Hypoxia induced non-apoptotic cellular changes during aerenchyma formation in rice (Oryza sativa L.) roots

The stress of low oxygen concentrations in a waterlogged environment is minimized in some plants that produce aerenchyma, a tissue characterized by prominent intercellular spaces. It is produced by the predictable collapse of root cortex cells, indicating a programmed cell death (PCD) and facilitates gas diffusion between root and the aerial environment. The objective of this study was to characterize the cellular changes take place during aerenchyma formation in root of rice that accompany PCD. Scanning electron microscopy and transmission electron microscopy were used for cellular analysis of roots. Aerenchyma development was observed in both aerobic and flooded conditions. Structural changes in membranes and organelles were examined during development of root cortex cells to compare with previous examples of PCD. There was an initial collapse which started at a specific position in the mid cortex, indicating loss of turgor, and the cytoplasm became more electron dense. These cells were distinct in shape from those located towards the periphery. Mitochondria and endoplasmic reticulum appeared normal at this early stage though the tonoplast lost its integrity. Subsequently it underwent further degeneration while the plasmalemma retracted from the cell wall followed by death of neighboring cells followed a radial path. However, pycnosis of the nucleus, blebbing of plasma membrane and production of apoptotic bodies were not found which in turn indicated nonapoptotic PCD during aerenchyma formation in rice.     

荔枝雄花性别决定过程中细胞超微结构的变化

荔枝雄花雌蕊原基在大孢子母细胞减数分裂后开始衰退.内质网历经增生扩展,穿壁相连,同心缠绕,多条平行弯曲,不规则堆叠.内质网和高尔基体产生许多囊泡,囊泡在细胞内含物的降解和运输过程中起着重要的作用.线粒体在雌蕊原基细胞衰败的前、中期数量增加,后期分批降解.过氧化物酶体在雌蕊原基细胞衰败的中期紧挨核短暂出现.细胞核的染色质凝集断裂;核周腔扩大,形成胀泡;染色质趋边,外泄.细胞原生质表现出有序的、在膜包裹下的降解,首先是核糖体,而后依次是:过氧化物酶体、内质网、高尔基体、线粒体、核.雌蕊原基的衰败历程可能是一种程序性细胞死亡的过程.     

A Transmission and Cryo-Scanning Electron Microscopy Study of the Formation of Aerenchyma (Cortical Gas-Filled Space) in Adventitious Roots of Rice (Oryza sativa)

The initiation and development ofaerenchyma in adventitiousroots of rice (Oryza sativa) was studied in tissuc up to approximately36-h-old using transmission electron microscopy (TEM) and cryoscanningelectron microscopy (cryo-SEM). Aerenchyma resulting from selective cortical cell collapse isa naturally occurring feature of rice roots. Evidence of somecortical cell disruption was noticeable by TEM in cells thatwere approximately 6-h-old; it became more advanced as the cellsaged. At 12 h, early stages of cell wall breakdown and lossof cell turgidity were seen. Complete collapse of columns ofcells had occurred by 24 h. In tissue that was 36-h-old, thesenescent cytoplasm of many remaining cells began to disperse.The visual evidence suggests that cell collapse was the resultof autolysis. This pattern of cortical degeneration in ricewas dissimilar to that reported elsewhere for Zea roots grownin an oxygen depleted environment. Cryo-SEM revealed the occurrence of small structures withinthe cortex with the external appearance of miniature, intactcells which are not preserved during conventional SEM preparativeprocedures. Key words: Rice (Oryza sativa L.), roots, aerenchyma, ultrastructure     

Regeneration of tissue following cavity formation in the vascular cylinders of Pisum sativum (Fabaceae) primary roots

The reorganization of vascular cylinders of pea (Pisum sativum, cv. Alaska) primary roots following the formation of vascular cavities was examined by light and electron microscopy. Cavities usually began forming ~20 mm from the root tip and were continuous to ~90 mm from the tips in roots 150 mm long, where they began filling with specialized parenchyma cells (SP cells). SP cells were usually produced by enlargement of parenchymous cells of the primary xylem at cavity margins. Depending on the extent and shape of the cavity, they were also sometimes produced by primary phloem parenchyma and early derivatives of the vascular cambium. Enlargement and some divisions of SP cells continued until a cavity was completely filled by them. SP cells proceeded through a series of cytoplasmic changes as they developed. First the cytoplasmic layer became thicker and more electron dense than surrounding cells. As SP cells enlarged there was an increase in vesicular traffic and the cytoplasm became less electron dense. Ultimately the cytoplasm thinned further, organelles degenerated, and the tonoplast sometimes broke down. SP cells did not form secondary walls. X-ray microanalysis revealed that SP cells accumulated potassium and rubidium to the same degree as cortical and xylem parenchyma cells and to a greater degree than immature secondary and late-maturing tracheary elements.     

Rapid changes in cell wall pectic polysaccharides are closely associated with early stages of aerenchyma formation, a spatially localized form of programmed cell death in roots of maize (Zea mays L.) promoted by ethylene

Aerenchyma formation in roots of maize (Zea mays L.) involves programmed death of cortical cells that is promoted by exogenous ethylene (1 µL L⁻¹) or by endogenous ethylene produced in response to external oxygen shortage (3%, v/v). In this study, evidence that degeneration of the cell wall accompanies apoptotic-like changes previously observed in the cytoplasm and nucleus (Gunawardena et al. Planta 212, 205–214, 2001), has been sought by examining de-esterified pectins (revealed by monoclonal antibody JIM 5), and esterified pectins (revealed by monoclonal antibody JIM 7). In controls, de-esterified wall pectins were found at the vertices of triangular junctions between cortical cells (untreated roots). Esterified pectins in control roots were present in the three walls bounding triangular cell-to-cell junctions. After treatment with 3% oxygen or 1 µL L⁻¹ ethylene, this pattern was lost but walls surrounding aerenchyma gas spaces became strongly stained. The results showed that cell wall changes commenced within 0·5 d and evidently were initiated by ethylene in parallel with cytoplasmic and nucleoplasmic events associated with classic intracellular processes of programmed cell death.     

Ultrastructural evidence for apoptosis of pavement cells, chloride cells, and hatching gland cells in the developing branchial area of the trout Salmo trutta

Death by apoptosis of branchial epithelial cells was studied in brown trout embryos by means of transmission electron microscopy. Superficial pavement cells are sloughed off for the renewal of the epithelium after an apoptotic degeneration with shrinkage of the cytoplasm and loss of desmosomal contacts. Chloride cells appear as immature, mature and degenerating cells. Degenerating chloride cells, which are separated from the ambient water by pavement cells, show condensation of the cytoplasm and structural alterations in the tubular system and the mitochondria. Hatching gland cells degenerate either into apoptotic bodies or into cellular debris, depending on the functional stage of the cell. There was no phagocytosis by macrophages or adjacent cells of the degenerating chloride and hatching gland cells, but an infiltration of leucocytes was always observed in the epithelium undergoing cellular degeneration. In some instances, secondary necrosis of apoptotic hatching gland cells was observed. Apoptosis occurs in the three types of cells since early stages of development. However, a massive wave of cellular death occurred in pavement and hatching gland cells during the hatching stage and in the chloride cells during post-hatching stages.     

Modulation of apoptosis by starvation: morphological and biochemical study of rat intestinal mucosa

Morphology at light and electron microscopic levels, expression and activation of transglutaminase and DNA fragmentation at internucleosomal sites were used as markers to study the effect of starvation on the apoptosis of small intestinal epithelial cells. The cells entering apoptotic programme in well-fed animals undergo many morphological changes in apical cytoplasm involving alterations in actin cytoskeleton organisation which may cause a discharge of microvilli. Some free floating cells in the intestinal lumen show characteristics of apoptotic cell death, e.g. shrinkage of cell and peripheral condensation of chromatin, while mitochondria and lysosomes remain unchanged. Apoptotic bodies are also seen in scanning electron micrographs. During progressive starvation, epithelial cells do not enter the apoptotic cell death programme. Biochemical markers for apoptosis such as increased transglutaminase activity and DNA fragmentation are clearly discernible in normally fed animals. The percentage of cells labelled immunohistochemically by antibody against transglutaminase decreased during starvation while DNA fragmentation was absent. The exact mechanism for suppressing apoptosis in intestinal cells under starvation is not known. However, the data presented here support the existence of such a regulatory process.     

Ultrastructure of Male Gametophyte in wheat I. The Formation of Generative and Vegetative Cells

The present study of the formation of the generative and vegetative cells in wheat has demonstrated some cytological details at the ultrastructural level. The phragmoplast formed in telophase of the first microsporic mitosis extended centrifugally until it connected with the intine of the pollen grain. A new cell wall was then formed to separate the generative and the vegetative cells. By unequal cytokinesis the former is small and the latter large. In early developmental stage of male gametophyte, the organelles in the cytoplasm of the generaVive cell and the vegetative cells are similar, including mitochondria, dictyosomes, rough endoplasmic retieulum, free and clustered ribosomes and plastids, but microtubules were observed only in the early cytokinesis stage. In the further developmental stage of the male gemetophyte, the generative cell gradually detached from the intine of pollen grain and grew inward to the cytoplasm of the vegetation cell. When the generative cell became round and free in the cytoplasm of the vegetative cell, the wall materials between plasma membranes of the cytoplasm of the generative and the vegetative cells disappeared completely, so that it was a naked cell with a double-layer membrane at this time. The heterogeneity between both cells was then very conspiceous. The organelles in the cytoplasm of the generative cell have hardly any changed besides the degeneration of plastids, but in vegetative cytoplasm the mitochondria and plastids increased dramatically both in number and size. The rapid deposition of starch in the plastids of the cytoplasm of the vegetative cell made the most conspicuous feature of the vegetative cell in mature pollen grain. The significance of the presence of a temporary cell wall in generative cell and heterogeneity between generative and vegetative cells are discussed.     

A new enveloped helical virus from the blue crab,Callinectes sapidus

Quite different ultrastructural changes were observed in the columnar cell and the goblet cell of the silkworm midgut after administration of the crystalline toxin of Bacillus thuringiensis. Shortly after the ingestion of the toxin, the deep infoldings of the basal cell membrane of some columnar cells became very irregular in shape and the mitochondria near the basal region were transformed into a condensed form. A few goblet cells showed relatively high electron density in the cytoplasm. The earliest pathological changes were slight and located in a region lying between the first and second thirds of the midgut. With the passage of time, they spread anteriorly and posteriorly to include the entire anterior two thirds of the midgut and became more profound. The cytoplasm of columnar cells became very electron transparent. Most mitochondria were transformed into a condensed form and the endoplasmic reticulum assumed a vacuole-like configuration. The basal infoldings of the cell membrane almost disappeared. On the other hand, the cytoplasm of the goblet cells became very electron dense and granular. The clear basal infoldings of the cell membrane were enlarged making a striking contrast with the dense cytoplasm. However, the mitochondria and the endoplasmic reticulum did not show any pathological deformation.     

Effect of zinc on nectar secretion of Hibiscus rosa-sinensis L

Zinc toxicity in secretory cells caused a range of effects, mainly depending on metal concentration. Low concentrations activated nectary function increasing nectar secretion but secretion was greatly inhibited or stopped entirely by ongoing concentration. Water loss rate of zinc treated flower parts was significantly reduced whereas green sepals were dehydrated more rapidly in comparison to colored petals. The content of zinc, calcium, magnesium and manganese increased mainly in sepals under excess of zinc, but in the secreted nectar this metal was not evident. Morphological changes were observed in mucilage cells concerning the mucilage structure and appearance. The parenchymatic, subglandular cells displayed an early vacuolarization and cytoplasm condensation. Secretory hairs appeared to be thinner, the apical cell folded inwards and plasmolytic shrinkage became severe in all cells. The waxy cuticula showed an increased electron density. A plasmalemma detachment from the external cell walls was observed creating a gap between cell wall and plasmalemma. ER cisterns of all treated nectary hairs dominated the cytoplasm and electron dense deposits were seen within its profiles. A great number of other organelles were also present, showing electron dense deposits in their membranes as well. The vacuome was drastically reduced in all cells, except in the subglandular ones and electron dense membrane remnants were observed.     

Ultrastructural aspects and programmed cell death in the tapetal cells of Lathyrus undulatus Boiss

Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporogenous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage.     

拟南芥花蜜腺筛分子及蜜腺组织发育过程中的细胞学研究

应用高压冷冻和低温替代技术,对拟南芥(Arabidopsis thaliana L.)花蜜腺发育过程中细胞的超微结构变化进行了研究.蜜腺组织中深色细胞的超微结构与筛分子早期分化的超微结构十分相似:细胞核中染色质逐渐出现凝集并且边缘化;细胞器分布异常;细胞质浓稠.这些超微结构特征与近年来报道的动植物细胞程序性死亡的超微结构相似.在筛分子和深色细胞分化中,细胞核及一些细胞器的逐渐解体与原蜜汁的运输、加工和蜜汁的分泌有直接联系.这反映了蜜腺发育过程中筛分子和蜜腺组织的细胞学变化是与蜜腺的生长、发育和生理功能的完善联系在一起的.     

Structural Changes Associated with Resistance of Soybean to Heterodera glycines

Subcellular responses to infection by Race 3 of Heterodera glycines in susceptible (''Lee'') and resistant (''Forrest'' and ''Bedford'') soybean cultivars were compared. Syncytial formation, initiated in susceptible as well as resistant soybean cultivars, was characterized by wall perforations, dense cytoplasm, and increased endoplasmic reticulum, In susceptible plants, syncytia developed continuously until nematode maturity. This included hypertrophy of nuclei, increase of rough endoplasmic reticulum in early stages of infection, and formation of wall ingrowths at a late stage of infection. In the resistant reaction in Forrest, a necrotic layer surrounded syncytium component cells demarcating them from surrounding normal cells and leading to syncytial necrosis. Wall appositions were prominently formed near the necrotic layer, and the cytoplasm of the syncytium component cells was extremely condensed. The whole syncytium became necrotic at a late stage of infection. Bedford had nuclear degeneration prior to cytoplasmic degradation. Chromatin was often scattered throughout the syncytial cytoplasm. Finally the whole syncytium became degenerated with plasmalemma completely detached from the syncytial cell walls. The differences in resistant responses reflect a difference in genetic composition of the soybean cultivars tested.     

Ultrastructure of the theca interna of ovarian follicles in sheep

Summary The theca interna of non-atretic ovarian follicles from 2.0 mm in diameter up to the stage shortly following ovulation was studied by light and electron microscopy.In follicles <3.0mm in diameter, the theca interna consisted of about 8–12 layers of flattened cells, together with many capillaries and small bundles of collagen. Two main forms of cellular differentiation were seen. These were towards either fibroblast-like cells or presumed steroidogenic cells whose cytoplasm contained large amounts of predominantly smooth tubular endoplasmic reticulum, to which some ribosomes were attached. The majority of cells were of relatively undifferentiated or intermediate structure.In larger follicles up to the early stages of oestrus the theca interna cells became larger and less flattened, and cells rich in tubular endoplasmic reticulum became proportionately more numerous. By 18 h after the onset of oestrus the theca interna was oedematous, and many cells possessed pseudopodia. Many cells also contained numerous lipid droplets, but there were no signs of thecal cell degeneration or death. Shortly after ovulation the basal lamina of the membrana granulosa was incomplete, and it became more difficult to distinguish between theca and granulosa layers. Structural heterogeneity, with two major cell types and cells of intermediate structure, was present at all stages.It was concluded that: (1) the theca interna of 2.0–2.9 mm follicles contained many cells whose structure was compatible with a steroidogenic capacity; (2) changes in the differentiated thecal cells up to the early stages of oestrus were quantitative rather than qualitative, and suggestive of an increased steroidogenic capacity; (3) the accumulation of lipid in many cells of the theca interna by 18 h after the onset of oestrus probably reflected a reduction in steroidogenic activity; and (4) there was no evidence of any structural specialization to facilitate the transport of steroids from the theca interna to the membrana granulosa.     

木姜子油细胞发育的超微结构研究

利用超薄切片法和透射电镜研究了木姜子（Litsea pungens Hemsl.）油细胞的发育过程。油细胞3层细胞壁的发育可分为4个阶段,阶段1：油细胞仅有初生纤维素壁层,又可分为原始细胞和细胞 泡化两个时期。此阶段质体具透明小泡和黑色嗜锇物质,并与液泡融合。阶段2：木栓质化壁层的形成,片层状木栓质不断叠加在初生纤维素壁内侧,其细胞结构与前期相似,阶段3：内纤维素壁层的形成,较厚而松散的内纤维素壁层叠加在木栓质化壁层的内侧,在内纤维素壁层中可见黑色嗜锇物质,胞间连丝成为被阻塞的特化结构,此时大液泡被嗜锇油脂充满,成为油囊。阶段4：油细胞成熟及细胞质解体,杯形构造由内纤维素壁层向细胞腔内突起形成,油囊由液泡膜包被连接到杯形构造上,油呈浅灰色嗜锇状态,其细胞质和细胞器解体,变得电子不透明或呈杂乱状态。     

Starvation induces apoptosis in the midgut nidi of <Emphasis Type="Italic">Periplaneta americana</Emphasis>: a histochemical and ultrastructural study

The effects of starvation on cell death in the midgut of Periplaneta americana were studied histochemically and ultrastructurally. TUNEL assays showed that cell death began to increase in the columnar cells and nidi, the nests of stem cells and newborn cells from 2 weeks of starvation. A significant increase in cell death occurred in the nidi after 4 weeks of starvation. Cockroaches starved for 4 weeks showed active-caspase-3-like immuno-reactivity both in the columnar cells and nidi, whereas control cockroaches that were fed for 4 weeks showed this reactivity only in the apical cytoplasm of columnar cells. Electron microscopy revealed no chromatin condensation in the nucleus of columnar cells of cockroaches, whether fed or starved for 4 weeks. Starved cockroaches exhibited many small vacuoles in the cytoplasm of some columnar cells and “floating” organelles including nuclei in the lumen. A 4-week starvation induced the appearance of cytoplasmic fragmentation and secondary lysosomes in the nidi. Each fragment contained nuclear derivatives with condensed chromatin, i.e. apoptotic bodies. Mitotic cells were found in some, but not all nidi, even within the same starved sample. Fragmentation was not observed in the nidi of control cockroaches. Thus, starvation increases cell death not only in the columnar cells, but also in the nidi. The cell death in the nidi is presumably apoptosis executed by caspase 3.     

Cell interaction at the maternal-embryonic interface during implantation in the mouse

Summary The interaction between the trophoblast and the maternal epithelium at early implantation was studied by means of light and electron microscopy. The uterine horns were fixed in situ and a double-embedding method was used to locate implantation sites. Observations were made on mice killed at 2 hour intervals 90–116 h. post coitum which covered the following stages: pre-attachment (i) with zona pellucida intact (ii) with zona pellucida in dissolution (iii) after loss of the zona; attachment; adherence; and invasion.The intact zona pellucida was electron opaque and of uniform density. In the stage of apparent dissolution it became electron dense and was trapped between trophoblast and epithelium.At preattachment the trophoblast cells were round. Subsequently they became long and attenuated, often with lysosomes in the cytoplasm proximal to the epithelial layer. Epithelial cells, which could be seen in various stages of degeneration were apparently phagocytosed by the trophoblast. Occasional pyknotic epithelial cells were seen, as well as some apparently normal ones which contained cytosegresomes. The possible reasons for their presence are discussed.The microvilli of the epithelial cells changed from regular and pointed at preattachment to short and irregular at adherence and invasion.Research supported by the Lalor Foundation, Wilmington, Del, U.S.A. to I.B.W.We are grateful to Dr. H. M. Beaumont, and Dr. L. L. Franchi of the Anatomy Department, Birmingham, for helpful discussion and to Mr. J. Wallington for photographic assistance.     

Morphological aspects on pancreatic islets of non-obese diabetic (NOD) mice

The pancreatic islets of female non-obese diabetic (NOD) mice (a model of insulin-dependent diabetes mellitus), have been examined by both light and electron microscopy. At about the age of 2 weeks, mononuclear cells began to infiltrate in or near the islets and some of these cells were in contact with the islet cells. Following this degeneration of islet B-cells took place, the process occurring in two ways. In many cells numerous secretory granules with extremely dense cores occupied the cytoplasm. Other cells, however, were filled with low-density secretory granules and the nuclei of these cells became pycnotic. After degeneration of B-cells, the islets were effaced by numerous mononuclear cells. With the onset of the diabetic state these mononuclear cells gradually disappeared, and thereafter small islets remained. By electron microscopy, retrovirus-like particles were observed in cisternae of the rough endoplasmic reticulum in islet B-cells at all stages. With an anti-retrovirus serum (goat anti-KiMSV-NIHxeno serum), positive immunofluorescence was observed in some pancreatic islet cells of NOD mice aged 1 day and 4, 6, 8, 9, 10 and 14 weeks. It is suggested that these virus particles may be intimately related to the inflammatory reaction occurring in the islets and to the development of diabetes mellitus.     

Structural aspects of bulge formation during root hair initiation

Using light and electron microscopy, the early stages of root hair initiation were investigated under control conditions and in a situation where F-actin polymerization was effectively inhibited by latrunculin B. Trichoblasts in their early stage of bulge formation possessed large vacuole traversed by cytoplasmic strands and enclosed within a narrow peripheral layer of cytoplasm. The nucleus was settled at the inner periclinal cell wall, typically opposite the site of bulge formation. Within the bulging area, dense cytoplasm and numerous ER elements, and other organelles were accumulated, together with pleiomorphic membrane-bound structures, the identity and nature of which will require further studies. These unusual structures, which were associated with the outer cell wall, contained material similar to that of the cell wall. Similar cell wall-like bodies were observed also in the cytoplasm and sometimes within vacuoles. The possible role of these novel organelles of plant cells in cell wall thinning/degradation or in the turgor pressure maintenance are discussed. Latrunculin B treatment allowed bulge formation but prevented the switch from the slow and diffuse expansion of bulge into the rapid tip-growth characteristic of the emerging root hair. Moreover, the cytoplasm of the bulging domain became extensively vacuolated and lacked abundant ER elements and other organelles including the membrane-bound structures. These results indicate important roles of F-actin in the switch from diffuse to highly polarized tip growth.