self-organizing system to control oscillating polar growth. Such a self-organizing design principle might also work in other highly efficient Rho GTPase-mediated cellular processes such as fungal hyphal growth, neuronal growth and cell movement.

RhoGDIs are also conserved and play an important role in the spatial regulation of ROP GTPase in plants.20,99 The Arabidopsis genome encodes three RhoGDI highly similar to mammalian RhoGDIs.101 Loss-of-function of SCN1/AtRhoGDI1 (scn1) causes a phenotype in root hair similar to that induced by ROP2 overexpression since it leads to the mislocalization of ROP2 during root hair initiation and growth.6,7,102 In wild type, ROP2::YFP localization is restricted to the PM at root hair growth sites or the apical PM of growing hairs; however in scn1 mutant, it accumulates at the cell surface in ectopic locations where numerous root hairs are initiated and remains at the cell surface in growing root hairs.102 RNA interference lines of RhoGDI2a (gdi2a-RNAi) cause the depolarization of pollen tube that is also observed in GFP-ROP1 overexpression lines. And the localization of ROPs in pollen tube is depolarized in gdi2a-RNAi mutant that is similar to mislocalization of ROP2 in scn1 mutant root hairs.99 In tabacco pollen tubes, transient expression of NtRhoGDI2 inhibits pollen tube growth while NtRac5 causes depolarization in pollen tube. But co-expression of both proteins produces normal pollen tube, which indicates that NtRhoGDI2 and NtRac5 are together to control the polar growth of pollen tubes. NtRhoGDI2 was localized to cytoplasm, and co-expression of NtRhoGDI2 and YFP:NtRac5 inhibits the accumulation of NtRac5 at PM and translocates NtRac5 to cytoplasm. A point mutation in NtRac5 (R69A) that abolished the specific binding between NtRhoGDI2 and NtRac5 caused the mislocalization of NtRac5 to the flanks of pollen tube apex and enhanced the ability of NtRac5 to depolarize pollen tube when overexpressed.20 These findings demonstrate the role of RhoGDI in control of polar localization of RhoGTPases by depleting them from PM.

Future Perspectives

Plant invention of novel Rho effector proteins to regulate fundamental cellular activities such as cytoskeletal organization and vesicular trafficking raises an intriguing question: what is the necessity for plants to reinvent wheels for old cars? It is likely that these old “cars” have some plant-specific features that would work most efficiently on the new “wheels”, e.g., RIC1 organization of branched MTs into parallel bundles (a plant-specific feature of MT organization). Understanding how these novel ROP effectors regulate these fundamental cellular activities will most likely reveal interesting features of these activities. On the other hand, the studies of conserved ROP regulators in plant cell systems such as pollen tubes have revealed new paradigms regarding the spatial control of Rho GTPases. More new paradigms could be uncovered as ROP signaling mechanisms in multicellular plant systems are being elucidated. Furthermore, future studies of ROP effectors and regulators in mosses may generate insights into the evolution of tip growth mechanisms (reviewed in ref. 103). Elucidation of mechanisms activating the plant-specific RopGEFs will generate important new insights into molecular and cellular mechanisms of plant development.

Figures and Tables

Figure 1 Phylogenetic relationships of Rho-family small GTPases. A unrooted phylogenetic tree for representative members of the Rho family of small GTPases from different kingdoms. Representative members of ROP proteins are included, and only some members of Rho, CDC42 and Rac subfamilies are shown.

Figure 2 ROP signaling pathways in pollen tube and pavement cells. (A) ROP1 is locally activated at the apex of pollen tube PM. ROP1 activates downstream effector RIC4 that can promote F-actin assembly. ROP1 also activates another effector RIC3 that promotes Actin disassembly through regulating calcium concentration. These two pathways coordinately regulate actin dynamic that is essential for exocytosis at the sub-apical region of growing pollen tubes. REN1, a RhoGAP in plant, is translocated to the sub-apical region by exocytosis that negatively regulate ROP1 activity. (B) Counteraction of RIC3 and RIC4 in pollen tubes. All tubes are expressing pLat52::mTalin that marks actin, with co-expression of indicated RICs, and incubated with indicated amount of Ca concentration. Note that actin cable are protruding at the extreme tip of tubes in RIC3 expressing tube, while meshwork of actin covers the tip of RIC4 expressing tubes. Depolarized growth and abnormal actin organization are suppressed by coexpressing RIC3 and RIC4. Increase of Ca concentration also suppresses abnormal phenotypes induced by RIC4 expression. Images are reproduced from Gu et al.13 (C) In leaf pavement cells that contain interdigitated lobes and indentations, ROP2 is locally activated at the apex of lobes by auxin through ABP1, which leads to the activation of RIC4 at lobe sites that promotes accumulation of F-Acin, which is essential for lobe outgrowth. Local active ROP2 also promotes PIN1 polar localization to apical PM of lobe. And PIN1-exported auxin in turn activates ROP2 at the cell surface to initiate a positive feedback loop which is essential for continuous outgrowth of lobe. PIN1-exported auxin at the lobe sites can diffuse across the cell wall to activate ROP6 pathway at adjacent cell. ROP6 activate RIC1 that can bind with MTs and regulate MT organization to restrict growth in this region. (D) Polar localization of ROP2, ROP6 and PIN1 in pavement cells. GFP-ROP6 signal is stronger in indenting region while GFP-ROP2 is stronger in lobe tip in adjacent lobes and indentations (arrows). Images are reproduced from Fu et al.12 PIN1 localizes to tip of lobes in WT cells (arrows). Polar localization of PIN1 is lost and cytoplsmic signal is increased in rop2 RNAi rop4-1 cells (arrowheads). Images are reproduced from Xu et al.68

Figure 3 Comparison of CRIB motifs from CRIB-containing proteins found in plants and other kingdoms. Comparison of CRIB motifs of RIC proteins and other CRIB motif containing proteins. ROPGAPs from Arabidopsis as well as CDC42/Rac effector proteins are included. Note the high conservation of RIC proteins.

Figure 4 Novel ROP-interactive motif found in the C-terminal region of ICR/RIP proteins from Arabidopsis. Comparison of C-terminal conserved region in ICR/RIP proteins from Arabidopsis. Highly conserved QWRKAA motif required for ROP bining are highlighted.

Figure 5 Rho mediated regulation of exocyst complex in different kingdoms. Models describing regulation of exocyst complex by Rho proteins in different kingdoms. Note the difference in subunit or adaptor/scaffolding protein in mediating Rho regulation.

Figure 6 A working model for the polarization of PIN1 by ROP-mediated local suppression of endocytosis and polarized recycling. A working model describing PIN1 polarization achieved by ROP2-mediated regulation of both endocytosis and recycling. ROP2 activated by auxin is speculated to suppress endocytosis of PIN1 by unknown mechanisms. Active ROP2 presumably promotes recycling of PIN1 through the promotion of the ICR1/RIP1-mediated exocyst pathway. As a consequence, PIN1 accumulates at the side where ROP2 is activated, and transport auxin out of cell there. This will further increase auxin levels at the side, and thus a positive feedback loop is formed.

Figure 7 Schematic structures for two types of RhoGAPs in Arabidopsis. The AtRopGAP family that has 6 family members contains a CRIB domain for binding with RhoGTPase and a conserved RhoGAP domain. AtRENs family contains an N-terminal PH domain and a RhoGAP domain, and two coiled-coil (CC) domains at the C-terminus.

Table 1 List of ROP effectors

Effector protein name	ROP interaction suggested in	Type of protein	Putative in vivo function for ROP-effector binding.	Key references
RICs	arabidopsis, barley	scaffold (?)	Regulation of cytoskeletal organization and dynamics. Pathogen response.	11, 13, 36, 45
ICRs/RIPs	arabidopsis	scaffold	Scaffold for exocyst complex.	15, 17, 52
RBKs/RKKs	arabidopsis, medicago	receptor-like cytoplasmic kinase	Unknown.	70, 71
NCRK	arabidopsis	cysteine-rich receptor kinase	Unknown.	70
Rboh	rice	NADPH oxidase	ROS production at defence response.	32
CCR	rice	Cinnamoyl-CoA reductase	Deposition of lignin at defence response.	18
RACK1	rice	scaffold	Scaffold for immune complex.	34