Cell differentiation in microsporangia of Pinus sylvestris. III. Late pachytene

Cycles of hyperactivity were observed in tapetal and microspore mother cells of Pinus sylvestris L. during the pachytene stage of meiosis in microspore mother cells. Hyperactive periods were characterized by dilated rough ER, hypersecretory dictyosomes, autophagic vesicles having one to two sequestration envelopes, and maze-like whor-les of the endomembrane system. The extent and sequence of differentiation differed between the two cell types. During distinct phases of development there was either fi-brillar flocculent or lipoidal material or both within dilations and at cell surfaces. Tapetal cell transfer of material involved endocytotic and exocytotic vesicles and channels opening directly to the cell surface. Dilations of the nuclear envelope of microspore mother cells in late pachytene intruded into the nucleus and, in conjunction with dilated ER, dominated cell profiles. Cellular morphogenesis in a microspo-rangium was seldom synchronous except for intervals of dedifferentiation when plas-modesma-like connections were formed between tapetal cells. Cycles of differentiation and dedifferentiation were correlated each spring season for five years with the progressive change of pachytene chromosomes, suggesting control by a genetic program rather than annual variations in the environment.     

芡实绒毡层细胞发育的超微结构变化

芡实（ Ｅｕｒｙａｌｅｆｅｒｏｘ Ｓａｌｉｓｂ） 绒毡层细胞在小孢子母细胞时期, 质体出现明显的变形期,细胞中二核常相互贴近或呈嵌合状态, 细胞壁间层中胞间连丝发达。减数分裂期, 绒毡层细胞壁融解消失, 胞间连丝断离, 细胞间发育出现不同步现象。质体开始积累淀粉, 部分质体呈空泡状, 并出现质体膜内陷, 这与液泡具相似的功能。四分体时期, 绒毡层细胞内部结构开始解体。单核小孢子时期, 绒毡层细胞解体消失, 使小孢子后期发育的营养来源受到影响,作者认为这是生产上成熟花粉囊中花粉粒少而且发育不正常的主要原因之一。     

The formation of cytoplasmic channels between tapetal cells inZea mays

Summary In this report we show that large cytoplasmic channels form between the tapetal cells ofZea mays (maize) during the period of tapetal cell differentiation. Tapetal cells are connected by plasmodesmata through their cellulosic cell walls prior to the first meiotic division of the meiocytes. As the tapetal cellulose wall is degraded at the onset of meiosis, both plasmodesmata and cytoplasmic channels measuring 50–200 nm are detectable between tapetal cells. By the time the meiotic tetrad is formed, the cytoplasmic channels are well-established and vary in size from 100–400 nm. The channels, with an average diameter of 200–300 nm, persist after the microspores are released from the callose wall and throughout the period of exine development in microsporogenesis. The channels could potentially allow for free exchange of cytoplasm and organelles. As the tapetal cells begin to pull apart and become vacuolate prior to microspore mitosis, the connecting channels are no longer detectable.     

Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features

Summary The ultrastructure of the secretory, binucleate tapetum of Brassica oleracea in the micro spore mother cell (MMC) stage through to the mature pollen stage is reported. The tapetal cells differentiate as highly specialized cells whose development is involved in lipid accumulation in their final stage. They start breaking down just before anther dehiscence. Nuclei with dispersed chromatin, large nucleoli and many ribosomes in the cytoplasm characterize the tapetal cells. The wall-bearing tapetum phase ends at the tetrade stage. The dissolution of tapetal walls begins from the inner tangential wall oriented towards the loculus and proceeds gradually along the radial walls to the outer tangential one. The plasmodesmata transversing the radial walls between tapetal cells persist until the mature microspore, long after loss of the inner tangential wall. After wall dissolution, the tapetal protoplasts retain their integrity and position within the anther locule. The tapetal cell membrane is in direct contact with the exine of the microspores/pollen grains and forms tubular evaginations that increase its surface area and appear to be involved in the translocation of solutes from the tapetal cells to the microspores/ pollen grains. The tapetal cells exhibit a polarity expressed by spatial differentiation in the radial direction.     

Transmission and Scanning Electron Microscopic Observations on the Plasmodesmatal Channels Between the Pollen Mother Cells of Lily

The regularity of the presence of plasmodesmata channels in the pollen mother cells of lily was studied by transmission and scanning electron microscopy. A few plasmodesmata channels can be recognized between the pollen mother cells at leptotene stage, which increase in number at zygotene and expand in width at synizesis and they lie in the range 0.5—1 μm. Massive chromatin substance are transferred from one pollen mother cell to another during synizesis. The pre-existing plasmodesmate channels close again at late pachytene. There are no channels from metaphase Ⅰ to tetrad stage. Finally, the relation between the presence of plasmodesmata channels, synizesis and cytomixis were discussed.     

Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves

Summary The sink-source transition in tobacco leaves was studied noninvasively using transgenic plants expressing the green-fluorescent protein (GFP) under control of theArabidopsis thaliana SUC2 promoter, and also by imaging transgenic plants that constitutively expressed a tobacco mosaic virus movement protein (MP) fused to GFP (MP-GFP). The sink-source transition was measured on intact leaves and progressed basipetally at rates of up to 600 m/h. The transition was most rapid on the largest sink leaves. However, leaf size was a poor indicator of the current position of the sink-source transition. A quantitative study of plasmodesmatal frequencies revealed the loss of enormous numbers of simple plasmodemata during the sink-source transition. In contrast, branched plasmodesmata increased in frequency during the sink-source transition, particularly between periclinal cell walls of the spongy mesophyll. The progression of plasmodesmal branching, as mapped by the labelling of plasmodesmata with MP-GFP fusion, occurred asynchronously in different cell layers, commencing in trichomes and appearing lastly in periclinal cell walls of the palisade layer. It appears that dividing cells retain simple plasmodesmata for longer periods than nondividing cells. The rapid conversion of simple to branched plasmodesmata is discussed in relation to the capacity for macromolecular trafficking in developing leaf tissues.     

Plasmodesmata between synchronously and asynchronously developing cells of the antheridial filaments ofChara vulgaris L.

Summary Plasmodesmata connecting synchronously developing cells are filled with electron-transparent, homogenous ground cytoplasm. At the middle lamella, their average diameter is about 67 nm; the relative area occupied by plasmodesmata is calculated to be about 8 to 9% of the wall.Plasmodesmata occurring between cells which develop asynchronously are plugged by an electron-dense homogenous material. The plug fits tightly to the plasmalemma inside the plasmodesmatal canal. Occasionally (in 8% of the walls), the closing plugs are also found between synchronously dividing cells. Generally, the plugging takes place in the walls formed at the first stages of development of the antheridial filaments and is probably an irreversible process.It is supposed that the plugging of plasmodesmata is the cause of the appearance of two or more synchronous cell groups within a single filament.     

Changes in ultrastructure of plasmodesmata during spermatogenesis in Chara vulgaris L.

Two types of plasmodesmata are found within an antheridium of Chara vulgaris: open plasmodesmata filled with electron-transparent cytoplasm, and plugged plasmodesmata, filled with an osmiophilic dense substance. Open plasmodesmata occur only between cells synchronized completely in respect of their advancement in cell-cycle progression or differentiation. Plugged plasmodesmata connect different types of cells or cells of the same type at various stages of the cell cycle. Open plasmodesmata may become plugged, and vice versa. These changes are connected with the limitation or extension of synchronization of cellular divisions and differentiation within the groups of cells in the antheridium.     

Small heat shock protein LimHSP16.45 protects pollen mother cells and tapetal cells against extreme temperatures during late zygotene to pachytene stages of meiotic prophase I in David Lily

Plant meiotic prophase I is a complicated process involving the late zygotene and pachytene stages, both crucial for completing synapsis and recombination. Using David Lily (Lilium davidii var. Willmottiae) as our research material, we performed suppression subtractive hybridization to construct EST library of anthers at various stages of development by the pollen mother cells. From this library, we identified small heat shock protein LimHSP16.45 was highly expressed during the late zygotene to pachytene stages. Our results also showed that LimHSP16.45 was almost specifically expressed in the anther compared with the root, stem, or leaf, and in situ expression of LimHSP16.45 mRNAs showed strong signals in the pollen mother cells and tapetal cells. LimHSP16.45 could be induced by heat and cold in lily anthers, and its ectopic expression enhanced the viability of E. coli cells under both high and low temperatures. In vitro, it acted as molecular chaperone and could help luciferase refolding after heat shock stress. All of these data suggest that LimHSP16.45, working as molecular chaperone, possibly protects pollen mother cells and tapetal cells against extreme temperatures during late zygotene to pachytene stages of meiotic prophase I in David Lily.     

Microsporogenesis inPinus sylvestris L. VIII. Tapetal and late pollen grain development

This last portion of our developmental study ofPinus sylvestris L. pollen grains extends from just prior to the first microspore mitosis to the microsporangial dehiscence preparatory to pollen shedding. In nine years of collecting each day the duration of the above period was 7 to 11 days. Tapetal cells extended into the loculus and embraced microspores during the initial part of the above period. Thereafter tapetal cells receded, became parallel to parietal cells and so imbricated that there appeared to be two or three layers of tapetal cells. Tapetal cells were present up to the day before pollen shedding, but only rER and some mitochondria appeared to be in good condition at that time. A callosic layer (outer intine) was initiated under the endexine before microspore mitosis. After the first mitosis the first prothallial cell migrated to the proximal wall and was covered on the side next to the pollen cytoplasm by a thin wall joining the thick outer intine. There are plasmodesmata between pollen cytoplasm and the prothallial cell. After the second mitosis the second prothallial cell became enveloped by the outer intine. The inner intine appears after formation of the two prothallial cells but before the third mitosis. During this two-prothallial cell period before the third mitosis, plastids had large and complex fibrillar assemblies shown to be modified starch grains. After the third mitosis plastids of the pollen cytoplasm contained starch and the generative cell (antheridial initial), the product of that mitosis, is enveloped by the inner intine. On the day of pollen shedding cells are removed from the microsporangial wall by what appears to be focal autolysis. The tapetal and endothecial cells for 10–15 µm on each side of the dehiscence slit are completely removed. One or more epidermal cells are lysed, but both a thin cuticle and the very thin sporopollenin-containing peritapetal membrane remain attached to the undamaged epidermal cells bordering the dehiscence slit. Our study terminates on the day of pollen shedding with mature pollen still within the open microsporangium. At that time there is no longer a clear morphological distinction between the outer and inner intine but, judging by stain reactions, there is a chemical difference. The exine of shed pollen grains was found to be covered by small spinules on the inner surface of alveoli. These had the same spacing as the Sporopollenin Acceptor Particles (SAPs) associated with exine initiation and growth.     

Ultrastructural characterization of apospory in Panicum maximum

The nucellar ultrastructure of apomictic Panicum maximum was analyzed during the meiocytic stage and during aposporous embryo sac formation. At pachytene the megameiocyte shows a random cell organelle distribution and sometimes only an incomplete micropylar callose wall. The chalazal nucellar cells are meristematic until the tetrad stage. They can turn into initial cells of aposporous embryo sacs. The aposporous initials can be recognized by their increased cell size, large nucleus, and the presence of many vesicles. The cell wall is thin with few plasmodesmata. If only a sexual embryo sac is formed, the nucellar cells retain their meristematic character. The aposporous initial cell is somewhat comparable to a vacuolated functional megaspore. It shows large vacuoles around the central nucleus and is surrounded by a thick cell wall without plasmodesmata. In the mature aposporous embryo sac the structure of the cells of the egg apparatus is similar to each other. In the chalazal part of the egg apparatus the cell walls are thin and do not hamper the transfer of sperm cells. Structural and functional aspects of nucellar cell differentiation and aposporous and sexual embryo sac development are discussed.     

A histochemical and ultrastructural study of the ontogeny and differentiation of pollen inPodocarpus macrophyllus D. Don

Summary Male cones ofPodocarpus macrophyllus D. Don enter a period of dormancy lasting almost a year after the differentiation of archesporial tissue. The cell walls of the sporogenous and tapetal cells are different in composition from those of the cells comprising the wall of the microsporangium. The walls of tapetal cells undergo complete dissolution but the naked protoplasts do not invade the cavity of the microsporangium, and eventually degeneratein situ. Sporopollenin-containing bodies are formed on the tapetal plasmalemma although no specific tapetal organelles can be singled out as sites of synthesis of sporopollenin precursors. The original walls of the microspore mother cells are broken down completely and replaced by a thin callose-like wall. No cytomictic channels are formed prior to or during early meiosis. The outer nuclear membrane of the sporogenous cells forms numerous vesicles which likely play an important role in preparing the cell for meiosis and in the breakdown of the original sporogenous cell wall and the formation of the new wall. Pronounced evaginations and invaginations of the nuclear envelope during the tetrad stage are seen which again indicate vital nucleo-cytoplasmic exchange at the time when species specific sexine layer is being laid down. The microspore protoplast synthesizes a portion of sporopollenin precursors. Sexine and part of nexine I are laid down during the tetrad stage on lamellae of unit membrane dimensions while nexines II and III are formed after the dissolution of the tetrads by the coalescence of small, electron dense particles. Cells of the male gametophyte are initially separated from each other by distinct cell walls often traversed by plasmodesmata. Mature pollen grains have appreciable reserves of protein, lipid and starch. Results of histochemical and scanning electron microscopical observations are also reported and discussed.     

Relationship between DNA cycle and growth rate in Synechococcus sp. strain PCC 6301.

Flow cytometry was used to examine cell cycle regulation in Synechococcus sp. strain PCC 6301 under a variety of growth conditions. The DNA frequency distributions of exponentially growing and dark-blocked populations confirmed that this cyanobacterium contains multiple chromosome copies even at very slow growth rates. Furthermore, the presence of major peaks corresponding to other than 2" chromosome copies strongly suggests that DNA replication is initiated asynchronously. Although this suggestion is at odds with the standard formulation of the procaryotic cell cycle model, it is similar to recent observations of asynchrony in Escherichia coli replication mutants.     

The C.elegans MAPK phosphatase LIP-1 is required for the G(2)/M meiotic arrest of developing oocytes

In the Caenorhabditis elegans hermaphrodite germline, spatially restricted mitogen-activated protein kinase (MAPK) signalling controls the meiotic cell cycle. First, the MAPK signal is necessary for the germ cells to progress through pachytene of meiotic prophase I. As the germ cells exit pachytene and enter diplotene/diakinesis, MAPK is inactivated and the developing oocytes arrest in diakinesis (G(2)/M arrest). During oocyte maturation, a signal from the sperm reactivates MAPK to promote M phase entry. Here, we show that the MAPK phosphatase LIP-1 dephosphorylates MAPK as germ cells exit pachytene in order to maintain MAPK in an inactive state during oocyte development. Germ cells lacking LIP-1 fail to arrest the cell cycle at the G(2)/M boundary, and they enter a mitotic cell cycle without fertilization. LIP-1 thus coordinates oocyte cell cycle progression and maturation with ovulation and fertilization.     

Cytoskeleton,cell surface and the development of invasive plasmodial tapetum inTradescantia virginiana L.

Summary The anther tapetum inTradescantia virginiana L. is of the invasive plasmodial type: the cells lose their walls during early spore meiosis and develop long invasion processes which invade the loculus to penetrate spaces between the sporogenous cells. Fusion to form a syncytium is delayed and conventional ultra-thin sections and the Thiéry reaction reveal the presence of a loose fibrillar extracellular cell coat on the free surfaces of tapetal cells and their invasion processes. Cell fusion involves formation of apposition areas characterized by an absence of cell coat and the local appearance of microtubular arrays. Conspicuous membrane sacs, associated closely with microtubules, were found to migrate to and accumulate at the plasma membranes near the fusion sites and sporogenous cells. Microtubules are always present in the cortical regions of the tapetal cells and their invasion processes. It is surmised that microtubules are not responsible either for initiating or guiding tapetal invasion of the loculus; instead they may help to sustain the form of the invasion processes, help in the migration of membrane sacs, and participate in cell fusion. The cell coat disappears with syncytium formation towards the end of meiosis, and the developing spore cells become surrounded by a perispore membrane, which, derived from the original plasma membranes and augmented by membrane sacs, forms labyrinthine membrane reservoirs that are described further in the accompanying paper.     

The effect of the wheat Ph1 locus on chromatin organisation and meiotic chromosome pairing analysed by genome painting

The Ph1 locus in wheat influences homo(eo)logous chromosome pairing. We have analysed its effect on the behaviour and morphology of two 5RL rye telosomes in a wheat background, by genomic in situ hybridisation (GISH), using rye genomic DNA as a probe. Our main objective was to study the effect of different alleles of the Ph1 locus on the morphology and behaviour of the rye telosomes in interphase nuclei of tapetal cells and in pollen mother cells at early stages of meiosis. The telosomes, easily detectable at all stages, showed a brightly fluorescing chromomere in the distal region and a constriction in the proximal part. These diagnostic markers enabled us to define the centromere and telomere regions of the rye telosomes. In the presence of functional copies of Ph1, the rye telosomes associated at pre-leptotene, disjoined and reorganised their shape at leptotene, and became fully homologously paired at zygotene – pachytene. In plants without functional alleles (ph1bph1b), the rye telosomes displayed an aberrant morphology, their premeiotic associations were clearly disturbed and their pairing during zygotene and pachytene was reduced and irregular. The Ph1 locus also influenced the behaviour of rye telosomes in the interphase nuclei of tapetal cells: in Ph1Ph1 plants, the rye telosomes occupied distinct, parallel-oriented domains, whereas in tapetal nuclei of ph1bph1b plants they were intermingled with wheat chromosomes and showed a heavily distorted morphology. The results shed new light on the effect of Ph1, and suggest that this locus is involved in chromosome condensation and/or scaffold organisation. Our explanation might account for various apparently contradictory and pleiotropic effects of this locus on both premeiotic associations of homologues, the regulation of meiotic homo(eo)logous chromosome pairing and synapsis, the resolution of bivalent interlockings and centromere behaviour. Received: 27 April 1998; in revised form: 5 August 1998 / Accepted: 11 August 1998     

Differences on the microsporogenesis and tapetal development of male fertile and cytoplasmic male sterile pepper (Capsicum annuum L.)

To clarify the time and cause of pollen abortion, differences on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and cytoplasmic male sterile (CMS) line pepper were studied using transmission electron microscopy. The results showed that CMS line anthers appeared to have much greater variability in developmental pattern than male fertile maintainer line ones. The earliest deviation from normal anther development occurred in CMS line anthers at prophase I was cytomixis in some microspore mother cells (MMCs), and vacuolisation in tapetal cells. Then, MMCs in CMS line anthers developed asynchronously and a small part of ones at the different stage degenerated in advance appearing to have typical morphological features of programmed cell death (PCD). Most MMCs could complete the meiosis, but formed non-tetrahedral tetrad microspores with irregular shape and different size and uncertain number of nuclei, and some degenerated ahead of time as well. Tapetal cells in CMS line anther degenerated during meiosis, and were crushed at the tetrad stage, which paralleled the collapse of pollens. Pollen abortion in CMS line anthers happened by PCD themselves, and the premature PCD of tapetal cells were closely associated with male sterility.     

Plasmodesmal changes are related to different developmental stages of antheridia of Chara species

Summary During the development of the antheridia of Chara species, dynamic changes in the occurrence and ultrastructure of plasmodesmata are observed which are closely correlated to particular developmental phases and presumably regulate the morphogenetic events in the antheridia. The disappearance of plasmodesmata between shield cells and between shied cells and the basal cell leads to a cessation in symplasmic transport around the antheridum and determines its concentric or centrifugal character via centrally situated capitular cells. Unplugged plasmodesmata are present between fully synchronously developing antheridial filament cells and obviously coordinate the development of the cells. In the middle phase of spermiogenesis, rough endoplasmic reticulum in antheridial filaments passes uncompressed through wide plasmodesmata and provides an additional transport pathway for developmental control factors. Plugged plasmodesmata link cells of different types or cells of the same type which are at different phases of cell cycle and guarantee their individual development. The plugging of plasmodesmata is a reversible process that depends on the morphogenetic situation. Plasmodesmata connecting the basal cell and the subbasal cell as well as the basal cell and capitular cells are transformed successively from the simple into the complex type and might be the pathways for an import of gibberellins and nutrients into the strong sink tissues of the developing antheridium. There is a symplasmic connection between the antheridum and the thallus via a basal cell. Prior to the initiation of spermatozoid differentiation (spermiogenesis), plasmodesmata connecting the basal cell with a subbasal cell and the basal cell with capitular cells are spontaneously broken, resulting in symplasmic isolation of the antheridium that is probably a signal which triggers the induction of spermatozoid differentiation. Premature plasmolytically evoked symplasmic isolation of the antheridium leads to the elimination of 1 to 2 cell cycles from the proliferative stage of spermatogenesis. Autoradiographic studies demonstrate that both natural and induced symplasmic isolation drastically decreases the entry of isotopically labeled gibberellic acid into antheridia of Chara species that may be the consequence of the elimination of the hormone's transport through plasmodesmata.Correspondence and reprints: Department of Cytophysiology, University of ód, ulica Pilarskiego 14, 90-231 ód, Poland.Received March 11, 2002; accepted September 19, 2002; published online August 26, 2003     

Chara tomentosa Antheridial Plasmodesmata at Various Stages of Spermatogenesis

Chara tomentosa antheridial plasmodesmata are described during proliferation and spermiogenesis. In antheridial filament cells which are cycling completely synchronously, unplugged plasmodesmata are filled with light cytoplasm. The same plasmodesmata are observed after cessation of mitotic division followed by the onset of synchronous spermiogenesis. Walls separating cells at different cell cycle stages and dividing antheridial filaments into asynchronous domains are plugged with a dense osmophilic substance. Similarly plugged plasmodesmata are present between antheridial cells of different types, e.g., capitular cells and antheridial filaments. In mid spermiogenesis when abundant endoplasmic reticulum (ER) appears temporarily it penetrates into plasmodesmata enabling cell-to-cell transport via ER cisternae. In late spermiogenesis there are no cisternae in plasmodesmata. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation,sorting, and characterization of uni- and binucleate tapetal protoplasts from anthers of normal and Texas cytoplasmic male-sterileZea mays L.

Summary A cytological study of Texas cytoplasmic male sterile (Tcms) and normal (N) anther tapetal protoplasts ofZea mays was undertaken to determine whether there were any differences prior to Tcms male cell abortion not noted in previous published studies. Squash preparations, tapetal protoplast separation via flow cytometry, image analysis, and electron microscopy were utilized. Chemically preserved tapetal protoplasts from both lines were prominently angular in shape and typically smaller than any other cell type in the anthers. The tapetum from both lines consisted of a mixture of uninucleate and binucleate protoplasts. The Tcms tapetum consistently had a higher proportion of binucleate protoplasts during all stages of microsporogenesis prior to abortion. The size of Tcms uniand binucleate tapetal protoplasts was more variable than the N tapetal protoplasts and was largest during the microspore stage when male cells abort. Tapetal nuclear size in both lines was less variable. Uni- and binucleate tapetal protoplasts from each line could be separated from the other anther cells and from each other by filtration and then by flow cytometry, based on intensity of nuclear fluorescence. These results suggest that Tcms uninucleate tapetal protoplasts have a higher level of DNA than N uninucleate tapetal protoplasts. Both fluorescence microscopy and electron microscopy confirmed pure populations of intact uni- and binucleate tapetal protoplasts using flow cytometry. The results from this study indicate that the methodology presented here could be used for a variety of further studies to better understand the cellular and molecular basis of male sterility in maize, and in other taxa, where the tapetum is the primary target that leads to male sterility.Abbreviations AO acridine orange - Bi binucleate protoplast - D dyad - DAPI 4,6-diamidino-2-phenylindole - FC flow cytometry - M meiocyte - MI microspore - MMC mithramycin - N normal anther tapetal protoplast - PI propidium iodide - PS protoplast sorting - RT room temperature - SM sporogenous mass - Tems Texas cytoplasmic male sterile anther tapetal protoplast - Uni uninucleate protoplast