The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth

Root hairs provide a model system for the study of cell polarity. We examined the possibility that one or more members of the distinct plant subfamily of RHO monomeric GTPases, termed Rop, may function as molecular switches regulating root hair growth. Specific Rops are known to control polar growth in pollen tubes. Overexpressing Rop2 (Rop2 OX) resulted in a strong root hair phenotype, whereas overexpressing Rop7 appeared to inhibit root hair tip growth. Overexpressing Rops from other phylogenetic subgroups of Rop did not give a root hair phenotype. We confirmed that Rop2 was expressed throughout hair development. Rop2 OX and constitutively active GTP-bound rop2 (CA-rop2) led to additional and misplaced hairs on the cell surface as well as longer hairs. Furthermore, CA-rop2 depolarized root hair tip growth, whereas Rop2 OX resulted in hairs with multiple tips. Dominant negative GDP-bound Rop2 reduced the number of hair-forming sites and led to shorter and wavy hairs. Green fluorescent protein-Rop2 localized to the future site of hair formation well before swelling formation and to the tip throughout hair development. We conclude that the Arabidopsis Rop2 GTPase acts as a positive regulatory switch in the earliest visible stage in hair development, swelling formation, and in tip growth.     

NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.     

The ROP2 GTPase controls the formation of cortical fine F-actin and the early phase of directional cell expansion during Arabidopsis organogenesis

Polar cell expansion in differentiating tissues is critical for the development and morphogenesis of plant organs and is modulated by hormonal and developmental signals, yet little is known about signaling in this fundamental process in plants. In contrast to tip-growing cells, such as pollen tubes and root hairs, cells in developing tissues are thought to expand by diffuse growth. In this study, we provide evidence that these cells expand in two phases with distinct mechanisms. In the early phase, cell expansion can occur in both longitudinal and radial or lateral directions and is mediated by Rop GTPase signaling, a mechanism known to control tip growth. The expression of a dominant-negative mutant for ROP2 (DN-rop2) inhibited polar cell expansion, whereas the expression of a constitutively active mutant (CA-rop2) caused isotropic expansion in the early phase. In the late phase, expansion occurs only in the longitudinal direction and is not affected by DN-rop2 or CA-rop2 expression. The transition from the early to the late phase coincides with the reorientation of cortical microtubules from random to transverse arrangements. Thus, cell expansion in the late phase is consistent with polar diffuse growth, in which polarity probably is defined by transverse cortical microtubules. We show that the direction of cell expansion in the early phase is associated with the localization of diffuse fine cortical F-actin in leaf epidermal cells. DN-rop2 expression specifically inhibited the formation of this F-actin, but not actin cables, whereas CA-rop2 expression caused delocalized distribution of this fine F-actin throughout the cell cortex. Furthermore, green fluorescent protein-ROP2 was localized preferentially to the cortical region of the cell, where expansion apparently occurs. These observations suggest that ROP2 control of the polar expansion of cells within tissues is analogous to the Rop control of tip growth and of tip-localized F-actin in pollen tubes and root hairs and that the tip growth mechanism also may modulate polar cell expansion in differentiating tissues.     

Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx.

We have shown that Rop1At, a pollen-specific Rop GTPase that is a member of the Rho family of small GTP binding proteins, acts as a key molecular switch controlling tip growth in Arabidopsis pollen tubes. Pollen-specific expression of constitutively active rop1at mutants induced isotropic growth of pollen tubes. Overexpression of wild-type Arabidopsis Rop1At led to ectopic accumulation of Rop1At in the plasma membrane at the tip and caused depolarization of pollen tube growth, which was less severe than that induced by the constitutively active rop1at. These results indicate that both Rop1At signaling and polar localization are critical for controlling the site of tip growth. Dominant negative rop1at mutants or antisense rop1at RNA inhibited tube growth at 0.5 mM extracellular Ca(2+), but growth inhibition was reversed by higher extracellular Ca(2+). Injection of anti-Rop antibodies disrupted the tip-focused intracellular Ca(2+) gradient known to be crucial for tip growth. These studies provide strong evidence for a Rop GTPase-dependent tip growth pathway that couples the control of growth sites with the rate of tip growth through the regulation of tip-localized extracellular Ca(2+) influxes and formation of the tip-high intracellular Ca(2+) gradient in pollen tubes.     

Active ROP2 GTPase inhibits ABA- and CO2-induced stomatal closure

ROP GTPases function as molecular switches in diverse cellular processes. Previously, we showed that ROP2 GTPase is activated upon light irradiation, and thereby negatively regulates light-induced stomatal opening. Here we studied the role of ROP2 during stomatal closure. The expression of a constitutively active form of ROP2 (CA-rop2) in Arabidopsis thaliana and Vicia faba resulted in slower and reduced stomatal closure in response to abscisic acid (ABA) and CO(2) . In contrast, the expression of a dominant-negative form of ROP2 (DN-rop2) and the knockout mutation of ROP2 (rop2 KO) promoted ABA-induced stomatal closure in Arabidopsis. As early as 10 min after ABA treatment, ROP2 was inactivated and translocated to the cytoplasm of the stomatal guard cells. To elucidate the mechanism by which active ROP2 suppresses stomatal closure, we monitored endocytotic membrane trafficking, which is regulated by Rho GTPases in animal cells. We found that the endocytosis of plasma membrane (PM), as tracked by FM4-64, was lower in CA-rop2-expressing guard cells than in those of wild-type plants, which suggests that active ROP2 suppresses the endocytotic internalization of PM, a process required for stomatal closure. Together, our results suggest that ROP2 is inactivated by ABA, and that this inactivation is required for the timely stomatal closure.     

The Rop GTPase: an emerging signaling switch in plants

G proteins are ubiquitous molecular switches in eukaryotic signal transduction, but their roles in plant signal transduction had not been clearly established until recent studies of the plant-specific Rop subfamily of RHO GTPases. Rop participates in signaling to an array of physiological processes including cell polarity establishment, cell growth, morphogenesis, actin dynamics, H₂O₂ generation, hormone responses, and probably many other cellular processes in plants. Evidence suggests that plants have developed unique molecular mechanisms to control this universal molecular switch through novel GTPase-activating proteins and potentially through a predominant class of plant receptor-like serine/threonine kinases. Furthermore, the mechanism by which Rop regulates specific processes may also be distinct from that for other GTPases. These advances have raised the exciting possibility that the elucidation of Rop GTPase signaling may lead to the establishment of a new paradigm for G protein-dependent signal transduction in plants.     

The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein-ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.     

Arabidopsis RopGAPs are a novel family of rho GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for rop-specific GTPase stimulation

The plant-specific Rop subfamily of Rho GTPases, most closely related to the mammalian Cdc42 and Rac GTPases, plays an important role in the regulation of calcium-dependent pollen tube growth, H(2)O(2)-mediated cell death, and many other processes in plants. In a search for Rop interactors using the two-hybrid method, we identified a family of Rho GTPase-activating proteins (GAP) from Arabidopsis, termed RopGAPs. In addition to a GAP catalytic domain, RopGAPs contain a Cdc42/Rac-interactive binding (CRIB) motif known to allow Cdc42/Rac effector proteins to bind activated Cdc42/Rac. This novel combination of a GAP domain with a CRIB motif is widespread in higher plants and is unique to the regulation of the Rop GTPase. A critical role for CRIB in the regulation of in vitro RopGAP activity was demonstrated using point and deletion mutations. Both types of mutants have drastically reduced capacities to stimulate the intrinsic Rop GTPase activity and to bind Rop. Furthermore, RopGAPs preferentially stimulate the GTPase activity of Rop, but not Cdc42 in a CRIB-dependent manner. In vitro binding assays show that the RopGAP CRIB domain interacts with GTP- and GDP-bound forms of Rop, as well as the transitional state of Rop mimicked by aluminum fluoride. The CRIB domain also promotes the association of the GAP domain with the GDP-bound Rop, as does aluminum fluoride. These results reveal a novel CRIB-dependent mechanism for the regulation of the plant-specific family of Rho GAPs. We propose that the CRIB domain facilitates the formation of or enhanced GAP-mediated stabilization of the transitional state of the Rop GTPase.     

Arabidopsis ROP9 and ROP10 GTPases differentially regulate auxin and ABA responses

Auxin and abscisic acid (ABA) are major plant hormones that act together to modulate numerous aspects of plant growth and development, including seed germination, primary root elongation, and lateral root formation. In this study, we analyzed the loss-of-function mutants of two closely related ROP (Rho of plants) GTPases, ROP9 and ROP10, and found that these ROP GTPases differentially regulate the auxin and ABA responses. rop9 and rop10 mutations enhanced the ABA-induced suppression of seed germination, primary root growth, and lateral root formation and the expression of ABA-responsive genes, whereas rop9 but not rop10 suppressed auxin-induced root phenotypes and auxin-responsive gene expression. These results suggest that both ROP9 and ROP10 function as negative regulators of ABA signaling, and that ROP9, but not ROP10, functions as a positive regulator of auxin signaling. Previously, ROPinteractive CRIB motif-containing protein 1 (RIC1) was reported to participate in auxin and ABA responses, and to have a similar effect as ROP9 and ROP10 on gene expression, root development, and seed germination. Because RIC proteins mediate ROP GTPase signaling, our results suggest that ROP9 and ROP10 GTPases function upstream of RIC1 in auxin- and ABA-regulated root development and seed germination.     

Rop1Ps promote actin cytoskeleton dynamics and control the tip growth of lily pollen tube

Rop, the small GTPase of the Rho family in plants, is believed to exert molecular control over dynamic changes in the actin cytoskeleton that affect pollen tube elongation characteristics. In the present study, microinjection of Rop1Ps was used to investigate its effects on tip growth and evidence of interaction with the actin cytoskeleton in lily pollen tubes. Microinjected wild type WT-Rop1Ps accelerated pollen tube elongation and induced actin bundles to form in the very tip region. In contrast, microinjected dominant negative DN-rop1Ps had no apparent effect on pollen tube growth or microfilament organization, whereas microinjection of constitutively active CA-rop1Ps induced depolarized growth and abnormal pollen tubes in which long actin bundles in the shank of the tube were distorted. Injection of phalloidin, a potent F-actin stabilizer that inhibits dynamic changes in the actin cytoskeleton, prevented abnormal growth of the tubes and suppressed formation of distorted actin bundles. These results indicate that Rop1Ps exert control over important aspects of tip morphology involving dynamics of the actin cytoskeleton that affect pollen tube elongation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro

Plants have evolved distinct mechanisms to link Rho-type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor-like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP-bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho-insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta.     

A cysteine-rich receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop GTPase interactors

In plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two-hybrid screening we have found three previously uncharacterized receptor-like protein kinases to be Rop GTPase-interacting molecules: a cysteine-rich receptor kinase, named NCRK, and two receptor-like cytosolic kinases from the Arabidopsis RLCK-VIb family, named RBK1 and RBK2. Uniquely for Rho-family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP-bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro , suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co-localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single-copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK-VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2 . We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross-talk between Rop GTPases and specific receptor-like kinases through direct molecular interaction.     

Characterization of GhRac1 GTPase expressed in developing cotton (Gossypium hirsutum L.) fibers

Cytoskeleton assembly plays an important role in determining cotton fiber cell length and morphology and is developmentally regulated. As in other plant cells, it is not clear how cytoskeletal assembly in fibers is regulated. Recently, several Rac/Rop GTPases in Arabidopsis were shown to regulate isotropic and polar cell growth of root hairs and pollen tubes by controlling assembly of the cytoskeleton. GhRac1, isolated from cottonseeds, is a member of the Rac/Rop GTPase family and is abundantly expressed in rapidly growing cotton tissues. GhRac1 shows the greatest sequence similarity to the group IV subfamily of Arabidopsis Rac/Rop genes. Overexpression of GhRac1 in E. coli led to the production of a functional GTPase as shown by in vitro enzyme activity assay. In contrast to other Rac/Rop GTPases found in cotton fiber, GhRac1 is highly expressed during the elongation stage of fiber development with expression decreasing dramatically when the rate of fiber elongation declines. The association of highest GhRac1 expression during stages of maximal cotton fiber elongation suggests that GhRac1 GTPase may be a potential regulator of fiber elongation by controlling cytoskeletal assembly.     

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

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.     

Localization of a Rho GTPase Implies a Role in Tip Growth and Movement of the Generative Cell in Pollen Tubes

The Rho family GTPases function as key molecular switches, controlling a variety of actin-dependent cellular processes, such as the establishment of cell polarity, cell morphogenesis, and movement in diverse eukaryotic organisms. A novel subfamily of Rho GTPases, Rop, has been identified in plants. Protein gel blot and RNA gel blot hybridization analyses indicated that one of these plant Rho GTPases, Rop1, is expressed predominantly in the male gametophyte (pollen and pollen tubes). Cell fractionation analysis of pollen tubes showed that Rop is partitioned into soluble and particulate fractions. The particulate Rop could be solubilized with detergents but not with salts, indicating that it is tightly bound to membranes. The membrane association appears to result from membrane anchoring via a geranylgeranyl group because an in vitro isoprenylation assay demonstrated that Rop1Ps is geranylgeranylated. Subcellular localization, using indirect immunofluorescence and confocal microscopy, showed that Rop is highly concentrated in the cortical region of the tube apex and in the periphery of the generative cell. The cortical Rop protein at the apex forms a gradient with decreasing concentration from tip to base and appears to be associated with the plasma membrane. These results suggest that the apical Rop GTPase may be involved in the signaling mechanism that controls the actin-dependent tip growth of pollen tubes. Localization of the Rop GTPase to the periphery of the generative cell is analogous to that of myosin, suggesting that the Rop GTPase plays an important role in the modulation of an actomyosin motor system involved in the movement of the generative cell.     

Overexpression of a tobacco small G protein gene NtRop1 causes salt sensitivity and hydrogen peroxide production in transgenic plants

The small GTPases of Rop/Rho family is central regulators of important cellular processes in plants. Tobacco small G protein gene NtRop1 has been isolated; however, its roles in stress responses were unknown. In the present study, the genomic sequence of NtRop1 was cloned, which has seven exons and six introns, similar to the Rop gene structure from Arabidopsis. The NtRop1 gene was constitutively expressed in the different organs whereas the other six Rop genes from tobacco had differential expression patterns. The expression of the NtRop1 gene was moderately induced by methyl viologen, NaCl, and ACC treatments, but slightly inhibited by ABA treatment, with no significant induction by NAA treatment. The transgenic Arabidopsis plants overexpressing the NtRop1 showed increased salt sensitivity as can be seen from the reduced root growth and elevated relative electrolyte leakage. The hydrogen peroxide production was also promoted in the NtRop1-trangenic plants in comparison with wild type plants. These results imply that the NtRop1 may confer salt sensitivity through activation of H2O2 production during plant response to salt stress.     

Arabidopsis NAP and PIR regulate actin-based cell morphogenesis and multiple developmental processes

The actin cytoskeleton mediates cellular processes through the dynamic regulation of the time, location, and extent of actin polymerization. Actin polymerization is controlled by several types of evolutionarily conserved proteins, including those comprising the ARP2/3 complex. In animal cells ARP2/3 activity is regulated by WAVE complexes that contain WAVE/SCAR proteins, PIR121, Nap125, and other proteins. The activity of the WAVE complex is regulated by Rho-GTPase-mediated signaling that leads to ARP2/3 activation by WAVE/SCAR proteins. We describe in this report Arabidopsis (Arabidopsis thaliana) genes encoding Nap and PIR proteins. Light-grown Atnap-1 and Atpir-1 mutant plants displayed altered leaf, inflorescence, silique, and seed set phenotypes. Dark-grown Atnap-1 and Atpir-1 seedlings also exhibited longer roots, enhanced skotomorphogenesis and Glc responses, and shorter thicker hypocotyls than those of wild type, showing that AtNAP and AtPIR participate in a variety of growth and developmental processes. Mutations in AtNAP and AtPIR caused cell morphology defects in cotyledon pavement cells and trichomes seen in mutants in ARP2/3 subunits and in plants expressing constitutively active Rop2 GTPase. The patterns and levels of actin polymerization observed in Atnap-1 and Atpir-1 mutant trichome cells and epidermal pavement cell morphology is consistent with Arabidopsis NAP and PIR proteins forming a WAVE complex that activates ARP2/3 activity. The multiple growth and developmental phenotypes of Atnap and Atpir mutants reveals these proteins are also required for a wider variety of cellular functions in addition to regulating trichome cell growth.