Localization of membrane-associated calcium following cytokinin treatment in Funaria using chlorotetracycline

We have investigated the changes in membrane-associated calcium that occur during cytokinin induced bud formation in Funaria hygrometrica Hedw. using the fluorescent Ca²⁺-chelate probe chlorotetracycline (CTC). In the target caulonema cells a localization of CTC fluorescent material becomes evident at the presumptive bud site 12 h after cytokinin treatment. By the time of the initial asymmetric division this region is four times as fluorescent as the entire caulonema cell. Bright CTC fluorescence remains localized in the dividing cells of the bud. To relate the changes in CTC fluorescence to changes in Ca²⁺ as opposed to membrane-density changes we employed the general membrane marker N-phenyl-1-naphthylamine (NPN). NPN fluorescence increases only 1.5 times in the initial bud cell. We conclude that the relative amount of Ca²⁺ per quantity of membrane increases in this localized area and is maintained throughout bud formation. We suggest that these increases in membrane-associated Ca²⁺ indicate a localized rise in intracellular free Ca²⁺ concentration brought about by cytokinin action.Abbreviations BA 6-benzyladenine - CTC chlorotetracycline - ER endoplasmic reticulum - NPN N-phenyl-1-naphthylamine     

Visualization of the Ca2+-gradient in growing pollen tubes ofLilium longiflorum with chlorotetracycline fluorescence

Summary Epifluorescence microscopy with chlorotetracycline (CTC) fluorescence was used to visualize the Ca²⁺ distribution in germinating pollen grains and growing pollen tubes ofLilium longiflorum. The intensity of the fluorescence shows a gradient with the highest fluorescence at the growing tip. Added external Ca²⁺ influences the intensity of the gradient in germinating grains. Ionophore A 23187 treated pollen tubes do not show the fluorescence gradient with CTC. These results are interpreted as evidence for the role of a Ca²⁺ gradient in pollen tube tip growth.     

Storage of the phytochrome-mediated phototropic stimulus of moss protonemal tip cells

A phytochrome-regulated phototropic response of the moss Ceratodon purpureus was investigated. Chlorotetracycline (CTC) was used to visualize membrane-associated calcium gradients in the tip cell of moss caulonemal filaments. A tip-to-base Ca²⁺ gradient was observed. The ionophore monensin rapidly inhibited the growth of the tip cell and abolished the CTC fluorescence. Six hours after transferring to inhibitor-free medium, protonemal growth resumed and reached the normal growth rate within 12 h. The growth was accompanied by a reappearance of the CTC-fluorescence gradient. Unilateral irradiation given during the monensin treatment or after the treatment during the period when growth inhibition persisted led, with the re-initiation of growth, to a typical positive phototropic bending in complete darkness. Far-red light applied just before the growth response started, or during growth inhibition, abolished the phototropic response. The phytochrome-mediated signal was qualitatively (position) and quantitatively (degree of bending) memorized. Signal perception and response could be separated temporally. This result indicates that at least under some circumstances, e.g. under the influence of monensin, the phytochrome-mediated signal can be stored for several hours in darkness. Calcium seems to be essential for the processing of polar growth only. A specific function (second messenger) in phytochrome-dependent signal transduction could not be confirmed.Abbreviation CTC chlorotetracycline     

Localization of membrane-associated calcium during development of fucoid algae using chlorotetracycline

During the first day of development, fertilized eggs of fucoid algae generate an embryonic axis and commence rhizoid growth at one pole. Using Fucus distichus (L.) Powell, F. vesiculosus L. and Pelvetia fastigiata (J.Ag.) DeTony we have investigated the role of calcium in axis formation and fixation as well as in tip growth. The intracellular distribution of membrane-associated calcium was visualized with the fluorescent calcium probe chlorotetracycline (CTC). Punctate fluorescence associated with organelle-like structures was found in conjunction with diffuse staining at all developmental stages. This membrane-associated calcium remained uniformly distributed throughout the cortical cytoplasm while the axis was established, but increased in the rhizoid protuberance at germination. In subsequent development, fluorescence was restricted to the cortical cytoplasm at the elongating tip and at sites of crosswall biosynthesis.The requirement for Ca²⁺ uptake during development was investigated through inhibition studies; influx was impaired with transport antagonists or by removal of extracellular calcium. Both treatments curtailed germination and tip elongation but had little effect on axis polarization. Reductions in external calcium that interfered with elongation also markedly reduced the apical CTC fluorescencence, indicating that calcium uptake and localization are prerequisites for tip growth. This apical Ca²⁺ is probably involved in the secretory process that sustains tip elongation. By contrast, calcium was not implicated in the generation of an embryonic axis.Abbreviations ASW artificial seawater - CTC chlorotetracycline - DU developmental unit - EGTA erhylene glycol bis(amino-ethyl ether) N,N,N¹,N¹–tetraacetic acid - NPN N-phenyl-1-napthylamine     

Local accumulation of membrane-associated calcium according to cell pattern formation inMicrasterias denticulata,visualized by chlorotetracycline fluorescence

Summary Developing cells ofMicrasterias denticulata Bréb. show a characteristic fluorescence of the plasma membrane (or cortical protoplasm) after treatment with chlorotetracycline (CTC), which is known to be an indicator for membrane-bound Ca²⁺. Depending on the stage of development the fluorescing sites of the young half cell are distributed in a specific pattern which corresponds to cell pattern formation. Therefore growth and thus cytomorphogenesis inMicrasterias seem to be mediated by a patterned accumulation of Ca²⁺ at the periphery of the differentiating cell. Participation of Ca²⁺ in a membrane-recognition process responsible for local vesicle incorporation is discussed.     

Studies ofNajas pollen tubes. Fine structure and the dependence of chlorotetracycline fluorescence on external free ions

Summary The distribution of membrane-associated calcium was investigated in pollen grains and tubes of the underwater pollinated angiospermNajas marina L. using chlorotetracycline (CTC). Tubes grown in distilled water (pH 6) showed the highest fluorescence in a subapical region that tapered basally into a fluorescent strand centrally located in the tube and extending back towards the pollen grain. The apical cap had low fluorescence as did the cytoplasm surrounding the fluorescent strand, the tube base and the pollen grain. Tubes grown in different pond waters (pH 8) revealed no intracellular CTC fluorescence. Instead there was an external fluorescence forming a distinct layer around the whole tube, frequently enhanced in a subapical region to form an external collar.Modification of the patterns of fluorescence could be induced by manipulation pH of the growth media and content of specific ions. For example tubes grown in distilled water with 10^–3 M Mg²⁺ salts showed a similar CTC fluorescence as those grown in pond water. In contrast, Ca²⁺ enrichment had no visible influence on the patterns of fluorescence. The pattern of fluorescence displayed by tubes grown in distilled water, could be reproduced in pond water if the pH was artificially reduced to pH 6.Ultrastructurally, there was no detectable difference in the markedly polar distribution of organelles between pollen tubes grown in the various growth media. The secretory vesicles found in the pollen grain prior to germination become distributed throughout the pollen tube but are least concentrated in regions that show highest internal CTC fluorescence. These regions appear to have large amounts of endoplasmic reticulum and include mitochondria.These results are discussed in relation to the significance of calcium gradients for tip growth and limitations in the use of CTC.Abbreviations CTC chlorotetracycline - SV secretory vesicle - ER endoplasmic reticulum - PIXE proton induced X-ray emissions     

Broad-range effects of ionophore X-537A on pollen tubes of Lilium longiflorum

The effects of the broad-range cationophore X-537A on pollen tubes of Lilium longiflorum were investigated, using both light and electron microscopy. Pollen tube growth is completely inhibited within 30 min after the application of 5·10^-5 M ionophore X-537A; cytoplasmic streaming is stopped only after 60 min of ionophore treatment. Ultrastructurally, X-537A effects are a vacuolation of Golgi cisternae and a general vacuolation. The wall is thickened at the very tip. Coated vesicles and coated regions are enriched close to and at the plasma membrane. The results indicate that pollen tube tip growth needs a specific ion distribution.Abbreviations CTC chlorotetracycline - DMSO dimethylsulfoxide     

Calcium localization in oat aleurone cells using chlorotetracycline and X-ray microanalysis

Calcium distribution was studied in oat caryopses. Using the chlorotetracycline method it was found that membrane-associated Ca²⁺ was present in the aleurone layer. X-ray microanalysis confirmed the presence of calcium in aleurone cells; it also demonstrated the presence of considerable amounts of calcium in the cell wall surrounding these cells.Abbreviation CTC chlorotetracycline     

Disoriented growth of pollen tubes of Lilium longiflorum Thunb. induced by prolonged treatment with the calcium-chelating antibiotic,chlorotetracycline

Pollen of Lilium longiflorum Thunb. was germinated for 12 h in growth medium containing 1·10^-4 M chlorotetracycline (CTC), or growing tubes were treated with 1·10^-4 M CTC for up to 2 h. These treatments have drastic effects: In the CTC-containing medium, out-growing tubes form only short tubes. Irregular wall thickenings are visible. Thirty minutes CTC-treatment cause growing tubes to bend and grow back toward the grain. Electron micrographs of CTC-treated tubes show that CTC affects the organelle distribution: The polar zonation of organelles is disturbed. Vesicle-and endoplasmic reticulum-accumulations are found in the wrong places, together with extensive wall thickenings and a very irregular plasma membrane. The structural details of most cell organelles look normal after CTC treatment, but the mitochondria possess unusual cristae, and microtubules are absent. The disoriented growth is interpreted as an effect of the ability of CTC to chelate intracellular calcium ions, to bind them to membranes, and thus to disturb the dynamics of the delicate Ca²⁺-equilibria thought to regulate oriented exocytosis.Abbreviations CTC chlorotetracycline - ER endoplasmic reticulum     

Membrane-bound Ca2+ distribution visualized by chlorotetracycline fluorescence during morphogenesis of soredia in a lichen

Summary In the lichenParmelia caperata (L.) Ach. the distribution pattern of membrane-bound Ca²⁺ is investigated in the symbionts by chlorotetracycline (CTC)-induced fluorescence during the development of propagative structures, the soredia. The results demonstrate that Ca²⁺ accumulation in the alga and the fungus is associated with this morphogenetic process; particularly, polarized hyphal growth involves a tip-to-base Ca²⁺ gradient.CTC fluorescence distribution is coincident with that of cholinesterase (ChE) activity during morphogenesis of soredia. A comparison is suggested with embryonic ChE of animal cells, where developmental events are regulated by a cholinergic mechanism that also modulates Ca²⁺ levels.     

Rise in chlorotetracycline fluorescence accompanies tracheary element differentiation in suspension cultures ofZinnia

Summary Developing tracheary elements in suspension cultures ofZinnia elegans fluoresce intensely relative to non-differentiating cells when stained with chlorotetracycline (CTC), a fluorescent chelate probe for membrane associated calcium. This suggests that a change in calcium uptake or subcellular distribution accompanies the onset of tracheary element differentiation. A few cells in early differentiating cultures were brightly fluorescent, but did not have visible cell wall thickenings, suggesting that a rise in sequestered calcium may precede visible differentiation. Diffuse CTC fluorescence in early differentiation most likely results from sequestration of calcium in the endoplasmic reticulum. Late in differentiation, CTC fluorescence becomes punctate in appearance, probably due to loss of plasma membrane integrity occurring at the onset of autolysis.Zinnia suspension culture cells were found to be very sensitive to CTC and low concentrations (10 M) were used to assure accurate localization of membrane-associated calcium in healthy cells.Abbreviations CTC chlorotetracycline - DIC differential interference contrast - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - EGTA ethylene glycol bis-(amino-ethyl ether) N,N,N¹N¹-tetraacetic acid - NPN n-phenylnaphthylamine - OsFeCN osmium tetroxide and potassium ferricyanide - TE tracheary element - TEM transmission electron microscopy     

Cllmodulin in tip-growing plant cells,visualized by fluorescing calmodulin-binding phenothiazines

Calmodulin (CaM) was visualized light-microscopically by the fluorescent CaM inhibitors fluphenazine and chlorpromazine, both phenothiazines, during polar tip growth of pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya spec. and in the alga Acetabularia mediterranea, as well as during multipolar tip growth in Micrasterias denticulata. Young pollen tubes and root hairs showed tip fluorescence; at later stages and in the growing parts of the other subjects the fluorescence was almost uniform. After treatment with cytochalasin B, punctuate fluorescence occurred in the clear zone adjacent to the tip of pollen tubes. The observations indicate that there is CaM in all our tested systems detectable with this method. It may play a key role in starting polar growth. As in pollen tubes, CaM might be in part associated with the microfilament network at the tip, and thus regulate vesicle transport and cytoplasmic streaming.Abbreviations CaM calmodulin - CB cytochalasin B - CTC chlorotetracycline     

Inhibition of exogenous NADH oxidation in plant mitochondria by chlorotetracycline in the presence of calcium ions

Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca²⁺. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca²⁺. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca²⁺. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn²⁺ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca²⁺ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca²⁺-chelators is not reversible or is enhanced by the addition of excess Ca²⁺. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca²⁺. It is suggested that a neutral 1:2 complex is formed between Ca²⁺ and chlorotetracycline which can substitute for Ca²⁺ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.     

Cytological indication of the involvement of calcium and calcium-related proteins in the early responses ofBryonia dioica to mechanical stimulus

Summary The distribution of membrane-bound calium, activated calmodulin, and callose synthesis was visualized inBryonia dioica internodes before and after mechanical stimulus, using fluorescent probes, respectively, chlorotetracycline, fluphenazine, and aniline blue. Bright chlorotetracycline fluorescence remains localized in the plasma membrane of control cells, 30 s after stimulation calcium left the plasmalemma. A delocalization of activated calmodulin was observed after wounding and deposition of callose, which could not be detected before, appeared in the same times in most cells. The callose formation and the decrease in membrane-associated calcium suggest a rapid influx of calcium in the cytosol and an intervention of this ion in the cascade of the early events underlyingBryonia dioica thigmomorphogenesis.Abbreviation CTC chlorotetracycline     

CHLOROTETRACYCLINE-ASSOCIATED FLUORESCENCE CHANGES DURING CALCIUM UPTAKE AND RELEASE BY RAT BRAIN SYNAPTOSOMES1

Abstract– The fluorescent divalent metal chelate-probe, chlorotetracycline (CTC), was used as a dynamic monitor of calcium association with rat brain snynaptosomes. The determined fluorescence excitation and emission maxima, 412 nm and 522 nm respectively, were used to monitor membrane-calcium interactions as a function of various parameters. Positive correlations were observed between increased or decreased fluorescence quantum yield and the uptake of both CTC and ⁴⁵Ca by synpatosomes. The divalent metal ionophore A23187 enhanced fluorescence as well as probe and ⁴⁵Ca uptake. Whereas, the polar chelator, EGTA, markedly reduced fluorescence, and the synaptosomal bound CTC and ⁴⁵Ca. The CTC fluorescence changes also demonstrated the saturable manner in which ⁴⁵Ca bound synaptosomes. At concentrations greater than 100μg/ml, CTC bound to the synaptosomes in a manner which quenched fluorescence at 522 nm. Also, CTC, at concentrations above 15 μg/ml, enhanced the uptake of ⁴⁵Ca. At CTC concentrations between 10 and 15 μg/ml the quenching and iono-phoretic properties of the probe were minimized without affecting the capability of using the probe to visualize calcium interactions with synaptosomal membranes. Also, at a low CTC concentration (12.5 μg/ml) the inhibition of calcium uptake by increasing monovalent ion concentrations was clearly demonstrated.     

X-ray microanalysis and chlorotetracycline staining of calcium vesicles in the green alga Mougeotia

Calcium ions have been proposed to play a key role in the sensory transduction of phytochrome-governed chloroplast movement in the green alga Mougeotia. To test this hypothesis, the intracellular pattern of calcium distribution was studied in this alga by two independent techniques, namely, X-ray microanalysis of fixed and of unfixed frozen-hydrated cells, as well as in vivo fluorescence by chlorotetracycline. Both methods of detection reveal a significant compartmentation of calcium in vesicles close to the chloroplast edge and, less frequently, in the cortical cytoplasm. Microfilaments, presumably actin, which could function in driving chloroplast movement, have been observed running between the chloroplast edge and the cortical cytoplasm (Wagner, G., Klein, K. (1978) Photochem. Photobiol. 27, 137). The vesicular calcium concentration is stable and decays only slowly in the absence of extracellular calcium much in the same way as the ability of the chloroplast to perform movements decreases. A functional relationship between vesicular calcium compartmentation and phytochrome-governed chloroplast movement in the green alga Mougeotia seems indicated.Abbreviation CTC chlorotetracycline     

Effect of Seasonal and Regional Variations on Phenolic Compounds of Deverra scoparia (Flowers/Seeds) Methanolic Extract and the Evaluation of Its in Vitro Antioxidant Activity

The widespread use of Deverra scoparia Coss. & Durieu in Algerian folk‐medicine as a remedy can be relatively attributed to its total phenolic compounds. The current study aimed to provide a scientific basis for optimal collection and usage of Deverra scoparia Coss. & Durieu plant. Hence, 37 samples were gathered from nine sites in Algeria during two seasons 2016 and 2017, then exposed to a green extraction. Total phenolic (TPC), flavonoid (FC) and condensed tannins (CTC) content were estimated spectrophotometrically. The antioxidant activity was measured using five different methods, DPPH^., ABTS^.+, FRAP, CUPRAC and Fe²⁺‐chelating. The results have revealed considerable amounts of TPC varied from 804 to 1544 mg GAE/100 g dry matter, FC started from 187 up to 410 mg QE/100 g dry matter and CTC varied from 111 to 394 mg CE/100 g dry matter. The best IC₅₀ values (μg/mL) of DPPH^., ABTS^?+, FRAP, CUPRAC and Fe²⁺‐chelating tests were 56.62, 5.41, 21.26, 52.93 and 78.10, respectively. Moreover, high correlations were found between CTC and most of the antioxidant tests. Hence, CTC are suggested to be the principal group of antioxidant activity in Deverra scoparia Coss. & Durieu extracts.     

A calcium-rich intraspindle membrane system in spermatocytes of wolf spiders

The meiotic spindle of spermatocytes of two wolf spiders contains a highly organized system of ER-like membranes. In cells observed ultrastructurally at early prometaphase, these membranes completely invest each bivalent and are present in the periphery of the spindle in association with the centrosomes. By metaphase each bivalent and its kinetochore fibers are completely encased in a tube of this membrane. We have treated living spermatocytes with the permeant, fiuorescent-chelate probe, chlorotetracycline (CTC) to determine whether or not the intraspindle membrane system is rich in associated Ca²⁺. Spider testes were dissected into PIPES-buffered saline containing 200 M CTC and were kept in this solution for 10 min. Autofluorescence controls were prepared by incubation in saline without CTC, and nonspecific effects of CTC were assessed by incubation for 10 min in 200 M oxytetracycline (OTC). Neither unstained nor OTC-treated spermatocytes emit significant fluorescence. In contrast, CTC treatment yields bright, punctate fluorescence, which coincides with the distribution of the mitochondria. The plasma membrane is only weakly fluorescent, while the nuclear envelope exhibits prominent fluorescence. The chromosomes are not fluorescent during prophase, but after nuclear envelope breakdown, they become outlined by dim, but distinct fluorescence. As spindle formation commences, the CTC signal from the intraspindle membrane system becomes strong. In some cells, thin lines of CTC fluorescence are apparent in the metaphase half spindle; this fluorescence pattern mimics the distribution of the intraspindle membrane system and suggests that it is rich in associated Ca²⁺. We suggest that the intraspindle membrane system functions in the regulation of cytosolic Ca²⁺during meiosis through sequestration of the cation.     

Ultrastructural Observations on CTC-Induced Callose Formation in Riella helicophylla

The Ca²⁺-chelator CTC binds to a specific site on both outer surfaces of all non-meristematic cells of the unistratose thallus of Riella, known to be rich in anionic wall components and calcium, and induces there the deposition of callose. Structural changes in this region during prolonged CTC treatment have been followed by light and transmission electron microscopy. With fluorescence microscopy punctate structures can be detected after 10 min, which upon longer incubation in CTC develop into large vesicular bodies, surrounded by a circular structure. The aniline blue-derived fluorescence intensity of these structures is highest in cells of the extension growth zone. At the ultrastructural level a mosaic of numerous smooth-surfaced vesicles, presumably containing callose, initially appears subjacent to the plasma membrane. These vesicles swell and fuse with each other, forming ultimately a circular fusion profile with the plasma membrane. This complex of callose-forming vesicles is thought to develop from elements of the partially coated reticulum (PCR), based on the presence of coated vesiculation profiles on the callose vesicles and numerous aggregates of coated vesicles in their immediate vicinity. After 30 min in CTC osmiophilic particles appear around these callose vesicles and at the cytoplasmic face of mitochondria. They are later (after 60 min) deposited in the periplasmic space between wall and plasma membrane and are also released into the surrounding medium. As judged by their reaction with FeCl₃, the osmiophilic particles appear to be phenolic in nature. We propose that upon binding of CTC a local increase of cytoplasmic calcium triggers callose synthesis in PCR-like compartments beneath the plasma membrane. However it remains to be shown as to why callose is synthesized exclusively in these intracellular compartments and not at the plasma membrane.     

Development of membrane- and calcium-gradients during pollen germination of Lilium longiflorum

Chlorotetracyclin (10^-4M) has been used to observe the distribution of membrane-associated calcium during pollen germination of Lilium longiflorum. For comparison, the general membrane distribution has been determined with 4·10^-5 M fluorescamine. The pollen grains show a calcium gradient with either weak or strong chlorotetracycline-fluorescence intensity, but always increasing toward the germination colpus. This gradient intensifies during germination, reaching a maximum before the pollen tube emerges. The typical tip-to-base calcium gradient of the tube does not change during growth. Independent of the developmental stage, the pollen grains show a flat fluorescamine-fluorescence gradient with the highest intensity in one half of the grain. Pollen tubes reveal a tip-to-base membrane gradient, independent of their length. As an additional marker for membrane distribution, the distribution of phosphorus, measured by proton-induced X-ray emission in chemically fixed tubes, has been used. A tip-to-base phosphorus gradient, distinct from the calcium gradient measured with the same method, was detected.Abbreviation CTC chlorotetracycline