Comparisons of tests for aneuploidy

The fundamental problems that face us in the development of suitable assay systems for the detection of potentially aneugenic (aneuploidy-inducing) chemicals include: (a) the diversity of cellular targets and mechanisms where perturbations of structure and function may give rise to changes in chromosome number, and (b) the phylogenetic differences that exist between species in their mechanism and kinetics of cell division and their metabolic profiles. A diverse range of assay systems have been developed, which have been shown to have potential for use in the detection of either changes in chromosome number or of perturbations of the events which may be causal in the induction of aneuploidy.

Chromosome number changes may be detected cytologically by karyotypic analysis, or by the use of specialised strains in which aneuploid progeny may be observed due to phenotypic differences with aneuploid parental cells or whole organisms. Techniques for the detection of cellular target modifications range from in vitro studies of tubulin polymerisation to observations of the behaviour of various cellular organelles and their fidelity of action during the division cycle.

The diversity of mechanisms which may give rise to aneuploidy and the qualitative relevance of events observed in experimental organisms compared to man make it unlikely that the detection and risk assessment of the aneugenic activity of chemicals will be possible using a single assay system. Optimal screening and assessment procedures will thus be dependent upon the selection of an appropriate battery of predictive tests for the measurement of the potentially damaging effects of aneuploidy induction.     

The Chlamydomonas cell cycle is regulated by a light/dark-responsive cell-cycle switch

Cultures of the unicellular green alga Chlamydomonas reinhardii can be synchronized by light/dark cycling not only under photoautotrophic but also under mixotrophic growth conditions. We observed that cultures synchronized in the presence of acetate continue to divide synchronously for one cell-cycle period when transferred to heterotrophic growth conditions. This finding enabled us to investigate the differential effects of light on cell growth and cell division. When cells were exposed to continuous light at the beginning of the growth period they entered the division phase earlier than dark-grown cells as a consequence of an increased growth rate. Illumination at the end of the growth period, however, caused a considerable delay in cell division and an extended growth period. The light-induced delay in cell division was also observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. This finding demonstrates that cell division is directly influenced by a light/dard-responsive cell-cycle switch rather than by light/dark-dependent changes in energy metabolism. The importance of this light/dark control to the regulation of the Chlamydomonas cell cycle was investigated in comparison with other control mechanisms (size control, time control). We found that the light/dard-responsive cell-cycle switch regulates the transition from G1-to S-phase. This control mechanism is effective in cells which have attained the commitment to at least one round of DNA replication and division but have not attained the maximal cell mass which initiates cell division in the light.Abbreviations dCTP deoxycytidine 5-triphosphate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Immuno-gold localization of α-L-arabinofuranosyl residues in pollen tubes of Nicotiana alata Link et otto

Immuno-gold labelling using a monoclonal antibody (PCBC3) with a primary specificity for -L-arabinofuranosyl residues was used to locate these residues in pollen tubes of Nicotiana alata grown in vivo. The antibody bound to the outer fibrillar layer of the pollen-tube wall: the inner, non-fibrillar wall layer was not labelled. Cytoplasmic vesicles (0.2 m diameter) were also labelled. The antibody may bind to an arabinan in the pollen-tube wall.     

The control of chloroplast number in wheat mesophyll cells

Chloroplast number per cell and mesophyll cell plan area were determined in populations of separated cells from the primary leaves of different wheat species representing three levels of ploidy. Mean chloroplast number per cell increases with ploidy level as mean cell size increases. But in addition the analysis of individual cells clearly shows that cells of a similar size but from species of different ploidies have similar numbers of chloroplasts. We conclude that the number of chloroplasts within a cell is closely correlated (P<0.001) with the size of the cell and this relationship is consistent for species of different ploidies over a wide range of cell sizes. These results are discussed in relation to the hypothesis that chloroplast number in leaf mesophyll cells is determined by the size of the cell.     

Auxin-induced rapid changes in translatable mRNAs in tobacco cell suspension

When 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell suspensions, one normal and one transformed by Agrobacterium tumefaciens, were subcultured on hormone-lacking medium the stationary phase of the cell cycle was reached earlier than on medium containing 2,4-D. Addition of the auxin 2,4-D could restore cell division activity within 10–12 h for the most rapidly reacting cell line. The cell-division response was characterized as being auxin-specific and optimal with 2,4-D at 2.2 10^-6 M. Although the cell lines used showed different characteristics, both reacted with a rapid increase in at least three mRNA species within 1 or 2 h after 2,4-D application. Two, 2,4-D-induced protein spots, seen after in-vitro translation, had the same characteristics (MWs 35 kilodaltons (kDa) and 25 kDa with isoelectric points of 7.1 and 6.3, respectively) in both cell lines. Water-treated controls did not show alterations in the translatable mRNA populations. This indicates that the accumulation of the corresponding mRNAs is an early hormone-induced event. Since cell division is the only measurable reaction found after auxin application, cell systems as described here offer excellent possibilities for studying early auxin-induced changes at the molecular level preceding mitosis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - kDa kilodalton     

Ribonuclease in plant vacuoles: purification and molecular properties of the enzyme from cultured tomato cells

A ribonuclease which was previously shown to be located in isolated vacuoles from suspension-cultured cells of tomato (Lycopersicon esculentum L.; Abel and Glund 1986, Physiol. Plant. 66, 79–86) has been purified to near homogeneity. Purification was up to 55000-fold with a yield of about 20%. The vacuolar origin of the protein was evidenced by comparing its electrophoretic mobility, isoelectric point, pH-optimum for activity and other properties with that of the RNA-degrading activity present in isolated vacuoles. The molecular weight of the native single polypeptide chain was estimated at 17500 and 20300 by gel filtration and sedimentation analysis, respectively. The enzyme hydrolyzed only single-stranded RNA with a mode of action that was endonucleolytic. The vacuolar ribonuclease had no requirement for divalent metal ions, and did not exhibit phosphomonoesterase (EC 3.1.3.1; EC 3.1.3.2) and phosphodiesterase (EC 3.1.15.1; EC 3.1.16.1) activity. The specificity of the enzyme has been studied by using homopolyribonucleotides as substrates. The end-products obtained were the respective nucleoside 2:3-cyclic monophosphates and, to minor extents, the corresponding nucleoside 3(2)-monophosphates. According to these observations, the vacuolar ribonuclease from tomato can be classified as ribonuclease I (EC 3.1.27.1).Abbreviations DEAE diethylaminoethyl - RNase ribonuclease - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Cooperation of epidermis and inner tissues in auxin-mediated growth of maize coleoptiles

The function of the epidermis in auxinmediated elongation growth of maize (Zea mays L.) coleoptile segments was investigated. The following results were obtained: i) In the intact organ, there is a strong tissue tension produced by the expanding force of the inner tissues which is balanced by the contracting force of the outer epidermal wall. The compression imposed by the stretched outer epidermal wall upon the inner tissues gives rise to a wall-pressure difference which can be transformed into a water-potential difference between inner tissues and external medium (water) by removal of the outer epidermal wall. ii) Peeled segments fail to respond to auxin with normal growth. The plastic extensibility of the inner-tissue cell walls (measured with a constant-load extensiometer using living segments) is not influenced by auxin (or abscisic acid) in peeled or nonpeeled segments. It is concluded that auxin induces (and abscisic acid inhibits) elongation of the intact segment by increasing (decreasing) the extensibility specifically in the outer epidermal wall. In addition, tissue tension (and therewith the pressure acting on the outer epidermal wall) is maintained at a constant level over several hours of auxin-mediated growth, indicating that the inner cells also contribute actively to organ elongation. However, this contribution does not involve an increase of cell-wall extensibility, but a continuous shifting of the potential extension threshold (i.e., the length to which the inner tissues would extend by water uptake after peeling) ahead of the actual segment length. Thus, steady growth involves the coordinated action of wall loosening in the epidermis and regeneration of tissue tension by the inner tissues. iii) Electron micrographs show the accumulation of striking osmiophilic material (particles of approx. 0.3 m diameter) specifically at the plasma membrane/cell-wall interface of the outer epidermal wall of auxin-treated segments. iv) Peeled segments fail to respond to auxin with proton excretion. This is in contrast to fusicoccin-induced proton excretion and growth which can also be readily demonstrated in the absence of the epidermis. However, peeled and nonpeeled segments show the same sensitivity to protons with regard to the induction of acid-mediated in-vivo elongation and cell-wall extensibility. The observed threshold at pH 4.5–5.0 is too low to be compatible with a second messenger function of protons also in the growth response of the inner tissues. Organ growth is described in terms of a physical model which takes into account tissue tension and extensibility of the outer epidermal wall as the decisive growth parameters. This model states that the wall pressure increment, produced by tissue tension in the outer epidermal wall, rather than the pressure acting on the inner-tissue walls, is the driving force of growth.Abbreviations and symbols E _el, E _pl elastic and plastic in-vitro cell-wall extensibility, respectively - E _tot E _el+E _pl - FC fusicoccin - IAA indole-3-acetic acid - IT inner tissue - ITW inner-tissue walls - OEW outer epidermal wall - osmotic pressure - P wall pressure - water potential     

The oxidation of extracellular NADH by sugarcane cells: Coupling to ferricyanide reduction,oxygen uptake and pH change

Suspension-cultured cells of sugarcane (Saccharum sp. hybrids) did not oxidize exogenously supplied NADH in the absence of ferricyanide (potassium hexacyanoferrate [III]), whereas they did at a low rate in the presence of ferricyanide. Concomitantly, ferricyanide was reduced at a slow rate. Neither a pH change nor a change in respiration was caused by the addition of NADH and-or ferricyanide, but ferricyanide was a strong inhibitor of sugar transport. In contrast to cells, protoplasts rapidly oxidized exogenous NADH. This oxidation was accompanied by an increase in oxygen consumption and a net proton disappearance from the medium. Exogenous ferricyanide was reduced only slowly by protoplasts. Simultaneous presence of NADH and ferricyanide produced two effects: 1) a very rapid stoichiometric oxidation of NADH and reduction of ferricyanide until one of the reaction compounds was exhausted, and 2) a nearly instantaneous inhibition of the slower phase of NADH oxidation, which was observed in the presence of NADH but absence of ferricyanide. The extra oxygen consumption and the alkalinization of the medium, as observed with NADH, were also immediately stopped by ferric ions and ferrous ions. The presence of NADH and ferricyanide caused a fast stoichiometric acidification of the medium. These results were taken as evidence that the oxidation of NADH in the absence of ferricyanide is not related to the NADH-ferricyanide-coupled redox reaction. Furthermore, addition of NADH caused some uncoupling of the protoplasts, an effect which would explain the strong acidification of the cell cytoplasm and the inhibition of various transport systems. The NADH-oxidizing systems oxidized both the -configurated pyridine nucleotide and the -configurated form. Since NADH-linked dehydrogenases usually do not work with -NADH (with the exception of the endoplasmic-reticulum-bound electron-transport system), the observed activities could have been derived from contaminating membranes and dying protoplasts in the suspension. All reported reactions partly or predominantly occurred in the supernatant of the protoplast suspension and increased considerably during incubation of the protoplasts. The rates and quantities of oxygen consumption, pH change, and ferricyanide reduction fitted with NADH oxidation in a stoichiometric ratio, which implied that all these reactions occurred in the extracellular space, without involving transmembrane steps. No evidence for a physiological role in energization of the plasmalemma was found.Abbreviation NADH -nicotinamide adenine dinucleotide reduced form     

Activation of fatty acids by non-glyoxysomal peroxisomes

Ethylene treatment (approx. 20 l ·1^-1 in air for 2 d) of tobacco (Nicotiana tabacum L. cv. Havana 425) plants markedly increases the endo--1,3-glucanase (EC 3.2.1.39) content of leaves. The antigenic form of the enzyme induced is the same one whose production is blocked by treating cultured cells with combinations of auxin (1.1 · 10^-5 M -naphthaleneacetic acid) and cytokinin (1.4 · 10^-6 M kinetin). Evidence is presented that cultured tobacco cells require ethylene for -1,3-glucanase accumulation: i) ethylene treatment increased the accumulation of \-1,3-glucanase in callus tissues >10 d after subculturing and in cell-suspension cultures; ii) callus tissues can produce ethylene; iii) conditions known to inhibit ethylene production (1 mM CoCl₂; 33° C treatment) or ethylene action (approx. 1.6 mmol · 1^-1 norbornadiene in air) inhibited -1,3-glucanase accumulation by callus tissues treated for 4 d following subculturing; and, these inhibitory effects were prevented by exogenous ethylene. Combinations of auxin and cytokinin blocked ethylene-induced accumulation of -1,3-glucanase by cell-suspension cultures. The results favor a model in which ethylene induces results favor a model in which ethylene induces 1,3-glucanase accumulation, and auxin and cytokinin inhibit this induction process.Abbreviations NAA -naphthaleneacetic acid - NDE norbornadiene     

Localization of mitochondria in plant cells by vital staining with rhodamine 123

The positively-charged fluorescent dye rhodamine 123 (r-123) specifically stains mitochondria in living plant protoplasts, suspensionculture cells, and root hairs. This dye functions as a vital stain and permits visualization of the localization, distribution and movement of the mitochondria. Dehydration of root hairs caused mitochondria to aggregate into clumps. Mitochondria were either homogenous or heterogeneous and were frequently seen to accumulate in the perinuclear regions of suspension-culture cells but not in those of protoplasts or root-hair cells. Dinitrophenol and high concentrations of ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid and KCl immediately eliminated fluorescence in r-123-stained mitochondria whereas ionomycin enhanced it. Treatment of seedlings with r-123 resulted in differential brightness of fluorescence in different tissues. Meristematic tissues, such as root and shoot tips, exhibited the brightest fluorescence. The cytotoxicity of r-123 in both germinating seedlings and suspension-culture cells was low. The specificity, sensitivity and low toxicity of r-123 should make it a useful tool in experiments designed to examine agents and conditions which affect the location, the physiological status or the viability of mitochondria.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - DAPI 46-diamidino-2-phenylindole - r-123 rhodamine 123