Pattern and process of wall formation in developing endosperm

Endosperm is emerging as a system for investigating the genetic control of wall placement and deposition in plant development. Development of endosperm progresses in distinct stages from a wall-less syncytial stage to a cellular stage that is entirely typical of plant meristems where the division plane is predicted by a preprophase band of microtubules (PPB) and cytokinesis is completed by formation of a cell plate in association with a phragmoplast. Four developmentally different types of walls, each associated with a different microtubule system, are sequentially produced: (1) free growing walls deposited in the absence of mitosis and phragmoplasts; (2) walls guided by cytoplasmic phragmoplasts formed adventitiously in the absence of mitosis; (3) walls formed by interzonal phragmoplasts in a cell cycle that lacks PPBs; and (4) wall deposition driven by interzonal phragmoplasts in a cycle that includes PPBs. We are using methods of differential screening to isolate cDNA clones corresponding in temporal and spatial pattern to the types of wall development, and are studying mutants for clues to the genetic controls of wall development.     

Phragmoplasts in the Absence of Nuclear Division

All land plants (embryophytes) use a phragmoplast for cytokinesis. Phragmoplasts are distinctive cytoskeletal structures that are instrumental in the deposition of new walls in both vegetative and reproductive phases of the life cycle. In meristems, the phragmoplast is initiated among remaining non-kinetochore spindle fibers between sister nuclei and expands to join parental walls at the site previously marked by the preprophase band of microtubules (PPB). The microtubule cycle and cell cycle are closely coordinated: the hoop-like cortical microtubules of interphase are replaced by the PPB just prior to prophase, the PPB disappears as the spindle forms, and the phragmoplast mediates cell plate deposition after nuclear division. In the reproductive phase, however, cortical microtubules and PPBs are absent and cytokinesis may be uncoupled from the cell cycle resulting in multinucleate cells (syncytia). Minisyncytia of 4 nuclei occur in microsporocytes and several (typically 8) nuclei occur in the developing megagametophyte. Macrosyncytia with thousands of nuclei may occur in the nuclear type endosperm. Cellularization of syncytia involves formation of adventitious phragmoplasts at boundaries of nuclear-cytoplasmic domains (NCDs) defined by radial microtubule systems (RMSs) emanating from non-sister nuclei. Once initiated in the region of microtubule overlap at interfaces of opposing RMSs, the adventitious phragmoplasts appear structurally identical to interzonal phragmoplasts. Phragmoplasts are constructed of multiple opposing arrays similar to what have been termed microtubule converging centers. The individual phragmoplast units are distinctive fusiform bundles of anti-parallel microtubules bisected by a dark mid-zone where vesicles accumulate and fuse into a cell plate.     

Development of the endosperm in rice (Oryza sativa L.): Cellularization

The syncytial endosperm of rice undergoes cellularization according to a regular morphogenetic plan. At 3 days after pollination (dap) mitosis in the peripheral synctium ceases. Radial systems of microtubules emanating from interphase nuclei define nuclear-cytoplasmic domains (NCDs) which develop axes perpendicular, to the embryo sac wall. Free-growing anticlinal walls between adjacent NCDs compart-mentalize the cytoplasm into open-ended alveoli which are overtopped by syncytial cytoplasm adjacent to the central vacuole. At 4 dap, mitosis resumes as a wave originating adjacent to the vascular bundle. The spindles are oriented parallel to the alveolar walls and cell plates formed in association with interzonal phragmoplasts result in periclinal walls that cut off a peripheral layer of cells and an inner layer of alveoli displaced toward the center. Polarized growth of the newly formed alveoli and elongation of the anticlinal walls occurs during interphase. The next wave of cell division in the alveoli proceeds as the first and a second cylinder of cells is cut off inside the peripheral layer. The periods of polarized growth/anticlinal wall elongation alternating with periclinal cell division are repeated 3–4 times until the grain is filled by 5 dap.     

Development of endosperm in Arabidopsis thaliana

 The process of endosperm development in Arabidopsis was studied using immunohistochemistry of tubulin/microtubules coupled with light and confocal laser scanning microscopy. Arabidopsis undergoes the nuclear type of development in which the primary endosperm nucleus resulting from double fertilization divides repeatedly without cytokinesis resulting in a syncytium lining the central cell. Development occurs as waves originating in the micropylar chamber and moving through the central chamber toward the chalazal tip. Prior to cellularization, the syncytium is organized into nuclear cytoplasmic domains (NCDs) defined by nuclear-based radial systems of microtubules. The NCDs become polarized in axes perpendicular to the central cell wall, and anticlinal walls deposited among adjacent NCDs compartmentalize the syncytium into open-ended alveoli overtopped by a crown of syncytial cytoplasm. Continued centripetal growth of the anticlinal walls is guided by adventitious phragmoplasts that form at interfaces of microtubules emanating from adjacent interphase nuclei. Polarity of the elongating alveoli is reflected in a subsequent wave of periclinal divisions that cuts off a peripheral layer of cells and displaces the alveoli centripetally into the central vacuole. This pattern of development via alveolation appears to be highly conserved; it is characteristic of nuclear endosperm development in angiosperms and is similar to ancient patterns of gametophyte development in gymnosperms. Received: 21 September 1998 / Revision accepted: 17 November 1998     

ENDOSPERM DEFECTIVE1 Is a Novel Microtubule-Associated Protein Essential for Seed Development in Arabidopsis

Early endosperm development involves a series of rapid nuclear divisions in the absence of cytokinesis; thus, many endosperm mutants reveal genes whose functions are essential for mitosis. This work finds that the endosperm of Arabidopsis thaliana endosperm-defective1 (ede1) mutants never cellularizes, contains a reduced number of enlarged polyploid nuclei, and features an aberrant microtubule cytoskeleton, where the specialized radial microtubule systems and cytokinetic phragmoplasts are absent. Early embryo development is substantially normal, although occasional cytokinesis defects are observed. The EDE1 gene was cloned using a map-based approach and represents the pioneer member of a conserved plant-specific family of genes of previously unknown function. EDE1 is expressed in the endosperm and embryo of developing seeds, and its expression is tightly regulated during cell cycle progression. EDE1 protein accumulates in nuclear caps in premitotic cells, colocalizes along microtubules of the spindle and phragmoplast, and binds microtubules in vitro. We conclude that EDE1 is a novel plant-specific microtubule-associated protein essential for microtubule function during the mitotic and cytokinetic stages that generate the Arabidopsis endosperm and embryo.     

Pollen development in orchids

Summary Cytokinesis in microsporocytes of moth orchids is unusual in that it occurs simultaneously after meiosis, the cytoplasm does not infurrow in the division planes, and cell plates are deposited in association with centrifugal expansion of phragmoplasts. Microtubules radiating from the nuclear envelopes appear to be of fundamental importance in establishment of division planes. Primary interzonal spindles develop between sister nuclei and interaction of radial microtubules triggers development of secondary interzonal spindles between non-sister nuclei. From three to six or more phragmoplasts, depending upon the arrangement of nuclei in the coenocyte, develop from these postmeiotic arrays. The phragmoplasts consist of co-aligned microtubules and F-actin organized into bundles that are broad proximal to the mid-plane and taper distally. Ultrastructure of the phragmoplast/cell plate reveals that abundant ER is associated with vesicle aggregation and coalescence. Cell plates are deposited in association with phragmoplasts as they expand centrifugally to join the parental wall and/or fuse with one another in the interior of the cell.Abbreviations CLSM confocal laser scanning microscope/microscopy - FITC flnorescein isothiocyanate - PPB preprophase band of microtubules - TEM transmission electron microscope/microscopy     

Cytoskeletal organization of the micropylar endosperm in Coronopus didymus L. (Brassicaceae)

Summary. The micropylar chamber of the mustard Coronopus didymus is a developmental domain distinct from the contiguous central chamber and the more extreme chalazal chamber. Early in syncytial development the micropylar endosperm surrounding the embryo becomes populated with unusual fusiform to multilobed nuclei. These nuclei are sheathed by unique parallel arrays of microtubules that focus at tips of the nuclei and flare to connect with a reticulate network in the common cytoplasm. F-actin does not closely invest the nuclei but instead forms an extensive but separate cytoplasmic reticulum. When the embryo is in the early heart stage, the cytoskeleton of the endosperm undergoes a remarkable transition in preparation for cellularization. Microtubules become reorganized into radial arrays emanating from the nuclei, which themselves become spherical. Radial microtubule systems (RMSs), which replace both the parallel microtubules and the cytoplasmic reticulum, organize the common cytoplasm into evenly spaced nuclear cytoplasmic domains (NCDs). F-actin gradually becomes coaligned with the RMSs. Phragmoplasts are initiated adventitiously at the interfaces of opposing RMSs in the absence of mitosis. Cell plate deposition, which is initiated at multiple sites, results in a network of walls formed more or less simultaneously around the densely packed NCDs. The walls, which are rich in 1–3-β-glucans, join with one another and with the existing walls of both the central cell and embryo to complete cellularization in the micropylar chamber. In the adjacent central chamber where the syncytium is restricted to a thin peripheral layer by the large central vacuole, basic organization of the syncytium into NCDs is followed by alternating cycles of alveolation and periclinal cell division resulting in cellularization. Received July 19, 2001 Accepted October 16, 2001     

Rice caryopsis development Ⅱ: Dynamic changes in the endosperm

The rice endosperm plays crucial roles in nourishing the embryo during embryogenesis and seed germination. Although previous studies have provided the general information about rice endosperm, a systematic investigation throughout the entire endosperm developmental process is still lacking. In this study, we examined in detail rice endosperm development on a daily basis throughout the 30‐day period of post‐fertilization development. We observed that coenocytic nuclear division occurred in the first 2 days after pollination (DAP), cellularization occurred between 3 and 5 DAP, differentiation of the aleurone and starchy endosperm occurred between 6 and 9 DAP, and accumulation of storage products occurred concurrently with the aleurone/starchy endosperm differentiation from 6 DAP onwards and was accomplished by 21 DAP. Changes in cytoplasmic membrane permeability, possibly caused by programmed cell death, were observed in the central region of the starchy endosperm at 8 DAP, and expanded to the whole starchy endosperm at 21 DAP when the aleurone is the only living component in the endosperm. Further, we observed that a distinct multi‐layered dorsal aleurone formed near the dorsal vascular bundle, while the single‐ or occasionally two‐cell layered aleurone was located in the lateral and ventral positions of endosperm. Our results provide in detail the dynamic changes in mitotic divisions, cellularization, cell differentiation, storage product accumulation, and programmed cell death that occur during rice endosperm development.     

Cell wall (1 → 3)- and (1 → 3, 1 → 4)-β-glucans during early grain development in rice (Oryza sativa L.)

Immunogold labeling was used to study the distribution of (1 → 3)-β-glucans and (1 → 3, 1 → 4)-β-glucans in the rice grain during cellularization of the endosperm. At approximately 3–5 d after pollination the syncytial endosperm is converted into a cellular tissue by three developmentally distinct types of wall. The initial free-growing anticlinal walls, which compartmentalize the syncytium into open-ended alveoli, are formed in the absence of mitosis and phragmoplasts. This stage is followed by unidirectional (centripetal) growth of the anticlinal walls mediated by adventitious phragmoplasts that form between adjacent interphase nuclei. Finally, the periclinal walls that divide the alveoli are formed in association with centripetally expanding interzonal phragmoplasts following karyokinesis. The second and third types of wall are formed alternately until the endosperm is cellular throughout. All three types of wall that cellularize the endosperm contain (1 → 3)-β-glucans but not (1 → 3, 1 → 4)-β-glucans, whereas cell walls in the surrounding maternal tissues contain considerable amounts of (1 → 3, 1 → 4)-β-glucans with (1 → 3)-β-glucans present only around plasmodesmata. The callosic endosperm walls remain thin and cell plate-like throughout the cellularization process, appearing to exhibit a prolonged juvenile state. Received: 7 January 1997 / Accepted: 11 February 1997     

Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo

The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizating organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self-reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubule-related processes.     

The Pleiomorphic Plant MTOC： An Evolutionary Perspective

γ-Tubulin is an essential component of the microtubule organizing center （MTOC） responsible for nucleating microtubules in both plants and animals. Whereas γ-tubulin is tightly associated with centrosomes that are inheritable organelles in cells of animals and most algae, it appears at different times and places to organize the myriad specialized microtubule systems that characterize plant cells. We have traced the distribution of γ-tubulin through the cell cycle in representative land plants （embryophytes） and herein present data that have led to a concept of the pleiomorphic and migratory MTOC. The many forms of the plant MTOC at spindle organization constitute pleiomorphism, and stage-specific “migration” is suggested by the consistent pattern of redistribution of γ-tubulin during mitosis. Mitotic spindles may be organized at centriolar centrosomes （only in final divisions of spermatogenesis）, polar organizers （POs）, plastid MTOCs, or nuclear envelope MTOCs （NE-MTOCs）. In all cases, with the possible exception of centrosomes in spermatogenesis, the γ-tubulin migrates to broad polar regions and along the spindle fibers, even when it is initially a discrete polar entity. At anaphase it moves poleward, and subsequently migrates from polar regions （distal nuclear surfaces） into the interzone （proximal nuclear surfaces） where interzonal microtubule arrays and phragmoplasts are organized. Following cytokinesis, γ-tubulin becomes associated with nuclear envelopes and organizes radial microtubule systems （RMSs）. These may exist only briefly, before establishment of hoop-like cortical arrays in vegetative tissues, or they may be characteristic of interphase in syncytial systems where they serve to organize the common cytoplasm into nuclear cytoplasmic domains （NCDs）.     

The cytoskeleton and spatial control of cytokinesis in the plant life cycle

Summary One of the intriguing aspects of development in plants is the precise control of division plane and subsequent placement of walls resulting in the specific architecture of tissues and organs. The placement of walls can be directed by either of two microtubule cycles. The better known microtubule cycle is associated with control of the future division plane in meristematic growth where new cells become part of tissues. The future daughter domains are determined before the nucleus enters prophase and the future site of cytokinesis is marked by a preprophase band (PPB) of cortical microtubules. The spindle axis is then organized in accordance with the PPB and, following chromosome movement, a phragmoplast is initiated in the interzone and expands to join with parental walls at the site previously occupied by the PPB. The alternative microtubule cycle lacks both the hooplike cortical microtubules of interphase and the PPB. Wall placement is determined by a radial microtubule system that defines a domain of cytoplasm either containing a nucleus or destined to contain a nucleus (the nuclear cytoplasmic domain) and controls wall placement at its perimeter. This more flexible system allows for cytoplasmic polarization and migration of nuclei in coenocytes prior to cellularization. The uncoupling of cytokinesis from karyokinesis is a regular feature of the reproductive phase in plants and results in specific, often unusual, patterns of cells which reflect the position of nuclei at the time of cellularization (e.g., the arrangement of spores in a tetrad, cells of the male and female gametophytes of angiosperms, and the distinctive cellularization of endosperm). Thus, both microtubule cycles are required for completion of plant life cycles from bryophytes to angiosperms. In angiosperm seed development, the two methods of determining the boundaries of domains where walls will be deposited are operative side by side. Whereas the PPB cycle drives embryo development, the radial-microtubule-system cycle drives the common nuclear type of endosperm development from the syncytial stage through cellularization. However, a switch to the PPB cycle can occur in endosperm, as it does in barley, when peripheral cells divide to produce a multilayered aleurone. The triggers for the switch between microtubule cycles, which are currently unknown, are key to understanding plant development.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

γ-Tubulin,microtubule arrays,and quadripolarity during sporogenesis in the hepatic<Emphasis Type="Italic"> Aneura pinguis</Emphasis> (Metzgeriales)

This is the first report on the organization of a quadripolar microtubule system (QMS) in polyplastidic meiosis of a hepatic with polar organizers (POs). Unlike the monoplastidic sporocytes of mosses and hornworts, in which meiotic quadripolarity can be traced to plastid division and migration, sporocytes of Aneura pinguis are polyplastidic and tetrahedrally lobed before the QMS is organized. Whereas the QMS in mosses and hornworts is plastid-based, the QMS of A. pinguis is focused at four POs where gamma tubulin (-tubulin) is concentrated. An aster of microtubules emanates from each PO centered in the four cytoplasmic lobes and the opposing radial microtubules interact to form the QMS that envelops the nucleus. A functionally bipolar spindle is gradually formed as the four poles converge in pairs on either side of opposite cleavage furrows. The resulting spindle remains quadripolar. Although -tubulin is most concentrated in the deeply concave poles straddling cleavage furrows, it also extends into the spindle itself. Telophase groups of chromosomes curve around the polar cleavage furrows and a phragmoplast that originates in the interzonal region guides a cell plate that extends to the equatorial cleavage furrows. Discrete POs are reformed at opposite tips of the elongated dyad nuclei in prophase II and microtubules radiating from them give rise to the spindles of second meiosis. Spindles remain sharply focused and -tubulin extends into distal portions of the spindle. Interzonal phragmoplasts that expand to join with pre-established cleavage furrows mediate cytokinesis resulting in a tetrad of spores. Each young tetrad member has a radial microtubule system emanating from the nucleus.     

Kinesins Are Indispensable for Interdigitation of Phragmoplast Microtubules in the Moss Physcomitrella patens

Microtubules form arrays with parallel and antiparallel bundles and function in various cellular processes, including subcellular transport and cell division. The antiparallel bundles in phragmoplasts, plant-unique microtubule arrays, are mostly unexplored and potentially offer new cellular insights. Here, we report that the Physcomitrella patens kinesins KINID1a and KINID1b (for kinesin for interdigitated microtubules 1a and 1b), which are specific to land plants and orthologous to Arabidopsis thaliana PAKRP2, are novel factors indispensable for the generation of interdigitated antiparallel microtubules in the phragmoplasts of the moss P. patens. KINID1a and KINID1b are predominantly localized to the putative interdigitated parts of antiparallel microtubules. This interdigitation disappeared in double-deletion mutants of both genes, indicating that both KINID1a and 1b are indispensable for interdigitation of the antiparallel microtubule array. Furthermore, cell plates formed by these phragmoplasts did not reach the plasma membrane in ∼20% of the mutant cells examined. We observed that in the double-deletion mutant lines, chloroplasts remained between the plasma membrane and the expanding margins of the cell plate, while chloroplasts were absent from the margins of the cell plates in the wild type. This suggests that the kinesins, the antiparallel microtubule bundles with interdigitation, or both are necessary for proper progression of cell wall expansion.     

Microtubules in early development of the megagametophyte of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Food storage tissue in the seeds of gymnosperms is female gametophyte (megagametophyte) that develops before fertilization, whereas, in seeds of angiosperms, food is stored as endosperm initiated by double fertilization. The megagametophyte is haploid, and endosperm is usually triploid, at least initially. Despite differences in origin, ploidy level, and developmental trigger, the early events of female gametophyte development in ginkgo are very similar to nuclear endosperm development in the seeds of angiosperms. In both, development begins as a single cell that undergoes multiple mitoses without cytokinesis, to produce a large syncytium. This study provided evidence that microtubule involvement in organization of the syncytium into nuclear cytoplasmic domains (NCDs) via nuclear-based radial microtubule systems is a critical developmental feature in the ginkgo megagametophyte, as it is in endosperm. Once the initial anticlinal walls have been deposited at the boundaries of NCDs, cellularization proceeds by the process of alveolation. Continued unidirectional growth of the alveolar walls is an outstanding example of polar cytokinesis. Ginkgo megagametophyte development appears to occur uniformly throughout the entire chamber, whereas nuclear type endosperm usually exhibits distinct developmental domains. These observations suggest that there is a fundamental pathway for the development and cellularization of syncytia in seed development.     

Temporal and spatial appearance of wall polysaccharides during cellularization of barley (Hordeum vulgare) endosperm

Barley endosperm begins development as a syncytium where numerous nuclei line the perimeter of a large vacuolated central cell. Between 3 and 6 days after pollination (DAP) the multinucleate syncytium is cellularized by the centripetal synthesis of cell walls at the interfaces of nuclear cytoplasmic domains between individual nuclei. Here we report the temporal and spatial appearance of key polysaccharides in the cell walls of early developing endosperm of barley, prior to aleurone differentiation. Flowering spikes of barley plants grown under controlled glasshouse conditions were hand-pollinated and the developing grains collected from 3 to 8 DAP. Barley endosperm development was followed at the light and electron microscope levels with monoclonal antibodies specific for (1→3)-β-d-glucan (callose), (1→3,1→4)-β-d-glucan, hetero-(1→4)-β-d-mannans, arabino-(1→4)-β-d-xylans, arabinogalactan-proteins (AGPs) and with the enzyme, cellobiohydrolase II, to detect (1→4)-β-d-glucan (cellulose). Callose and cellulose were present in the first formed cell walls between 3 and 4 DAP. However, the presence of callose in the endosperm walls was transient and at 6 DAP was only detected in collars surrounding plasmodesmata. (1→3,1→4)-β-d-Glucan was not deposited in the developing cell walls until approximately 5 DAP and hetero-(1→4)-β-d-mannans followed at 6 DAP. Deposition of AGPs and arabinoxylan in the wall began at 7 and 8 DAP, respectively. For arabinoxylans, there is a possibility that they are deposited earlier in a highly substituted form that is inaccessible to the antibody. Arabinoxylan and heteromannan were also detected in Golgi and associated vesicles in the cytoplasm. In contrast, (1→3,1→4)-β-d-glucan was not detected in the cytoplasm in endosperm cells; similar results were obtained for coleoptile and suspension cultured cells.     

Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.     

{gamma}-Tubulin and microtubule organization during meiosis in the liverwort Ricciocarpus natans (Ricciaceae)

Extant liverworts are "living fossils" considered sister to all other plants and as such provide clues to the evolution of the microtubule organizing center (MTOC) in anastral cells. This report is the first on microtubule arrays and their γ-tubulin-nucleating sites during meiosis in a member of the Ricciales, a specialized, species-rich group of complex thalloid (marchantioid) liverworts. In meiotic prophase, γ-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci give rise to a multipolar prometaphase spindle. By metaphase I, the spindle has matured into a bipolar structure with truncated poles. In both first and second meiosis, γ-tubulin forms box-like caps at the spindle poles. γ-Tubulin moves from spindle poles to the proximal surfaces of telophase chromosomes where interzonal microtubules are nucleated. Although a phragmoplast is organized, no cell plate is deposited, and second division occurs simultaneously in the undivided sporocyte. γ-Tubulin surrounds each of the tetrad nuclei, and phragmoplasts initiated between both sister and nonsister nuclei direct simultaneous cytokinesis. The overall pattern of meiosis (unlobed polyplastidic sporocytes, nuclear envelope MTOC, multipolar spindle origin, spindles with box-like poles, and simultaneous cytokinesis) more closely resembles that of Conocephalum than other marchantiod liverworts.     

Gamma-tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells

Cortical microtubules are considered to regulate the direction of cellulose microfibril deposition. Despite their significant role in determining cell morphology, cortical microtubules completely disappear from the cell cortex during M phase and become reorganized at G1 phase. The mechanism by which these microtubules become properly formed again is, however, still unclear. We have proposed that the origin of cortical microtubules is on the daughter nuclear surface, but further cortical microtubule reorganization occurs at the cell cortex. Hence it is probable that the locations of microtubule organizing centers (MTOCs) are actively changing. However, the actual MTOC sites of cortical microtubules were not clearly determined. In this paper, we have examined the distribution of gamma-tubulin, one of the key molecules of MTOCs in various organisms, during cortical microtubule reorganization using both immunofluorescence and a GFP reporter system. Using a monoclonal antibody (clone G9) that recognizes highly conserved residues in y-tubulin, y-tubulin was found to be constitutively expressed and to be clearly localized to microtubule structures, such as the preprophase bands, spindles, and phragmoplasts, specific to each cell cycle stage. This distribution pattern was confirmed by the GFP reporter system. During cortical microtubule reorganization at the M to G1 transition phase, gamma-tubulin first accumulated at the daughter nuclear surfaces, and then seemed to spread onto the cell cortex along with microtubules elongating from the daughter nuclei. Based on the results, it was confirmed that daughter nuclear surfaces acted as origins of cortical microtubules, and that further reorganization occurred on the cell cortex.     

Organisation of the endosperm and endosperm–placenta syncytia in bladderworts (Utricularia, Lentibulariaceae) with emphasis on the microtubule arrangement

Multinucleate cells play an important role in higher plants, especially during reproduction; however, the configurations of their cytoskeletons, which are formed as a result of mitosis without cytokinesis, have mainly been studied in coenocytes. Previous authors have proposed that in spite of their developmental origin (cell fusion or mitosis without cytokinesis), in multinucleate plant cells, radiating microtubules determine the regular spacing of individual nuclei. However, with the exception of specific syncytia induced by parasitic nematodes, there is no information about the microtubular cytoskeleton in plant heterokaryotic syncytia, i.e. when the nuclei of fused cells come from different cell pools. In this paper, we describe the arrangement of microtubules in the endosperm and special endosperm–placenta syncytia in two Utricularia species. These syncytia arise from different progenitor cells, i.e. cells of the maternal sporophytic nutritive tissue and the micropylar endosperm haustorium (both maternal and paternal genetic material). The development of the endosperm in the two species studied was very similar. We describe microtubule configurations in the three functional endosperm domains: the micropylar syncytium, the endosperm proper and the chalazal haustorium. In contrast to plant syncytia that are induced by parasitic nematodes, the syncytia of Utricularia had an extensive microtubular network. Within each syncytium, two giant nuclei, coming from endosperm cells, were surrounded by a three-dimensional cage of microtubules, which formed a huge cytoplasmic domain. At the periphery of the syncytium, where new protoplasts of the nutritive cells join the syncytium, the microtubules formed a network which surrounded small nuclei from nutritive tissue cells and were also distributed through the cytoplasm. Thus, in the Utricularia syncytium, there were different sized cytoplasmic domains, whose architecture depended on the source and size of the nuclei. The endosperm proper was isolated from maternal (ovule) tissues by a cuticle layer, so the syncytium and chalazal haustorium were the only way for nutrients to be transported from the maternal tissue towards the developing embryo.