The anti-microtubule drug carbetamide stopsNicotiana sylvestris pollen tube growth in the style

Summary Recently, we found that the anti-microtubule drugs colchicine and propham caused the absence of microtubules and thus loss of cytoplasmic zonation in in vitro growing pollen tubes ofNicotiana sylvestris, but did not seriously affect growth. In the present study we used the herbicide carbetamide as an anti-microtubule drug. It had the same effect as colchicine and propham: the cytoplasm, including the generative cell, was no longer concentrated in the tip but was distributed randomly. In addition, ultrastructural investigations have shown that even the vesicle zone, usually found at the very tip of pollen tubes, had disappeared in some tubes. Nonetheless, in vitro growth was not inhibited by more than 20% over a period of 22 h.In contrast, tube growth in plants ceased 1 cm down in the style when carbetamide was applied to the stigma before pollination. At the lowest concentration causing this effect, microtubules of the vegetative cell had disappeared and the cytoplasm was distributed randomly, as it was for in vitro grown tubes. It can be concluded that microtubules of the vegetative cell are essential for pollen tube growth in the style.Abbreviations DAPI 4,6-diamidmo-2-phenylindole - EGTA ethyleneglycerol-bis-(aminoethyl ether) tetraacetic acid - DIC differential interference contrast - GC generative cell - IC₅₀ inhibition concentration 50% - MF microfilament - MT microtubule - PEM-buffer 50 mM PIPES 1 mM EGTA, 2 mM MgSO₄, pH 6.9 - PBS phosphate buffered saline - PIPES piperazine-bis-ethanesulphonic acid - PTG-Test pollen tube growth test - SAM substrate adhesion molecule - VC vegetative cell     

Ultrastructural investigations on Lycopersicum peruvianum pollen activation and pollen tube organization after self-and cross-pollination

No differences have been observed in vivo between Lycopersicum peruvianum compatible and incompatible pollen during activation and pollen tube emission and organization, that is until 4 h and 30 min after pollination. During pollen activation the main events are the setting free of rough endoplasmic reticulum (RER) cisterns which were stacked in the mature pollen, the increase in the number of polysomes, and a great activity of the dictyosomes. Immediately after germination of the vegetative nucleus and the generative cell move into the tube, the generative cell diviting to form the male gametes; the tube then becomes organized in four zones. This series of changes is similar to what has already been observed in vitro except that in vitro the generative cell remains undivided and the whole process from seeding to tube organization takes 3 h instead of 4 h and 30 min after pollination, as it does in vivo. Our findings are compatible with the main models of the tube inhibition mechanism proposed till now.Abbreviations RER rough endoplasmic reticulum - GC generative cell - VN vegetative nucleus - GP germinative pore Research performed under C.N.R. (Italian National Research Council) program Biology of Reproduction     

Microtubule organization in sperm cells in the pollen tubes ofBrassica oleracea L.

Summary Microtubules tightly cross-linked into bundles are described in the sperm cells ofBrassica oleracea pollen tubes. The sperm cells are lobed and tailed and the microtubule bundles are often located in these parts of the cells. In the present paper we suggest that the cross-linked microtubule organization could determine an intertubular sliding, probably generating a motility system that propels the sperm cells through the tube.Abbreviations GC generative cell - Mfs microfilaments - Mts microtubules - SC sperm cell - VC vegetative cell - VN vegetative nucleus     

Microtubules in pollen tube subprotoplasts: organization during protoplast formation and protoplast outgrowth

Summary Microtubules inNicotiana tabacum pollen tube subprotoplasts reassembled in wave-like to concentric cortical arrays. Crosslinks between microtubules were either 15 or 80 nm in length. Cortical actin filaments showed different distributions; no colocalization like that in pollen tubes was observed. Degradation of actin filaments by cytochalasin D had no influence on microtubule organization. Degradation of microtubules and/or actin filaments did not affect outgrowth of the subprotoplasts. Organization of the microtubules occurred independent of the presence of the generative cell and/or the vegetative nucleus. No relation of actin filament and microtubule organization with organelle distribution could be detected.Abbreviations AFs actin filaments - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol bis (2-amino ethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MTs microtubules - SPPs subprotoplasts - TRITC tetramethyl rhodamine B isothiocyanate     

γ-Tubulin in tobacco pollen tubes: association with generative cell and vegetative microtubules

-Tubulin was localized in tobacco pollen tubes using an antibody raised against a peptide conserved in all known -tubulins. Antibody staining occurs in a primarily punctate pattern along the length of the microtubule bundles in generative cells and along cortical microtubules in the vegetative cytoplasm. During generative cell division, -tubulin is localized in the forming mitotic apparatus. By metaphase, it is present along kinetochore fibers except at their plus ends located at the kinetochores. By telophase, staining is observed in the phragmoplast, where it again avoids the plus ends of microtubules at the cell plate. -Tubulin is also present at the periphery of the sperm nuclei. A patch of intense staining on the distal side of each nucleus marks the site of assembly of a new population of sperm microtubules. No specific fluorescence is present in control pollen tubes treated with preimmune IgG. These localization patterns bear similarities to those seen in somatic cells and in addition may help explain changes in microtubule arrays between generative cells and sperm.     

The arrangement of F-actin and microtubules during germination of Mucor rouxii sporangiospores

The distribution of F-actin microfilaments and microtubules was analyzed in germinating sporangiospores of Mucor rouxii by labeling with rhodamine-tagged phalloidin and by immunofluorescence microscopy. The transition from isodiametrical to apical growth was accompanied by a switch from uniform distribution of F-actin patches to a polarized accumulation of F-actin material at the germ tube tips. Immunoblotting of cell-free extracts of M. rouxii with a monoclonal anti-porcine -tubulin antibody (TU-01) disclosed two discrete bands of -tubulin suggesting the existence of two -tubulin genes in this fungus. Immunofluorescence microscopy of germinating cells stained with the same antibody revealed an elaborate network of cytoplasmic microtubules that persisted during the entire germination process and extended into the apex of the germ tube. Although their precise roles remain undetermined, the observed arrangement of cytoskeletal elements during germination is consistent with their presumed involvement in cell wall morphogenesis: the long axial microtubules serving as long-distance conveyors of wall-building vesicles to the apical region while the concentrated F-actin patches mark the participation of microfilaments in the zone of intense vesicle exocytosis at the hyphal apex.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DTT dithiothreitol - EGTA Ethylene glycol-bis (beta-aminoethyl ether) - N,N,N,N tetraacetic acid - F-actin Filamentous actin - MES 2-(N0morpholino)-ethanesulfonic acid - PIPES Piperazine-N,N-bis-2-ethanesulfonic acid - PMSF Phenyl-methylsulphonyl fluoride - TBS Tris-buffered saline     

Ultrastructure of freeze-substituted pollen tubes ofLilium longiflorum

Summary In view of the importance of the lily pollen tube as an experimental model and the improvements in ultrastructural detail that can now be attained by the use of rapid freeze fixation and freeze substitution (RF-FS), we have reexamined the ultrastructure of these cells in material prepared by RF-FS. Several previously unreported details have been revealed: (1) the cytoplasm is organized into axial slow and fast lanes, each with a distinct structure; (2) long, straight microtubule (MT) and microfilament (MF) bundles occur in the cytoplasm of the fast lanes and are coaligned with every organelle present; (3) the cortical cytoplasm contains complexes of coaligned MTs, MFs, and endoplasmic reticulum (ER); (4) the cortical ER is arranged in a tight hexagonal pattern and individual elements are closely appressed to the plasma membrane with no space between; (5) mitochondria and ER extend into the extreme apex along the flanks of the pollen tube, and vesicles and ER are packed into an inverted cone-shaped area at the center of the apex; (6) MF bundles in the tip region are fewer, finer, and in random orientation in comparison to those of the fast lanes; (7) the generative cell (GC) cell wall complex contains patches of plasmodesmata; (8) The GC cytoplasm contains groups of spiny vesicles that are closely associated with and seem to be fusing with or pinching off from mitochondria, and (9) the vegetative nucleus (VN) contains internal MT-like structures as well as numerous cytoplasmic MTs associated with its membrane and also located between the VN and GC.Abbrevations CF chemical fixation - ER endoplasmic reticulum - GC generative cell - MF microfilament - MT microtubule - PD plasmodesmata - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution - VN vegetative nucleus     

Partial purification of myosin from lily pollen tubes by monitoring with in vitro motility assay

Summary Myosin in pollen tubes ofLilium longiflorum was partially purified, using an in vitro motility assay as a monitor. The main components in the partially purified preparation had molecular masses of 110, 120, and 140 kDa in SDS-PAGE. They became bound to actin filaments in an ATP-dependent manner. Among the components, only that of 120 kDa became bound to ATP and was concluded to be the heavy chain of pollen tube myosin.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EB extraction buffer - EGTA ethyleneglycol-bis-(-aminoethylether) N, N, N, N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate - TBS Tris buffered saline - TEB Tris-EGTA buffer     

Juxtaposition of the generative cell and vegetative nucleus in the mature pollen grain of amaryllis (Hippeastrum vitatum)

Summary Computer-generated, three-dimensional reconstructions from serial ultrathin sections were used to investigate the spatial organization and extent of association between the generative cell and vegetative nucleus within the mature pollen grain of amaryllis. In all cases examined, the highly lobed vegetative nucleus was found in close proximity and positioned laterally to the elongated, oval shaped generative cell. Numerous projections of the vegetative nucleus come to within 53 nm of the inner vegetative cell plasma membrane which surrounds the generative cell. These areas of close association may continue transversely around the generative cell for a distance of up to 4 m. Although an association exists between the generative cell and vegetative nucleus of the mature pollen grain, it is apparent that several changes must take place after pollination in order to achieve the high amount of close contact that occurs between the vegetative nucleus and the numerous terminal cell extensions of the leading sperm in the pollen tube of amaryllis (Mogensen 1986). Thus, this study demonstrates that the spatial organization among components of the male germ unit in the mature pollen grain does not necessarily reflect relationships that ultimately exist among these components within the pollen tube.     

Characterization of the translocator associated with pollen tube organelles

Summary In pollen tubes, the motive force of cytoplasmic streaming is assumed to be generated by the sliding of the translocator associated with cell organelles along actin filaments. In the present study, the characteristics of the translocator were studied by reconstituting the movement of pollen tube organelles along characean actin bundles. Movement of pollen tube organelles proceeded from the pointed end to the barbed end of the actin filaments of the characean cells. The reconstituted movement was not inhibited by vanadate. KCL at higher concentrations inhibited the movement. Furthermore, heavy meromyosin (HMM) prepared from rabbit skeletal muscle myosin partially inhibited the reconstituted movement and pCMB-modified HMM inhibited it completely. The present results strongly support our previous conclusion that the translocator which generates the motive force of cytoplasmic streaming in pollen tube is myosin.Abbreviations AMP-PNP adenylyl-imidodiphosphate - ATP adenosine-5-triphosphate - ATP--S adenosine-5-0-(3-thiotriphosphate) - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - HB homogenization buffer - HMM heavy meromyosin - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PPi pyrophosphate     

Pollen development in orchids 4. Cytoskeleton and ultrastructure of the unequal pollen mitosis inPhalaenopsis

Summary The unequal first mitosis in pollen ofPhalaenopsis results in a small generative cell cut off at the distal surface of the microspore and a large vegetative cell. No preprophase band of microtubules is present, but polarization of the microspore prior to this critical division is well marked. A generative pole microtubule system (GPMS) marks the path of nuclear migration to the distal surface, and the organelles become unequally distributed. Mitochondria, plastids and dictyosomes are concentrated around the vegetative pole in the center of the microspore and are almost totally excluded from the generative pole. The prophase spindle is multipolar with a dominant convergence center at the GPMS site. The metaphase spindle is disc-shaped with numerous minipoles terminating in broad polar regions. In anaphase, the spindle becomes cone-shaped as the spindle elongates and the vegetative pole narrows. These changes in spindle architecture are reflected in the initial shaping of the telophase chromosome groups. F-actin is coaligned with microtubules in the spindle and is also seen as a network in the cytoplasm. An outstanding feature of orchid pollen mitosis is the abundance of endoplasmic reticulum (ER) associated with the spindle. ER extends along the kinetochore fibers, and the numerous foci of spindle fibers at the broad poles terminate in a complex of ER.Abbreviations CLSM confocal laser scanning microscope/microscopy - DMSO dimethyl sulfoxide - ER endoplasmic reticulum - FITC fluorescein isothiocyanate - GPMS generative pole microtubule system - MBS m-maleimidobenzoic acidN-hydroxysuccinimide ester - PPB preprophase band of microtubules - RhPh rhodamine palloidin - TEM transmission electron microscope/microscopy     

Cytoplasmic streaming in the cell model ofNitella

Summary The plasmalemma ofNitella internode was made freely permeable to solutes by treating the cell with detergent and EGTA under plasmolysis. After the treatment, the cytoplasmic streaming was stopped by bathing the cell in a medium lacking ATP. The streaming was reactivated by perfusing the exterior of the permeabilized cell with a medium containing both Mg²⁺ and ATP. The reactivated streaming could be reversibly stopped by depletion of ATP. However, depletion of Mg²⁺ irreversibly inhibited the streaming.Cytochalasin B at 5 g/ml irreversibly inhibited the reactivated streaming within a minute, showing that microfilaments are involved in the streaming.Abbreviations ATP adenosine-5-triphosphoric acid - CB cytochalasin B - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DMSO dimethylsulfooxide - DTT dithiothreitol - EGTA ethyleneglycol-bis(-aminoethylether)-N,N tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl-sulfonylfluoride     

The organization of microtubules during generative-cell division inConvallaria majalis

Summary The organization of the microtubule cytoskeleton in the generative cell ofConvallaria majalis has been studied during migration of the cell through the pollen tube and its division into the two sperm cells. Analysis by conventional or confocal laser scanning microscopy after tubulin staining was used to investigate changes of the microtubule cytoskeleton during generative-cell migration and division in the pollen tube. Staining of DNA with 4,6-diamidino-2-phenylindole was used to correlate the rearrangement of microtubules with nuclear division during sperm cell formation. Before pollen germination the generative cell is spindle-shaped, with microtubules organized in bundles and distributed in the cell cortex to form a basketlike structure beneath the generative-cell plasma membrane. During generative-cell migration through the pollen tube, the organization of the microtubule bundles changes following nuclear division. A typical metaphase plate is not usually formed. The generative-cell division is characterized by the extension of microtubules concomitant with a significant cell elongation. After karyokinesis, microtubule bundles reorganize to form a phragmoplast between the two sperm nuclei. The microtubule organization during generative-cell division inConvallaria majalis shows some similarities but also differences to that in other members of the Liliaceae.Abbreviations CLSM confocal laser scanning microscopy - EM electron microscopy - GC generative cell - GN generative nucleus - MT microtubule - SC sperm cell - SN sperm nucleus - VN vegetative nucleus     

Centromere sizes,positions, and movements in the interphase nucleus

Each microspore of the onion Allium fistulosum (n=8) has 8 chromosomes. It is shown that in the microspore the 8 centromeres aggregate to form 2 or 3 centromeric structures. Subsequently, at early mitotic prophase, these aggregates are resolved into 8 separate centromeres and each becomes structurally double. After mitosis the pollen grain contains 2 nuclei, each with 8 separate and distinct centromeres, clustered at the nuclear envelope. As interphase progresses the centromeres of the vegetative nucleus are no longer at the nuclear envelope and they aggregate into 3 or 4 centromeric masses. In the generative nucleus there is less movement. The interphase centromere movements occur in the absence of microtubules. The centromeres range in size from about 0.10 to 0.17 m³ with an average of 0.14 m³ per centromere.     

Isolation and characterization of plant myosin from pollen tubes of lily

Summary A plant myosin was isolated from pollen tubes of lily,Lilium longiflorum. Pollen tubes were homogenized in low ionic strength solution containing casein, and myosin from this crude extract was purified by co-precipitation with F-actin prepared from chicken breast muscle, followed by hydroxylapatite column and gel filtration column chromatography. Upon SDS-PAGE on 6% polyacrylamide gel, only 170 kDa polypeptide was detected in the purified myosin fraction. Furthermore, with immunoblotting using antiserum raised against 170 kDa polypeptide, only the 170 kDa component crossreacted in the crude sample of pollen tube proteins. This antiserum did not crossreact with the heavy chain of skeletal muscle myosin. The ATPase activity of pollen tube myosin was stimulated up to 60-fold by F-actin prepared from chicken breast muscle. The translocation velocity of rhodamine-phalloidin-labeled F-actin on a glass surface covered with pollen tube myosin ranged from 6.0 to 9.8 m/s with an average of 7.7 m/s. This velocity was similar to or a little faster than that of the cytoplasmic streaming that occurred in pollen tubes. These results suggested that myosin composed of a 170 kDa heavy chain produces the motive force for cytoplasmic streaming in pollen tube of lily.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazin-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate     

Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes ofNicotiana alata

Summary The ultrastructure of the cytoskeleton inNicotiana alata pollen tubes grownin vitro has been examined after rapid freeze fixation and freeze substitution (RF-FS). Whereas cytoplasmic microtubules (MTs) and especially microfilaments (MFs) are infrequently observed after conventional chemical fixation, they occur in all samples prepared by RF-FS. Cortical MTs are oriented parallel to the long axis of the pollen tube and usually appear evenly spaced around the circumference of the cell. They are always observed with other components in a structural complex that includes the following: 1. a system of MFs, in which individual elements are aligned along the sides of the MTs and crossbridged to them; 2. a system of cooriented tubular endoplasmic reticulum (ER) lying beneath the MTs, and 3. the plasma membrane (PM) to which the MTs appear to be extensively linked. The cortical cytoskeleton is thus structurally complex, and contains elements such as MFs and ER that must be considered together with the MTs in any attempt to elucidate cytoskeletal function. MTs are also observed within the vegetative cytoplasm either singly or in small groups. Observations reveal that some of these may be closely associated with the envelope of the vegetative nucleus. MTs of the generative cell, in contrast to those of the vegetative cytoplasm, occur tightly clustered in bundles and show extensive cross-bridging. These bundles, especially in the distal tail of the generative cell, are markedly undulated. MFs are observed commonly in the cytoplasm of the vegetative cell. They occur in bundles oriented predominantly parallel to the pollen tube axis. Although proof is not provided, we suggest that they are composed of actin and are responsible for generating the vigorous cytoplasmic streaming characteristic of living pollen tubes.Abbreviations EGTA ethylene glycol bis-(-aminoethyl ether), N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - MF microfilament - MT microtubule - PEG polyethylene glycol - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution     

Different effects of inorganic and triethyl lead on growth and ultrastructure of lily pollen tubes

Summary Pollen tubes ofLilium longiflorum growingin vitro were treated for 1 h with inorganic lead (Pb) and with triethyl lead (TriEL) and studied by light and electron microscopy. Pb was considerably more toxic in relation to inhibition of pollen tube growth (EC₅₀=6 M Pb) than was TriEL (EC₅₀=60 M TriEL). On the other hand, at almost the entire concentration range tested (25-500 M) TriEL caused aberrant tubes and tube swellings. Pb did not cause tube swellings, even at highly growth-impairing concentrations. Pb (60 M) predominantly affected the ultrastructure of the growing cell walls without impairing the distribution of the cell organelles in the tube tips. In contrast, 50 and 100 M TriEL did not visibly influence cell wall ultrastructure but it severely damaged dictyosomes; 100 M TriEL also disturbed the original order of cell organelles in the tube tips. Cortical microtubules were selectively and completely destructed by TriEL at concentrations (50 M) where no effect on polar organization of the tube tips occurred but they remained unimpaired by 60 M Pb, indicating selective and effective interaction of TriEL with these cell organelles.Abbreviations EC⁵⁰ effective lead concentration causing 50% inhibition of pollen tube growth - MTs microtubules - Pb inorganic lead - TriAL trialkyl lead - TriEL triethyl lead     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

The actin microfilament network within elongating pollen tubes of the gymnospermPicea abies (Norway spruce)

Summary Actin microfilaments form a dense network within pollen tubes of the gymnosperm Norway spruce (Picea abies). Microfilaments emanate from within the pollen grain and form long, branching arrays passing through the aperture and down the length of the pollen tube to the tip. Pollen tubes are densely packed with large amyloplasts, which are surrounded by branching microfilament bundles. The vegetative nucleus is suspended within the elongating pollen tube within a complex array of microfilaments oriented both parallel to and perpendicular with the growing axis. Microfilament bundles branch out along the nuclear surface, and some filaments terminate on or emanate from the surface. Microfilaments in the pollen tube tip form a 6 m thick, dense, uniform layer beneath the plasma membrane. This layer ensheathes an actin depleted core which contains cytoplasm and organelles, including small amyloplasts, and extends back 36 m from the tip. Behind the core region, the distinct actin layer is absent as microfilaments are present throughout the pollen tube. Organelle zonation is not always maintained in these conifer pollen tubes. Large amyloplasts will fill the pollen tube up to the growing tip, while the distinct layer of microfilaments and cytoplasm beneath the plasma membrane is maintained. The distinctive microfilament arrangement in the pollen tube tips of this conifer is similar to that seen in tip growth in fungi, ferns and mosses, but has not been reported previously in seed plants.