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.     

Microtubules are involved in maintaining the cellular polarity in pollen tubes ofNicotiana sylvestris

Summary The polarity of a growing pollen tube is clearly reflected by a distinct zonation of the cytoplasmic content. The vegetative nucleus and the generative cell (GC) are located in the tip region of the tube, and the basal cytoplasmic portion is highly vacuolated. Using pollen tubes ofNicotiana sylvestris Spegazz. & Comes grown in vitro, we examined the effects of varying concentrations of the microtubule inhibitors colchicine and propham. The depolymerization of the cortical microtubules by 25 M colchicine led to a disorganization of the cytoplasm, i.e., vacuolization of the tip region, and to a deranged position of both the vegetative nucleus and the generative cell. The same concentration of colchicine inhibited tube growth by 10–20% of the control. Mitosis of the GC was not affected. Only from concentrations of 200 M the configuration of the GC's microtubules was altered and an inhibition of mitosis was observed. At this concentration the disorganization of the cytoplasm was always reversible, but neither inhibition of mitosis nor derangement of the nuclear positioning was. At 1,800 M colchicine, pollen tube growth was inhibited by 50% of the control. Using propham, the same three steps of action were observed, although propham proved to be about a hundred times more effective than colchicine. We conclude that the cortical microtubules of the pollen tube are involved in maintaining cellular polarity, probably as a part of a heterogeneous cytoskeletal network including also microfilaments and membranous elements. Nuclear positioning seems to be dependent on both, the tube's cortical and the GC's microtubules. A possible involvement of the extracellular matrix in maintaining intracytoplasmic polarity is suggested.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EGTA ethyleneglycol-bis-(aminoethyl ether) tetraacetic acid - GC generative cell - 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 - VN vegetative nucleus Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

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     

Isolation of generative cells and their protoplasts from pollen ofLilium longiflorum

Summary Methods are described for the isolation of large quantities of generative cells and their protoplasts from the pollen ofLilium longiflorum. First, large numbers of pollen protoplasts were enzymatically isolated from immature pollen grains. When they were gently disrupted mechanically, the pollen contents including spindle-shaped generative cells were released. The generative cells were separated from other structures by Percoll density gradient centrifugation. They were nearly spherical, but had a callosic cell wall. The isolated generative cells were then re-treated in enzyme solution to yield authentic protoplasts. The generative cell protoplasts, gametoplasts, were uniform in size and contained a condensed haploid nucleus with relatively little cytoplasm.     

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     

Ultrastructure of the sperms ofPlumbago zeylanica

Summary Male gametes ofPlumbago zeylanica were examined in pollen grains and tubes using light and electron microscopy of chemically and physically fixed tissues, and Nomarski interference microscopy of isolated, living sperm cells. Male gametes are elongate, spindleshaped cells containing a nucleus, mitochondria, ER, ribosomes, vesicles, dictyosomes, probable microfilaments, and a variable number of plastids. In mature pollen grains ofP. zeylanica, the two sperm cells are directly linked; they share a transverse cell wall with plasmodesmata and are enclosed together by the inner vegetative cell plasma membrane. One of these two sperms is also associated with the vegetative nucleus as a consistent feature of pollen grain organization. The basis of this association appears to be a long, narrow projection of the sperm cell (averaging < 1 m wide and about 30 m long) which wraps around the periphery of the vegetative nucleus and occupies embayments of that nucleus. This association is maintained throughout pollen tube growth but becomes less extensive near the completion of tube growth and is severed following tube discharge. The consistent occurrence of the sperm-vegetative nucleus association in pollen grains, tubes and isolated pollen cytoplasm suggests that the two structures may be directly connected, but attempts to visualize this type of connection were unsuccessful. Possibly, the entwining nature and extent of complementary interfaces between vegetative nucleus and sperm may have a role in stabilizing their association. Functionally, the two sperms and vegetative nucleus appear to travel as a linked unit within the pollen tube, possibly increasing the effectiveness of gamete delivery and helping to ensure nearly simultaneous transmission of sperms into the receptive megagametophyte.     

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     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

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.     

土麦冬离体萌发花粉管中生殖细胞与营养核的动态变化

主要报道了土麦冬人工培养萌发花粉管中生殖细胞与营养核的动态变化。多数花粉管中,生殖细胞与营养核贴合后,开始进行有丝分裂,贴合时,营养核略呈弥散状态。在分裂早中期,生殖细胞与营养核分开,从贴合到分开大约经历３－５ｈ,精子形成后,不与营养核连接。ＤＡＰＩ对生殖细胞的有丝分裂有抑制作用。少数花粉管中,生殖细胞核进行无丝分裂,有缢裂和劈裂两种方式。生殖细胞核发生缢裂的花粉管中,未观察到生殖细胞与营养核的贴     

Immunogold localization of arabinogalactan proteins,unesterified and esterified pectins in pollen grains and pollen tubes ofNicotiana tabacum L.

Summary The monoclonal antibodies JIM 5 (against unesterified pectin), JIM 7 (against methyl esterified pectin), MAC 207 (against arabinogalactan proteins, AGPs), and JIM 8 (against a subset of AGPs) were utilized singly or in combinations for immunogold labelling of germinated pollen grains and pollen tubes ofNicotiana tabacum. Pectins were localized in the inline of pollen grain, unesterified pectin being more abundant than the esterified one. AGPs were co-localized with pectin in the inline, but were present preferably close to the plasma membrane. In pollen tubes, AGPs, unesterified and esterified pectins were co-localized in the outer and middle layers of the cell wall. The density of the epitopes was not uniform along the length of the pollen tube, but showed alterations. In the pollen tube tip wall esterified pectin was abundantly present, but not AGPs. In the cytoplasm esterified pectin and AGPs were detected in Golgi derived vesicles, indicating their role in the pathway of the cell wall precursors. In the cell wall of generative cell only AGPs, but no pectins were localized. The co-localization of pectins and AGPs in the cell wall of pollen grain and pollen tube might play an important role, not only in maintenance of the cell shape, but also in cell-cell interaction during pollen tube growth and development.Abbreviations AGP arabinogalactan protein - BSA bovine serum albumin - GA glutaraldehyde - MAb monoclonal antibody - NGS normal goat serum - PFA paraformaldehyde     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

玉竹雄配子的发育——着重阐明质体在生殖细胞和营养细胞中的分布和变化

玉竹(Polygonatum simizui Kitag)小孢子在分裂前,质体极性分布导致分裂后形成的生殖细胞不含质体,而营养细胞包含了小孢子中全部的质体。生殖细胞发育至成熟花粉时期,及在花粉管中分裂形成的两个精细胞中始终不含质体。虽然生殖细胞和精细胞中都存在线粒体,但细胞质中无DNA类核。玉竹雄性质体的遗传为单亲母本型。在雄配子体发育过程中,营养细胞中的质体发生明显的变化。在早期的营养细胞质中,造粉质体增殖和活跃地合成淀粉。后期,脂体增加而造粉质体消失。接近成熟时花粉富含油滴。对百合科的不同属植物质体被排除的机理及花粉中贮藏的淀粉与脂体的转变进行了讨论。     

Computer-assisted reconstruction of the male germ unit in pollen ofBrassica campestris

Summary In mature tricellular pollen of rapeseed,Brassica campestris L., the pair of sperm cells are held together within the common plasma membrane of the vegetative cell and are closely associated with the vegetative nucleus. Serial thin sections were cut of entire sperm cell associations of 7 pollen grains, and 3-dimensional coordinated information obtained by digitization. Precise lengths and placement of the sperm cells and vegetative nucleus in three dimensions were computed and stereoscopic images generated and confirmed by two manually constructed 3-dimensional models. The sperm cell most closely associated with the vegetative nucleus, possessed a long tail, > 10 m in length, that penetrated through a passage in the highly convoluted nucleus. This long tail contained a forked array of microtubules in all 7 grains examined. Arrays of microtubules occurred in the second sperm cell, aligned within plasma-membrane evaginations or ridges.     

Changes in volume,surface area,and frequency of nuclear pores on the vegetative nucleus of tobacco pollen in fresh,hydrated, and activated conditions

The vegetative nucleus (VN) of Nicotiana tabacum L. has been qualitatively and quantitatively studied in fresh, hydrated, and activated pollen. Techniques included the use of optical sectioning by confocal scanning laser microscopy to obtain volume and surface area measurements, and stereoscopic pairs; and freeze-etch electron microscopy to estimate the frequency of nuclear pores per m² in the vegetative nucleus. Several morphological changes were observed to occur in pollen grain nuclei during the early processes of tube growth. In freshly dehisced pollen grain, the vegetative and generative nuclei were side by side, but following hydration and activation of the grain, the elongated generative nucleus became partially surrounded by the vegetative nucleus. It was found that during hydration, the surface area of the vegetative nucleus increased and there was a decrease in the frequency of nuclear pores. The calculated total number of pores remained similar. After activation and pollen-tube growth, the vegetative nucleus retained the same surface area as in the hydrated state but the frequency of nuclear pores decreased; therefore, the calculated total number of pores was significantly lowered. When considered alongside complementary biochemical data, these morphological results indicate that RNA production in the vegetative nucleus decreases following germination.Abbreviations VN vegetative nucleus (nuclei) - GN generativenucleus - GC generative cell - CSLM confocal scanning laser microscope We acknowledge research support by the Biotechnology Action Programm of the Commission of European Communities, and CNR for the fellowship awarded to Dr. Wagner. We would also like to thank Mrs. C. Faleri for the expert technical help.     

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     

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     

Histone H4 acetylation and DNA methylation dynamics during pollen development

Summary The first pollen mitosis results in generative and vegetative cells which are characterised by a striking difference in their chromatin structure. In this study, histone H4 acetylation and DNA methylation have been analysed during pollen development inLilium longiflorum. Indirect immunofluorescence procedures followed by epifluorescence and laser scanning microscopy enabled a relative quantification of H4 acetylation and DNA methylation in microspores, immature binucleate pollen, mature pollen, and pollen tubes. The results show that histone H4 of the vegetative nucleus, in spite of its decondensed chromatin structure, is strongly hypoacetylated at lysine positions 5 and 8 in comparison with both the original microspore nucleus and the generative-cell nucleus. These H4 terminal lysines in the vegetative nucleus are, however, progressively acetylated during the following pollen tube growth. The DNA methylation analysis inversely correlates with the histone acetylation data. The vegetative nucleus in mature pollen grains is heavily methylated, but a dramatic nonreplicative demethylation occurs during the pollen tube development. Changes neither in H4 acetylation nor in DNA methylation have been found during development of the generative nucleus. The results obtained indicate that the vegetative nucleus enters the quiescent state (accompanied by DNA hypermethylation and H4 underacetylation) during the maturation of pollen grain which enables pollen grains a long-term survival without external source of nutrients until they reach the stigma.