Regulation of swelling of etiolated-wheat-leaf protoplasts by phytochrome and gibberellic acid

The effect of light on the size of intact protoplasts isolated from the primary leaves of etiolated Triticum aestivum was studied. A 2-min red-light irradiation in the presence of 1 mM KCl was sufficient to cause a swelling of protoplasts compared with those maintained in darkness. The effect was photoreversible by far-red light over two light cycles, indicating the involvement of phytochrome. At 4°C, escape from reversibility occurred between 2 and 5 min after the exposure to red light. In exposure-response experiments, 20 s red light at 27 μmol m^-2s^-1 was sufficient to saturate the response. Exogenous gibberellic acid added in darkness in the presence of KCl also induced protoplast swelling. Gibberellins may act as an intermediate in the phytochrome-induced swelling of protoplasts.     

Isolation and sugar uptake characteristics of protoplasts from maize endosperm suspension cultures

The isolation and sugar uptake characteristics of protoplasts from maize ( Zea mays L.) endosperm-derived suspension cultures are described. In contrast with protoplasts from intact developing endosperm, which by virtue of their large size and high starch content are too fragile for sugar uptake experiments, suspension cultures yielded protoplasts capable of withstanding the necessary handling and centrifugations. Intactness of the protoplasts was demonstrated by dye exclusion or accumulation and latency of malate dehydrogenase activity. Uptake of radioactivity from [³H]-inulin did not increase with time, but that from [¹⁴C]-sugars increased over a wide range of external concentrations. Kinetics of fructose, glucose and sucrose uptake were biphasic, and the saturable components of uptake were eliminated by p -chloromercuribenzene sulfonate (PCMBS). Rates of uptake of sucrose and 1'-fluorosucrose were similar, confirming that hydrolysis by cell wall invertase contributes to sucrose uptake by the suspension cultures. The isolation of protoplasts from this tissue source will enable experimental access to plasma membrane sugar carriers which may exist in the intact maize endosperm.     

Concomitant regulation of hippocampal calcium antagonist receptors and calcium uptake by substance P

The interaction of various neuropeptides with calcium antagonist binding was investigated in rat hippocampus. Among the peptides examined Substance P selectively increased the binding of phenylalkylamine and dihydropyridine calcium antagonists; this action was receptor mediated. No effect was observed with Substance P in other brain areas and with neurotensin and met-enkephalin in all the areas examined. The modification in calcium antagonist binding is functionally paralleled by an area specific increase in voltage-dependent calcium uptake. These data suggest that in hippocampus Substance P may be an endogenous regulator of voltage sensitive calcium channels.     

Contribution of damaged protoplasts to DNA uptake by purified plant protoplasts

Purified protoplast preparations contained few (5–9%) but significant numbers of non-spherical damaged protoplasts. Such damaged protoplasts rapidly saturated with relatively large amounts of exogenous DNA which was largely associated with their nuclei. The relative fraction of damaged protoplasts was found to increase with DNA uptake time and DNA concentration. Discontinuous gradient centrifugation provided a convenient method for selective removal of damaged protoplasts and reliable evaluation of exogenous DNA uptake by intact spherical protoplasts.     

Liposome-mediated uptake of chloroplasts by plant protoplasts

Summary Liposomes, artificial lipid vesicles, have been used to induce the uptake of isolated spinach chloroplasts into protoplasts of bothNeurospora crassa andDatura innoxia. Chloroplasts showed high oxygen evolution rates preceding uptake and for extended periods of time after uptake. Survival, as measured by pigment retention and oxygen evolution, was much improved over polyethylene glycol induce uptake. Possible future application of liposome-mediated uptake in plant protoplast studies are discussed.     

Evidence for the involvement of carboxyl groups in passive calcium uptake by liver plasma membrane vesicles and in agonist-induced calcium uptake by hepatocytes

The hydrophobic reagents DCCD and EEDQ, each of which reacts with protein carboxyl groups, were found to inhibit both passive Ca2+ uptake by plasma membrane vesicles isolated from rat liver and agonist-induced Ca2+ uptake by hepatocytes. The data raise the possibility that the Ca2+ inflow pathway(s) in liver has a specific requirement for a reactive carboxyl group or groups.     

Factors influencing stable transformation of maize protoplasts by electroporation

Two chimaeric genes, containing the promoter of the 35S gene of cauliflower mosaic virus coupled to neomycin phosphotransferase (35S-NPT-NOS) or to hygromycin phosphotransferase (35S-HPT-NOS) have been stably transferred to maize (Zea mays cv. Black Mexican sweet) cells by electroporation. Transformation frequencies of 7.6×10^-4 and 8×10^-4, respectively, (based on the number of surviving cells that divided) were obtained with four pulses of 1 ms duration using 400 V capacitive discharge. Cells transformed to kanamycin-resistance and hygromycin-resistance subsequently multiplied to form callus. Southern blot analysis demonstrated the integration of the selectable marker genes, neomycin or hygromycin phosphotransferase, with single or multiple copy numbers. The blots from DNA of hygromycin-resistant calli also suggested the formation of plasmid concatemers.     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Photoreversible calcium fluxes induced by phytochrome in oat coleoptile cells

The chromometallic dye murexide was used to measure photoreversible Ca fluxes in apical tips of etiolated oat coleoptiles and in suspension cultures of protoplasts derived from the coleoptile segments. Phytochrome presence in the protoplasts was indicated by a repeatably photoreversible ΔA(725 - 800 nm) of >0.001 A centimeters⁻¹, recorded on a dual wavelength spectrophotometer. Concentrations of Ca in the solution bathing the cells were observed to change photoreversibly, red irradiation inducing an increase in the medium Ca concentration and subsequent farred irradiation inducing a decrease down to near dark control levels. These changes could be measured in media with or without exogenously added Ca. Protoplasts from green primary leaves of oat, which had no spectro-photometrically detectable phytochrome, showed no photoreversible Ca fluxes when measured by the same method. These data imply that red light induces an efflux of Ca from phytochrome-containing cells and that far red light can reverse this change by promoting a Ca reentry into these cells.     

Growth and ion uptake by maize seedlings on solutions variable in calcium and flow rate

Summary Growth and ion uptake by maize seedlings was studied as functions of calcium concentration and rate of flow of nutrient solutions. With calcium concentrations established at 1, 10, and 100 ppm Ca, all other nutrients being kept at constant concentration, the solutions passed through narrow culture trays at velocities of 2, 8, and 24 liter/day.Fresh and dry weights of seedlings increased as the concentration of calcium increased from 1 to 10 or 100 ppm for any value ofV.Positive response in the plant calcium content was particularly noticed when Ca increased to 100 ppm in the nutrient solution. Phosphorus content of tops decreased with increasing (Ca) in the nutrient solution, while the other elements were more or less unaffected. In the case of roots, phosphorus was generally increased, iron decreased and potassium showed inconsistent trends.Estimates of the uptake efficiency relative to calcium were made by calculating the ratio of the amount of calcium taken up by the entire seedling to the product of calcium concentration and daily flow rate of the solution. It appears that the uptake efficiency diminishes with increasing flow rate. Other instances points to the maintenance of the same uptake efficiency as (Ca) increased with the decreasingV or vice versa. These observations point to the critical nature of environmental factors on the effective contacts between supply and uptake belts that have been adopted as model in this study.     

Tomato seed germination: regulation of different response modes by phytochrome B2 and phytochrome A

Lycopersicon esculentum seeds germinate after rehydration in complete darkness. This response was inhibited by a far-red light (FR) pulse, and the inhibition was reversed by a red light (R) pulse. Comparison of germination in phytochrome-deficient mutants (phyA, phyB1, phyB2, phyAB1, phyB1B2 and phyAB1B2) showed that phytochrome B2 (PhyB2) mediates both responses. The germination was inhibited by strong continuous R (38 micromol m(-2) s(-1)), whereas weak R (28 nmol m(-2) s(-1)) stimulated seed germination. Hourly applied R pulses of the same photon fluence partially replaced the effect of strong continuous R. This response was called 'antagonistic' because it counteracts the low fluence response (LFR) induced by a single R pulse. This antagonistic response might be an adaptation to a situation where the seeds sit on the soil surface in full sunlight (adverse for germination), while weak R might reflect that situation under a layer of soil. Unexpectedly, the effects of continuous R or repeated R pulses were mediated by phytochrome A (PhyA). We therefore suggest that low levels of PhyA in its FR-absorbing form (Pfr) cause inhibition of seed germination produced either by extended R irradiation (by degradation of PhyA-Pfr) or by extended FR irradiation [keeping a low Pfr/R-absorbing form (Pr) ratio].     

Role of membrane potential in the regulation of lectin-induced calcium uptake

Incubation of lymphocytes with mitogenic lectins triggers Ca2+ uptake. This increase in free cytoplasmic Ca2+ is postulated to be an important signal in the initiation of DNA synthesis. Transmembrane fluxes of monovalent ions and changes in membrane potential are also associated with lectin-induced activation of lymphocytes. We have examined the relationship between extra-cellular monovalent ion substitution, the associated electrical potential changes (measured with cyanine dyes), phytohemagglutinin-induced Ca2+ uptake (measured with Quin-2) and proliferation in human T cells. The results show that (1) the magnitude of the increase in free cytoplasmic Ca2+ concentration is correlated with the extent of the lymphoproliferative response, (2) lectin-induced Ca2+ fluxes are sensitive to membrane potential, decreasing with depolarization, and are likely conductive, and (3) the presence of extra-cellular Na+ during incubation with phytohemagglutinin is not essential to mitogenic triggering.     

Inhibition of nitrate uptake by aluminium in maize

Experiments with two maize (Zea mays L.) hybrids were conducted to determine (a) if the inhibition of nitrate uptake by aluminium involved a restriction in the induction (synthesis/assemblage) of nitrate transporters, and (b) if the magnitude of the inhibition was affected by the concurrent presence of ambient ammonium. At pH 4.5, the rate of nitrate uptake from 240 μM NH₄NO₃ was maximally inhibited by 100 μM aluminium, but there was little measurable effect on the rate of ammonium uptake. Presence of ambient aluminium did not eliminate the characteristic induction pattern of nitrate uptake upon first exposure of nitrogen-depleted seedlings to that ion. Removal of ambient aluminium after six hours of induction resulted in recovery within 30 minutes to rates of nitrate uptake that were similar to those of plants induced in absence of aluminium. Addition of aluminium to plants that had been induced in absence of aluminium rapidly restricted the rate of nitrate uptake to the level of plants that had been induced in the presence of aluminium. The data are interpreted as indicating that aluminium inhibited the activity of nitrate transporters to a greater extent than the induction of those transporters. When aluminium was added at initiation of induction, the effect of ambient ammonium on development of the inhibition by aluminium differed between the two hybrids. The responses indicate a complex interaction between the aluminium and ammonium components of high acidity soils in their influence on nitrate uptake. ei]{gnA C}{fnBorstlap}     

Evidence for bound phytochrome in oat seedlings

Phytochrome is consistently observed in pellets centrifuged from homogenates of etiolated, 5-day-old oat seedlings. The majority of pigment associated with the pellet cannot be removed by buffer washes, nor can appreciable quantities of additional phytochrome be adsorbed onto the sedimented material. Over 70% of phytochrome in the pellet is released by 1% Triton X-100.