Cell division versus cell elongation: the control of radicle elongation during thermoinhibition of Tagetes minuta achenes

Endogenous embryo factors, which act mainly in the radicle, prevent germination in Tagetes minuta at high temperatures. These factors act to prevent cell elongation, which is critical for radicle protrusion under optimal conditions. Once the radicle has emerged both cell elongation and cell division are required for post-germination growth. Germination can be induced at high temperatures by fusicoccin, which rapidly stimulates cell elongation. In addition, priming seeds at 25 °C on polyethylene glycol (PEG) 6000 and mannitol could also induce germination on water at 36 °C, indicating that priming prevents radicle protrusion at a point subsequent to the point of control in thermoinhibited achenes. Flow cytometry studies revealed that DNA synthesis occurs during thermoinhibition and the inhibition of DNA synthesis during this process inhibits subsequent germination on water under optimal conditions, suggesting a protective role for DNA synthesis in thermoinhibited achenes of T. minuta.     

β-Tubulin accumulation and DNA synthesis are sequentially resumed in embryo organs of cucumber (Cucumis sativus L.) seeds during germination

Summary Resumption of DNA synthetic activities and -tubulin accumulation was studied in embryo organs of germinating cucumber (Cucumis sativus L.) seeds. Flow-cytometric analysis indicated the existence of 2C, 4C, and 8C nuclei in the radicle of mature embryos, whereas in cotyledons most of the cells contained nuclei with 2C DNA content. Upon imbibition of water, nuclear DNA replication was initiated in the radicle within 15 h, subsequently spreading towards the cotyledons. Bromodeoxyuridine incorporation preceded detectable changes in the relative amounts of DNA, implying the occurrence of putative DNA repair. Organellar DNA synthesis occurred independently of the nuclear DNA synthetic cycle. Western blotting and immunohistochemical localization demonstrated that the constitutive level of -tubulin originated from preserved -tubulin granules. During imbibition, disappearance of fluorescent tubulin granules, accumulation of -tubulin, and formation of microtubular cytoskeleton were found in the radicle, but not in the cotyledon areas. Mitosis only occurred after radicle protrusion at 21 h of imbibition. It is concluded that the differences in the initiation and progress of these cellular and molecular events are associated with the discrete behaviors of the radicle and the cotyledons upon imbibition. The formation of cortical microtubular cytoskeleton and the accumulation of tubulins are important features in preparation of radicle protrusion, whereas DNA synthesis may contribute to postgerminative growth.     

ABA inhibits embryo cell expansion and early cell division events during coffee (Coffea arabica 'Rubi') seed germination

Background and Aims

Coffee seed germination represents an interplay between the embryo and the surrounding endosperm. A sequence of events in both parts of the seed determines whether germination will be successful or not. Following previous studies, the aim here was to further characterize the morphology of endosperm degradation and embryo growth with respect to morphology and cell cycle, and the influence of abscisic acid on these processes.

Methods

Growth of cells in a fixed region of the axis was quantified from light micrographs. Cell cycle events were measured by flow cytometry and by immunocytochemistry, using antibodies against β-tubulin. Aspects of the endosperm were visualized by light and scanning electron microscopy.

Key Results

The embryonic axis cells grew initially by isodiametric expansion. This event coincided with reorientation and increase in abundance of microtubules and with accumulation of β-tubulin. Radicle protrusion was characterized by a shift from isodiametric expansion to elongation of radicle cells and further accumulation of β-tubulin. Early cell division events started prior to radicle protrusion. Abscisic acid decreased the abundance of microtubules and inhibited the growth of the embryo cells, the reorganization of the microtubules, DNA replication in the embryonic axis, the formation of a protuberance and the completion of germination. The endosperm cap cells had smaller and thinner cell walls than the rest of the endosperm. Cells in the endosperm cap displayed compression followed by loss of cell integrity and the appearance of a protuberance prior to radicle protrusion.

Conclusions

Coffee seed germination is the result of isodiametric growth of the embryo followed by elongation, at the expense of integrity of endosperm cap cells. The cell cycle, including cell division, is initiated prior to radicle protrusion. ABA inhibits expansion of the embryo, and hence subsequent events, including germination.Key words: Abscisic acid, β-tubulin, Coffea arabica, coffee seed, cell morphology, germination, microtubules     

Multiple DNA contents in the haploid protonema of the mossFunaria hygrometrica sibth

Summary Cytofluorometry with fluorochrome Hoechst 33258 shows that caulonema cells in the protonema of the haploid mossFunaria hygrometrica endoreduplicate their DNA up to 8 or 16 C during normal development. The increase in DNA content depends on age and position of the cells in the filament and occurs in those cells, which normally do not undergo any further growth or division.Single cells from different positions between the tip and the tenth cell of the filament were isolated and regenerated to new protonema. Groth and differentiation of these regenerates depend on the age and position of the original cell. Cytofluorometry of the tip cells of these regenerates shows, that the higher DNA content is maintained during regeneration.     

Activities of Antioxidant Systems During Germination of Chenopodium rubrum Seeds

The activities of superoxide-dismutase (SOD), catalase (CAT) and peroxidase (POD), and concentrations of glutathione and ascorbate have been studied during the first stages of germination in Chenopodium rubrum L. seeds. The highest CAT and SOD activity was found prior to radicle protrusion, while POD activity was maximal at the time of radicle protrusion and seedling development, new POD isozymes simultaneously appearing. The concentrations of total, reduced and oxidized glutathione showed similar changes during germination, the highest values being detected at the time of radicle protrusion. Ascorbic acid was present in the seeds in a detectable concentration only at the time preceding radicle protrusion, while its oxidized form dehydroascorbic acid was detected during the whole germination period studied. Gibberellic acid (GA₃, 160 M) had no effect on germination percentage, but in presence of GA₃, SOD and CAT activity notably increased prior to radicle protrusion, and oxidized glutathione concentration decreased in further germination.     

Endo-β-mannanase isoforms are present in the endosperm and embryo of tomato seeds, but are not essentially linked to the completion of germination

A current hypothesis is that endo--mannanase activity in the endosperm cap of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds is induced by gibberellin (GA) and weakens the endosperm cap thus permitting radicle protrusion. We have tested this hypothesis. In isolated parts, the expression of endo--mannanase in the endosperm after germination is induced by GAs, but the expression of endo--mannanase in the endosperm cap prior to radicle protrusion is not induced by GAs. Also, abscisic acid (ABA) is incapable of inhibiting endo--mannanase activity in the endosperm cap, even though it strongly inhibits germination. However, ABA does inhibit enzyme activity in the endosperm and embryo after germination. There are several isoforms in the endosperm cap and embryo prior to radicle protrusion that are tissue-specific. Tissue prints showed that enzyme activity in the embryo spreads from the radicle tip to the cotyledons with time after the start of imbibition. The isoform and developmental patterns of enzyme activity on tissueprints are unaffected when seeds are incubated in ABA, even though germination is inhibited. We conclude that the presence of endo--mannanase activity in the endosperm cap is not in itself sufficient to permit tomato seeds to complete germination.Abbreviations ABA cis/trans-abscisic acid - GA(s) gibberellin(s) - IEF isoelectric focussing - pI(s) isoelectric point(s) We thank Dr. Bruce Downie for the seemingly endless but inspiring discussions.     

Loricate choanoflagellates of the Southern Ocean with new observations on cell division in Bicosta spinifera (Throndsen, 1970) from Antarctica and Saroeca attenuata Thomsen, 1979, from the Baltic Sea

Summary The loricate choanoflagellate Bicosta spinifera was observed frequently in Antarctic samples collected along Scotia/Weddell Sea transects. The entire population showed a conspicuous bimodal size distribution. Large forms were predominant in Scotia Sea samples, while only small specimens were found in the Confluence area. Prior to cell division Bicosta spinifera produces a complete set of costal strips in a temporary tail-like protrusion. Small specimens sometimes possess a tail similar to that found in large specimens. The fact that B. spinifera may increase considerably in size following cell division, in connection with the finding of aberrant minute forms, have prompted us to hypothesize a polymorphic life history in B. spinifera. The term caudiform division is introduced to describe division in B. spinifera. Saroeca attenuata also produces a tail prior to cell division, but is otherwise shown to undergo a mixed caudiform/tectiform type of division.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundatin     

Molecular organization of the human CD3 gene family on chromosome 11q23

The genes encoding three invariant components of the human T-cell antigen receptor, the CD3 , , and chains, are located on human chromosome 11 at band q23. We isolated cosmid clones containing the human CD3 and chain genes in vectors designed for rapid and efficient chromosome walking. The human CD3 gene was located in the region immediately downstream of the CD3 and genes using synthetic oligonucleotide probes and the localization of this gene confirmed by DNA sequencing. Detailed restriction mapping of the CD3 locus demonstrated that all three CD3 subunits are encoded within 60 kb of DNA with the CD3 gene located 26 kb downstream of the CD3 and genes. Analysis of genomic DNA on pulsed field gels using probes isolated from these cosmid clones defined a physical map of 750 kb spanning the CD3 locus on human chromosome 11g23. The CD3 genes thus comprise a multigene family encoding cell surface components important for transmembrane signaling on T lymphocytes. The arrangement of these genes suggest that they may share common regulatory elements for the control of gene expression during T-cell ontogeny.     

Expression of the T antigen on a T-lymphoid cell line, SupT1

We have measured glycosyltransferase activities of SupT1 cells, a T-lymphoid cell line shown to react with autoantibodies in the sera of many HIV patients. Since considerable -N-acetylgalactosaminyl-transferase and 1, 3 galactosyl-transferase activities were found in SupT1 cells, at least the O-glycan core 1 structure can probably be synthesized. FACS analysis using an anti-T monoclonal antibody showed expression of the T antigen (Gal 1-3 GalNAc). Glycoproteins with the T antigen were isolated by immunoprecipitation with the anti-T antibody from a SupT1 cell lysate labelled metabolically with³H-glucosamine and then analysed by SDS-PAGE. It was revealed that the precipitate contained a glycoprotein with a molecular weight corresponding to that of leukosialin. O-glycans were prepared from the immunoprecipitate by alkaline-borohydride treatment and then fractionated on Bio-Gel P-2, GalNAcOH and Gal-GalNAcOH being identifiedinter alia. These results suggest that an anti-T antibody may be included in the autoantibodies found in HIV-1 infected individuals.     

Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

Summary The localization of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.Supported by grants from the Ministry of Education Science and Culture, Japan     

Lack of correlation between overall protein synthesis and the onset of cell elongation in germinating embryos of Haplopappus gracilis

10⁻⁵M abscisic acid (ABA) completely inhibits germination or (if seeds deprived of integuments are used) embryo elongation in Haplopappus gracilis (Nutt.) Gray. Nevertheless, considerable rates of protein and RNA synthesis were found in embryos grown in abscisic acid, at least during the early hours after sowing. On the contrary, seeds grown in cycloheximide + fusicoccin (a powerful promoter of cell expansion), where protein synthesis is almost completely inhibited, show full protrusion of radicle, thus simulating a "germination" process. These results suggest that some of the most important events involved in seed germination, i.e. protein and RNA synthesis, and cell elongation which leads to radicle protrusion, may not necessarily be linked together and are possibly regulated by different control mechanisms. Moreover, when seeds or embryos are grown in abscisic acid + fusicoccin, protein synthesis is considerable, cell elongation is greater than in water controls at least for 12 h, and germination in its early stages appears to be normal; but DNA synthesis and cell division are not resumed, possibly since some other factor is required. All these findings propose a reevaluation of criteria for defining successful germination.     

Improved production of ginseng polysaccharide by adding conditioned medium to Panax notoginseng cell cultures

By adding 50% (v/v) filtered culture broth to fresh MS medium, the specific growth rate of Panax notoginseng was increased from 0.046 d–1 to 0.068 d–1, and the polysaccharide production and productivity reached 1.21 g l–1 and 61 mg/(ld), respectively, which were 1.3- and 2.3-fold of the control. Further supplementation of the conditioned medium with sucrose, ammonium, nitrate and phosphate gave a cell density of 13.7 g l–1 and a specific growth rate of 0.086 d–1. Polysaccharide production was 1.65 g l–1 and the productivity was 78 mg/(ld).     

Cell elongation and cell division in elongating lettuce hypocotyl sections

The roles of cell division and cell elongation in the growth of sections excised from hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Elongation of sections incubated in the light is inhibited compared to dark-grown sections and this inhibition is reversed by gibberellic acid (GA₃). The elongation of both dark-grown and GA₃-treated, light-grown sections can be enhanced by 10mM KCl. Under all conditions of incubation, elongation growth is greatest in the uppermost quarter of the hypocotyl section while the basal quarter does not elongate. In darkness the two apical segments of sections marked into four equal parts grow at the same rate, while in light, growth of the apical segment exceeds that of the second segment. Cell division in cortical or epidermal cells, as measured by mitotic index or cell number, is not affected by illumination conditions nor by GA₃ or KCl treatments. Although -irradiation and FUDR pretreatment eliminate or cause a marked reduction in cell division in the excised hypocotyl, sections from seeds irradiated with -rays or incubated in 5-fluorodeoxyuridine elongate in response to GA₃ and KCl treatment as do sections from non-pretreated controls. Therefore, since neither GA₃ nor darkness affect celldivision activity and since treatments which eliminate or significantly reduce cell division do not affect growth, we conclude that the effect of GA₃ and darkness in this material is to increase cell elongation.Abbreviations FUDR 5-fluorodeoxyuridine - GA(s) gibberellin(s) - GA₃ gibberellic acid     

Measurement of viscoelastic properties of root cell walls affected by low pH in lateral roots of Pisum sativum L.

Mechanical extensibility of the cell wall limits the elongation growth of roots. Low pH, ranging from pH 3–4.5, induces rapid elongation of excised roots, a phenomenon known as acid growth. The creep-extension analysis was carried out to measure and elucidate the viscoelastic properties of root cell walls in the acidic environment in vitro. The viscoelastic properties were determined at the elongation zone of the lateral roots of pea (Pisum sativumL. cv. Alaska) and described by the physical parameters of three elastic (E₀, E₁, E₂) and three viscosity (₀, ₁, ₂) parameters using a Kelvin–Voigt–Burgers' model. The present method could measure the viscoelasticity of 1-mm long root zones from 2 to 9 mm behind the tip. Among the parameters, E₀ and ₀ were the most significant parameters to represent the whole extensibility of the roots. The parameter ₀ markedly declined in response to the environmental low pH (acid growth), whereas other parameters were not much affected by low pH. Relationship between the change in these physical parameters and the change in cell wall extensibility under low pH was discussed in order to elucidate the rheological processes taking place in the elongating cell walls.     

The skeletal isotopic composition as an indicator of ecological and physiological plasticity in the coral genus<Emphasis Type="Italic"> Madracis</Emphasis>

Three species of the reef coral genus Madracis display skeletal isotopic characteristics that relate to depth, colony topography, and consequently to coral physiology. The joint interpretation of skeletal ¹³C and ¹⁸O provides information on the ecological plasticity and adaptation to depth of a coral species. Isotopic results are most easily understood in terms of kinetic effects, which reduce both ¹⁸O and ¹³C below isotopic equilibrium values, and metabolic effects, which only influence the skeletal ¹³C. Madracis mirabilis is adapted to depths shallower than 20 m, and shows the greatest range in kinetic effects and the strongest metabolic ¹³C enrichments caused by symbiont photosynthesis. Madracis formosa lives deeper than 40 m, and shows a reduced range of kinetic effects and relatively weak metabolic ¹³C enrichments. Madracis pharensis inhabits depths from 5 to >60 m, and does not attain the strength of kinetic effects of either of the other two species, apparently because it is not quite as well adapted to rapid growth at either extreme.     

The responses of one class of neurons in the optic tract of crayfish (Procambarus) to monochromatic light

Summary Spectral sensitivity curves for four sustaining neurons in the optic tracts of Procambarus clarkii were determined under dark-adapted and chromatic light-adapted conditions. The _max in the dark-adapted state is at 570 to 575 nm, and shifts to longer wavelengths in the violet-light-adapted state (Fig. 4). Red-light-adaptation suppresses the sensitivity of the yellow-green receptors of the eye and alters the discharge pattern of the sustaining neurons, thereby exposing an input with a _max at 445 nm from the blue-sensitive receptors (Fig. 5). Such data raise the possibility that sustaining neurons may carry information that functions in color vision.This work was supported by a predoctoral fellowship FO1-GM-31,230 and then a training grant 2TO1-GM00836 to D.L.T. and grant NB-05423 to J.L.L. and Biomedical Sciences Support Grant 5-S05 FR-07091, all from the U.S.P.H.S.     

Ion-Exchange Properties of Cell Walls Isolated from Lupine Roots

Acid–base properties of cell walls isolated from various root tissues of 7-day-old lupine seedlings and 14-day-old lupine plants grown in various media were studied. The ion-exchange capacity of root cell walls was estimated at various pH values (from 2 to 12) and constant ionic strength (10 mM). The parameters determining the qualitative and quantitative composition of cell wall ionogenic groups along the root length and in its radial direction were estimated using Gregor's model. This model fits the experimental data reasonably well. Four types of ionogenic groups were found in the cell walls: an amino group (pK _a 3), two types of carboxylic groups (pK _a 5 and 7.3, the first being the carboxylic group of galacturonic acid), and a phenolic group (pK _a 10). The number of functional groups of each type was estimated, and the corresponding ionization constant values were calculated. It is shown that the chemical composition of the ionogenic groups was constant along the root length as well as in its radial direction and did not depend on either physiological state or root nutrition, while the number of different groups varied. The content of carboxylic groups of -D-polygalacturonic acid in the root cell walls of 14-day-old plants was shown to depend on the distance from the root tip, being maximal in the zone of lateral roots. The number of these groups was 10- and 2-fold less in the central cylinder compared to that of cortex for 14-day-old plants and 7-day-old seedlings, respectively.     

Gibberellin-induced hydrolysis of endosperm cell walls in gibberellin-deficient tomato seeds prior to radicle protrusion

The weakening of the mechanical restraint of the endosperm layer in tomato (Lycopersicon esculentum Mill.) seeds, a prerequisite for germination, has been studied with the use of seeds of the gibberellin (GA)-deficientgib-1 mutant. Incubation ofgib-1 endosperms, including part of the testa, in 10 M GA₄₊₇, resulted within 12 h in the release of fructose, glucose, galactose and mannose into the incubation medium. Only small amounts of sugars diffused out of thegib-1 endosperms during incubation in water. Chemical hydrolysis of endosperm cell walls ofgib-1 seeds showed that they are mainly composed of mannose, and smaller quantities of glucose and galactose. Treatment with GA₄₊₇ induced in the endosperms the production of endo--mannanase activity that was not detectable during incubation in water, and also increased the activities of mannohydrolase and -galactosidase as compared with the water controls. No cellulase activity was found. It is concluded that in tomato seeds the weakening of endosperms prior to radicle protrusion is mediated by a GA-induced enzymatic degradation of the mannan-rich cell walls.Abbreviation GA(s) gibberellin(s)     

On induction of cell differentiation by cyclic AMP pulses in Dictyostelium discoideum

Repeated pulses of cyclic AMP, applied at intervals of 5 min, efficiently induced differentiation in cells of agip 53, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. In contrast, pulses applied at intervals of 2 min did not induce cell differentiation. To analyze this phenomenon the hydrolysis of cyclic AMP between the pulses as well as the effect of the pulses on the intracellular concentration of cyclic GMP were investigated. Experiments performed in the presence of added cyclic AMP phosphodiesterase revealed that incomplete hydrolysis of cyclic AMP was not the reason for the inefficiency of the pulses applied with a 2-min rhythm. Cyclic AMP pulses applied at intervals of 2 min induced discrete increases of the cyclic GMP concentration. Limited time resolution at the level of cyclic GMP cannot account for the inefficiency of the 2-min pulses.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Effect of osmotic pressure on root growth,cell cycle and cell elongation

Summary The paper reports a study of root growth, the duration of the cell division cycle and cell size, inAllium cepa roots grown in mannitol solution, with osmotic pressures of 0–16 atm., at 25° C, with aeration. Root growth decreases markedly as the osmotic pressure rises, at 12 atm. being about 35% of what it is at 0 atm. The rate of the cell cycle, , expressed as the percentage of cells passing through any given point in the cycle in one hour, is a roughly linear function of the osmotic pressure, and at 12 atm. is reduced to 80% of what it is at 0 atm. The reaction of the cell size to osmotic pressure is similar to that of the growth. The relative values of the two diverge progressively as the osmotic pressure increases and at 12 atm. the elongation of the cells has dropped to 40% of normal.The data obtained agree with those given by the equationG = K · · L, in whichK is a constant, is the rate of the cycle (reciprocal of its duration) andL the average size of the mature epidermal cells.