Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth

The dynamic activity of tip-localized filamentous actin (F-actin) in pollen tubes is controlled by counteracting RIC4 and RIC3 pathways downstream of the ROP1 guanosine triphosphatase promoting actin assembly and disassembly, respectively. We show here that ROP1 activation is required for both the polar accumulation and the exocytosis of vesicles at the plasma membrane apex. The apical accumulation of exocytic vesicles oscillated in phase with, but slightly behind, apical actin assembly and was enhanced by overexpression of RIC4. However, RIC4 overexpression inhibited exocytosis, and this inhibition could be suppressed by latrunculin B treatment or RIC3 overexpression. We conclude that RIC4-dependent actin assembly is required for polar vesicle accumulation, whereas RIC3-mediated actin disassembly is required for exocytosis. Thus ROP1-dependent F-actin dynamics control tip growth through spatiotemporal coordination of vesicle targeting and exocytosis.     

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.     

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.     

Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis

Coordinating growth and communication between adjacent cells is a critical yet poorly understood aspect of tissue development and organ morphogenesis. We report a Rho GTPase signaling network underlying the jigsaw puzzle appearance of Arabidopsis leaf pavement cells, in which localized outgrowth in one cell is coordinated with localized inhibition of outgrowth of the adjacent cell to form interdigitating lobes and indentations. Locally activated ROP2, a Rho-related GTPase from plants, activates RIC4 to promote the assembly of cortical actin microfilaments required for localized outgrowth. Meanwhile, ROP2 inactivates another target RIC1, whose activity promotes well-ordered cortical microtubules. RIC1-dependent microtubule organization not only locally inhibits outgrowth but in turn suppresses ROP2 activation in the indentation zones. Thus, outgrowth-promoting ROP2 and outgrowth-inhibiting RIC1 pathways antagonize each other. We propose that the counteractivity of these two pathways demarcates outgrowing and indenting cortical domains, coordinating a process that gives rise to interdigitations between adjacent pavement cells.     

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.     

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.     

Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca²⁺ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth.     

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.     

RISAP Is a TGN-Associated RAC5 Effector Regulating Membrane Traffic during Polar Cell Growth in Tobacco

RAC/ROP GTPases coordinate actin dynamics and membrane traffic during polar plant cell expansion. In tobacco (Nicotiana tabacum), pollen tube tip growth is controlled by the RAC/ROP GTPase RAC5, which specifically accumulates at the apical plasma membrane. Here, we describe the functional characterization of RISAP, a RAC5 effector identified by yeast (Saccharomyces cerevisiae) two-hybrid screening. RISAP belongs to a family of putative myosin receptors containing a domain of unknown function 593 (DUF593) and binds via its DUF593 to the globular tail domain of a tobacco pollen tube myosin XI. It also interacts with F-actin and is associated with a subapical trans-Golgi network (TGN) compartment, whose cytoplasmic position at the pollen tube tip is maintained by the actin cytoskeleton. In this TGN compartment, apical secretion and endocytic membrane recycling pathways required for tip growth appear to converge. RISAP overexpression interferes with apical membrane traffic and blocks tip growth. RAC5 constitutively binds to the N terminus of RISAP and interacts in an activation-dependent manner with the C-terminal half of this protein. In pollen tubes, interaction between RAC5 and RISAP is detectable at the subapical TGN compartment. We present a model of RISAP regulation and function that integrates all these findings.     

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.     

Nuclear dynamics during the simultaneous and sustained tip growth of multiple root hairs arising from a single root epidermal cell

Nuclear dynamics in root hairs, which depends upon the actin cytoskeleton, appears to be an important factor in root-hair tip growth. Previous evidence suggests that there is an absolute requirement for the nucleus to be a fixed distance from the growing root-hair tip for tip growth to proceed. To test this hypothesis, nuclear dynamics were examined in root-hair cells bearing multiple root hairs. The majority of root-hair cells of transgenic plants overexpressing the ROP2 GTPase (ROP2 OX) bear multiple root hairs. Simultaneous and sustained fast tip growth occurred in multiple root hairs of ROP2 OX, with the continual presence of tip-localized cytoplasm in these growing hairs. Nuclear dynamics were imaged in ROP2 OX by co-expressing a transgene encoding a nuclear localization signal (NLS)-green fluorescent protein (GFP) fusion protein. The nucleus was in continual proximity to one of the growing root-hair tips, whilst the other tip elongated at a similar rate but in the absence of the nucleus from the shank of that root hair. To test whether this phenomenon was an artefact of ROP2 overexpression, nuclear dynamics were examined in wild-type and NLS-GFP transgenic plants. Multiple root hairs on the same cell underwent simultaneous and sustained fast tip growth, with the nucleus lying deep within the shank of only one of these hairs. The nucleus was also moved into the root-hair tip during the severe root-hair tip branching which is characteristic of ROP2 OX transgenic plants. These results suggest that fast tip growth can proceed in some multiple root hairs at extreme distances from the nucleus.     

F-actin organization and pollen tube tip growth in Arabidopsis are dependent on the gametophyte-specific Armadillo repeat protein ARO1

The signal-mediated and spatially controlled assembly and dynamics of actin are crucial for maintaining shape, motility, and tip growth of eukaryotic cells. We report that a novel Armadillo repeat protein in Arabidopsis thaliana, ARMADILLO REPEAT ONLY1 (ARO1), is of fundamental importance for polar growth and F-actin organization in tip-growing pollen tubes. ARO1 is specifically expressed in the vegetative cell of pollen as well as in the egg cell. ARO1-GFP (for green fluorescent protein) fusion proteins accumulate most notably in pollen tube tips and partially colocalize with F-actin in the shank of pollen tubes. ARO1 knockout results in a highly disorganized actin cytoskeleton, growth depolarization, and ultimately tube growth arrest. Tip-localized ARO1-GFP is spatially shifted toward the future site of tip growth, indicating a role of ARO1 in the signaling network controlling tip growth and regulating actin organization. After the pollen tube discharges its contents into the receptive synergid, ARO1-GFP colocalizes with emerging F-actin structures near the site of sperm cell fusion, suggesting additional participation in the mechanism of sperm cell tracking toward the female gametes. The variable localization of ARO1 in the cytoplasm, the nucleus, and at the plasma membrane, however, indicates a multifunctional role like that of beta-catenin/Armadillo and the p120 catenins.     

An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes

Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.     

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.     

Members of a novel class of Arabidopsis Rho guanine nucleotide exchange factors control Rho GTPase-dependent polar growth

Rho family small GTPases are signaling switches controlling many eukaryotic cellular processes. Conversion from the GDP- to GTP-bound form is catalyzed by guanine nucleotide exchange factors (GEFs). Rho GEFs in animals fall into two structurally distinct classes containing DH and DOCKER catalytic domains. Using a plant Rho GTPase (ROP1) as bait in yeast two-hybrid screens, we identified a family of Rho GEFs, named RopGEFs. The Arabidopsis thaliana RopGEF family of 14 members contains a conserved central domain, the domain of unknown function 315 (DUF315), and variable N- and C-terminal regions. In vitro GEF assays show that DUF315 but not the full-length version of RopGEF1 has high GEF activity toward ROP1. Our data suggest that the variable regions of RopGEF1 are involved in regulation of RopGEF through an autoinhibitory mechanism. RopGEF1 overexpression in pollen tubes produced growth depolarization, as does a constitutively active ROP1 mutant. The RopGEF1 overexpression phenotype was suppressed by expression of a dominant-negative mutant of ROP1, probably by trapping RopGEF1. Deletion mutant analysis suggested a requirement of RopGEF activity for the function of RopGEF1 in polar growth. Green fluorescent protein-tagged RopGEF1 was localized to the tip of pollen tubes where ROP1 is activated. These results provide strong evidence that RopGEF1 activates ROP1 in control of polar growth in pollen tubes.     

Latrunculin B has different effects on pollen germination and tube growth

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics

The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. We show that in PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)-LIM kinase (LIMK)-cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.     

Actin-depolymerizing factor mediates Rac/Rop GTPase-regulated pollen tube growth

Pollen tube elongation is a rapid tip growth process that is driven by a dynamic actin cytoskeleton. A ubiquitous family of actin binding proteins, actin-depolymerizing factors (ADFs)/cofilins, bind to actin filaments, induce severing, enhance depolymerization from their slow-growing end, and are important for maintaining actin dynamics in vivo. ADFs/cofilins are regulated by multiple mechanisms, among which Rho small GTPase-activated phosphorylation at a terminal region Ser residue plays an important role in regulating their actin binding and depolymerizing activity, affecting actin reorganization. We have shown previously that a tobacco pollen-specific ADF, NtADF1, is important for maintaining normal pollen tube actin cytoskeleton organization and growth. Here, we show that tobacco pollen grains accumulate phosphorylated and nonphosphorylated forms of ADFs, suggesting that phosphorylation could be a regulatory mechanism for their activity. In plants, Rho-related Rac/Rop GTPases have been shown to be important regulators for pollen tube growth. Overexpression of Rac/Rop GTPases converts polar growth into isotropic growth, resulting in pollen tubes with ballooned tips and a disrupted actin cytoskeleton. Using the Rac/Rop GTPase-induced defective pollen tube phenotype as a functional assay, we show that overexpression of NtADF1 suppresses the ability of NtRac1, a tobacco Rac/Rop GTPase, to convert pollen tube tip growth to isotropic growth. This finding suggests that NtADF1 acts in a common pathway with NtRac1 to regulate pollen tube growth. A mutant form of NtADF1 with a nonphosphorylatable Ala substitution at its Ser-6 position [NtADF1(S6A)] shows increased activity, whereas the mutant NtADF1(S6D), which has a phospho-mimicking Asp substitution at the same position, shows reduced ability to counteract the effect of NtRac1. These observations suggest that phosphorylation at Ser-6 of NtADF1 could be important for its integration into the NtRac1 signaling pathway. Moreover, overexpression of NtRac1 diminishes the actin binding activity of green fluorescent protein (GFP)-NtADF1 but has little effect on the association of GFP-NtADF1(S6A) with actin cables in pollen tubes. Together, these observations suggest that NtRac1-activated activity regulates the actin binding and depolymerizing activity of NtADF1, probably via phosphorylation at Ser-6. This notion is further supported by the observation that overexpressing a constitutively active NtRac1 in transformed pollen grains significantly increases the ratio of phosphorylated to nonphosphorylated ADFs. Together, the observations reported here strongly support the idea that NtRac1 modulates NtADF1 activity through phosphorylation at Ser-6 to regulate actin dynamics.     

Screening and characterization of apple Rho-like GTPase (MdROPs) genes related to S-RNase mediated self-incompatibility

ROP/Rac GTPase is a conserved class of proteins which play diverse signalling roles in plants. They regulate many fundamental cellular processes such as F-actin dynamics, cell polarity and polar growth. Using apple genomic database analyses, nine ROP family members were cloned for the first time in a fruit tree (apple). Phylogenetic analyses indicated that the nine MdROPs were distributed into two groups, as previously described in the literature for model plants. Expression analyses show all MdROPs were highly expressed in pollen, in particular MdROP1, 3, 4 and 8. Yeast two hybrid and bimolecular fluorescence complementation analyses indicated MdROP8 interacts with S-RNase, a pistil determinate factor in gametophyte self-incompatibility. The pollen tube microtubule is shown to depolymerize in response to S-RNase treatment, during which the expression of MdROP8 rapidly decreased. These results indicate MdROP8 is related to S-RNase mediated self-incompatibility, and gives some useful evidence in modeling the relationship between cytoskeleton depolymerization and pollen tube growth inhibition during the apple SI reaction.