Microtubules and microfilaments coordinate to direct a fountain streaming pattern in elongating conifer pollen tube tips

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-m-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations APM Amiprophosmethyl - FITC Fluorescein isothiocyanate - LATB Latrunculin B     

Growing Pollen Tubes Possess a Constitutive Alkaline Band in the Clear Zone and a Growth-dependent Acidic Tip

Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H⁺) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 μM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.     

Arabidopsis Formin3 Directs the Formation of Actin Cables and Polarized Growth in Pollen Tubes

Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785–amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes'' apices. Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains. This led to the disruption of the reverse fountain streaming pattern in pollen tubes, confirming a role for actin cables in the regulation of cytoplasmic streaming. Furthermore, these tubes became wide and short and swelled at their tips, suggesting that actin cables may regulate growth polarity in pollen tubes. Thus, AFH3 regulates the formation of actin cables, which are important for cytoplasmic streaming and polarized growth in pollen tubes.     

Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca2+ concentrations lower than 0.18 microM (pCa 6.8) but was inhibited at concentration higher than 4.5 microM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca2+ through "myosin" inactivation.     

Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization

Prevention of actin polymerization with low concentrations of latrunculin B (Lat-B; 2 nm) exerts a profound inhibitory effect on pollen tube growth. Using flow-through chambers, we show that growth retardation starts after 10 min treatment with 2 nm Lat-B, and by 15 to 20 min reaches a basal rate of 0.1 to 0.2 microm/s, during which the pollen tube exhibits relatively few oscillations. If treated for 30 min, complete stoppage of growth can occur. Studies on the intracellular Ca(2+) concentration indicate that the tip-focused gradient declines in parallel with the inhibition of growth. Tubes exhibiting nonoscillating growth display a similarly reduced and nonoscillating Ca(2+) gradient. Studies on the pH gradient indicate that Lat-B eliminates the acidic domain at the extreme apex, and causes the alkaline band to move more closely to the tip. Removing Lat-B and returning the cells to control medium reverses these effects. Phalloidin staining of F-actin reveals that 2 nm Lat-B degrades the cortical fringe; it also disorganizes the microfilaments in the shank causing the longitudinally oriented elements to be disposed in swirls. Cytoplasmic streaming continues under these conditions, however the clear zone is obliterated with all organelles moving into and through the extreme apex of the tube. We suggest that actin polymerization promotes pollen tube growth through extension of the cortical actin fringe, which serves as a track to target cell wall vesicles to preferred exocytotic sites on the plasma membrane.     

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.     

Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth

Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes.     

Calcium gradients in conifer pollen tubes; dynamic properties differ from those seen in angiosperms

Pollen tubes are an established model system for examining polarized cell growth. The focus here is on pollen tubes of the conifer Norway spruce (Picea abies, Pinaceae); examining the relationship between cytosolic free Ca2+, tip elongation, and intracellular motility. Conifer pollen tubes show important differences from their angiosperm counterparts; they grow more slowly and their organelles move in an unusual fountain pattern, as opposed to reverse fountain, in the tip. Ratiometric ion imaging of growing pollen tubes, microinjected with fura-2-dextran, reveals a tip-focused [Ca2+]i gradient extending from 450 nM at the extreme apex to 225 nM at the base of the tip clear zone. Injection of 5,5' dibromo-BAPTA does not dissipate the apical gradient, but stops cell elongation and uniquely causes rapid, transient increases of apical free Ca2+. The [Ca2+]i gradient is, however, dissipated by reversible perfusion of extracellular caffeine. When the basal cytosolic free Ca2+ concentration falls below 150 nM, again a large increase in apical [Ca2+]i occurs. An external source of calcium is not required for germination but significantly enhances elongation. However, both germination and elongation are significantly inhibited by the inclusion of calcium channels blockers, including lanthanum, gadolinium, or verapamil. Modulation of intracellular calcium also affects organelle position and motility. Extracellular perfusion of lanthanides reversibly depletes the apical [Ca2+]i gradient, altering organelle positioning in the tip. Later, during recovery from lanthanide perfusion, organelle motility switches direction to a reverse fountain. When taken together these data show a unique interplay in Picea abies pollen tubes between intracellular calcium and the motile processes controlling cellular organization.     

Circular F-actin bundles and a G-actin gradient in pollen and pollen tubes of Lilium davidii

The distribution of and relationship between F-actin and G-actin were investigated in pollen grains and pollen tubes of Lilium davidii Duch. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. Circular F-actin bundles were found to be the main form of microfilament cytoskeleton in pollen grains and pollen tubes. Consistent with cytoplasmic streaming in pollen tubes, there were no obvious F-actin bundles in the 10- to 20-microm tip region of long pollen tubes, only a few short F-actin fragments. Labeling with fluorescein isothiocyanate (FITC)-DNase I at first established the presence of a tip-focused gradient of intracellular G-actin concentration at the extreme apex of the tube, the concentration of G-actin being about twice as high in the 10- to 20-microm region of the tip as in other regions of the pollen tube. We also found that the distribution of G-actin was related negatively to that of the F-actin in pollen tubes of L. davidii. Caffeine treatment caused the G-actin tip-focused gradient to disappear, and F-actin to extend into the pollen tube tip. Based on these results, we speculate that the circular F-actin bundles may be the track for bidirectional cytoplasmic streaming in pollen tubes, and that in the pollen tube tip most of the F-actin is depolymerized into G-actin, leading to the absence of F-actin bundles in this region.     

Differential organelle movement on the actin cytoskeleton in lily pollen tubes

We have examined the arrangement and movement of three major compartments, the endoplasmic reticulum (ER), mitochondria, and the vacuole during oscillatory, polarized growth in lily pollen tubes. These movements are dependent on the actin cytoskeleton, because they are strongly perturbed by the anti-microfilament drug, latrunculin-B, and unaffected by the anti-microtubule agent, oryzalin. The ER, which has been labeled with mGFP5-HDEL or cytochalasin D tetramethylrhodamine, displays an oscillatory motion in the pollen tube apex. First it moves apically in the cortical region, presumably along the cortical actin fringe, and then periodically folds inward creating a platform that transects the apical domain in a plate-like structure. Finally, the ER reverses its direction and moves basipetally through the central core of the pollen tube. When subjected to cross-correlation analysis, the formation of the platform precedes maximal growth rates by an average of 3 s (35-40 degrees ). Mitochondria, labeled with Mitotracker Green, are enriched in the subapical region, and their movement closely resembles that of the ER. The vacuole, labeled with carboxy-dichlorofluorescein diacetate, consists of thin tubules arranged longitudinally in a reticulate network, which undergoes active motion. In contrast to the mitochondria and ER, the vacuole is located back from the apex, and never extends into the apical clear zone. We have not been able to decipher an oscillatory pattern in vacuole motion. Because this motion is dependent on actin and not tubulin, we think this is due to a different myosin from that which drives the ER and mitochondria.     

Overexpression of an Arabidopsis formin stimulates supernumerary actin cable formation from pollen tube cell membrane

Formins, actin-nucleating proteins that stimulate the de novo polymerization of actin filaments, are important for diverse cellular and developmental processes, especially those dependent on polarity establishment. A subset of plant formins, referred to as group I, is distinct from formins from other species in having evolved a unique N-terminal structure with a signal peptide, a Pro-rich, potentially glycosylated extracellular domain, and a transmembrane domain. We show here that overexpression of the Arabidopsis formin AFH1 in pollen tubes induces the formation of arrays of actin cables that project into the cytoplasm from the cell membrane and that its N-terminal structure targets AFH1 to the cell membrane. Pollen tube elongation is a polar cell growth process dependent on an active and tightly regulated actin cytoskeleton. Slight increases in AFH1 stimulate growth, but its overexpression induces tube broadening, growth depolarization, and growth arrest in transformed pollen tubes. These results suggest that AFH1-regulated actin polymerization is important for the polar pollen cell growth process. Moreover, severe membrane deformation was observed in the apical region of tip-expanded, AFH1-overexpressing pollen tubes in which an abundance of AFH1-induced membrane-associated actin cables was evident. These observations suggest that regulated AFH1 activity at the cell surface is important for maintaining tip-focused cell membrane expansion for the polar extension of pollen tubes. The cell surface-located group-I formins may play the integrin-analogous role as mediators of external stimuli to the actin cytoskeleton, and AFH1 could be important for mediating extracellular signals from female tissues to elicit the proper pollen tube growth response during pollination.     

Polarized cell growth, organelle motility, and cytoskeletal organization in conifer pollen tube tips are regulated by KCBP, the calmodulin-binding kinesin

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin 14 family, is a minus end directed C-terminal motor unique to plants and green algae. Its motor activity is negatively regulated by calcium/calmodulin binding, and its tail region contains a secondary microtubule-binding site. It has been identified but not functionally characterized in the conifer Picea abies. Conifer pollen tubes exhibit polarized growth as organelles move into the tip in an unusual fountain pattern directed by microfilaments but uniquely organized by microtubules. We demonstrate here that PaKCBP and calmodulin regulate elongation and motility. PaKCBP is a 140 kDa protein immunolocalized to the elongating tip, coincident with microtubules. This localization is lost when microtubules are disrupted with oryzalin, which also reorganizes microfilaments into bundles. Colocalization of PaKCBP along microtubules is enhanced when microfilaments are disrupted with latrunculin B, which also disrupts the fine network of microtubules throughout the tip while preserving thicker microtubule bundles. Calmodulin inhibition by W-12 perfusion reversibly slows pollen tube elongation, alters organelle motility, promotes microfilament bundling, and microtubule bundling coincident with increased PaKCBP localization. The constitutive activation of PaKCBP by microinjection of an antibody that displaces calcium/calmodulin and activates microtubule bundling repositions vacuoles in the tip before rapidly stopping organelle streaming and pollen tube elongation. We propose that PaKCBP is one of the target proteins in conifer pollen modulated by calmodulin inhibition leading to microtubule bundling, which alters microtubule and microfilament organization, repositions vacuoles and slows organelle motility and pollen tube elongation.     

Low concentration of LatB dramatically changes the microtubule organization and the timing of vegetative nucleus/generative cell entrance in tobacco pollen tubes

Low concentration of LatB inhibits not only the actin polymerization, but also induces profound alteration of MT distribution in pollen tubes of Nicotiana tabacum. The short randomly oriented MTs in the apical and subapical regions, became organized as bundles forming subapical rings or basket-like structures, surrounding the apex. Moreover, the depolymerization of AFs in the cortical regions of the apex and subapical region affects the timing of entrance of the vegetative nucleus and generative cell into the pollen tube.     

Arabidopsis FIMBRIN5, an actin bundling factor, is required for pollen germination and pollen tube growth

Actin cables in pollen tubes serve as molecular tracks for cytoplasmic streaming and organelle movement and are formed by actin bundling factors like villins and fimbrins. However, the precise mechanisms by which actin cables are generated and maintained remain largely unknown. Fimbrins comprise a family of five members in Arabidopsis thaliana. Here, we characterized a fimbrin isoform, Arabidopsis FIMBRIN5 (FIM5). Our results show that FIM5 is required for the organization of actin cytoskeleton in pollen grains and pollen tubes, and FIM5 loss-of-function associates with a delay of pollen germination and inhibition of pollen tube growth. FIM5 decorates actin filaments throughout pollen grains and tubes. Actin filaments become redistributed in fim5 pollen grains and disorganized in fim5 pollen tubes. Specifically, actin cables protrude into the extreme tips, and their longitudinal arrangement is disrupted in the shank of fim5 pollen tubes. Consequently, the pattern and velocity of cytoplasmic streaming were altered in fim5 pollen tubes. Additionally, loss of FIM5 function rendered pollen germination and tube growth hypersensitive to the actin-depolymerizing drug latrunculin B. In vitro biochemical analyses indicated that FIM5 exhibits actin bundling activity and stabilizes actin filaments. Thus, we propose that FIM5 regulates actin dynamics and organization during pollen germination and tube growth via stabilizing actin filaments and organizing them into higher-order structures.     

Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes

Pollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4‐64 and charged nanogold. Time‐lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co‐localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin‐dependent and actin‐indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin‐dependent secretory activity occurs in the shank of pollen tubes.     

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.     

Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of lily

Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events.     

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.     

Monomeric G‐actin is uniformly distributed in pollen tubes and is rapidly redistributed via cytoplasmic streaming during pollen tube growth

Dynamic assembly and disassembly of the actin cytoskeleton has been implicated in the regulation of pollen germination and subsequent tube growth. It is widely accepted that actin filaments are arrayed into distinct structures within different regions of the pollen tube. Maintenance of the equilibrium between monomeric globular actin (G‐actin) and filamentous actin (F‐actin) is crucial for actin assembly and array construction, and the local concentration of G‐actin thus directly impacts actin assembly. The localization and dynamics of G‐actin in the pollen tube, however, remain to be determined conclusively. To address this question, we created a series of fusion proteins between green fluorescent protein (GFP) and the Arabidopsis reproductive actin ACT11. Expression of a fusion protein with GFP inserted after methionine at position 49 within the DNase I‐binding loop of ACT11 (GFP_Met49–ACT11) rescued the phenotypes in act11 mutants. Consistent with the notion that the majority of actin is in its monomeric form, GFP_Met49–ACT11 and GFP fusion proteins of four other reproductive actins generated with the same strategy do not obviously label filamentous structures. In further support of the functionality of these fusion proteins, we found that they can be incorporated into filamentous structures in jasplakinolide (Jasp)‐treated pollen tubes. Careful observations showed that G‐actin is distributed uniformly in the pollen tube and is rapidly redistributed via cytoplasmic streaming during pollen tube growth. Our study suggests that G‐actin is readily available in the cytoplasm to support continuous actin polymerization during rapid pollen tube growth.     

Microfilament Orientation Constrains Vesicle Flow and Spatial Distribution in Growing Pollen Tubes

The dynamics of cellular organelles reveals important information about their functioning. The spatio-temporal movement patterns of vesicles in growing pollen tubes are controlled by the actin cytoskeleton. Vesicle flow is crucial for morphogenesis in these cells as it ensures targeted delivery of cell wall polysaccharides. Remarkably, the target region does not contain much filamentous actin. We model the vesicular trafficking in this area using as boundary conditions the expanding cell wall and the actin array forming the apical actin fringe. The shape of the fringe was obtained by imposing a steady state and constant polymerization rate of the actin filaments. Letting vesicle flux into and out of the apical region be determined by the orientation of the actin microfilaments and by exocytosis was sufficient to generate a flux that corresponds in magnitude and orientation to that observed experimentally. This model explains how the cytoplasmic streaming pattern in the apical region of the pollen tube can be generated without the presence of actin microfilaments.