Generative Cell Research Articles

ARID1 is required to regulate and reinforce H3K9me2 in sperm cells in Arabidopsis

Heterochromatin de-condensation in companion gametic cells is conserved in both plants and animals. In plants, microspore undergoes asymmetric pollen mitosis (PMI) to produce a vegetative cell (VC) and a generative cell (GC). Subsequently, the GC undergoes pollen mitosis (PMII) to produce two sperm cells (SC). Consistent with heterochromatin de-condensation in the VC, H3K9me2, a heterochromatin mark, is barely detected in VC. However, how H3K9me2 is differentially regulated during pollen mitosis remains unclear. Here, we show that H3K9me2 is gradually evicted from the VC since PMI but remain unchanged in the GC and SC. ARID1, a pollen-specific transcription factor that facilitates PMII, promotes H3K9me2 maintenance in the GC/SC but slows down its eviction in the VC. The genomic targets of ARID1 mostly overlaps with H3K9me2 loci, and ARID1 recruits H3K9 methyltransferase SUVH6. Our results uncover that differential pattern of H3K9me2 between two cell types is regulated by ARID1 during pollen mitosis.

The male germ unit association is independently regulated of GUM in Arabidopsis thaliana.

Cytoplasmic projections (CPs) formed by the generative and sperm cells link the male gametes with the vegetative cell (VC) nucleus, which are required to build the male germ unit (MGU) assemblage in the angiosperm pollen grain. As molecular and genetic controls underlying CP development and formation of the MGU are unknown, it was hypothesized that physical association between germ cells and the VC nucleus might be lost in germ unit malformed (gum) mutants or in those which either block generative cell (GC) division or that additionally prevent gamete differentiation. In vivo, analysis of marked cellular components demonstrated a linkage of sperm cells (SCs) and the VC nucleus in gum mutant alleles despite their increased physical separation. Similarly, for several independent classes of bicellular pollen mutants, undivided GCs were associated with the VC nucleus like GCs in wild-type pollen. We conclude that the early formation of GC CPs to establish the MGU is regulated independently of DUO1-DAZ1 and DUO3 transcription factors as well as cyclin-dependent kinase function (CDKA;1). As the absence of cytoplasmic protrusion was expected in the gum mutants in Arabidopsis, early histological studies reported temporal disappearance of cytoplasmic protrusion in several organisms. Our findings demonstrated the striking importance of live imaging to verify the broad conservation of the persistent MGU contact in all the angiosperms and its important role in successful double fertilization.

Genetic control of generative cell shape by DUO1 in Arabidopsis

Key messageThe main features of generative cell morphogenesis, formation of a cytoplasmic projection and elongation of the GC body, operate through independent genetic pathways.Male gametogenesis in developing angiosperm pollen involves distinctive changes in cell morphogenesis. Re-shaping and elongation of the generative cell (GC) are linked to the formation of a GC cytoplasmic projection connected to the vegetative cell nucleus. Although genetic control of GC morphogenesis is unknown, we suspected the involvement of the germline-specific MYB transcription factor DUO POLLEN1 (DUO1). We used light and fluorescence microscopy to examine male germline development in pollen of wild-type Arabidopsis and in four allelic duo1 mutants expressing introduced cell markers. Our analysis shows that the undivided GC in duo1 pollen forms a cytoplasmic projection, but the cell body fails to elongate. In contrast GCs of cyclin-dependent kinase function mutants, which fail to divide like duo1 mutants, achieve normal morphogenesis. We conclude that DUO1 has an essential role in the elongation of the GC, but DUO1-independent pathways control the development of the GC cytoplasmic projection. The two main features of GC morphogenesis therefore operate through independently regulated genetic pathways.

Proteomic Analysis of Generative and Vegetative Nuclei Reveals Molecular Characteristics of Pollen Cell Differentiation in Lily.

In plants, the cell fates of a vegetative cell (VC) and generative cell (GC) are determined after the asymmetric division of the haploid microspore. The VC exits the cell cycle and grows a pollen tube, while the GC undergoes further mitosis to produce two sperm cells for double fertilization. However, our understanding of the mechanisms underlying their fate differentiation remains limited. One major advantage of the nuclear proteome analysis is that it is the only method currently able to uncover the systemic differences between VC and GC due to GC being engulfed within the cytoplasm of VC, limiting the use of transcriptome. Here, we obtained pure preparations of the vegetative cell nuclei (VNs) and generative cell nuclei (GNs) from germinating lily pollens. Utilizing these high-purity VNs and GNs, we compared the differential nucleoproteins between them using state-of-the-art quantitative proteomic techniques. We identified 720 different amount proteins (DAPs) and grouped the results in 11 fate differentiation categories. Among them, we identified 29 transcription factors (TFs) and 10 cell fate determinants. Significant differences were found in the molecular activities of vegetative and reproductive nuclei. The TFs in VN mainly participate in pollen tube development. In comparison, the TFs in GN are mainly involved in cell differentiation and male gametogenesis. The identified novel TFs may play an important role in cell fate differentiation. Our data also indicate differences in nuclear pore complexes and epigenetic modifications: more nucleoporins synthesized in VN; more histone variants and chaperones; and structural maintenance of chromosome (SMC) proteins, chromatin remodelers, and DNA methylation-related proteins expressed in GN. The VC has active macromolecular metabolism and mRNA processing, while GC has active nucleic acid metabolism and translation. Moreover, the members of unfolded protein response (UPR) and programmed cell death accumulate in VN, and DNA damage repair is active in GN. Differences in the stress response of DAPs in VN vs. GN were also found. This study provides a further understanding of pollen cell differentiation mechanisms and also a sound basis for future studies of the molecular mechanisms behind cell fate differentiation.

DNA methylation dynamics of sperm cell lineage development in tomato.

During the sexual reproduction of higher plants, DNA methylation and transcription are broadly changed to reshape a microspore into two sperm cells (SCs) and a vegetative cell (VC). However, when and how the DNA methylation of SCs is established remains not fully understood. Here we investigate the DNA methylation (5mC) dynamics of SC lineage and the VC in tomato using whole-genome bisulfite sequencing. We find the asymmetric division of the microspore gives its two daughter cells differential methylome. Compared with the generative cell (GC), the VC is hypomethylated at CG sites while hypermethylated at CHG and CHH sites, with the majority of differentially methylation regions targeted to transposable elements (TEs). SCs have a nearly identical DNA methylome to the GC, suggesting that the methylation landscape in SCs may be pre-established following the asymmetric division or inherited from the GC. The random forest classifier for predicting gene and TE expression shows that methylation within the gene body is a more powerful predictor for gene expression. Among all tested samples, gene and TE expression in the microspore may be more predictable by DNA methylation. Our results depict an intact DNA methylome landscape of SC lineage in higher plants, and reveal that the impact of DNA methylation on transcription is variant in different cell types.

Transcriptomics analyses reveal the molecular roadmap and long non-coding RNA landscape of sperm cell lineage development.

Sperm cell (SC) lineage development from the haploid microspore to SCs represents a unique biological process in which the microspore generates a larger vegetative cell (VC) and a smaller generative cell (GC) enclosed in the VC, then the GC further develops to functionally specified SCs in the VC for double fertilization. Understanding the mechanisms of SC lineage development remains a critical goal in plant biology. We isolated individual cells of the three cell types, and characterized the genome-wide atlas of long non-coding (lnc) RNAs and mRNAs of haploid SC lineage cells. Sperm cell lineage development involves global repression of genes for pluripotency, somatic development and metabolism following asymmetric microspore division and coordinated upregulation of GC/SC preferential genes. This process is accompanied by progressive loss of the active marks H3K4me3 and H3K9ac, and accumulation of the repressive methylation mark H3K9. The SC lineage has a higher ratio of lncRNAs to mRNAs and preferentially expresses a larger percentage of lncRNAs than does the non-SC lineage. A co-expression network showed that the largest set of lncRNAs in these nodes, with more than 100 links, are GC-preferential, and a small proportion of lncRNAs co-express with their neighboring genes. Single molecular fluorescence insitu hybridization showed that several candidate genes may be markers distinguishing the three cell types of the SC lineage. Our findings reveal the molecular programming and potential roles of lncRNAs in SC lineage development.

Proteomic analysis reveals the differential histone programs between male germline cells and vegetative cells in Lilium davidii

In flowering plants, male germline fate is determined after asymmetric division of the haploid microspore. Daughter cells have distinct fates: the generative cell (GC) undergoes further mitosis to generate sperm cells (SCs), and the vegetative cell (VC) terminally differentiates. However, our understanding of the mechanisms underlying germline development remains limited. Histone variants and modifications define chromatin states, and contribute to establishing and maintaining cell identities by affecting gene expression. Here, we constructed a lily protein database, then extracted and detailed histone entries into a comprehensive lily histone database. We isolated large amounts of nuclei from VCs, GCs and SCs from lily, and profiled histone variants of all five histone families in all three cell types using proteomics approaches. We revealed 92 identities representing 32 histone variants: six for H1, 11 for H2A, eight for H2B, five for H3 and two for H4. Nine variants, including five H1, two H2B, one H3 and one H4 variant, specifically accumulated in GCs and SCs. We also detected H3 modification patterns in the three cell types. GCs and SCs had almost identical histone profiles and similar H3 modification patterns, which were significantly different from those of VCs. Our study also revealed the presence of multiple isoforms, and differential expression patterns between isoforms of a variant. The results suggest that differential histone programs between the germline and companion VCs may be established following the asymmetric division, and are important for identity establishment and differentiation of the male germline as well as the VC.

Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for "omics" analysis.

The development of sperm cells (SCs) from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput “omics” approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cells of different types, especially generative cells (GCs), from the pollen. Here, we optimized the conditions of tomato pollen germination and pollen tube growth to allow for long-term growth of pollen tubes in vitro with SCs generated in the tube. Using this culture system, we developed methods for isolating GCs, SCs and vegetative cell nuclei (VN) from just-germinated tomato pollen grains and growing pollen tubes and their purification by Percoll density gradient centrifugation. The purity and viability of isolated GCs and SCs were confirmed by microscopy examination and fluorescein diacetate staining, respectively, and the integrity of VN was confirmed by propidium iodide staining. We could obtain about 1.5 million GCs and 2.0 million SCs each from 180 mg initiated pollen grains, and 10 million VN from 270 mg initiated pollen grains germinated in vitro in each experiment. These methods provide the necessary preconditions for systematic biology studies of SC development and differentiation in higher plants.

Comparative Proteomic Analysis of Generative and Sperm Cells Reveals Molecular Characteristics Associated with Sperm Development and Function Specialization

In flowering plants, two sperm cells (SCs) are generated from a generative cell (GC) in the developing pollen grain or growing pollen tube and are then delivered to the embryo sac to initiate double fertilization. SC development and function specialization involve the strict control of the protein (gene) expression program and coordination of diverse cellular processes. However, because methods for collecting a large amount of highly purified GCs and SCs for proteomic and transcriptomic studies from a plant are not available, molecular information about the program and the interconnections is lacking. Here, we describe a method for obtaining a large quantity of highly purified GCs and SCs from just-germinated lily pollen grains and growing pollen tubes for proteomic analysis. Our observation showed that SCs had less condensed chromatin and more vacuole-like structures than GCs and that mature SCs were arrested at the G2 phase. Comparison of SC and GC proteomes revealed 101 proteins differentially expressed in the two proteomes. These proteins are involved in diverse cellular and metabolic processes, with preferential involvement in metabolism, the cell cycle, signaling, the ubiquitin/proteasome pathway, and chromatin remodeling. Impressively, almost all proteins in SCF complex-mediated proteolysis and the cell cycle were up-regulated in SCs, whereas those in chromatin remodeling and stress response were down-regulated. Our data also reveal the coordination of SCF complex-mediated proteolysis, cell cycle progression, and DNA repair in SC development and function specialization. This study revealed for the first time a difference in protein profiles between GCs and SCs.

Gametophytic development of Brassica napus pollen in vitroenables examination of cytoskeleton and nuclear movements

Isolated microspores and pollen suspension of Brassica napus “Topas” cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a “comet-tail” form. These GC “tail” MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a “whole-mount” immunodetection technique and confocal laser-scanning microscopy was essential.Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-011-0287-0) contains supplementary material, which is available to authorized users.

Ultrastructure and cytochemistry of Eucalyptus globulus (Myrtaceae) pollen grain

The morphology, ultrastructure and cytochemistry of Eucalyptus globulus mature pollen were investigated using light (LM), scanning electron (SEM) and transmission electron microscopy (TEM). The pollen morphology is typically myrtaceous with a suite of characters that allows its distinction from the pollen of other Eucalyptus species. The exine consists of a thick endexine and a massive ectexine with hardly distinguishable columellae. The endexine is 2‐layered towards the apertural regions where the inner and spongy‐granulate layer forms a continuous colpus membrane and a thinner pore membrane. Under the pores the intine is 3‐layered forming complex onci. The spindle‐shaped generative cell (GC) is deeply undulated and located in a cup‐shaped depression of the vegetative cell (VC) nucleus. In the dense VC cytoplasm the main storage reserves are lipid bodies and insoluble carbohydrates in the cytosol, although proteins are also present. The most characteristic feature of the VC cytoplasm is the extremely well‐developed rough endoplasmic reticulum (RER), which forms extensive stacks filling large areas of the central cytoplasm. Single RER cisternae are also scattered throughout the cytoplasm, most of them establishing an intimate association with lipid bodies, storage vacuoles, the VC plasmalemma and a few proplastids. The physiological significance of the RER stacks and of the RER cisternae association with other cell components, as well as the structure and function of an endomembrane compartment, found only in the freeze‐fixed pollen, are discussed.

Uptake of heavy metal salt solutions by pollen grains ofMagnolia×soulangeana(Magnoliaceae)

To investigate the role of the plasmalemmic cord in the pollen grains of members of the Magnoliaceae, anthers of Magnolia × soulangeana Soul.-Bod. were incubated for 1, 2.5, and 5 h in lead and lanthanum salt solutions. Ultrastructural analysis revealed differences in the labelling of the pollen grains with the time of exposure to the heavy metal salt, which is likely related to metal tolerance mechanisms operating in the pollen grains. Within 1 h of treatment, both tracers produced a fine precipitate lining the entire vegetative cell (VC) plasmalemma (including the plasmalemmic cord) as well as the generative cell (GC) plasmalemma. Heavy deposits were not found inside both the VC and the GC. At later times, the amount of heavy deposits increased on the pollen wall surface and, particularly, in the two outer intine layers. Also, heavy deposits were found in membrane-bound cell components of the VC and the GC, but never in the cytosol. In the VC, the cell components more frequently labelled were the protein storage vacuoles. In both pollen cells, multivesicular bodies, dictyosome cisternae, and small vesicles were also labelled. We hypothesize that the latter organelles participate in the metal ions accumulation (end of the trip), together with the protein vacuoles, or in the transport of metal ions to the vacuoles following the endocytotic uptake of these ions. The presence of both coated and uncoated pits and vesicles in the treated and the untreated pollen reinforces the latter hypothesis. Another significant result was that the tracers, after diffusing through the microchannels in the exine and crossing the intine layers, moved through the plasmalemmic cord to reach the GC periplasm. We suggest that the plasmalemmic cord may provide a system of communication for the flux of solutes from the anther loculus to both pollen cells and (or) from the VC to the GC.Key words: pollen grain, ultrastructure, metal uptake, metal tolerance, lead nitrate, lanthanum nitrate.

Angiosperm species that produce sperm cell pairs or generative cells with polarized distribution of DNA-containing organelles

We examined the distribution of DNA-containing organelles (plastids or mitochondria) in pollen from 115 angiosperm species (104 genera, 56 families) to determine whether there was polarized distribution of sperm cells (SC) and generative cells (GC). Using an improved method of fixation and squashing, we found polarization in the GC or SC pairs in mature pollen (obtained from the flower at anthesis) from five species (Abelia spathulata, Campsis grandiflora, Erythrina variegata, Limonium sinuatum, and Tecoma capensis). Including Plumbago, polarization is now known to occur in six genera representing four unrelated families. The results show that the production of pairs of dimorphic SC occurs in species other than those in the genus Plumbago, in which this phenomenon has already been well characterized. These results suggest that differentiation of SC and preferential double fertilization are not restricted to Plumbago and may occur in a variety of angiosperms. In species with significant polarization in GC or SC pairs, polarization of GC occurs at an especially early stage when GC are freed and become spherical, or just after microspore mitosis. This suggests that the distribution of DNA-containing organelles is strictly controlled during the early development of the GC.

Movement of generative cell and vegetative nucleus in tobacco pollen tubes is dependent on microtubule cytoskeleton but independent of the synthesis of callose plugs

The role of microtubules (MTs) in vegetative nucleus (VN) and generative cell (GC) transport was investigated by comparing VN and GC distribution with callose plug formation in tobacco pollen grains germinated and grown for 12 h with the plant-specific anti-MT drug oryzalin. The VN-GC complex or VN alone was located close to the tube tip in 100% of controls, but in only 5% of oryzalin-treated tubes. Instead, in 38% of oryzalin tubes, the complex or VN occurred close to the last-formed callose plug; in 40% between or in the middle of plugs; and in 17%, in or near the grain. An aberrant microfilament (MF) cytoskeleton was revealed by expression of a green fluorescent protein-talin fusion protein in living oryzalin-treated tubes. The abnormal MF structures probably resulted from the absence of MTs and impaired – or were a consequence of – VN and GC movement into the tube tip. In oryzalin tubes with several callose plugs, the VN and GC could be in or near the grain, indicating that callose plug synthesis is not dependent on the movement of VN and GC into the tube. VN and GC movement and callose plug formation are apparently independent events, in which the transport of the VN-GC complex must precede callose plug synthesis. Maintenance of the correct developmental program requires an intact MT cytoskeleton, otherwise no fertile pollen tubes are formed.

Pollen Tube Cytoskeleton: Structure and Function

This review deals with the structure and function of the pollen tube cytoskeleton from pollen germination to the formation of two sperm cells by division of the generative cell in the tube. The structure and function of microfilaments (MFs) and microtubules (MTs) are described in the binucleate/bicellular pollen tubes of Nicotiana tabacum. In this species the pollen grain and tube are formed of two cells; the vegetative cell (VC) enclosing the generative cell (GC). After pollen germination the VC forms the pollen tube, and the vegetative nucleus (VN) and GC move in the early phase of tube elongation to the tube tip, where they stay in tight association until division of the GC takes place. In this review we discuss the function of MFs, actin binding proteins (ABPs), MTs, and microtubule-associated proteins (MAPs and motor molecules) in growth and in the movement of cell organelles in the tube. Special attention is paid to the description of GC division and to consideration of factors that could cause GC cell cycle arrest during the early phase and induce GC division in the late phase of tube elongation. The movement of the VN and GC close to the tube tip can be thought to divide the function of the tube roughly into two parts: a tip part that contains the secretion machinery for primary wall formation and growth, with highly efficient exo- and endocytotic pathways, and a basal part behind the VN and GC, where secretion is more directed to secondary wall thickening, callose plug formation, and vacuolar biogenesis. The local calcium concentration seems to be an important factor determining the structure and function of the cytoskeletal elements in the tip region and in the basal part of the tube. In the future, in vivo or semi- in vivo cultured pollen tubes need to be used to test whether cytoskeletal functions deduced from the in vitro experiments occur also during in vivo tube growth.

Ultrastructural study of the relationship between generative and vegetative cells inMagnolia �soulangeana Soul.-Bod. pollen grains

InMagnolia ×soulangeana pollen grains the generative cell (GC) does not become totally free within the vegetative cell (VC), at least until the pollen tube emergence. Due to a deviation in its detachment process from the sporoderm, the opposing ends of the VC plasmalemma do not fuse themselves when the GC moves away from the intine. Consequently, the interplasmalemmic space surrounding the GC does not become isolated but rather maintains continuity with the sporoderm through a complex formation that we have called plasmalemmic cord. The real existence of this formation was confirmed through serial sectioning showing the plasmalemmic cord to consist of the VC plasmalemma. In its initial portion it is occupied by a reasonably accentuated wall ingrowth of the inner layer of the intine (intine 3). In the remainder portion, neither of the cytochemical tests used in this work have revealed the presence of a significant amount of wall material. However, ultrathin sections of samples processed either chemically or by cryofixation showed the existence of an intricate system of tubules and vesicles, some of which are evaginations of the VC plasmalemma. The hypothesis that the plasmalemmic cord may have a role in the complex interactions between the two pollen cells is discussed.

Microtubules and the positioning of the vegetative nucleus and generative cell in the angiosperm pollen tube: a quantitative study

In germinating pollen of the liliaceous angiosperm Endymion nonscriptus, the vegetative nucleus (VN) leaves the grain first, followed by the generative cell (GC), a sequence maintained during the linear period of pollen-tube growth. The VN and GC undergo some random repositioning within the protoplast, but during the interval of extension preceding GC division they move slowly, but consistently, towards the apex. In a colchicine concentration eliminating microtubules (MTs) from the vegetative cell (VC) germination is not affected, but the sequence of departure of the VC and the GC from the grain is reversed in most instances, and the two bodies are more widely dispersed in the tube thereafter. We interpret the results as implying that the positioning of VN and GC in the VC is governed by two forces: (i) that resulting from the balancing stresses placed on their myosin-bearing surfaces in interaction with the two polarities of actin fibrils in the cytoplasm; and (ii) a second tension imposed by an endoplasmic MT system which links the bodies with microtubule organizing centres (MTOCs) at the cell membrane just proximal to the growing point. The nature of the interaction of the MT system with the VN and GC remains to be established, but it may be significant that the presence of a dynein-like molecule in the angiosperm pollen tube has recently been reported.

Actin and tubulin expression and isotype pattern during tobacco pollen tube growth

The hybridization of total RNA isolated fromNicotiana tabacum pollen tubes with probes for tubulin and actin mRNA revealed a high level of tubulin and actin transcripts in pollen tubes 1.5–18 h old. A weak signal for tubulin and a strong signal for actin mRNAs were still detected in tubes 24 h old. The results suggest that the presynthesized tubulin and actin mRNAs inN. tabacum pollen tubes are very stable or that transcription of genes encoding cytoskeletal proteins may take place during growth of the pollen tubes. The increase of the signal for α- and β-tubulin and actin in the immunoblots of one-dimensional gels between 1.5 and 6 h indicated that the transcripts observed in the northern blots were translated. The increase in the cytoskeletal protein levels correlated well with previous cytological observations which show that inN. tabacum the movement of the vegetative nucleus (VN) and generative cell (GC) from the grain into the tube is most efficient between 3 and 6 h after germination. In the two-dimensional immunoblots it was observed that the signal for three out of four α-tubulin isotypes present in pollen tubes increased between 1.5 and 3 h. The time of the increase of α-tubulins was in good agreement with the start of movement of the VN and GC from the grain into the tube, and the synthesis of the α-tubulins could be a prerequisite for the beginning of the movement. These observations lend support to the idea that the microtubule cytoskeleton, in addition to microfilaments, is involved in the transport of the VN and GC in pollen tubes.

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 α-tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 4′6-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.

Pollen and Tapetum Development in Male FertileRosmarinus OfficinalisL. (Lamiaceae)

Abstract The development of microspores/pollen grains and tapetum was studied in fertile Rosmarinus officinalis L. (Lamiaceae). Most parts of the cell walls of the secretory anther tapetum undergo modifications before and during meiosis: the inner tangential and radial cell walls, and often also the outer tangential and radial wall, acquire a fibrous appearance; these walls become later transformed into a thin poly-saccharidic film, which is finally dissolved after microspore mitosis. Electron opaque granules found within the fibrous/lamellated tapetal walls consist of sporopollenin-like material, but cannot be interpreted as Ubisch bodies. The middle lamella and the primary wall of the outer tangential and radial tapetal walls remain unmodified, but get covered by an electron opaque, sporopollenin-like layer. Pollenkitt is formed only by lipid droplets from the ground plasma and/or ER profiles, the plastids do not form pollenkitt precursor lipids. Tapetum maturation (“degeneration”) does not take place before late vacuolate stage. The apertures are determined during meiosis by vesicles or membrane stacks on the surface of the plasma membrane. The procolumellae are conical, but at maturity the columellae are more cylindrical in shape. The columellar bases often fuse, but a genuine foot layer is lacking. The formation of the endexine starts with sporopollenin-accumulating white lines adjacent to the columellar bases. Later, the endexine grows more irregularly by the accumulation of sporopollenin globules. In mature pollen the intine is clearly bilayered. Generative cells (GCs) and sperm cells contain a comparatively large amount of cytoplasm, and organelles like mitochondria, dictyosomes, ER, and multi-vesicular bodies, but no plastids; GCs and sperms are separated from the vegetative cell only by two plasma membranes.