The cytological changes of tobacco zygote and proembryo cells induced by beta-glucosyl Yariv reagent suggest the involvement of arabinogalactan proteins in cell division and cell plate formation

ABSTRACT: BACKGROUND: In dicotyledonous plant, the first asymmetric zygotic division and subsequent several cell divisions are crucial for proembryo pattern formation and later embryo development.. Arabinogalactan proteins (AGPs) are a family of extensively glycosylated cell surface proteins that are thought to have important roles in various aspects of plant growth and development, including embryogenesis. Previous results from our laboratory show that AGPs are concerned with tobacco egg cell fertilization and zygotic division. However, how AGPs interact with other factors involved in zygotic division and proembryo development remains unknown. RESULTS: In this study, we used the tobacco in vitro zygote culture system and series of meticulous cell biology techniques to investigate the roles of AGPs in zygote and proembryo cell division. For the first time, we examined tobacco proembryo division patterns detailed to every cell division. The bright-field images and statistical results both revealed that with the addition of an exogenous AGPs inhibitor, beta-glucosyl Yariv (beta-GlcY) reagent, the frequency of aberrant division increased remarkably in cultured tobacco zygotes and proembryos, and the cell plate specific locations of AGPs were greatly reduced after beta-GlcY treatment. In addition, the accumulations of new cell wall materials were also significantly affected by treating with beta-GlcY. Detection of cellulose components by Calcofluor white stain showed that strong fluorescence was located in the newly formed wall of daughter cells after the zygotic division of in vivo samples and the control samples from in vitro culture without beta-GlcY treatment; while there was only weak fluorescence in the newly formed cell walls with beta-GlcY treatment. Immunocytochemistry examination with JIM5 and JIM7 respectively against the low- and high-esterified pectins displayed that these two pectins located in opposite positions of zygotes and proembryos in vivo and the polarity was not affected by beta-GlcY. Furthermore, FM4-64 staining revealed that endosomes were distributed in the cell plates of proembryos, and the localization pattern was also affected by beta-GlcY treatment. These results were further confirmed by subsequent observation with transmission electron microscopy. Moreover, the changes to proembryo cell-organelles induced by beta-GlcY reagent were also observed using fluorescent dye staining technique. CONCLUSIONS: These results imply that AGPs may not only relate to cell plate position decision, but also to the location of new cell wall components. Correlated with other factors, AGPs further influence the zygotic division and proembryo pattern establishment in tobacco.     

Localization of arabinogalactan proteins in egg cells, zygotes, and two-celled proembryos and effects of beta-D-glucosyl Yariv reagent on egg cell fertilization and zygote division in Nicotiana tabacum L

Arabinogalactan proteins (AGPs) have been implicated in a variety of plant development processes including sexual plant reproduction. As a crucial developmental event, plant sexual reproduction generally occurs inside an ovule embedded in an ovary. The inaccessibility of the egg cells, zygotes, and embryos has hindered our understanding of the importance of AGPs in the early events involving fertilization, zygotic division, and early embryogenesis. In this study, the well-established in vitro zygote and ovary culture systems, together with immunofluorescence and immunogold labelling techniques, were employed to investigate the role of AGPs in the early events of sexual reproduction in Nicotiana tabacum. Dramatic changes in AGP content during ovule development were evidenced by western blotting. Subcellular localization revealed that AGPs are localized in the plasma membrane, cell wall, and cytoplasm of pre- and post-fertilized egg cells, and cytoplasm and vacuoles of two-celled proembryos. Abundant AGPs were detected in unfertilized egg cells; however, the level of AGPs substantially decreased in fertilized egg cells. Polar distribution of AGPs in elongated zygotes was observed. The early two-celled proembryos just from zygote division displayed accumulation of AGPs at a low level, while in the elongated two-celled proembryos at the late stage, the AGP content clearly increased. Provision of betaGlcY, a synthetic phenylglycoside that specifically binds AGPs, to the in vitro cultures of isolated zygote and fertilized ovaries increased abnormal symmetrical division of zygotes. In the culture of pollinated but unfertilized ovaries, addition of betaGlcY resulted in arrest of fertilization of the egg cells, but had no effect on fertilization of the central cells. The possible roles of AGPs in fertilization, zygotic division, and proembryo development are discussed.     

Reproduction and sexual development in a freshwater gastrotrich

Summary Post-embryonic development of parthenogenic eggs of Lepidodermella squammata was studied by light and electron microscopy in animals of known age and reproductive history. Each bilateral gonad initially contains eight cells. No mitotic proliferation occurs during parthenogenic egg development. Germ cells are tightly clustered, have smooth plasma membranes with no interconnections, and are uninucleate. There is no surrounding ovary or oviduct. At hatching, two cells in each gonad are identifiable as parthenogenic eggs. The enlarged nucleolus of the most mature egg has already attained the morphology that persists throughout vitellogenesis, with intertwined granular and fibrillar threads. Less mature eggs have earlier stages of nucleolar development, and lack indications of meiotic events. Parthenogenic eggs enter vitellogenesis singly, with formation of RER and active Golgi complexes, and the accumulation of lipid, yolk, and various granules. The shell is formed in situ, whereas the spines elongate after egg deposition. Most animals produce four parthenogenic eggs, which undergo immediate development (tachyblastic eggs). Resting (opsiblastic) eggs are rare in isolation culture. Both types of eggs are produced only prior to the formation of sperm and primary oocytes. The absence of synaptonemal complexes, which would indicate synapsis of homologous chromosomes in prophase of meiosis I, implies that parthenogenesis is by apomixis in L. squammata.     

Ultrastructural Observations on Parthenogenesis and Antipodal Apogamy of Allium tuberosum Roxb

Alliurn tuberosum Roxb is a species characterized by spontaneous parthenogenesis and antipodal apogamy. This paper deals with the ultrastructural changes during these processes. Before pollination, the mature egg cell contained abundant mitochondria with well developed cristae, spherical or ellipsoidal plastids and some polyribosomes, which suggested a relatively high metabolic activity. After fertilization, in zygotes the mitochondria changed to irregular shape and their cristae degenerated, the plastids elongated or became cup-shaped, the polyribosomes decreased and the free ribosomes increased in number. Some unfertilized egg ceils, two days after anthesis, showed similar ultrastructural changes as those taking place in zygote, which. seemed to be a sign of triggering to parthenogensis. In Allium tuberosum Roxb, the three antipodai cells bore a close resemblance to the egg apparatus: Among them two antipodal cells were similar to the synergids with a filiform apparatus-like structure and plentiful organelles at their chalazal end. The other was an egg-like antipodal cell which could undergo similar ultrastructural changes as those happened in zygote, leading to divide into apogamic proembryos two days after anthesis. The problems of parthenogenesis and antipodal apogamy in Allium tuberosum Roxb is diicussed in view of ultrastructural features of the egg and egg-like antipodal cell.     

Histological comparison between wheat embryos developing in vitro from isolated zygotes and those developing in vivo

There is currently great interest shown in understanding the process of embryogenesis and, due to the relative inaccessibility of these structures in planta, extended studies are carried out in various in vitro systems. The culture of isolated zygotes in particular provides an excellent platform to study the process of in planta embryogenesis. However, very few comparisons have been made between zygotic embryos grown entirely in cultures and those grown in vivo. The present study analyses the differences and similarities between the in vitro and in vivo development of wheat zygotic embryos at the level of morphology and histology. The study was possible thanks to an efficient culture system and an appropriate method of preparing isolated wheat zygotes for microscopy. The in vitro embryos were fixed, embedded and sectioned in the two-celled, globular, club-shaped and fully differentiated stages. Embryos developing in vitro closely followed the morphology of their in planta counterparts and their cell types and tissues were also similar, demonstrating the applicability of the present culture system for studying the process of zygotic embryogenesis. However, some important differences were also detected in the case of in vitro development: the disturbance of or lack of initial polarity led to changes in the division symmetry of the zygotes and subsequently to the formation of uniform cells in the globular structures. Presumably, differences between the in vitro and in planta environments resulted in a lower level of differentiation and maturation in in vitro embryos and in abundant starch and protein accumulation in the scutellum.     

千里光合子胚和胚乳形成及其早期发育的细胞学特征（英文）

千里光(Senecio scandens Buch.-Ham. ex D. Don)是传统中草药,抗菌功效显著。本研究从细胞学角度对千里光合子胚和胚乳的形成与发育进行观察研究。结果显示,结构和功能迥异的基细胞和顶细胞源自细胞质不均一分布的合子所致,推测合子的极性与胚囊的极性和生殖核分裂为"二态"精细胞有关;基细胞在合子胚胎"球型期"末期出现分化,早期胚胎的组织分化始于"三角期",可辨别的结构差异直到"鱼雷期"才出现。此外,胚乳形成遵循无细胞壁核化模型。本研究对千里光细胞分化、组织分化和结构差异各发育阶段特征的观察结果,不仅可为深入分析胚胎发育过程功能基因的时空表达提供依据,也为相关近缘物种的系统植物学研究提供参考资料。     

Young microspore-derived maize embryos show two domains with defined features also present in zygotic embryogenesis

In this work we report the existence of two domains in early microspore maize proembryos displaying similar features of zygotic embryogenesis. The large or so-called endosperm-like domain exhibits specific features: coenocytic organisation, synchronous mitosis, vacuolated cytoplasm, starch granules, incomplete walls containing callose and differential tubuline organisation. The small or embryo-like domain displays small polygonal uninucleate cells with typical organisation of proliferating cells. The structural organisation and the subcellular localization of specific cytoplasmic and cell wall components (starch, tubuline and callose) in both proembryo domains have been determined by using specific cytochemical and immunocytochemical methods. Four morphological types of proembryos containing both domains have been characterised. Taking into account their relative size, the high asynchrony of the culture and the homologies between structural features of endosperm-like domain and zygotic endosperm development, they could represent different stages in microspore embryogenesis development. The ZmAE1 and ZmAE3 genes expressed in the Embryo Surrounding Region of the endosperm during zygotic embryogenesis (Magnard et al., 2000), were revealed to be expressed in early microspore proembryos by in situ hybridization at light and electron microscopy levels. This data supports the existence of an endosperm-like function during early microspore embryogenesis and provides new insights into the onset of microspore embryogenesis in maize, and its parallelism with zygotic embryogenesis.     

Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation

We have developed a reliable in vitro zygotic embryogenesis system in tobacco. A single zygote of a dicotyledonous plant was able to develop into a fertile plant via direct embryogenesis with the aid of a co-culture system in which fertilized ovules were employed as feeders. The results confirmed that a tobacco zygote could divide in vitro following the basic embryogenic pattern of the Solanad type. The zygote cell wall and directional expansion are two critical points in maintaining apical-basal polarity and determining the developmental fate of the zygote. Only those isolated zygotes with an almost intact original cell wall could continue limited directional expansion in vitro, and only these directionally expanded zygotes could divide into typical apical and basal cells and finally develop into a typical embryo with a suspensor. In contrast, isolated zygote protoplasts deprived of cell walls could enlarge but could not directionally elongate, as in vivo zygotes do before cell division, even when the cell wall was regenerated during in vitro culture. The zygote protoplasts could also undergo asymmetrical division to form one smaller and one larger daughter cell, which could develop into an embryonic callus or a globular embryo without a suspensor. Even cell walls that hung loosely around the protoplasts appeared to function, and were closely correlated with the orientation of the first zygotic division and the apical-basal axis, further indicating the essential role of the original zygotic cell wall in maintaining apical-basal polarity and cell-division orientation, as well as subsequent cell differentiation during early embryo development in vitro.     

Early cytological events after induction of cell division in egg cells and zygote development following in vitro fertilization with angiosperm gametes

Early events, such as formation of the cell wall, first nuclear division and first unequal division of the zygote, were examined following in vitro fusion of single egg and sperm protoplasts of maize ( Zea mays L.). The time course of these events was determined. The formation of cell wall components was observed 30 sec following egg—sperm fusion and proceeded continuously thereafter. Within 15 h after fusion most of the organelles became more densely grouped around the nucleus of the zygote. In the in vitro produced zygote the location of the cell organelles and of the dividing nucleus showed polarity. Two nucleoli were first observed 18 h after gamete fusion. The zygotic nucleus remained undivided for about 40 h. The first cell division was observed 40–60 h, generally 42–46 h, after egg—sperm fusion. The non-fused egg cell could be triggered to sporophytic development in vitro by pulses of high amounts of 2,4-D. Without such a treatment, cultured egg cells of different maize lines did not divide. Although nuclear fusion seemed to occur, fusion products of two egg cells also did not divide. Cell wall formation was incomplete and non-uniform, showing a polarity of cultured egg cells and fusion products of two egg protoplasts. Cell division was also induced after fusion of maize egg with sperms of genetically remote species, such as Coix, Sorghum, Hordeum or Triticum . These gametic heterologous fusion products developed to microcalli. Moreover, cell division occurred in fusion products of an egg and a diploid somatic cell-suspension protoplast from maize.     

Cytological Events during Zygote Formation of the Fern Ceratopteris thalictroides

The cytological events, including nuclear fusion, digestion of male organelles and rebuilding of the plasmalemma and cell wall, during zygote formation of the fern Ceratopteris thalictroides (L.) Brongn. are described based on the observations of transmission electron microscopy. When the spermatozoid enters the egg and contacts the cytoplasm, the male chromatin relaxes continually. The microtubular ribbon (MTr) is separated from the male nucleus and then an envelope reappears around the male nucleus. During nuclear fusion, the egg nucleus becomes highly irregular and extends some nuclear protrusions. It is proposed that the protrusions fuse with the male nucleus actively. After nuclear fusion the irregular zygotic nucleus contracts gradually. It becomes spherical before the zygote divides. The male chromatin is identifiable as fibrous structure in the zygotic nucleus in the beginning, but it gradually becomes diffused completely. The male organelles, including the MTr, multilayered structure, flagella and the male mitochondria are finally digested in the zygotic cytoplasm. Finally a new plasmalemma and cell wall are formed outside the protoplast. The organelles in the zygote are rearranged, which produces a horizontal polarity zygote. The zygote divides with an oblique-vertical cell plate facing the apical notch of the gametophyte.     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

Cytological investigation of the mechanism of parthenogenesis in Drosophila mercatorum

Thelytokous parthenogenesis (female progeny only) in animals is believed to arise initially in unfertilized eggs produced by bisexual females via the fusion of two haploid nuclei following meiosis, to produce diploid female progeny. The transition from sexual to parthenogenetic mechanisms of reproduction requires that the egg replace the paternal contributions of a haploid genetic complement and the basal body, which is thought to be essential for centrosome formation. The transitional facultative parthenogenetic stage is usually associated with a high rate of failed or abortive development, but the molecular and mechanistic reasons for this failure remain unclear. We show that a facultative parthenogenetic strain of Drosophila mercatorum produces a high percentage of unfertilized eggs competent to restore diploidy and form centrosomes de novo following meiosis. The female meiotic products replicate and divide by an acentrosomal mechanism in most oocytes and cytoplasmic centrosomes form in 35% of the oocytes. However, after pronuclear replication the cytoplasmic centrosomes must "capture" two haploid nuclei in order to restore diploidy. In practice, this process frequently fails due to centrosome-mediated capture events of single or more than two haploid nuclei, as well as multiple nuclear capture events in a single embryo when excess free centrosomes are not inactivated following formation of the first zygotic nucleus. Additionally, as development proceeds, many of the centrosomes that initiate syncytial development do not remain functional, possibly due to centrosome maturation defects, and later stages of syncytial development fail. The combined effect of the high error rate associated with nuclear capture and the failure of centrosome maturation during later developmental prevents successful parthenogenesis in most of the eggs that initiate development. This shows that the high rate of failed development associated with the transition from sexual to parthenogenetic reproduction is limited by the low probability of the formation of a diploid zygotic nucleus with the correct complement of centrosomes in D. mercatorum.     

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.     

千里光合子胚和胚乳形成及其早期发育的细胞学特征(英文稿)

千里光（Senecio scandens Buch.-Ham. ex D. Don）是传统中草药, 抗菌功效显著。本研究从细胞学角度对千里光合子胚和胚乳的形成与发育进行观察研究。结果显示,结构和功能迥异的基细胞和顶细胞源自细胞质不均一分布的合子所致,推测合子的极性与胚囊的极性和生殖核分裂为“二态”精细胞有关;基细胞在合子胚胎“球型期”末期出现分化,早期胚胎的组织分化始于“三角期”,可辨别的结构差异直到“鱼雷期”才出现。此外,胚乳形成遵循无细胞壁核化模型。本研究对千里光细胞分化、组织分化和结构差异各发育阶段特征的观察结果,不仅可为深入分析胚胎发育过程功能基因的时空表达提供依据,也为相关近缘物种的系统植物学研究提供参考资料。     

Embryo initiation from Pinus sibirica megagametophytes in in vitro culture

Megagametophytes of Siberian pine were cultured on an in vitro culture medium 1/2 LV supplemented with growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and benzylaminopurine (6-BAP) to form embryos. The competency of somatic cell of explants to embryogenesis manifested itself in an organized growth and polarity. A coenocyte consisting of long vacuolated cells was formed in the megagametophyte culture. Then, the migration of the nuclei to one of the poles of the cell, their division, and formation of embryoids was observed. The megagametophyte culture of the Siberian pine differed from the zygotic embryo culture by the absence of asymmetric division in the vacuolated cell.     

Cytological Investigation of the Mechanism of Parthenogenesis in Drosophila mercatorum

Thelytokous parthenogenesis (female progeny only) in animals is believed to arise initially in unfertilized eggs produced by bisexual females via the fusion of two haploid nuclei following meiosis, to produce diploid female progeny. The transition from sexual to parthenogenetic mechanisms of reproduction requires that the egg replace the paternal contributions of a haploid genetic complement and the basal body, which is thought to be essential for centrosome formation. The transitional facultative parthenogenetic stage is usually associated with a high rate of failed or abortive development, but the molecular and mechanistic reasons for this failure remain unclear. We show that a facultatively parthenogenetic strain of Drosophila mercatorum produces a high percentage of unfertilized eggs competent to restore diploidy and form centrosomes de novo following meiosis. The female meiotic products replicate and divide by an acentrosomal mechanism in most oocytes and cytoplasmic centrosomes form in 35% of the oocytes. However, after pronuclear replication the cytoplasmic centrosomes must "capture" two haploid nuclei in order to restore diploidy. In practice, this process frequently fails due to centrosome-mediated capture events of single or more than two haploid nuclei, as well as multiple nuclear capture events in a single embryo when excess free centrosomes are not inactivated following formation of the first zygotic nucleus. Additionally, as development proceeds, many of the centrosomes that initiate syncytial development do not remain functional, possibly due to centrosome maturation defects, and later stages of syncytial development fail. The combined effect of the high error rate associated with nuclear capture and the failure of centrosome maturation during later developmental prevents successful parthenogenesis in most of the eggs that initiate development. This shows that the high rate of failed development associated with the transition from sexual to parthenogenetic reproduction is limited by the low probability of the formation of a diploid zygotic nucleus with the correct complement of centrosomes in D. mercatorum.     

Low-temperature treatment of tobacco ovaries improves the isolation, viability, and embryogenic potential of two-celled proembryos

The influence of low-temperature treatment of ovaries on the isolation and subsequent culture of two-celled proembryo in vitro was explored in tobacco. The efficiency of isolation of two-celled proembryos was significantly improved, by an average of 23.47?% after treatment of ovaries at 4°C for 5?C24?h. Cell viability and polarity of two-celled proembryos, as assessed by organelle distribution, growth pattern, and cell wall component distribution, were all well maintained after cold treatment. Furthermore, in vitro cultivated two-celled proembryos divided normally, with timing paralleling that of two-celled proembryos isolated from fresh ovaries. Unexpectedly, the division frequency of two-celled proembryos was significantly improved by cold treatment. These results demonstrate that low-temperature treatment of tobacco ovaries was beneficial for two-celled proembryo manipulation in vitro, by not only improving isolation efficiency but also by promoting cell division and improving the potential for further cultivation.