Cytoplasmic motors and pollen tube growth

The growth of pollen tubes is characterized by an intense cytoplasmic streaming, during which the movements of smaller organelles (like secretory vesicles) and larger ones (including the generative cell and vegetative nucleus) are precisely coordinated. A well-characterized cytoskeletal apparatus is likely responsible for these intracellular movements. In recent years both microfilament and microtubule-based motor proteins have been identified and assumed to be the translocators of the several organelle categories. Their precise function during pollen tube growth is not yet clear, but apparently an actomyosin-based system is mainly responsible for pollen tube elongation. On the other hand, microtubules and microtubule-based motors have been thought to play a role in the maintenance of cell polarity. Both cytoskeletal systems (and their respective motor activities) could cooperate to ensure a precise regulation of pollen 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.     

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.     

Reversible protein tyrosine phosphorylation affects pollen germination and pollen tube growth via the actin cytoskeleton

Summary. Phenylarsine oxide (PAO) and genistein are two well-known specific inhibitors of tyrosine phosphatases and kinases, respectively, that have been used in the functional analysis of the status of protein phosphotyrosine in different cell types. Our experiments showed that both PAO and genistein arrested pollen germination and pollen tube growth and led to the malformation of the pollen tubes, although genistein had a lesser effect. The malformations of the pollen tubes caused by PAO and genistein were, however, quite different. In addition, it was found that the rate of pollen germination and tube growth recovered to a certain extent when phalloidin was present during PAO treatment, but not when it was present during genistein treatment. Furthermore, PAO treatment also had a great effect on the dynamic organization of filamentous actin in the pollen grain and pollen tube, while genistein only caused reorganization of actin at the turning point of the pollen tube. Our results suggest that reversible protein tyrosine phosphorylation is a crucial step in pollen germination and pollen tube growth, but that tyrosine kinases and phosphatases may have different effects which may function through the reorganization of the actin cytoskeleton. Correspondence and reprints: Key Laboratory of Cell Proliferation and Regulation Biology of the Ministry of Education, College of Life Science, Beijing Normal University, Beijing 100875, People’s Republic of China.     

Cytochalasin effects on structure and movement in the pollen tube of Iris

Summary Cytochalasins B and D in the concentration range 0.5–10 g ml^–1 produced similar effects on growth, movement and cytoplasmic structure in the pollen tubes of Iris spp. cultured in vitro. Continuous video recording showed that at 5 g ml_–1, CB was capable of stopping organelle circulation in as short a period as 20 s. The usually elongated vegetative nuclei were also arrested, and subsequently contracted irregularly. Generative cells were not radically changed in shape, but occasionally moved erratically before being halted. Detailed examination of CD- and CB-treated tubes regarded as being capable of recovering growth upon transfer to normal medium revealed several characteristic effects on cytoplasmic structure. Fibrils presumed to consist of, or contain, microfilament bundles are readily visible in the older parts of the living tube where they form the pathways of organelle movement; these were either condensed into amorphous columns or fragmented by treatment. In the distal parts of the tube, the cytoplasm had contracted into amorphous masses which continued to show very slow shape changes. With the arrest of extension growth, pectin accumulated over the tube tip and in patches along the flanks. In a medium containing 1 mM ATP, recovery from treatment was achieved in some instances within l min. Organelle circulation in the younger tubes was resumed, and fresh adventive tube tips were formed. The fibrillar system of the older tubes was not restored, however; instead, the cytoplasm in these zones formed aggregates which underwent continuous amoeboid movement, the organelles within moving rapidly in irregular trajectories with no indication of the resumption of the original long-range cyclotic flux. Some possible implications of the results are briefly discussed.     

Tocopherols and carotenes are differently distributed in subfractions of high-density lipoprotein

An apolipoprotein (apo) E-rich and an apo E-poor fraction of high-density lipoprotein (HDL) were isolated from four healthy men by heparin-Sepharose affinity chromatography. On a cholesterol basis, the apo E-poor HDL fraction contained a third more α- and γ-tocopherol and about a third less α- and β-carotene than the apo E-rich HDL fraction. Plasma concentrations of HDL cholesterol were highly correlated with the contribution of the apo E-rich HDL subfraction to total HDL α-tocopherol (r = − 0.990, P < 0.001).     

Molecular motors are differentially distributed on Golgi membranes from polarized epithelial cells

Microtubules (MT) are required for the efficient transport of membranes from the trans-Golgi and for transcytosis of vesicles from the basolateral membrane to the apical cytoplasm in polarized epithelia. MTs in these cells are primarily oriented with their plus ends basally near the Golgi and their minus-ends in the apical cytoplasm. Here we report that isolated Golgi and Golgi-enriched membranes from intestinal epithelial cells possess the actin based motor myosin-I, the MT minus- end-directed motor cytoplasmic dynein and its in vitro motility activator dynactin (p150/Glued). The Golgi can be separated into stacks, possessing features of the Golgi cisternae, and small membranes enriched in the trans-Golgi network marker TGN 38/41. Whereas myosin-I is present on all membranes in the Golgi fraction, dynein is present only on the small membrane fraction. Dynein, like myosin-I, is associated with membranes as a cytoplasmic peripheral membrane protein. Dynein and myosin-I coassociate with membranes that bind to MTs and cross-link actin filaments and MTs in a nucleotide-dependent manner. We propose that cytoplasmic dynein moves Golgi membranes along MTs to the cell cortex where myosin-I provides local delivery through the actin- rich cytoskeleton to the apical membrane.     

Dispersal and recruitment limitation contribute differently to community assembly

Aims The neutral theory of biodiversity has been criticized for being fragile with even slight deviations from its basic assumption of equal fitness among species. In response to this criticism, Hubbell ((2001) The Unified Neutral Theory of Biodiversity and Biogeography. Princeton, NJ: Princeton University Press) proposed that competitive exclusion can be infinitely delayed by dispersal and recruitment limitation, thus making species effectively neutral. But the theoretical foundation for this claim still remains unclear and controversial, and the effects of dispersal and recruitment limitation are often confounded, especially in field studies. This study aims to provide an affirmative theoretical answer to the question of whether dispersal limitation and recruitment limitation can separately or jointly overwhelm the effects of fitness differences among species and lead to neutral community dynamics.Methods Computer simulations were used to investigate the effects of dispersal and recruitment limitation on delaying competitive exclusion in a homogeneous habitat in a spatially explicit context.Important findings We found that even a slight competitive asymmetry would require extremely strong dispersal and recruitment limitation for neutrality to emerge. Most importantly, when the effects of dispersal and recruitment limitation were set apart, it is found that recruitment limitation is more effective in delaying competitive exclusion, whereas dispersal limitation tends to have a stronger impact on the general shape of both species abundance distributions and species–area relationships.     

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.     

Intermediate filaments of the vimentin-type and the cytokeratin-type are distributed differently during mitosis

Immunofluorescence microscopy has been used to follow the rearrangement of intermediate-sized filaments during mitosis in rat kangaroo PtK2 cells. These epithelial cells express two different intermediate filament systems: the keratin-related tonofilament-like arrays typical of epithelial cells, and the vimentin-type filaments characteristic of mesenchymal cells in vivo, and of many established cell lines. The two filament systems do not appear to depolymerize extensively during mitosis, but show differences in their organization and display which may indicate different functions. The most striking rearrangements have been seen with the vimentin filaments, and in particular in prometaphase a transient cage-like structure of vimentin fibers surrounding the developing spindle is formed. In metaphase, this cage disappears, and vimentin fibers are found in an elliptical band surrounding the chromosomes and the interzone. In telophase, these bands separate, usually breaking first on the side closest to where the cleavage furrow has started to form. Double label experiments with tubulin and vimentin antibodies have indicated that the microtubules and the chromosomes are contained within the thick crescents of vimentin filaments and suggest that the vimentin intermediate filaments may be involved in the orientation of the spindle and/or the chromosomes during mitosis. In contrast, extensive arrays of cytokeratin filaments are present throughout mitosis on the substrate-attached side of the cell and also in other cellular areas, although they are usually not present in the spindle region. Thus the cytokeratin filaments probably continue to play a cytoskeletal role during mitosis and may be responsible for the flat shape that certain epithelial cells such as PtK2 cells continue to maintain during mitosis.     

Organelle movement and fibrillar elements of the cytoskeleton in the angiosperm pollen tube

Summary Continuous observation of organelles and other cytoplasmic inclusions in the older stretches of living pollen tubes of Iris pseudacorus shows that in the more attentuated parts of the protoplast they move along single, mainly longitudinally oriented fibrils, corresponding to those previously isolated from other species and shown to contain bundles of uniformly polarised actin microfilaments. The traffic associated with each fibril is unidirectional, but organelles move along them independently, sometimes with conspicuously different velocities. Larger columns of cytoplasm passing along the tube are associated with several such fibrils, as revealed in occasional discontinuities and also in columns isolated from the tube in suitable medium without fixation. The dimensions of the individual fibrils suggest that the bundles of actin microfilaments are not likely to be enclosed in a unit membrane corresponding to a tonoplast. If so, the nature of the continuous cavities traversed by numerous fibrils in the older parts of the pollen tube requires reappraisal, since these are more likely to be volumes of attentuated cytoplasm comparable with that of the central cavity of the sieve tube than vacuoles of the normal plant-cell type.     

Arabidopsis ACT11 modifies actin turnover to promote pollen germination and maintain the normal rate of tube growth

Actin is an ancient conserved protein that is encoded by multiple isovariants in multicellular organisms. There are eight functional actin genes in the Arabidopsis genome, and the precise function and mechanism of action of each isovariant remain poorly understood. Here, we report the characterization of ACT11, a reproductive actin isovariant. Our studies reveal that loss of function of ACT11 causes a delay in pollen germination, but enhances pollen tube growth. Cytological analysis revealed that the amount of filamentous actin decreased, and the rate of actin turnover increased in act11 pollen. Convergence of actin filaments upon the germination aperture was impaired in act11 pollen, consistent with the observed delay of germination. Reduction of actin dynamics with jasplakinolide suppressed the germination and tube growth phenotypes in act11 pollen, suggesting that the underlying mechanisms involve an increase in actin dynamics. Thus, we demonstrate that ACT11 is required to maintain the rate of actin turnover in order to promote pollen germination and maintain the normal rate of pollen tube growth.     

Effects of anion channel blockers NPPB and DIDS on tobacco pollen tube growth and its mitochondria state

The influence of anion channel blockers NPPB and DIDS on pollen tube growth and its mitochondria functioning was studied by means of fluorescence microscopy and flow cytometry. NPPB (40 μM) blocked pollen tube growth completely, but didn’t change its diameter. DIDS (20–80 μM) caused pollen tube swelling and bursting, suggesting that DIDS-sensitive channels take part in the regulation of pollen tube osmotic balance. The osmotic effect of low DIDS concentration (20 μM) wasn’t accompanied by changes in the tube growth rate. The mapping of plasma membrane potential of pollen tubes using Di-4-ANEPPS revealed the involvement of NPPB-sensitive but not DIDS-sensitive anion channels in the maintenance of the longitudinal membrane potential gradient along the tube surface. The study of isolated pollen mitochondria showed that DIDS increased their capacity to take up potential-dependent dye DiOC₅(3), i.e. caused hyperpolarization of mitochondrial membranes. At the same time DIDS influenced on intramitochondrial ROS content and ROS release from mitochondria. Thus, NPPB and DIDS in different ways influenced on plasma membrane potential distribution along pollen tube, on its osmotic balance, and on mitochondria functioning. This set of data suggests that pollen tube growth is dependent on activity of anion channels that differ in localization and functions.     

Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2(AA)). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2(AA) mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2(AA). Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.     

Gibberellins are required for seed development and pollen tube growth in Arabidopsis

Gibberellins (GAs) are tetracyclic diterpenoids that are essential endogenous regulators of plant growth and development. GA levels within the plant are regulated by a homeostatic mechanism that includes changes in the expression of a family of GA-inactivating enzymes known as GA 2-oxidases. Ectopic expression of a pea GA 2-oxidase2 cDNA caused seed abortion in Arabidopsis, extending and confirming previous observations obtained with GA-deficient mutants of pea, suggesting that GAs have an essential role in seed development. A new physiological role for GAs in pollen tube growth in vivo also has been identified. The growth of pollen tubes carrying the 35S:2ox2 transgene was reduced relative to that of nontransgenic pollen, and this phenotype could be reversed partially by GA application in vitro or by combining with spy-5, a mutation that increases GA response. Treatment of wild-type pollen tubes with an inhibitor of GA biosynthesis in vitro also suggested that GAs are required for normal pollen tube growth. These results extend the known physiological roles of GAs in Arabidopsis development and suggest that GAs are required for normal pollen tube growth, a physiological role for GAs that has not been established previously.     

Relationships of pollen size,pistil length and pollen tube growth rates in Rhododendron and their influence on hybridization

Summary Pollen size and pistil length data have been collected for 93 species of Rhododendron (Ericaceae) belonging to a number of different subgeneric taxa. For a sample of eight species in section Vireya, pollen tube growth in the style after selfor interspecific pollination has been quantified. Pollen volume and the time taken for pollen tubes to reach the ovary were both related to pistil length. Pollen-tube growth rates were generally greater for species with longer pistils and larger pollen. Increasing temperature increased the rate of pollen-tube growth. There was no detectable effect of pollen tube density on tube growth rate in the style. After interspecific pollinations tube growth rates in foreign styles could be faster or slower than in self styles. A semisterile individual with two viable pollen grains per tetrad and a plant grafted as scion to a longer-styled stock both showed more rapid pollen-tube growth than expected on the basis of pistil size. Data collected for 26 species in section Vireya showed that where extreme disparity of pollen/pistil size causes failure of interspecific crosses, one or more bridging species with intermediate pollen/pistil size can generally be selected.     

alpha4-Fucosyltransferase is regulated during flower development: increases in activity are targeted to pollen maturation and pollen tube elongation

alpha4-Fucosylation represents a final step of protein N- glycosylation. alpha4-fucosylated N-glycans are thought to be involved in cell-to-cell communication and recognition in primates and plants. Nevertheless, in the plant life cycle, the function of alpha4-fucosylation remains largely unknown. To gain an insight into the role of alpha4-fucosylation during development, the study focused on tobacco flowers. It is shown that an increase in alpha(1,4)fucosyltransferase (Fuc-T) activity is only observed during anther development, whereas it remains at a constant but low level (around 20 pmol Fuc h(-1) mg(-1) protein) in the gynoecium and perianth. At least a 4-fold higher activity is detected in mature pollen grains. These data suggest that alpha(1,4)Fuc-T activity is regulated during anther development. Furthermore, alpha(1,4)Fuc-T activity could be required during pollen tube elongation where the activity level peaks at 350 pmol h(-1) mg(-1) protein. Based on enzyme profile and cycloheximide effects on pollen germination and activity, it is hypothesized that the gene encoding alpha4-Fuc-T could be regulated late during pollen development. A potential role of alpha4- fucosylation during pollen tube elongation is also discussed.     

The identification of F actin in the pollen tube and protoplast of Amaryllis belladonna

The production of protoplasts from the pollen of Amaryllis belladonna has facilitated a more direct investigation of the physiological and mechano-chemical basis of streaming. Cytoplasm is removed from an actively streaming protoplast or intact pollen tube and layered on a coated grid in a solution containing a low free calcium ion concentration. Under these conditions 6 nm thin filaments are observed. The thin filaments are morphologically identical with F actin and bind rabbit muscle HMM, forming characteristic arrowhead complexes that are dissociated by subsequent treatment with MgATP.     

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.     

Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce, increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H₂O with ²H₂O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing ²H₂O with H₂O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 μm distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180°. Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.