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.     

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.     

Actin filament organization and polarity in pollen tubes revealed by myosin II subfragment 1 decoration

The actin cytoskeleton plays a crucial role in pollen tube growth. In elongating pollen tubes the organization and arrangement of actin filaments (AFs) differs between the shank and apical region. However, the orientation of AFs in pollen tubes has not yet been successfully demonstrated. In the present work we have used myosin II subfragment 1 (S1) decoration to determine the polarity of AFs in pollen tubes. Electron microscopy studies revealed that in the shank of the tube bundles of AFs exhibit uniform polarity with those close to the cell cortex having their barbed ends oriented towards the tip of the pollen tube while those in the cell center have their barbed ends oriented toward the base of the tube. At the subapex, some AFs are organized in closely packed and longitudinally oriented bundles and some form curved bundles adjacent to the cell membrane. In contrast, few AFs are dispersed with random orientation in the extreme apex of the pollen tube. Our results confirm that the direction of cytoplasmic streaming within pollen tubes is determined by the polarity of AFs in the bundles.     

Involvement of the ubiquitin/proteasome pathway in the organisation and polarised growth of kiwifruit pollen tubes

We recently reported the involvement of the ubiquitin pathway in microgametophyte development, and a direct role for the 26S proteasome in regulating pollen tube emergence in kiwifruit. Here we show that the ubiquitin/proteasome proteolytic pathway is involved not only in early kiwifruit pollen tube organisation, but also in maintaining polarised growth of tubes. By immunofluorescence analysis we show that ubiquitin and ubiquitin-protein conjugates are distributed mainly at the apex of emerging tubes, in both untreated pollen grains and pollen grains treated with MG132, an inhibitor of proteasome function. In the latter case, polysiphonous germination occurred and all the emerging areas were highly fluorescent. By adding MG132 to pollen when normal tube growth had already been established, accumulation of ubiquitin-protein conjugates, as well as a drastic reduction in tube growth and dramatic modifications of tube tip morphology were observed. Significantly, differential interference contrast microscopy analysis demonstrated that the clear zone was largely reduced or absent, and the nuclei were disconnected in their movements, reaching, in some cases, the extreme apex of the tip. These findings provide evidence that the ubiquitin- and proteasome-dependent proteolytic system could modulate the abundance and/or activity of key regulatory proteins involved in pollen tube emergence and polarised growth.     

Arabidopsis LIM proteins PLIM2a and PLIM2b regulate actin configuration during pollen tube growth

The pollen tube grows rapidly, exclusively at its tip, to deliver its sperm for fertilization. The polarized tip growth of pollen tubes is dependent on the highly dynamic actin cytoskeleton. Plant LIM proteins (named after initials of containing proteins Lin11, Isl-1, and Mec-3) have been shown to regulate actin bundling in different cells, however, their roles in pollen tube growth have remained obscure. Here, we report the function of Arabidopsis LIM proteins PLIM2a and PLIM2b in pollen tube growth. The PLIM2a mutation resulted in short and swollen Arabidopsis pollen tube with defective actin bundles. The expression of the construct green fluorescent protein (GFP)-PLIM2b led to fluorescence of the actin bundles in germinating pollen and also the long actin bundles along the growing pollen tubes in Arabidopsis, but not of the short and sparse actin bundles that characterize the tip regions of the pollen tubes. There is a partially redundant function between PLIM2a and PLIM2b in the shank actin bundle organization during Arabidopsis pollen tube growth, as PLIM2b could rescue for the defective shank actin bundles in PLIM2a mutation pollen tubes. This report suggests critical roles of PLIM2a/PLIM2b in actin configuration during Arabidopsis pollen germination and tube growth.     

Still life: Pollen tube growth observed in millisecond resolution

Our recent work used novel methods to localize and track discrete vesicle populations in pollen tubes undergoing oscillatory growth. The results show that clathrin-dependent endocytosis occurs along the shank of the pollen tube, smooth vesicle endocytosis occurs at the tip, and exocytosis occurs in the subapical region. Here, growth of tobacco and lily pollen tubes is examined in greater temporal resolution using refraction-free high-resolution time-lapse differential interference contrast microscopy. Images were collected at 0.21 s intervals for 10 min, sequentially examined for millisecond details, compressed into video format and then examined for details of growth dynamics. The subapical growth zone is structurally fluid, with vesicle insertion into the plasma membrane, construction of new cell surface and cellular expansion. Incorporation of new membrane and wall materials causes localized disruption at the cell surface that precedes the start of the growth cycle by 3.44 ± 0.39 s in tobacco, and 1.02 ± 0.01 s in lily pollen tubes. Vesicle deposition increases after the start of the growth cycle and supports expansion of the growth zone. Growth reorientation involves a shift in the position and angle of the growth zone. In summary, these results support a new model of pollen tube growth.Key words: growth zone, oscillation, exocytosis, growth reorientation, differential interference contrast microscopy, refraction-free     

A model for the mechanism of tip extension in pollen tubes

Three main mechanisms are proposed to account for the tip growth of pollen tubes. (1) The tip region is supported against the internal osmotic pressure of the cell by a fibrillar network, composed mainly of microfilaments, that is stabilized by calcium ions. Tip extension is promoted by a lowering of the local cytoplasmic calcium ion concentration, through uptake by the mitochondria and/or endoplasmic reticulum, which leads to a weakening of the fibrillar network. (2) Vesicles, derived from dictyosomes in the main body of the tube, fuse with the apical plasma membrane, providing new membrane and further carbohydrate for the wall. The rate of fusion is proportional to the rate of diffusion of calcium ion across the plasma membrane at the tip. (3) The callose lining present in the pollen tube wall, except at the tip, renders the wall impermeable and restricts entry of calcium ions to the apical plasma membrane. This restriction limits the rate of vesicle fusion, and tube growth, to the tip.This model is discussed in the light of previous observations on the growth and structure of pollen tubes under normal and experimental conditions.     

The regulation of actin organization by actin-depolymerizing factor in elongating pollen tubes

Pollen tube elongation is a polarized cell growth process that transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actomyosin-driven intracellular trafficking and active actin remodeling in the apical and subapical regions of pollen tubes are both important aspects of this rapid tip growth process. Actin-depolymerizing factor (ADF) and cofilin are actin binding proteins that enhance the depolymerization of microfilaments at their minus, or slow-growing, ends. A pollen-specific ADF from tobacco, NtADF1, was used to dissect the role of ADF in pollen tube growth. Overexpression of NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes and in the inhibition of pollen tube growth in a dose-dependent manner. Thus, the proper regulation of actin turnover by NtADF1 is critical for pollen tube growth. When expressed at a moderate level in pollen tubes elongating in in vitro cultures, green fluorescent protein (GFP)-tagged NtADF1 (GFP-NtADF1) associated predominantly with a subapical actin mesh composed of short actin filaments and with long actin cables in the shank. Similar labeling patterns were observed for GFP-NtADF1-expressing pollen tubes elongating within the pistil. A Ser-6-to-Asp conversion abolished the interaction between NtADF1 and F-actin in elongating pollen tubes and reduced its inhibitory effect on pollen tube growth significantly, suggesting that phosphorylation at Ser-6 may be a prominent regulatory mechanism for this pollen ADF. As with some ADF/cofilin, the in vitro actin-depolymerizing activity of recombinant NtADF1 was enhanced by slightly alkaline conditions. Because a pH gradient is known to exist in the apical region of elongating pollen tubes, it seems plausible that the in vivo actin-depolymerizing activity of NtADF1, and thus its contribution to actin dynamics, may be regulated spatially by differential H(+) concentrations in the apical region of elongating pollen tubes.     

Rab11 GTPase-regulated membrane trafficking is crucial for tip-focused pollen tube growth in tobacco

Pollen tube growth is a polarized growth process whereby the tip-growing tubes elongate within the female reproductive tissues to deliver sperm cells to the ovules for fertilization. Efficient and regulated membrane trafficking activity incorporates membrane and deposits cell wall molecules at the tube apex and is believed to underlie rapid and focused growth at the pollen tube tip. Rab GTPases, key regulators of membrane trafficking, are candidates for important roles in regulating pollen tube growth. We show that a green fluorescent protein-tagged Nicotiana tabacum pollen-expressed Rab11b is localized predominantly to an inverted cone-shaped region in the pollen tube tip that is almost exclusively occupied by transport vesicles. Altering Rab11 activity by expressing either a constitutive active or a dominant negative variant of Rab11b in pollen resulted in reduced tube growth rate, meandering pollen tubes, and reduced male fertility. These mutant GTPases also inhibited targeting of exocytic and recycled vesicles to the pollen tube inverted cone region and compromised the delivery of secretory and cell wall proteins to the extracellular matrix. Properly regulated Rab11 GTPase activity is therefore essential for tip-focused membrane trafficking and growth at the pollen tube apex and is pivotal to reproductive success.     

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.     

Lead Stress Disrupts the Cytoskeleton Organization and Cell Wall Construction During <Emphasis Type="Italic">Picea wilsonii</Emphasis> Pollen Germination and Tube Growth

Lead is a widespread pollutant and has been reported to inhibit pollen tube development, but the mechanism of toxicity involved remains unclear. Here, we report that lead stress significantly prevented Picea wilsonii pollen germination and tube growth and also dramatically altered the tube morphology in a concentration-dependent manner. Fluorescence labeling with JIM 5 (anti-acidic pectin antibody) and Calcofluor white revealed the lead-induced decline of acidic pectin and cellulose, especially in the subapical region. Decolorized aniline blue staining showed the marked accumulation of callose in the apical and subapical regions of lead-treated tubes. Fluorescence labeling with Alexa Fluor 568 phalloidin and anti-tubulin antibody revealed that the distribution of the cytoskeleton in P. wilsonii pollen grains and tubes were developmentally regulated and that lead disturbed the cytoskeleton organization, especially in the shank of the pollen tubes. Taken together, our experiments revealed a link between the dynamics of cytoskeleton organization and the process of P. wilsonii pollen tube development and also indicated that lead disturbed the cytoskeleton assembly and, consequently, cell wall construction. These findings provide new insights into the mechanism of lead toxicity in the tip growth of pollen tubes.     

Chloride fluxes in lily pollen tubes: a critical reevaluation

Microelectrodes, made from a Cl(-)-selective liquid ion exchanger previously used to measure putative Cl- fluxes in Lilium longiflorum pollen tubes, were characterized. The electrodes were poorly selective, possessing only about 10-fold selectivity for Cl- over other anions tested. They had only 2.4-fold selectivity for Cl- over the anionic form of the H+ buffer, MES, indicating that the electrode can indirectly detect H+ gradients. Apparent anion influx was detected along the pollen tube shafts and at the grains while apparent anion efflux was detected near the tip of the tube. During oscillating growth, the peak of the oscillating apparent anion efflux at the tip occurred, on average, 7.9 sec after the peak of the growth oscillations. Consideration of the previously characterized H+ fluxes in lily pollen grains and tubes, as well as the poor anion selectivity of the Cl- electrodes, indicates that the putative Cl- fluxes are in fact changes in the anionic concentration of the buffer resulting from H+ gradients and not changes in Cl- concentration. The claim of a central role for Cl- in lily pollen tube growth is further undermined by the fact that these tubes grow at the same rate if the Cl- content of the growth medium is reduced to trace levels (< or =31 microM), and that the grains have only small reserves of Cl-. These results lead to the conclusion that Cl- fluxes are not a significant component of pollen tube growth and Cl- itself is not required for growth.     

Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro

Summary. Pears (Pyrus pyrifolia L.) have an S-RNase-based gametophytic self-incompatibility system, and S-RNases have also been implicated in self-pollen or genetically identical pollen rejection. Tip growth of the pollen tube is dependent on a functioning actin cytoskeleton. In this study, configurations of the actin cytoskeleton in P. pyrifolia pollen and effects of stylar S-RNases on its dynamics were investigated by fluorescence and confocal microscopy. Results show that actin filaments in normal pollen grains exist in fusiform or circular structures. When the pollen germinates, actin filaments assembled around one of the germination pores, and then actin bundles oriented axially throughout the shank of the growing tube. There was a lack of actin filaments 5–15 μm from the tube tip. When self-stylar S-RNase was added to the basal medium, pollen germination and tube growth were inhibited. The configuration of the actin cytoskeleton changed throughout the culturing time: during the first 20 min, the actin configurations in the self-pollen and tube were similar to the control; after 20 min of treatment, the actin filaments in the pollen tube gradually moved into a network running from the shank to the tip; finally, there was punctate actin present throughout the whole tube. Although the actin filaments of the self-pollen grain also disintegrated into punctate foci, the change was slower than in the tube. Furthermore, the alterations to the actin cytoskeleton occurred prior to the arrest of pollen tube growth. These results suggest that P. pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen grains and tubes. Correspondence: Shao-ling Zhang, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China.     

Plasma membrane H+‐ATPases‐mediated cytosolic proton gradient regulates pollen tube growth

The polar growth of pollen tubes is essential for the delivery of sperm cells during fertilization in angiosperms. How this polar growth is regulated has been a long‐standing question. An in vitro pharmacological assay previously implicated proton flux in pollen tube growth, although genetic and cellular supporting evidence was lacking. Here, we report that protons form a gradient from the pollen tube tip to the shank region and this gradient is generated by three members of Arabidopsis H⁺‐ATPases (AHAs). Genetic analysis suggested that these AHAs are essential for pollen tube growth, thus providing new insight into the regulation of polar growth.     

Calcium Channel Activity during Pollen Tube Growth and Reorientation

We have shown previously that the inhibition of pollen tube growth and its subsequent reorientation in Agapanthus umbellatus are preceded by an increase in cytosolic free calcium ([Ca2+]c), suggesting a role for Ca2+ in signaling these processes. In this study, a novel procedure was used to measure Ca2+ channel activity in living pollen tubes subjected to various growth reorienting treatments (electrical fields and ionophoretic microinjection). The method involves adding extracellular Mn2+ to quench the fluorescence of intracellular Indo-1 at its ca2+-insensitive wavelength (isosbestic point). The spatial and temporal kinetics of Ca2+ channel activity correlated well with measurements of [Ca2+]c dynamics obtained by fluorescence ratio imaging of Indo-1. Tip-focused gradients in Ca2+ channel activity and [Ca2+]c were observed and quantified in growing pollen tubes and in swollen pollen tubes before reoriented growth. In nongrowing pollen tubes, Ca2+ channel activity was very low and [Ca2+]c gradients were absent. Measurements of membrane potential indicated that the growth reorienting treatments induced a depolarization of the plasma membrane, suggesting that voltage-gated Ca2+ channels might be activated.     

Cytochalasin B Treatment of Apple (<Emphasis Type="Italic">Malus pumila</Emphasis> Mill.) Pollen Tubes Alters the Cytoplasmic Calcium Gradient and Causes Major Changes in the Cell Wall Components

It is well established that the actin cytoskeleton is absolutely essential to pollen germination and tube growth. In this study we investigated the effects of cytochalasin B (CB), which affects actin polymerization by binding to the barbed end of actin filaments, on apple (Malus pumila Mill.) pollen tube growth. Results showed that CB altered the morphology of pollen tubes, which had a larger diameter than control tubes beside inhibiting pollen germination and tube growth. Meantime CB also caused an abnormal distribution of actin filaments in the shank of the treated pollen tubes. Fluo-3/AM labeling indicated that the gradient of cytosolic calcium ([Ca²⁺]c) in the pollen tube tip was abolished by exposure to CB, which induced a much stronger signal in the cytoplasm. Cellulose and callose distribution in the tube apex changed due to the CB treatment. Immunolabeling with different pectin and arabinogalactan protein (AGP) antibodies illustrated that CB induced an accumulation of pectins and AGPs in the tube cytoplasm and apex wall. The above results were further supported by Fourier-transform infrared (FTIR) analysis. The results suggest the disruption of actin can result in abnormal growth by disturbing the [Ca²⁺]c gradient and the distribution of cell wall components at the pollen tube apex.     

Localized Apical Increases of Cytosolic Free Calcium Control Pollen Tube Orientation

To reach the ovule, pollen tubes must undergo many changes in growth direction. We have shown in previous work that elevation of cytosolic free calcium ([Ca2+]c) can manipulate orientation in growing pollen tubes, but our results suggested that [Ca2+]c changes either in the tip or in more distal regions might regulate the critical orienting mechanism. To identify the spatial location of the orienting motor, we combined the techniques of ion imaging with confocal microscopy and localized photoactivation of loaded caged Ca2+ (nitr-5) and diazo-2 (a caged Ca2+ chelator) to manipulate [Ca2+]c in different pollen tube domains. We found that increasing [Ca2+]c on one side of the pollen tube apex induced reorientation of the growth axis toward that side. Similarly, a decrease in [Ca2+]c promoted bending toward the opposite side. These effects could be mimicked by imposing localized external gradients of an ionophore (A23187) or a Ca2+ channel blocker (GdCl3); the pollen tubes bend toward the highest concentration of A23187 and away from GdCl3. Manipulation of [Ca2+]c in regions farther back from the apical zone also induced changes in growth direction, but the new orientation was at random. We observed communication of these distal events to the tip through a slow-moving [Ca2+]c wave. These data show that localized changes of [Ca2+]c in the tip, which could result from asymmetric channel activity, control the direction of pollen tube growth.     

TipChip: a modular,MEMS‐based platform for experimentation and phenotyping of tip‐growing cells

Large‐scale phenotyping of tip‐growing cells such as pollen tubes has hitherto been limited to very crude parameters such as germination percentage and velocity of growth. To enable efficient and high‐throughput execution of more sophisticated assays, an experimental platform, the TipChip, was developed based on microfluidic and microelectromechanical systems (MEMS) technology. The device allows positioning of pollen grains or fungal spores at the entrances of serially arranged microchannels equipped with microscopic experimental set‐ups. The tip‐growing cells (pollen tubes, filamentous yeast or fungal hyphae) may be exposed to chemical gradients, microstructural features, integrated biosensors or directional triggers within the modular microchannels. The device is compatible with Nomarski optics and fluorescence microscopy. Using this platform, we were able to answer several outstanding questions on pollen tube growth. We established that, unlike root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, pollen tubes were found to be able to elongate in air, raising the question of how and where water is taken up by the cell. The platform opens new avenues for more efficient experimentation and large‐scale phenotyping of tip‐growing cells under precisely controlled, reproducible conditions.