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Characterization of three Rop GTPase genes of alfalfa (Medicago sativa L.).

Three cDNA clones coding for Medicago sativa Rop GTPases have been isolated. The represented genes could be assigned to various linkage groups by genetic mapping. They were expressed in all investigated plant organs, although at different level. Relative gene expression patterns in response to Sinorhizobium infection of roots as well as during somatic embryogenesis indicated their differential participation in these processes. DNA sequences coding for altogether six different Medicago sp. Rop GTPases could be identified in sequence databases. Based on their homology to each other and to their Arabidopsis counterparts, a unified nomenclature is suggested for Medicago Rop GTPases.

Chromosome Mapping↗

A genome-wide analysis of Arabidopsis Rop-interactive CRIB motif-containing proteins that act as Rop GTPase targets.

The plant-specific Rop family GTPases are versatile molecular switches in many processes during plant growth, development, and responses to the environment. To understand how Rop achieves its functional versatility in signaling, we performed a genome-wide identification of putative Rop targets using a combination of the yeast two-hybrid method, bioinformatic tools, and a robust functional assay in pollen. In this study, we have identified 11 Arabidopsis genes encoding novel proteins, termed RICs (for Rop-interactive CRIB motif-containing proteins), that contain a CRIB (for Cdc42/Rac-interactive binding) motif required for their specific interaction with GTP-bound Rop1. RICs are divergent and classified into five groups that share little sequence homology outside of the conserved Rop-interactive domain. Overexpression in tobacco pollen tubes of the nine Ric genes that are expressed in Arabidopsis pollen causes distinct phenotypes, implying distinct functions for various RICs. RIC3 (group III) and RIC4 (group V) both cause depolarized growth like Rop1 and display Rop1-enhanced localization to the tip of pollen tubes, suggesting that these RICs may be two distinct targets of Rop1. In contrast, RIC10 (group I) promotes pollen tube elongation but does not affect pollen tube growth polarity and shows Rop1-independent localization to the cytoplasm, suggesting that RIC10 may participate in a Rop1-independent pathway probably controlled by a different Rop. Expression of all other RICs causes various degrees of growth inhibition in pollen tubes. Furthermore, these inhibitory RICs also exhibit distinct patterns of localization in pollen tubes. Our results suggest that various RICs have evolved to interact with Rops differentially and to perform distinct functions in pollen tubes. Reverse transcriptase-mediated polymerase chain reaction analysis showed that six of the nine RICs are expressed in various parts of Arabidopsis plants. On the basis of these observations, we propose that RICs function as Rop GTPase targets that control various Rop-dependent signaling pathways in plants.

Amino Acid Motifs↗

The Rop GTPase switch controls multiple developmental processes in Arabidopsis.

G proteins are universal molecular switches in eukaryotic signal transduction. The Arabidopsis genome sequence reveals no RAS small GTPase and only one or a few heterotrimeric G proteins, two predominant classes of signaling G proteins found in animals. In contrast, Arabidopsis possesses a unique family of 11 Rop GTPases that belong to the Rho family of small GTPases. Previous studies indicate that Rop controls actin-dependent pollen tube growth and H(2)O(2)-dependent defense responses. In this study, we tested the hypothesis that the Rop GTPase acts as a versatile molecular switch in signaling to multiple developmental processes in Arabidopsis. Immunolocalization using a general antibody against the Rop family proteins revealed a ubiquitous distribution of Rop proteins in all vegetative and reproductive tissues and cells in Arabidopsis. The cauliflower mosaic virus 35S promoter-directed expression of constitutively active GTP-bound rop2 (CA-rop2) and dominant negative GDP-bound rop2 (DN-rop2) mutant genes impacted many aspects of plant growth and development, including embryo development, seed dormancy, seedling development, lateral root initiation, morphogenesis of lateral organs in the shoot, shoot apical dominance and growth, phyllotaxis, and lateral organ orientation. The rop2 transgenic plants also displayed altered responses to the exogenous application of several hormones, such as abscisic acid-mediated seed dormancy, auxin-dependent lateral shoot initiation, and brassinolide-mediated hypocotyl elongation. CA-rop2 and DN-rop2 expression had opposite effects on most of the affected processes, supporting a direct signaling role for Rop in regulating these processes. Based on these observations and previous results, we propose that Rop2 and other members of the Rop family participate in multiple distinct signaling pathways that control plant growth, development, and responses to the environment.

Arabidopsis↗

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.

Amino Acid Sequence↗

Rop GTPase: a master switch of cell polarity development in plants.

Cell polarity is fundamentally important to plant growth and development, yet the mechanism governing its development is understood poorly. Several studies have revealed a role for Rop GTPases in pollen polar tip growth. Rop is also localized to the future site of root hair development and the tip of root hairs, and expression of constitutively active Rop mutants impacts on the morphogenesis of tip-growing root hairs as well as on non-tip-growing cells. These findings highlight the importance of Rop as a common switch in cell polarity control in plants.

Cell Polarity↗

Dynamic localization of rop GTPases to the tonoplast during vacuole development.

Vacuoles are essential pleomorphic organelles that undergo dynamic changes during cell growth and differentiation in plants. How developmental signals are linked to vacuole biogenesis and development is poorly understood. In this report, we show that a Rop GTPase is localized to developing vacuoles in pea (Pisum sativum cv Extra Early Alaska). Rop belongs to the RHO family of Ras-related small GTP-binding proteins that are key molecular switches in a wide variety of eukaryotic signal transduction pathways. Using indirect immunofluorescence and an anti-Rop antibody, we showed that Rop proteins accumulate to high levels in rapidly growing tapetal cells of pea anthers. In these cells, Rop is localized to an endomembrane system that exists as dynamic pleomorphic networks: a perinuclear fine network decorated with punctate dots, a network composed of small spheres and tubules, and interconnected chambers. Colocalization with a tonoplast annexin VCaB42 shows that these dynamic networks represent the tonoplast. Our results suggest that the dynamic Rop-containing tonoplast networks represent a unique stage of vacuole development. The specific localization of Rop to developing vacuoles supports a role for Rop in signal transduction that mediates vacuole development in plants.

Endoplasmic Reticulum↗

Brassica napus Rop GTPases and their expression in microspore cultures.

Androgenesis in plants involves a shift in development that causes cultured microspore cells to form embryos rather than continue to develop pollen. In Brassica napus microspore culture a mild heat stress is used to switch on embryo development. An early hallmark of embryogenesis in this system is a symmetrical division of the nucleus instead of the asymmetric division that occurs during pollen formation. ROP GTPases act as molecular switches in a variety of developmental processes; therefore, the current study was initiated to examine whether they might be involved in androgenesis. Five distinct Rop genes with nucleic acid similarities ranging from 82 to 93% to Arabidopsis Rop1 were isolated from B. napus cv Topas. A Southern blot hybridization with a BnRop sequence probe suggested that there are 11-15 ROP gene family members in B. napus. RT-PCR reactions with PCR primers specific to BnRop5, BnRop6, BnRop9 and BnRop10 showed that expression of the BnRop5 was restricted to pollen but the others were detected in leaf, root, stem and pollen tissue. Pollen-like cells obtained from 3-day-old cultures by flow cytometric sorting had BnRop5 transcript levels that were 2.8 times higher than in flow sorted embryogenic microspores. Conversely, the BnRop9 transcript levels were 2.5-fold higher in the embryogenic cells than in the pollen-like cells. The potential involvement of specific ROPs in early stage microspore culture responses is discussed.

Amino Acid Sequence↗

Profilin and Rop GTPases are localized at infection sites of plant cells.

We have found 5 profilin cDNAs in cultured parsley cells, representing a small gene family of about 5 members in parsley. Specific antibodies were produced using heterologously expressed parsley profilin as antigen. Western blot analysis revealed the occurrence of similar amounts of profilin in roots and green parts of parsley plants. Immunocytochemical staining of parsley cells infected with the oomycetous plant pathogen Phytophthora infestans clearly revealed that profilin accumulates at the site on the plasma membrane subtending the oomycetous appressorium, where the actin cables focus. We also observed the accumulation of Rop GTPases around this site, which might point to a potential function in signaling to the cytoskeleton.

Actins↗

Small GTPase 'Rop': molecular switch for plant defense responses.

The conserved Rho family of GTPases (Rho, Rac, and Cdc42) in fungi and mammals has emerged as a key regulator of diverse cellular activities, such as cytoskeletal rearrangements, programmed cell death, stress-induced signaling, and cell growth and differentiation. In plants, a unique class of Rho-like proteins, most closely related to mammalian Rac, has only been found and termed 'Rop' (Rho-related GTPase from plant [Li et al. (1998) Plant Physiol. 118, 407-417; Yang (2002) Plant Cell 14, S375-S388]). ROPs have been implicated in regulating various plant cellular responses including defense against pathogens. It has been shown that ROPs, like mammalian Rac, trigger hydrogen peroxide production and hence the 'oxidative burst', a crucial component associated with the cell death, most likely via activation of nicotinamide adenine dinucleotide phosphate oxidase in both monocotyledonous and dicotyledonous species. Recent studies have established that ROPs also function as a molecular switch for defense signaling pathway(s) linked with disease resistance. As discerning the defense pathway remains one of the priority research areas in the field of plant biology, this review is therefore particularly focused on recent progresses that have been made towards understanding the plant defense responses mediated by ROPs.

Amino Acid Sequence↗

RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance.

Transient soil flooding limits cellular oxygen to roots and reduces crop yield. Plant response to oxygen deprivation involves increased expression of the alcohol dehydrogenase gene (ADH) and ethanolic fermentation. Disruption of the Arabidopsis gene that encodes Rop (RHO-like small G protein of plants) guanosine triphosphatase (GTPase) activating protein 4 (ROPGAP4), a Rop deactivator, elevates ADH expression in response to oxygen deprivation but decreases tolerance to stress. Rop-dependent production of hydrogen peroxide via a diphenylene iodonium chloride-sensitive calcium-dependent reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is necessary for induction of both ADH and RopGAP4 expression. Tolerance to oxygen deprivation requires Rop activation and RopGAP4-dependent negative feedback regulation. This Rop signal transduction rheostat balances the ability to increase ethanolic fermentation with survival.

Alcohol Dehydrogenase↗

ROP GTPase regulation of pollen tube growth through the dynamics of tip-localized F-actin.

Pollen tubes expand by tip growth and extend directionally toward the ovule to deliver sperms during pollination. They provide an excellent model system for the study of cell polarity control and tip growth, because they grow into uniformly shaped cylindrical cells in culture. Mechanisms underlying tip growth are poorly understood in pollen tubes. It has been demonstrated that ROP1, a pollen-specific member of the plant-specific Rop subfamily of Rho GTPases, is a central regulator of pollen tube tip growth. Recent studies in pollen from Arabidopsis and other species have revealed a ROP-mediated signalling network that is localized to the apical PM region of pollen tubes. The results provide evidence that the localization of this signalling network establishes the site for tip growth and the localized activation of this signalling network regulates the dynamics of tip F-actin. These results have shown that the ROP1-mediated dynamics of tip F-actin is a key cellular mechanism behind tip growth in pollen tubes. Current understanding of the molecular basis for the regulation of the tip actin dynamics will be discussed.

Actins↗

Oscillatory ROP GTPase activation leads the oscillatory polarized growth of pollen tubes.

Oscillation regulates a wide variety of processes ranging from chemotaxis in Dictyostelium through segmentation in vertebrate development to circadian rhythms. Most studies on the molecular mechanisms underlying oscillation have focused on processes requiring a rhythmic change in gene expression, which usually exhibit a periodicity of >10 min. Mechanisms that control oscillation with shorter periods (<10 min), presumably independent of gene expression changes, are poorly understood. Oscillatory pollen tube tip growth provides an excellent model to investigate such mechanisms. It is well established that ROP1, a Rho-like GTPase from plants, plays an essential role in polarized tip growth in pollen tubes. In this article, we demonstrate that tip-localized ROP1 GTPase activity oscillates in the same frequency with growth oscillation, and leads growth both spatially and temporally. Tip growth requires the coordinate action of two ROP1 downstream pathways that promote the accumulation of tip-localized Ca2+ and actin microfilaments (F-actin), respectively. We show that the ROP1 activity oscillates in a similar phase with the apical F-actin but apparently ahead of tip-localized Ca2+. Furthermore, our observations support the hypothesis that the oscillation of tip-localized ROP activity and ROP-dependent tip growth in pollen tubes is modulated by the two temporally coordinated downstream pathways, an early F-actin assembly pathway and a delayed Ca2+ gradient-forming pathway. To our knowledge, our report is the first to demonstrate the oscillation of Rho GTPase signaling, which may be a common mechanism underlying the oscillation of actin-dependent processes such as polar growth, cell movement, and chemotaxis.

Actin Cytoskeleton↗

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

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.

Arabidopsis↗

Inhibition of Pollen Tube Elongation by Microinjected Anti-Rop1Ps Antibodies Suggests a Crucial Role for Rho-Type GTPases in the Control of Tip Growth.

Microinjection of anti-Rop1Ps antibodies was used to assess the function of a tip-localized Rho-type GTPase, Rop, in controlling pollen tube growth. Injected antibodies induced sustained growth arrest within 1 to 2 min after injection but did not affect cytoplasmic streaming. Coinjection with Rop rescued antibody-induced growth inhibition, indicating that injected antibodies specifically block the activity of Rop GTPases. Antibody-induced inhibition was significantly enhanced in the presence of a lower threshold of extracellular [Ca2+] or a subinhibitory dosage of caffeine. In contrast, injection of the C3 toxin, which inactivates a different Rho-type GTPase, arrested tube elongation 10 to 20 min after injection. C3-induced growth arrest was accompanied by the cessation of cytoplasmic streaming. These data suggest that Rho-type GTPases play a pivotal role in the control of pollen tube elongation. We propose that Rop may regulate a Ca2+-dependent pathway involved in vesicle docking/fusion, whereas a C3-sensitive Rho GTPase may mediate cytoplasmic streaming.

Journal Article↗

Endo/exocytosis in the pollen tube apex is differentially regulated by Ca2+ and GTPases.

Pollen tube growth relies on an extremely fast delivery of new membrane and wall material to the apical region where growth takes place. Despite the obvious meaning of this fact, the mechanisms that control this process remain very much unknown. It has previously been shown that apical growth is regulated by cytosolic free calcium ([Ca(2+)](c)) so it was decided to test how changes in [Ca(2+)](c) affect endo/exocytosis in pollen tube growth and reorientation. The endo/exocytosis was assayed in living cells using confocal imaging of FM 1-43. It was found that growing pollen tubes exhibited a higher endo/exocytosis activity in the apical region whereas in non-growing cells FM 1-43 is uniformly distributed. During pollen tube reorientation, a spatial redistribution of exocytotic activity was observed with the highest fluorescence in the side to which the cell will bend. Localized increases in [Ca(2+)](c) induced by photolysis of caged Ca(2+) increased exocytosis. In order to find if [Ca(2+)](c) changes were modulating endo/exocytosis directly or through a signalling cascade, tests were conducted to find how changes in GTP levels and GTPase activity (primary regulators of the secretory pathway) affect the apical [Ca(2+)](c) gradient and endo/exocytosis. It was found that increases in GTP levels could promote exocytosis (and growth). Interestingly, the increase in [GTP] did not significantly affect [Ca(2+)](c) distribution, thus suggesting that the apical endo/exocytosis is regulated in a concerted but differentiated manner by the Ca(2+) gradient and the activity of GTPases. Rop GTPases are likely candidates to mediate the Ca(2+)/GTP cross-talk as shown by knock-down experiments in growing pollen tubes.

Amino Acid Sequence↗

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.

Aluminum Compounds↗

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.

Arabidopsis↗

Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes.

Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein-tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase-activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.

Actins↗