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 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.     

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.     

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.     

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.     

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.     

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.     

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 PRK6 interacts specifically with AtRopGEF8/12 and induces depolarized growth of pollen tubes when overexpressed

The pollen receptor kinases (PRK) are critical regulators of pollen tube growth. The Arabidopsis genome encodes eight PRK genes, of which six are highly expressed in pollen tubes. The potential functions of AtPRK1 through AtPRK5, but not of AtPRK6,in pollen growth were analyzed in tobacco. Herein, AtPRK6 was cloned, and its function was identified. AtPRK6 was expressed specifically in pollen tubes. A yeast two-hybrid screen of AtPRK6 against 14 Arabidopsis Rop guanine nucleotide exchange factors (RopGEFs) showed that AtPRK6 interacted with AtRopGEF8 and AtRopGEF12. These interactions were confirmed in Arabidopsis mesophyll protoplasts. The interactions between AtPRK6 and AtRopGEF8/12 were mediated by the C-termini of AtRopGEF8/12 and by the juxtamembrane and kinase domain of AtPRK6, but were not dependent on the kinase activity. In addition, transient overexpression of AtPRK6::GFP in Arabidopsis protoplasts revealed that AtPRK6 was localized to the plasma membrane. Tobacco pollen tubes overexpressing AtPRK6 exhibited shorter tubes with enlarged tips. This depolarized tube growth required the kinase domain of AtPRK6 and was not dependent on kinase activity. Taken together, the results show that AtPRK6,through its juxtamembrane and kinase domains (KD), interacts with AtRopGEF8/12 and plays crucial roles in polarized growth of pollen tubes.     

Regulation of pollen tube polarity: Feedback loops rule

Targeted delivery of immotile sperm through growing pollen tubes is a crucial step in achieving sexual reproduction in angiosperms. Unlike diffuse-growing cells, the growth of a pollen tube is restricted to the very apical region where targeted exocytosis and regulated endocytosis occur. The plant-specific Rho GTPases, Rops, are central organizers for pollen tube polarity. Through effector binding, Rops regulate the tip-focused Ca²⁺ gradient and the actin cytoskeleton during pollen tube growth. Therefore, understanding the spatiotemporal regulation of Rop activity would reveal how establishment and maintenance of tube polarity as well as re-orientation of the growth axis are accomplished. Recent findings indicated that two feedback loops may be fundamental in maintaining a fine-tuned and dynamic Rop activity. The concerted activities of RopGAP and RopGDI prevent lateral diffusion of activated Rop, restricting Rop activity to the apical plasma membrane. Conversely, pollen receptor kinases (PRKs) and RopGEFs positively feedback regulate Rop activity through protein binding and membrane recruitment. Feedback loops would also be essential for pollen tube re-orientation. Shallow extracellular cues amplified by concerted activities of feedback loops would lead to asymmetric activation of Rop and result in tube re-orientation.Key words: Rho GTPases, Rop, GEF, GAP, GDI, receptor kinase, feedback loop     

A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes

Tip growth in neuronal cells, plant cells, and fungal hyphae is known to require tip-localized Rho GTPase, calcium, and filamentous actin (F-actin), but how they interact with each other is unclear. The pollen tube is an exciting model to study spatiotemporal regulation of tip growth and F-actin dynamics. An Arabidopsis thaliana Rho family GTPase, ROP1, controls pollen tube growth by regulating apical F-actin dynamics. This paper shows that ROP1 activates two counteracting pathways involving the direct targets of tip-localized ROP1: RIC3 and RIC4. RIC4 promotes F-actin assembly, whereas RIC3 activates Ca(2+) signaling that leads to F-actin disassembly. Overproduction or depletion of either RIC4 or RIC3 causes tip growth defects that are rescued by overproduction or depletion of RIC3 or RIC4, respectively. Thus, ROP1 controls actin dynamics and tip growth through a check and balance between the two pathways. The dual and antagonistic roles of this GTPase may provide a unifying mechanism by which Rho modulates various processes dependent on actin dynamics in eukaryotic cells.     

Regulation of pollen tube growth by Rac-like GTPases

Plant Rac-like GTPases have been classified phylogenetically into two major groups-class I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen- and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP-AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction.     

Tobacco RhoGTPase ACTIVATING PROTEIN1 spatially restricts signaling of RAC/Rop to the apex of pollen tubes

Regulation by Rho-type small GTPases, such as RAC5, is important for the maintenance of polarity in tobacco (Nicotiana tabacum) pollen tubes. We previously showed that RhoGDI2 is necessary for RAC5 localization. Here, we describe the GTPase activating protein RhoGAP1 that controls the area of RAC5 activity. RhoGAP1 N-terminal and CRIB (for Cdc42/Rac-interactive binding) domains are both necessary for targeting yellow fluorescent protein-RhoGAP1 fusions to the plasma membrane close to, but not in, pollen tube apices. We propose that this localization restricts apical Rho-type GTPase activity from spreading toward the flanks, which ensures the maintenance of RAC signaling at the apex. The CRIB domain is not required but enhances in vitro RhoGAP1 activity toward the pollen tube-specific-RAC5. A mutation reducing GAP activity of RhoGAP1 leads to ballooning pollen tubes resembling those overexpressing RAC5. To ascertain the specific targeting mechanism of RhoGAP1, we isolated a 14-3-3 protein interacting with RhoGAP1. When overexpressed with RhoGAP1, it counteracts the growth-retarding effect of RhoGAP1 overexpression and attenuates RhoGAP1 membrane localization but, overexpressed alone, induces only small architectural changes. We propose that inactivation of RAC5 by the subapically localized RhoGAP1, together with dynamic relocalization of inactivated RAC5 from flanks to tip by RhoGDI2, leads to spatial restriction of RAC5 to pollen tube apices, thereby sustaining polar growth.     

Pectins in the cell wall of Arabidopsis thaliana pollen tube and pistil

Plant sexual reproduction involves the growth of tip-polarized pollen tubes through the female tissues in order to deliver the sperm nuclei to the egg cells. Despite the importance of this crucial step, little is known about the molecular mechanisms involved in this spatial and temporal control of the tube growth. In order to study this process and to characterize the structural composition of the extracellular matrix of the male gametophyte, immunocytochemical and biochemical analyses of Arabidopsis pollen tube wall have been carried out. Results showed a well-defined localization of cell wall epitopes with highly esterified homogalacturonan and arabinogalactan-protein mainly in the tip region, weakly methylesterified homogalacturonan back from the tip and xyloglucan and (1→5)-α-L-arabinan all along the tube. Here, we present complementary data regarding (1) the ultrastructure of the pollen tube cell wall and (2) the immunolocalization of homogalacturonan and arabinan epitopes in 16-h-old pollen tubes and in the stigma and the transmitting tract of the female organ. Discussion regarding the pattern of the distribution of the cell wall epitopes and the possible mechanisms of cell adhesion between the pollen tubes and the female tissues is provided.Key words: arabinan, cell adhesion, cell wall, homogalacturonan, pistil, pollen tube growth, transmitting tractFertilization of flowering plants requires the delivery of the two sperm cells, carried by the fast growing tip-polarized pollen tube, to the egg cell. At every stage of the pollen tube development within the stigma, style and ovary, pollen tubes are guided to the ovules via multiple signals that need to pass through the cell wall of the pollen tube to reach their targets.^1–6The analysis of Arabidopsis pollen tube cell wall has recently been reported.⁷ Results showed a well-defined localization of cell wall epitopes with highly methylesterified homogalacturonan (HG) and arabinogalactan-protein (AGP) mainly in the tip region, weakly methylesterified HG back from the tip and xyloglucan and arabinan all along the tube. In addition, according to the one letter nomenclature of xyloglucan,⁸ the main motif of Arabidopsis pollen tube xyloglucan was XXFG harboring one O-acetyl group. In order to bring new information regarding the possible interaction between the pollen tubes and the female tissues, the ultrastructural organization of the pollen tube cell wall, the cytological staining and immunolocalization of the cell wall epitopes of the pistil and especially the transmitting tract (TT), a specialized tissue where pollen tubes grow, were carried out.     

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.     

Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments

The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca²⁺, in vitro. Analysis of a mutant that bears a point mutation at the Ca²⁺ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca²⁺ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.     

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.     

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.     

Overexpression of the Tomato Pollen Receptor Kinase LePRK1 Rewires Pollen Tube Growth to a Blebbing Mode

The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane–localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP), a Rop guanine nucleotide exchange factor. Here, we show that pollen tubes overexpressing LePRK1 or a truncated LePRK1 lacking its extracellular domain (LePRK1ΔECD) have enlarged tips but also extend their leading edges by producing “blebs.” Coexpression of LePRK1 and tomato PLIM2a, an actin bundling protein that interacts with KPP in a Ca²⁺-responsive manner, suppressed these LePRK1 overexpression phenotypes, whereas pollen tubes coexpressing KPP, LePRK1, and PLIM2a resumed the blebbing growth mode. We conclude that overexpression of LePRK1 or LePRK1ΔECD rewires pollen tube growth to a blebbing mode, through KPP- and PLIM2a-mediated bundling of actin filaments from tip plasma membranes. Arabidopsis thaliana pollen tubes expressing LePRK1ΔECD also grew by blebbing. Our results exposed a hidden capability of the pollen tube cell: upon overexpression of a single membrane-localized molecule, LePRK1 or LePRK1ΔECD, it can switch to an alternative mechanism for extension of the leading edge that is analogous to the blebbing growth mode reported for Dictyostelium and for Drosophila melanogaster stem cells.