Developmental studies on the loricate choanoflagellateStephanoeca diplocostata Ellis VI. Effects of silica replenishment on silica impoverished cells

Summary Stephanoeca diplocostata has a facultative requirement for silica in that silica starvation does not inhibit growth as measured by increase in cell numbers. In spite of the absence of a lorica silica impoverished protoplasts still divide in the characteristic tectiform manner and a juvenile protoplast, when released from the parent cell, still extends its lorica assembling tentacles despite the absence of costal strips with which to produce a lorica. Replenishment of silica to silica starved cells in mid to late exponential phase cultures results in a decrease in the growth rate but at the same time silica is taken up and utilised for the deposition of costal strips. Mature costal strips are extruded and accumulated in bundles of 5–8 on the surface of the protoplast but are not passed to the top of the collar as would be expected in silica enriched loricate cells. Eventually silica replenished protoplasts use the bundles of costal strips to assemble loricae for themselves. In early exponential phase cultures naked protoplasts are capable of division whilst at the same time depositing costal strips in preparation for subsequent lorica assembly. An undamaged protoplast deprived of its lorica by ultrasonic treatment also ultimately replaces the lost lorica. The manner in which the tectiform mode of costal strip accumulation and lorica assembly is modified to allow a cell to produce its own lorica is discussed.Abbrevations SDV silica deposition vesicle     

Costal strip production and lorica assembly in the large tectiform choanoflagellate Diaphanoeca grandis Ellis

Diaphanoeca grandis posseses a voluminous flask-shaped lorica comprising an outer layer of 12 longitudinal costae and an inner layer of four transverse costae. The cell is suspended just above the centre of the lorica chamber by tentacles that are attached to the anterior transverse ring. The component costal strips are superficially similar although four different strip categories can be distinguished on the basis of length and morphology. Costal strips are produced ‘upside-down’ within the parent cell and accumulated in a close-packed horizontal ring at the top of the inner surface of the collar. The order in which costal strips are produced is consistent, starting with those for the transverse rings, basal to anterior, and then the longitudinal costae, again with the posterior first and the anterior later. Cell division is of the classical tectiform variety with the juvenile cell being inverted and pushed backwards out of the parent lorica. Lorica assembly entails firstly the rotation of the anterior vertical strips so they become horizontal and then their movement backwards under the posterior layer of longitudinal strips. From this time onwards, lorica assembly proceeds in a standard manner with the lorica-assembling tentacles providing a forward and left-handed rotational movement.     

Choanoflagellate lorica construction and assembly: the nudiform condition. I. Savillea species

The lorica of Savillea spp. (Choanoflagellida) comprises a two layered arrangement of siliceous costae, the inner layer consists of helical costae and the outer layer longitudinal. In Savillea micropora, the helical costae, in a left-handed conformation, undergo 1.5 turns and extend from a short distance above the base of the lorica to the anterior opening. In S. parva the helical costae undergo two turns from base to anterior opening. Ratios of the numbers of helical to longitudinal costae vary from 1:1 in S. parva to 1:1-1:4 in S. micropora. Cell division in Savillea is of the nudiform type, whereby a cell divides to produce a 'naked' flagellated juvenile that swims away from the parent lorica, settles on to a surface and produces a complete set of costal strips. The first formed strips are those that will form the longitudinal costae, this is followed by the slightly thicker strips which will form the inner helical costae. Lorica assembly occurs as a single continuous process and is mediated by the forward movement of the collar tentacles and a rotational movement by the cell. The longitudinal and helical costae are thereby moved in this one combined movement to their respective positions. The longitudinal costae must rotate freely during assembly whilst the helical costae are held at their front end by the respective longitudinal costae and their rear end on the surface of the cell sheath. The concluding inference, based on lorica construction, that there must be a rotational as well as the observed forward movement during lorica assembly is of seminal importance to understanding the basic pattern of lorica construction and the mechanism of lorica assembly in all choanoflagellates.     

Developmental studies on the loricate choanoflagellateStephanoeca diplocostata ellis

Summary Cells ofStephanoeca diplocostata comprise a colourless, flagellated, protoplast lodged in a lorica made of siliceous costae. The single anterior flagellum creates a water current from which bacteria and other food particles are filtered by the collar and ingested by linguiform pseudopodia that arise from the protoplast at the base of the collar. A waist divides the lorica into two chambers, the anterior of which contains three transverse and 17–20 longitudinal costae whereas the posterior chamber comprises two systems of spirally deflected costae and on some cells a pedicel at the hind end. Between 150–185 costal strips of similar length form the lorica. A thin investment covers the inner surface of the posterior chamber and lower part of the anterior chamber and joins with the protoplast at the level of the waist. Costal strips are produced within membrane-bounded vesicles in the peripheral cytoplasm and, although the origin of these vesicles is unknown, there is usually a close association with the Golgi apparatus. Once complete, strips are apparently released sideways through the plasmalemma into the cavity of the posterior lorica chamber. Later, bundles of strips are transferred to the top of the inner surface of the collar where they collectively form a horizontal ring. When sufficient strips to form a lorica have been accumulated at the top of the collar, cell division proceeds.     

Costal Strip Accumulation and Lorica Assembly In the Marine Choanoflagellate Diplotheca Costata Valkanov

ABSTRACT The lorica of the tectiform choanoflagellate D. costata contains five categories of costal strips distinguishable from each other on the basis of morphology and patterning. Categories of strips include those forming the anterior transverse costa; the anterior, intermediate, and posterior costal strips, respectively, of the longitudinal costae and those constituting the posterior transverse costa. the distinctive morphology of each class of strips makes it possible to observe their location and orientation within the overall accumulation of strips at the top of the parent cell collar. In Diplotheca costata the orientation and positioning of the different categories of strips in an accumulation anticipates their orientation and imbrication in the mature lorica. Assembly of the lorica from an accumulation of strips involves lateral sliding of costal strips to constitute transverse costae and longitudinal sliding of strips to constitute longitudinal costae. the motive force for lorica assembly is provided by extension of the anterolateral tentacles.     

Developmental and ultrastructural observations on two stalked marine choanoflagellates, Acanthoecopsis spiculifera Norris and Acanthoeca spectabilis Ellis.

Acanthoecopsis spiculifera and Acanthoeca spectabilis are stalked, loricate choanoflagellates found in littoral sea water pools. The two taxa are distinguished from each other by the arrangement of costae forming the lorica chamber. In addition, Acanthoecopsis spiculifera usually has a longer stalk and may be colonial, consisting of two or more attached individuals. Division in Acanthoeca results in the production of a juvenile, flagellated, protoplast without a lorica. After separation, the juvenile protoplast swims away, settles down and produces an accumulation of costal strips. When sufficient strips have been produced the lorica is rapidly assembled.     

Role of the cytoskeleton in choanoflagellate lorica assembly

ABSTRACT. Cell division in Acanthoeca spectabilis produces a "naked" motile daughter cell (juvenile) that settles onto a surface and deposits siliceous costal strips that are stored extracellularly in bundles. When complete, the bundles of strips are assembled in a single continuous movement to form a basket-like lorica. Assembly can be divided into four overlapping stages. Stage 1 entails the left-handed rotation of strips at the anterior end while the posterior end remains stationary. Stage 2 includes the posterior protrusion of the cell to form a stalk. Stage 3 involves the anterior extension of the spines, and Stage 4 the dilation of the lorica chamber and deposition of the organic investment. Scanning electron microscopic images reveal a one-to-one association between the moving bundles of strips and the anterior ring of lorica-assembling tentacles. Treatment with microtubule inhibitors produces "dwarf" cells that lack stalks, have their spines extended, and possess collars but lack flagella. Treatment with microfilament (actin) inhibitors prevents extension of the anterior spines. These experiments demonstrate that posterior cell extension is primarily mediated by microtubules whereas extension of the spines is controlled by the actin cytoskeleton. The processes of cytoskeletal rotation and extracellular costal strip movement are compared, respectively, with rotation of nuclei in animal embryos and movement of mammalian cells over surfaces.     

Kakoeca antarctica gen. et sp.n., a loricate choanoflagellate (Acanthoecidae, Choanoflagellida) from Antarctic sea ice with a unique protoplast suspensory membrane

A new genus and species of loricate choanoflagellate, Kakoeca aniarctica Buck & Marchant gen. et sp.n. grown in rough culture from an Antarctic sea ice innoculum is described. This organism has a distinctive lorica morphology consisting of more than 200 costal strips arranged in transverse and longitudinal costae that arc perpendicular to one another in the posterior portion of the lorica. The transverse costae show declination with respect to the lorica axis in the anterior part of the lorica. The cell is suspended in the lorica by a robust protoplast suspensory membrane. This membrane blocks water flow from the posterior of the lorica necessitating water entry through the side of the lorica, an area where the maximum sized apertures in the lorica are found. Terminology (lorica lining and protoplast suspensory) is suggested for the two types of lorica membranes which have been found associated with loricas.     

Genetic control of early events in protoplast division and regeneration pathways in sunflower

Experiments were conducted to identify the genetic factors controlling protoplast division and to determine eventual relations between genetic factors involving organogenesis, somatic embryogenesis and protoplast division in sunflower. The present study involved protoplast culture and two traits: total division per 100 protoplasts (TOTD) and asymmetric division per 100 protoplasts (ASYD) were scored in 52 recombinant inbred lines (RILs) from a cross between PAC-2 and RHA-266. Asymmetric division is an early event in the formation of embryoids from protoplasts. Analysis of variance indicated the existence of highly significant differences among parental genotypes and their RILs. Heritability for the two protoplast division parameters (TOTD and ASYD) was high (0.87 and 0.89, respectively) and genetic gain expressed as percentage of the best parent for 10% of the selected RILs was significant. Twelve putative loci associated with total division per 100 protoplasts were identified. Eleven QTLs were also detected for asymmetric division per 100 protoplasts. The QTLs present high significant LOD scores and sum to a high percentage of phenotypic variance. The percentage of phenotypic variation explained by each QTL ranged from 2% to 24%. Some segments of the linkage groups I, XV and XVII are likely to contain genes important for organogenesis, somatic embryogenesis and protoplast division, as clustering of QTLs for these characters were described. The QTLs identified in these three linkage groups should be involved in cell division and in early events associated with cell differenciation. Received: 15 December 1999 / Accepted: 30 December 1999     

Cell division of Giardia intestinalis: assembly and disassembly of the adhesive disc, and the cytokinesis

Trophozoites of Giardia are equipped with a special organelle of attachment, essential for parasite survival and pathogenicity, the ventral disc. Although its basic structure is well established, its reorganization and assembly during cell replication is poorly understood. We addressed some of these problems with aid of conventional, confocal and electron microscopy. We found that dividing Giardia alternates attached and free swimming phases in accordance with functional competence of the parent or newly assembled discs. The division started in attached cells by detachment of the disc microtubules from basal bodies. Shortening and eventual loss of the giardin microribbons, and unfolding of the microtubular layer resulting in collapse of the disc chamber and parasite detachment underlined gradual disassembly of the parent disc skeleton. Two daughter discs assembled on the dorsal side of the attached cell, with their ventral sides exposed on the parent cell surface and their microtubular skeletons growing in counter-clockwise direction. A depression between the assembling discs marked the cleavage plane. The splitting continued during the free-swimming phase with ventral-ventral axial symmetry in a plane of the daughter discs. Finally, the daughter cells with fully developed discs but still connected tail to tail by a cytoplasmic bridge, attached to a substrate and terminated the division by a process resembling adhesion-dependent cytokinesis. The mode of assembly of the daughter discs and plane of the division is compatible with maintenance of the left-right asymmetry of the Giardia cytoskeleton in progeny, which cannot be satisfactorily explained by alternative models proposed so far.     

草木樨状黄芪和木本霸王的科间体细胞杂交

在成功培养原生质体的基础上, 用改进的PEG-高pH高钙法诱导草木樨状黄芪(Astragalus melilotoides)和木本霸王(Zygophyllum xanthoxylum)原生质体融合, 得到了科间体细胞杂种融合细胞。采用罗丹明-6G预处理草木樨状黄芪原生质体以及UV-B辐照霸王原生质体, 使双亲原生质体及其同源融合产物均不能持续分裂而死亡, 融合后的杂种细胞由于生理互补可恢复持续分裂能力而被筛选出来。融合产物经培养分裂获得了2个杂种细胞系, 其中1个分化出芽。染色体计数和分子鉴定证明了杂种的真实性。初步比较了杂种细胞系及亲本对盐分和水分胁迫的耐受性, 结果表明杂种细胞系对盐分和水分胁迫的耐受性介于两个亲本之间。     

An Ultrastructural Study on Triggering of Cell Division in Young Pollen Protoplast Culture of Hemerocallis fulva L.

The ultrastructural changes of young pollen protoplasts under culture condition in Hemerocallis fulva were studied. In comparison with the original pollen grains, the pollen protoplasts had been completely deprived of pollen wall, but kept the internal structure intact, including a large vacuole, a thin layer of cytoplasm and a peripherally located nucleus. After 8 days of culture a few pollen protoplasts were triggered to cell division: some of them were just undergoing mitosis with clearly visible chromosomes and spindle fibers; the others already divided into 2-celled units. The two daughter cells were equal or unequal in size but with similar distribution of organelles inside. Besides cell division, there were also free nuclear division, amitosis and formation of micronuclei indicating a diversity of division modes in pollen protoplast culture, A series of changes occurred during the process of induction of cell division, such as locomotion of the nucleus toward the central position, disappearence of the large vacuole, increase of electron density of cytoplasm, increase and activation of organelles, diminishing of starch granules in plastids, etc. However, the regeneration of surface wall was not sufficient it contained mostly vesicles with only a few microfibrits. The wall separating the two daughter cells were either complete or incomplete. The weak capability of wall formation is supposed to be one of the major obstacles which has so far restricted sustained cell divisions of young pollen protoplasts under current culture condition.     

草木樨状黄芪和木本霸王的科间体细胞杂交

在成功培养原生质体的基础上,用改进的PEG-高pH高钙法诱导草木樨状黄（Astragalus melilotoides）和木本霸王（Zygophyllum xanthoxylum）原生质体融合,得到了科间体细胞杂种融合细胞。采用罗丹明-6G预处理草木樨状黄芪原生质体以及UV-B辐照霸王原生质体,使双亲原生质体及其同源融合产物均不能持续分裂而死亡,融合后的杂种细胞由于生理互补可恢复持续分裂能力而被筛选出来。融合产物经培养分裂获得了2个杂种细胞系,其中1个分化出芽。染色体计数和分子鉴定证明了杂种的真实性。初步比较了杂种细胞系及亲本对盐分和水分胁迫的耐受性,结果表明杂种细胞系对盐分和水分胁迫的耐受性介于两个亲本之间。     

Three-dimensional images of choanoflagellate loricae

Choanoflagellates are unicellular filter-feeding protozoa distributed universally in aquatic habitats. Cells are ovoid in shape with a single anterior flagellum encircled by a funnel-shaped collar of microvilli. Movement of the flagellum creates water currents from which food particles are entrapped on the outer surface of the collar and ingested by pseudopodia. One group of marine choanoflagellates has evolved an elaborate basket-like exoskeleton, the lorica, comprising two layers of siliceous costae made up of costal strips. A computer graphic model has been developed for generating three-dimensional images of choanoflagellate loricae based on a universal set of 'rules' derived from electron microscopical observations. This model has proved seminal in understanding how complex costal patterns can be assembled in a single continuous movement. The lorica, which provides a rigid framework around the cell, is multifunctional. It resists the locomotory forces generated by flagellar movement, directs and enhances water flow over the collar and, for planktonic species, contributes towards maintaining cells in suspension. Since the functional morphology of choanoflagellate cells is so effective and has been highly conserved within the group, the ecological and evolutionary radiation of choanoflagellates is almost entirely dependent on the ability of the external coverings, particularly the lorica, to diversify.     

萱草幼嫩花粉原生质体培养启动细胞分裂的超微结构研究

萱草(Hemerocallis fulva L.)幼嫩花粉,即后期小孢子原生质体在培养8天时进入有丝分裂或已形成二个细胞。此外,还观察到游离核分裂、无丝分裂、微核形成等现象。这显示了花粉原生质体分裂方式的多样性。在启动分裂时发生一系列变化:如细胞核移位、大液泡消失、细胞质电子密度增加、细胞器增多、质体不含淀粉等。再生的细胞壁含许多小泡,很少纤丝,表现出现有培养条件下壁的形成能力薄弱。这是今后改进培养技术需要特别注意的问题。     

PROTOPLAST ISOLATION AND CELL DIVISION IN THE AGAR-PRODUCING SEAWEED GRACILARIA (RHODOPHYTA)1

Methods were developed for the isolation of large numbers of healthy protoplasts from two species of the agarophyte Gracilaria; G. tikvahiae McLachlan and G. lemaneiformis (Bory) Weber-van Bosse. This is the first report of protoplast isolation and cell division in a commercially important, phycocolloid-producing red seaweed, as well as for a member of the Florideophycidae. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 3% Onozuka R-10, 3% Macerozyme R-10, 1% agarase and 0.5% Pectolyase Y- 23 dissolved in a 60% seawater osmoticum containing 1.0 M mannitol. The complete removal of the cell wall was confirmed by several different methods, including electron microscopic examination, and the absence of Calcofluor White (for cellulose) and TBO (for sulfated polysaccharide) staining. Spontaneous protoplast fusion was observed on several occasions. Protoplast viability was dependent upon the strain and age of the parent material, as well as the mannitol concentration of the enzyme osmoticum. Cell wall regeneration generally occurred in 2-6 days; cell division in 5-10 days. Protoplast-produced cell masses up to the 16-32 cell stage have been grown in culture. However, efforts to regenerate whole plants have been unsuccessful to date.     

Reconciling the bizarre inheritance of microtubules in complex (euglenid) microeukaryotes

We introduce a hypothetical model that explains how surface microtubules in euglenids are generated, integrated and inherited with the flagellar apparatus from generation to generation. The Euglenida is a very diverse group of single-celled eukaryotes unified by a complex cell surface called the "pellicle", consisting of proteinaceous strips that run along the longitudinal axis of the cell and articulate with one another along their lateral margins. The strips are positioned beneath the plasma membrane and are reinforced with subtending microtubules. Euglenids reproduce asexually, and the two daughter cells inherit pellicle strips and associate microtubules from the parent cell in a semi-conservative pattern. In preparation for cell division, nascent pellicle strips develop from the anterior end of the cell and elongate toward the posterior end between two parent (mature) strips, so that the total number of pellicle strips and underlying microtubules is doubled in the predivisional cell. Each daughter cell inherits an alternating pattern of strips consisting of half of the nascent strips and half of the parent (mature) strips. This observation combined with the fact that the microtubules underlying the strips are linked to the flagellar apparatus created a cytoskeletal riddle: how do microtubules associated with an alternating pattern of nascent strips and mature strips maintain their physical relationship to the flagellar apparatus when the parent cell divides? The model of microtubular inheritance articulated here incorporates known patterns of cytoskeletal semi-conservatism and two new inferences: (1) a multigenerational "pellicle microtubule organizing center" (pMTOC) extends from the dorsal root of the flagellar apparatus, encircles the flagellar pocket, and underpins the microtubules of the pellicle; and (2) prior to cytokinesis, nascent pellicle microtubules fall within one of two "left/right" constellations that are linked to one of the two new dorsal basal bodies.     

草木樨状黄芪甲硫氨酸抗性系的原生质体培养及植株再生

建立了草木樨状黄芪(Astragalus melilotoides Pall．)甲硫氨酸抗性系原生质体再生植株的实验体系。以茎切段诱导的松软愈伤组织为材料,通过酶法分离出大量有活力的原生质体。原生质体经培养持续分裂形成了愈伤组织,并高频率地分化出再生苗。比较了不同培养基、培养方法和培养密度对原生质体分裂和再生的影响。结果表明,原生质体以3×10⁵/mL的植板密度,采用琼脂糖岛法培养在附加1．0mg／L 2,4-二氯苯氧乙酸(2,4-D)、0．5mg／L 6-苄氨基嘌呤(6BA)、500mg／L水解酪蛋白、3％蔗糖、0．3mol／L甘露醇的KM_8p培养基中,可获得最佳效果,其细胞分裂频率达38％左右。原生质体培养后仍然保持对甲硫氨酸的抗性,同时对乙硫氨酸表现交叉抗性。     

Enhanced protoplast division by encapsulation in droplets: An advance towards somatic hybridisation in recalcitrant white lupin

Lupins are highly nutritious fodder and pulse crops but the greatest challenge in their genetic enhancement is the difficulty in obtaining hybrids through conventional sexual approaches. To bypass this, a procedure for the culture of hitherto recalcitrant lupin protoplasts is now being developed so that the somatic hybrids can be regenerated. This study provides a basis for a regime to culture lupin protoplasts. Cotyledonary protoplasts of white lupin (Lupinus albus) were plated in two diverse media for the evaluation of various plating regimes. The protoplasts divided in agarose as well as in Gelrite? but embedding in agarose at 6 g L^?1 concentration resulted in a higher rate of mitosis. Sodium alginate embedding inhibited protoplast division. Protoplast plating in the form of liquid suspension was significantly inferior to embedding. A filter paper substratum was clearly noxious to protoplast division. Vis‐à‐vis other designs of plating, a 400% improvement in protoplast elongation and division was achieved by plating in the form of 25 μL droplets at the base of 60 mm × 15 mm Nunclon? dish and overlaying with liquid medium. Better results in terms of protoplast elongation and division were obtained with K8p medium as compared to the AS medium. This report on lupin protoplast culture represents a significant breakthrough in the genus in which morphogenesis has not been described to date.     

LIFE HISTORY AND TAXONOMY OF RHIZOOCHROMONAS ENDOLORICATA GEN. ET SP. NOV., A NEW FRESHWATER CHRYSOPHYTE INHABITING DINOBRYON LORICAE1

The life cycle of a previously undescribed chrysophyte, assigned to the new genus Rhizoochromonas, is described. It includes a small motile stage with heterokont flagellation which invades a Dinobryon lorica. Reproduction by cell division of the nearly spherical rhizopodial vegetative stage frequently leads to expulsion of the host protoplast through overcrowding of the lorica. Endogenous cysts (stomatocysts) are also formed within the Dinobryon lorica. The new family, Brehmiellaceae, is established to accommodate pseudopodial/rhizopodial chrysophytes with heterokont flagellation in the motile stage. Rhizoochromonas endoloricata gen. et sp. nov. has been found at two widely separated softwater locations in Ontario, and at one it constituted a major component of the planktonic flora during the autumns of six successive years.