Imaging elongating pollen tubes by green fluorescent protein

Pollen tube elongation is a dynamic process in which pollen tubes navigate and respond to female tissues to accomplish their mission of delivering the sperm cells for fertilization. The tube growth process itself is driven by regulated intracellular conditions that maintain the appropriate ionic environment, actin dynamics and a balance level of exocytosis and endocytosis to support growth at the tube apex. However, the interactive process within the pistil has not rendered itself accessible for direct observation. The contribution by individual cytosolic constituents of the pollen tube growth machinery remains to be determined. With the development of the green fluorescent protein reporter system, many of these questions can be addressed in live pollen tubes that elongate within the pistil and inchemically defined media. Analyses of the mechanisms that underlie pollen tube growth will be significantly facilitated. Received: 15 March 2001 / Accepted: 30 May 2001     

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     

Dynein-related polypeptides in pollen and pollen tubes

 Microtubules in pollen tubes are evident within the vegetative and generative cell cytoplasm. This observation led to the formulation of several hypotheses regarding the role of microtubules in cytoplasmic movement and the migration of the vegetative nucleus/generative cell along the pollen tube. The study of microtubular motor proteins in pollen tubes followed the discovery and characterization of an immunoreactive homolog of mammalian kinesin in tobacco pollen tubes. Recent identification of dynein-related polypeptides in pollen tubes of Nicotiana tabacum and pollen of Ginkgo biloba is a significant step in the definition of the role of microtubule function within pollen and pollen tubes. Received: 31 May 1996 / Revision accepted: 26 July 1996     

Disruption of actin filaments by latrunculin B affects cell wall construction in Picea meyeri pollen tube by disturbing vesicle trafficking

The involvement of actin filaments (AFs) in vesicle trafficking, cell wall construction and tip growth was investigated during pollen tube development of Picea meyeri. Pollen germination and tube elongation were inhibited in a dose-dependent manner by the latrunculin B (LatB) treatment. The fine AFs were broken down into disorganized fragments showing a tendency to aggregate. FM4-64 labeling revealed that the dynamic balance of vesicle trafficking was perturbed due to F-actin disruption and the fountain-like cytoplasmic pattern changed into disorganized Brownian movement. The configuration and/or distribution of cell wall components, such as pectins, callose and cellulose, as well as arabinogalactan proteins changed in obvious ways after the LatB application. Fourier transform infrared (FTIR) analysis further established significant changes in the chemical composition of the wall material. Our results indicate that depolymerization of AFs affects the distribution and configuration of cell wall components in Picea meyeri pollen tube by disturbing vesicle trafficking.     

The local cytomechanical properties of growing pollen tubes correspond to the axial distribution of structural cellular elements

Morphological studies of pollen tubes have shown that the configuration of structural cellular elements differs between the growing apex and the distal part of the cell. This polarized cellular organization reflects the highly anisotropic growth behavior of this tip growing cell. Accordingly, it has frequently been postulated that physical properties of pollen tubes such as cell wall plasticity should show anisotropic distribution, but no experimental evidence for this has been published hitherto. Using micro-indentation techniques, we quantify pollen tube resistance to lateral deformation forces and analyze its visco-elasticity as a function of distance from the growing apex. Our studies reveal that cellular stiffness is significantly higher at the distal portion of the cell. This part of the cell is also completely elastic, whereas the apex shows a visco-elastic component upon deformation. To relate these data to the architecture of the particular pollen tube investigated in this study, Papaver rhoeas, we analyzed the distribution of cell wall components such as pectin, callose, and cellulose as well as the actin cytoskeleton in this cell using fluorescence label. Our data revealed that, in particular, the degree of pectin methyl esterification and the configuration of the actin cytoskeleton correlate well with the distribution of the physical properties on the longitudinal axis of the cell. This suggests a role for these cellular components in the determination of the cytomechanics of pollen tubes.     

Distribution of calmodulin protein and mRNA in growing pollen tubes

 Pollen tube growth is a vital process for angiosperm fertilisation and is dependent on the presence of a tip-focused gradient of cytosolic free calcium ([Ca²⁺]_c). In order to clarify some of the target molecules which convey the Ca²⁺ signal information, we investigated calmodulin distribution during tube growth. Fluorescently labelled calmodulin was pressure microinjected into pollen tubes and its distribution monitored by confocal microscopy. Calmodulin distributes evenly throughout the cell, but some of its binding sites form a V-shaped collar behind the apical region. This specific association dissipates upon growth arrest, and suggests an interaction of calmodulin with cytoskeletal-bound target proteins. The distribution of calmodulin mRNA was also analysed by microinjection of fluorescently labelled mRNA. No specific pattern was observed, with an even localisation in the body of tube and a lower concentration in the cell apex. Studies with localised application of inhibitors/activators indicate that calmodulin plays a crucial role in tip elongation but does not direct tube orientation. Received: 6 March 1998 / Revision accepted: 20 April 1998     

Antisense perturbation of protein function in living pollen tubes

During the past few years pollen tubes grown in vitro became a popular model system for cell biology studies of signal transduction in plant cells. Here we report a simple and fairly inexpensive way of studying protein function by transiently perturbing expression of the target gene in living pollen tubes. The ability of antisense oligodeoxynucleotides (ODNs) to bind to complementary mRNA sequences was used to selectively inhibit gene expression and thus assess the putative function of specific proteins in tip growth. The delivery of ODNs to growing pollen tubes was accomplished with the help of a liposomal formulation, originally developed for transfection assays in animal cells. The limitations and potentialities of this technique are discussed. Received: 23 November 2000 / Revision accepted: 29 May 2001     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998     

Role of microtubules in the movement of the vegetative nucleus and generative cell in tobacco pollen tubes

The germination and growth of pollen grains of Nicotiana tabacum and N. alata with the anti-microtubule drug oryzalin retarded significantly the movement of the vegetative nucleus (VN) and the generative cell (GC) from the grain to the tube apex but had no effect on pollen tube elongation. In N. tabacum, only 11% and 48% of the pollen tubes treated with oryzalin for 6 h and 12 h, respectively, had the VN and GC in the tube mainly in its middle part. In corresponding control materials, 79% and 99% of pollen tubes contained the VN and GC close to the apex. Indirect immunofluorescence microscopy and related studies of the tubes grown in the presence of oryzalin revealed complete absence of microtubules (MTs) but apparently intact microfilaments (MFs). These results suggested that the movement of VN and GC from the grain into the tube is possible when no MTs but only MFs are present, but the movement is then slow. In control tubes, the parallel orientation of MT bundles and extensions of VN were interpreted to represent the structural organization needed for the MT-dependent movement of VN.     

Dynein heavy chain-related polypeptides are associated with organelles in pollen tubes of Nicotiana tabacum

 It is known that pollen tubes contain two high molecular weight polypeptides which share some biochemical and immunological properties with dynein heavy chains. This paper reports data on the subcellular localization of the two dynein heavy chain-related polypeptides during pollen tube growth. Immunofluoresence studies using a purified antibody (Dy-1) raised against a synthetic peptide reproducting the P-loop conserved sequence of dynein heavy chains showed spot-like structures, with a characteristic distribution pattern that depended on the tube length. Biochemical evidence confirmed the presence of dynein heavy chain-related bands in the pollen tube membrane fraction. The association of proteins carrying dynein heavy chain-related polypeptides to cell membranes was affected by detergent (Triton×100), whereas other stripping agents, like NaCl and Na₂CO₃, did not significantly influence the interaction of dynein heavy chain-related doublet with their cytoplasmic targets. These data suggest that dynein heavy chain-related polypeptides associate with membranous organelles within the vegetative cell of Nicotiana tabacum pollen tubes, implying their involvement in the cytoplasmic distribution of these organelles. Received: 22 May 1997 / Revision accepted: 11 November 1997     

Adhesion of lily pollen tubes on an artificial matrix

 We proposed that pollination in lily is a case of cell adhesion and cell movement, but experimental evidence for the adhesion event is lacking. In this study, we developed an artificial extracellular matrix that mimics the in vivo lily stylar transmitting tract. This artificial matrix was created by applying the transmitting tract exudate extracted from lily styles onto a nitrocellulose membrane. When in vitro-grown pollen tubes were applied to the matrix, they adhered by their tips to the area of the stylar exudate which is rich in arabinogalactan proteins. Once they adhered, they grew on the in vitro artificial matrix at rates faster than normal. This is the first experimental evidence demonstrating the adhesion of in vitro-grown pollen tubes, an event that has been described as common in vivo. The adhesion event is stylar exudate specific, concentration dependent, and is affected by the developmental age of the pollen tube. This bioassay for pollen tube adhesion will be used to isolate the adhesive molecules from the stylar exudate. Received: 9 December 1996 / Revision accepted: 5 May 1997     

Behavior of storage lipids during development and germination of olive ( Olea europaea L.) pollen

Summary.  The presence of abundant oil bodies in the mature olive pollen grain has led us to focus on the behavior of these lipid bodies during pollen development and in vitro pollen germination. The appearance, increase, and accumulation of lipid bodies have been determined by following the sequential development of the pollen grain. Semithin slices of anthers and pollen grains were stained with Sudan Black B in order to identify neutral lipids. Ultrastructural studies were also carried out. Our results show a notable increase in lipid bodies between the young-pollen-grain stage and the mature-pollen-grain stage. Substantial polarization of lipid bodies was observed after 1 or 2 h of pollen incubation in germination medium. During pollen tube growth, the lipid bodies are located near the germinative aperture after 3 h of incubation, as well as inside the pollen tube, thus suggesting that the lipid bodies move from the pollen grain to the pollen tube. After 7 h of germination the presence of lipid bodies inside the pollen tube is no longer substantial. Our results support the idea that lipid bodies are involved in pollen germination, stigma penetration, and pollen tube growth. These results are discussed in connection with their implications for the pollen germination process. Received June 4, 2002; accepted October 29, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain.     

Role of a callose synthase zymogen in regulating wall deposition in pollen tubes of Nicotiana alata Link et Otto

The callose synthase (CalS) activity of membrane preparations from cultured Nicotiana alata Link & Otto pollen tubes is increased several-fold by treatment with trypsin in the presence of digitonin, possibly due to activation of an inactive (zymogen) form of the enzyme. Active and inactive forms of CalS are also present in stylar-grown tubes. Callose deposition was first detected immediately after germination of pollen grains in liquid medium, at the rim of the germination aperture. During tube growth the 3-linked glucan backbone of callose was deposited at an increasing rate, reaching a maximum of 65 mg h⁻¹ in tubes grown from 1 g pollen. Callose synthase activity was first detected immediately after germination, and then also increased substantially during tube growth. Trypsin caused activation of CalS throughout a 30-h time course of tube growth, but the degree of activation was higher for younger pollen tubes. Over a 10-fold range of callose deposition rates, the assayed CalS activity was sufficient to account for the rate of callose deposition without trypsin activation, implying that the form of CalS active in isolated membranes is responsible for callose deposition in intact pollen tubes. Sucrose-density-gradient centrifugation separated a lighter, intracellular membrane fraction containing only inactive CalS from a heavier, plasma-membrane fraction containing both active and inactive CalS, with younger pollen tubes containing relatively more of the inactive intracellular enzyme. The increasing rate of callose deposition during pollen-tube growth may thus be caused by the transport of inactive forms of CalS from intracellular membranes to the plasma membrane, followed by the regulated activation of these inactive forms in this final location. Received: 1 December 1998 / Accepted: 21 January 1999     

Ultrastructural immunolocalization of periodic pectin depositions in the cell wall ofNicotiana tabacum pollen tubes

Summary The monoclonal antibody (MAb) JIM5, marking acidic pectins, was used to localize ultrastructurally pectin molecules in the pollen tube wall ofNicotiana tabacum. Longitudinal sections of LR-White embedded pollen tubes were exposed to antibody treatment; accumulations of pectins were identified by counting the density of the gold particles representing the pectin epitopes along the pollen tube wall. Significant accumulations of gold grains were marked and the distances between them were measured. In many pollen tubes a more or less regular distribution of the accumulations was observed along the tube indicating a periodical deposition of pectin. The distances between the accumulations were 4–6 m. Most of the label was found in the inner part of the outer layer of the bilayered cell wall. These findings correspond to and confirm the earlier observation by our group reporting ring-shaped periodical deposits in pollen tubes after immunofluorescence labelling with the MAb JIM5 under the confocal laser scanning microscope.Abbreviations Ab antibody - MAb monoclonal antibody     

Reversible protein phosphorylation regulates the dynamic organization of the pollen tube cytoskeleton: effects of calyculin A and okadaic acid

We investigated the cytoskeleton of Lilium longiflorum pollen tubes and examined the effects of the type 2A protein phosphatase (PP2A) inhibitors calyculin A and okadaic acid. An improved method for actin visualization, the simultaneous fixation and staining with rhodamine-labelled phalloidin during microscopical observation, revealed abundant actin filaments of no preferential orientation in the apical clear zone. Microtubules, visualized by indirect immunofluorescence, were mostly absent from the apices of straight-growing pollen tubes but present in those with irregular shape. Double labelling showed that both actin bundles and microtubules had a similar longitudinal or slightly helical orientation in the pollen tube shaft. In the presence of 30 nM calyculin A or okadaic acid, pollen tubes grew very slowly, branched frequently, and contained isolated, randomly oriented, curved actin bundles and microtubules. Treating pollen tubes with calyculin A or okadaic acid after germination arrested growth immediately, reversibly altered the alignment of actin bundles from axial to transverse, and disassembled microtubules. The changes in actin organization caused by the PP2A inhibitors were similar to those observed upon overexpression of AtRop1 (Y. Fu, G. Wu, Z. Yang, Journal of Cell Biology 152: 1019-1032, 2001), suggesting that hyperphosphorylation interferes with the signalling pathway of small GTPases. The effects of the PP2A inhibitors could be ameliorated with nanomolar concentrations of latrunculin B.     

In vitro fertilization from co-cultured pollen tubes and female gametophytes of Douglas fir (Pseudotsuga menziesii)

 Our previous attempt on in vitro fertilization (IVF) in conifers resulted in pollen tube penetration of female gametophytes, but because of the rapid decline in egg viability, no further interaction occurred. In this report, we describe for the first time that IVF has been achieved in conifers. Using Douglas fir (Pseudotsuga menziesii), we describe a two-step process which involved induction of pollen tubes in culture followed by introduction of isolated female gametophytes at the tips of growing pollen tubes. Pollen tubes penetrated the introduced isolated female gametophytes at various places, but a number of tubes entered the egg cell through the neck cells similar to the in vivo condition. Under our current culture conditions, longevity of pollen tubes and eggs has been improved resulting in the release of sperms, fusion of gametes, and initial formation of the proembryo. Continued plasmolysis of the egg limited the number of successful gametic interactions. IVF has been accomplished in flowering plants in several ways, but the gametophyte-gametophyte IVF system described in this paper is unique. IVF offers a novel breeding technology that takes advantage of the sexual reproductive route. When coupled with hybridization and genetic transformation, IVF could result in the development of stable novel genotypes of economically superior trees. Received: 28 October 1997 / Accepted: 9 December 1997     

Quantitative analysis of the distribution of organelles in tobacco pollen tubes: implications for exocytosis and endocytosis

Summary The present study provides the first quantitative analysis on the distribution of organelles in pollen tubes ofNicotiana tabacum L. Organelles were studied on living pollen tubes by means of fluorescence confocal laser scanning microscopy and on cryo-fixed, freeze-substituted and serially sectioned material by electron microscopy. In the tip a 300 nm to 400 nm thick wall was secreted that proximately gradually separated into a wall with an opaque inner side and a more translucent, layered outer side. Tubular endoplasmic reticulum was particularly abundant in the tip of the tube, surrounding the region where secretory vesicles (SV) accumulated. Mitochondria were randomly distributed throughout the cytoplasm, no accumulations were present. Dictyosomes, however, showed an increased abundance at 25–30 m behind the tip. The accumulation of coated pits (CP) in a zone 6–15 m behind the tip identifies this zone as the major site of endocytosis: 50% of all CP occur in this zone. Quantification of exo- and endocytosis showed that only part of the membrane material of the SV can be retrieved after exocytosis. The typical zonation in endocytotic activity may serve to maintain a difference in membrane protein composition between the tip and the tube.     

Involvement of microtubules in rhizoid differentiation of Spirogyra species

Summary.  Some species of Spirogyra form rosette-shaped or rod-shaped rhizoids in the terminal cell of the filaments. In the present study, we analyzed an involvement of microtubules (MTs) in rhizoid differentiation. Before rhizoid differentiation, cortical MTs were arranged transversely to the long axis of cylindrical cells, reflecting the diffuse growth. At the beginning of rhizoid differentiation, MTs were absent from the extreme tip of the terminal cell. In the other area of the cell, however, MTs were arranged transversely to the long axis of the cell. In the fully differentiated rosette-shaped rhizoid, MTs were randomly organized. However, at a younger stage of rosette-shaped rhizoids, MTs were sometimes arranged almost transversely in the lobes of the rosette. In the rod-shaped rhizoid, MTs were arranged almost transversely. MT-destabilizing drugs (oryzalin and propyzamide) induced swelling of rhizoids, and neither rosette-shaped nor rod-shaped rhizoids were formed. The role of MTs in rhizoid differentiation was discussed. Received June 17, 2002; accepted November 11, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678-1297, Japan.     

Selective inhibition of the growth of incompatible pollen tubes by S-protein in the Japanese pear

The S-allele-associated proteins (S-proteins) in the styles of the Japanese pear (Pyrus serotina Rehd. var. culta Rehd.) were purified by cation exchange chromatography. Their inhibitory action on the growth of incompatible pollen tubes (pollen tubes bearing the same S- allele as in the style from which the S-proteins were prepared) was characterized in vitro. Germination and tube growth of self-pollen (pollen from the same cultivar from which the S-proteins were prepared) decreased dose-dependently when the S-protein was added to the medium. Tube length was reduced to 10% that of compatible pollen tubes (pollen tubes bearing the S-allele different from that in the style from which the S-proteins were prepared) at 1.5 μg μl¹. S-proteins from Shinsui (S ₄ S ₅ ) also inhibited growth of cross-incompatible Kosui (S ₄ S ₅ ) pollen tubes, but not of compatible Chojuro (S ₂ S ₃ ) pollen tubes. After inactivation of RNase of the S- protein, the inhibitory action of the S-protein disappeared. These results indicate that the S-protein acts directly to inhibit growth of incompatible pollen tubes in Japanese pear styles, and that the RNase activity of the protein is essential for the biological function. However, small amounts of proteins that co-migrated with the S-protein may also play some roles in the inhibition. This is the first report on the selective inhibitory action of S-proteins in Rosaceae. Received: 11 April 2000 / Revision accepted: 28 September 2000